Structure, Function and Modulation of Neuronal Voltage-Gated Ion Channels

STRUCTURE, FUNCTION, AND MODULATION OF NEURONAL VOLTAGEGATED ION CHANNELS STRUCTURE, FUNCTION, AND MODULATION OF NEUR...

Author: Valentin K. Gribkoff | Leonard K. Kaczmarek

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STRUCTURE, FUNCTION, AND MODULATION OF NEURONAL VOLTAGEGATED ION CHANNELS

STRUCTURE, FUNCTION, AND MODULATION OF NEURONAL VOLTAGEGATED ION CHANNELS VALENTIN K. GRIBKOFF, PhD Knopp Neurosciences Inc., and The Department of Pharmacology, Yale University School of Medicine

LEONARD K. KACZMAREK, PhD The Department of Pharmacology Yale University School of Medicine

Copyright Ó 2009 by John Wiley & Sons, Inc. All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www. wiley.com. Library of Congress Cataloging-in-Publication Data: Gribkoff, Valentin K. Structure, function, and modulation of neuronal voltage-gated ion channels / Valentin K. Gribkoff, Leonard K. Kaczmarek. p. cm. Includes index. ISBN 978-0-470-42989-1 (cloth) 1. Ion channels. 2. Ion channels–Effect of drugs on. 3. Drug development. 4. Molecular pharmacology. I. Kaczmarek, Leonard K. II. Title. QH603.I54.G75 2009 6150 .10724–dc22 2008046707 ISBN: 9780471930136

Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

CONTENTS

Preface

ix

Contributors

xi

PART I 1

NEURONAL VOLTAGE-GATED ION CHANNEL FUNCTIONS

1

Neuronal L-Type Voltage-Gated Calcium Channels

3

Alexander Scriabine and David J. Triggle

2

Voltage-Gated N-Type and T-Type Calcium Channels and Excitability Disorders

35

Elizabeth Tringham and Terrance P. Snutch

3

Voltage-Gated Sodium Channels: Multiple Roles in the Pathophysiology of Pain

67

Sulayman D. Dib-Hajj, Bryan C. Hains, Joel A. Black, and Stephen G. Waxman

4

The Role of Ion Channels in the Etiology and Development of Gliomas

105

Amy K. Weaver and Harald Sontheimer

5

Shaker Family Kv1 Voltage-Gated Potassium Channels in Mammalian Brain Neurons

127

Helene Vacher and James S. Trimmer

v

vi

6

CONTENTS

Unique Mitochondrial Ion Channels: Roles in Synaptic Transmission and Programmed Cell Death

155

Elizabeth A. Jonas

7

Regulation of Neuronal Excitability by the Sodium-Activated Potassium Channels Slick (SLO2.1) and Slack (SLO2.2)

193

Valentin K. Gribkoff and Leonard K. Kaczmarek

PART II MODULATORY MECHANISMS AND INFLUENCES ON NEURONAL VOLTAGE-GATED ION CHANNEL FUNCTION 8

Alternative Splicing of Neuronal Cav2 Calcium Channels

217 219

Diane Lipscombe, Summer E. Allen, Annette C. Gray, Spiro Marangoudakis, and Jesica Raingo

9

Effect of Hypoxia/Ischemia on Voltage-Dependent Channels

251

Xiang Q. Gu, Hang Yao, and Gabriel G. Haddad

10

In Vivo Roles of Ion Channel Regulatory Protein Complexes in Neuronal Physiology and Behavior

279

Smitha Reddy, Mohammad Shahidullah, and Irwin B. Levitan

11

Regulation of Neuronal Ion Channels by G-Protein-Coupled Receptors in Sympathetic Neurons

291

Mark S. Shapiro and Nikita Gamper

12

BK Channels: Regulation of Expression and Physiological Impact

317

Pallob Kundu, Abderrahmane Alioua, Yogesh Kumar, Rong Lu, Jimmy W. Ou, Enrique Sanchez-Pastor, Min Li, Enrico Stefani, and Ligia Toro

13

Structural Basis for Auxiliary KChIP Modulation of Kv4 Channels

343

KeWei Wang and Jijie Chai

PART III 14

DRUG DISCOVERY TARGETS AND TECHNOLOGY

Sodium Channel Blockers for the Treatment of Chronic Pain

363 365

Mark R. Bowlby and Edward Kaftan

15

Neuronal Kv7 Potassium Channels as Emerging Targets for the Treatment of Pain Steven I. Dworetzky and Valentin K. Gribkoff

393

CONTENTS

16

Small-Molecule Modulators of Large-Conductance, Calcium-Activated (BK) Channels

vii

423

John E. Starrett Jr.

17

High-Throughput Screening Technologies in Ion Channel Drug Discovery

443

Edward B. Stevens, Andrew D. Whyment, and J. Mark Treherne

Index

467

PREFACE

This book began as an idea in 2000 at a Keystone Conference on potassium channels that was organized by us, and the idea was subsequently expanded to include contributions on neuronal voltage-gated ion channels of all types. In 2005, after hosting a symposium on ion channels as drug discovery targets, one of us (V.G.) was invited by the editors at John Wiley & Sons to organize a book primarily focusing on ion channels as drug targets. We later decided that we would follow through on our original plan to publish a book that looked at neuronal voltage-gated ion channels from multiple perspectives, including the translational perspective of drug discovery and development. This was an ambitious project, and from the outset our plan was not to cover every facet of research in those areas encompassed by the title of this book, but to include chapters that were in-depth reviews of selected topics relevant to the structure, function, and modulation of these important ion channel families. We hoped that while the individual chapters would prove interesting and useful to established ion channel scientists, our priority was to provide students of this important and exciting ﬁeld glimpses into a number of interesting areas worthy of their further study. What we have arrived at after more than 2 years of organizing, inviting, cajoling (gently), writing, and editing are 17 chapters from leading authorities in their respective ﬁelds, either in academia or industry. Each of these is either a comprehensive review of a particular voltage-gated ion channel member, such as the review by Scriabine and Triggle on “Neuronal L-type voltage-gated calcium channels” (Chapter 1), or is an in-depth review of a topic relevant to multiple voltage-gated ion channel subfamilies, such as the chapter by Shapiro and Gamper on the “Regulation of ion channels by G-proteincoupled receptors” (Chapter 11). The book is divided into three general sections: (1) Neuronal voltage-gated ion channel functions, (2) Modulatory mechanisms and

ix

x

PREFACE

inﬂuences on voltage-gated ion channel activity, and (3) Drug discovery targets and technology. There is some overlap in both chapter content and section assignment, but these divisions should serve to orient the reader to groups of chapters of similar scope. We trust that the reader will ﬁnd these contributions interesting, useful for future reference and helpful in stimulating further discussion and research.

VALENTIN K. GRIBKOFF, PhD Knopp Neurosciences Inc., and The Department of Pharmacology, Yale University School of Medicine

LEONARD K. KACZMAREK, PhD The Department of Pharmacology, Yale University School of Medicine

CONTRIBUTORS

Abderrahmane Alioua, UCLA, Department of Anesthesiology, Division of Molecular Medicine, USA Summer E. Allen, Department of Neuroscience, Brown University, Providence, RI 02912, USA Joel A. Black, Department of Neurology and Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New Haven, CT 06510, USA; Rehabilitation Research Center, VA Connecticut Healthcare System, West Haven, CT 06516, USA Mark R. Bowlby, Discovery Neuroscience, Wyeth Research, Princeton, NJ 08543, USA Jijie Chai, National Institute of Biological Sciences, No. 7 Science Park Road, Beijing 102206, China Sulayman D. Dib-Hajj, Department of Neurology and Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New Haven, CT 06510, USA; Rehabilitation Research Center, VA Connecticut Healthcare System, West Haven, CT 06516, USA Steven I. Dworetzky, Knopp Neurosciences Inc., 2100 Wharton Street, Suite 615, Pittsburgh, PA 15203, USA Nikita Gamper, Institute of Membrane and Systems Biology, Faculty of Biological Sciences, The University of Leeds, Leeds LS2 9JT, UK

xi

xii

CONTRIBUTORS

Annette C. Gray, Department of Biology, Brandeis University, Waltham, MA 02454, USA Valentin K. Gribkoff, Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA; Discovery Research, Knopp Neurosciences Inc., 2100 Wharton Street, Suite 615, Pittsburgh, PA 15203, USA Xiang Q. Gu, Department of Pediatrics, 9500 Gilman Drive, University of California at San Diego, San Diego, CA 92093-0735, USA Gabriel G. Haddad, Department of Pediatrics, 9500 Gilman Drive, University of California at San Diego, San Diego, CA 92093-0735, USA; Department of Neuroscience, 9500 Gilman Drive, University of California at San Diego, San Diego, CA 92093-0735, USA; The Rady Children’s Hospital-San Diego, 3020 Children’s Way, San Diego, CA 92123, USA Bryan C. Hains, Department of Neurology and Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New Haven, CT 06510, USA; Rehabilitation Research Center, VA Connecticut Healthcare System, West Haven, CT 06516, USA Elizabeth A. Jonas, Department of Internal Medicine, Yale University, New Haven, CT 06520, USA Leonard K. Kaczmarek, Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA Edward Kaftan, Discovery Neuroscience, Wyeth Research, Princeton, NJ 08543, USA Yogesh Kumar, UCLA, Department of Anesthesiology, Division of Molecular Medicine, USA Pallob Kundu, UCLA, Department of Anesthesiology, Division of Molecular Medicine, USA Irwin B. Levitan, Department of Neuroscience, University of Pennsylvania School of Medicine, 215 Stemmler Hall, 3450 Hamilton Walk, Philadelphia, PA 191046074, USA Min Li, UCLA, Department of Anesthesiology, Division of Molecular Medicine, USA Diane Lipscombe, Department of Neuroscience, Brown University, Providence, RI 02912, USA Rong Lu, UCLA, Department of Anesthesiology, Division of Molecular Medicine, USA Spiro Marangoudakis, Department of Neuroscience, Brown University, Providence, RI 02912, USA

CONTRIBUTORS

xiii

Jimmy W. Ou, UCLA, Department of Anesthesiology, Division of Molecular Medicine, USA Jesica Raingo, Department of Neuroscience, Brown University, Providence, RI 02912, USA Smitha Reddy, Department of Neuroscience, University of Pennsylvania School of Medicine, 215 Stemmler Hall, 3450 Hamilton Walk, Philadelphia, PA 191046074, USA Enrique Sanchez-Pastor, UCLA, Department of Anesthesiology, Division of Molecular Medicine, USA Alexander Scriabine, Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA Mohammad Shahidullah, Department of Neuroscience, University of Pennsylvania School of Medicine, 215 Stemmler Hall, 3450 Hamilton Walk, Philadelphia, PA 19104-6074, USA Mark S. Shapiro, Department of Physiology, University of Texas Health Science Center at San Antonio, MS7756, San Antonio, TX 78229, USA Terrance P. Snutch, Neuromed Pharmaceuticals, Rm 301, 2389 Health Sciences Mall, Vancouver, BC, Canada; Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada Harald Sontheimer, Department of Neurobiology and Center for Glial Biology in Medicine, The University of Alabama, Birmingham, AL 35294, USA John E. Starrett Jr., Discovery Chemistry, Bristol-Myers Squibb Co., 5 Research Parkway, Wallingford, CT 06492, USA Enrico Stefani, UCLA, Department of Anesthesiology, Division of Molecular Medicine, USA; UCLA, Department of Physiology, USA; UCLA, Department of Molecular and Medical Pharmacology, USA; UCLA, Cardiovascular Research Laboratory, USA Edward B. Stevens, Pfizer Global Research and Development, Sandwich Laboratories (IPC 351), Sandwich, Kent, CT13 9NJ, UK David J. Triggle, School of Pharmacy and Pharmaceutical Sciences, State University of New York, Buffalo, NY 14260, USA James S. Trimmer, Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California, Davis, CA 95616, USA; Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis, CA 95616, USA Elizabeth Tringham, Neuromed Pharmaceuticals, Rm 301, 2389 Health Sciences Mall, Vancouver, BC, Canada

xiv

CONTRIBUTORS

Ligia Toro, UCLA, Department of Anesthesiology, Division of Molecular Medicine, USA; UCLA, Department of Molecular and Medical Pharmacology, USA; UCLA, Cardiovascular Research Laboratory, USA; UCLA, Brain Research Institute, USA J. Mark Treherne, Pfizer Global Research and Development, Sandwich Laboratories (IPC 351), Sandwich, Kent, CT13 9NJ, UK Stephen G. Waxman, Department of Neurology and Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New Haven, CT 06510, USA; Rehabilitation Research Center, VA Connecticut Healthcare System, West Haven, CT 06516, USA Amy K. Weaver, Department of Neurobiology and Center for Glial Biology in Medicine, The University of Alabama, Birmingham, AL 35294, USA Helene Vacher, Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California, Davis, CA 95616, USA KeWei Wang, Neuroscience Research Institute and Department of Neurobiology, Key Laboratory for Neuroscience of the Ministry of Education, Center for Protein Sciences, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100083, China Andrew D. Whyment, Pfizer Global Research and Development, Sandwich Laboratories (IPC 351), Sandwich, Kent, CT13 9NJ, UK Hang Yao, Department of Neuroscience, 9500 Gilman Drive, University of California at San Diego, San Diego, CA 92093-0735, USA

FIGURE 4.6 (a) Model representing glioma cell shape and volume-adaptive changes that occur during invasion in spatially restricted conditions. These changes, accompanied by water loss and cytoskeletal rearrangements, are mediated by ion fluxes through Cl and Kþ ion channels and other ion transport mechanisms. (See text for full caption.)

FIGURE 4.7 Axial view of T1-Wc, (a) coregistered (b) and SPECT (c) (day 8) images. Longterm retention of 131I-TM-601 radioactivity at tumor site was observed in all patient scans (Hockaday et al., 2005).

FIGURE 5.1 Immunofluorescence staining of Kv1a and Kvb subunits in mammalian brain. (a) Staining for Kv1.1 (blue), Kv1.2 (green), and Kvb2 (red) in adult rat cerebellar cortex. Note overlap of all three fluors yielding white signal in basket cell terminals (arrowheads). (b) Staining for Kv1.2 (blue) in adult rat optic nerve (green ¼ Nav channels, red ¼ Caspr). Image courtesy of Dr. Matthew Rasband. (c) Staining for Kv1.4 (green) in the axon initial segment of a cultured hippocampal neuron (blue ¼ MAP2). Image courtesy of Drs. Yasuhiro Ogawa and Matthew Rasband. (d) Kv1.2 (red) in adult rat hippocampus (green ¼ Kv2.1). Arrowheads point to prominent Kv1.2 staining in perforant path presynaptic terminals in the middle molecular layer of the dentate gyrus.

FIGURE 5.2 Structure of Kv1a and Kvb subunits. (a) Cartoon of the predicted topology of Kv1a subunits. The predicted transmembrane topology of a Kv1a subunit and sites defined as important determinants of intracellular trafficking and polarized expression are shown schematically. T1, tetramerization domain important for Kvb-mediated intracellular and polarized trafficking of Kv1a subunits; NLG, N-linked glycosylation site of Kv1.1–Kv1.5; ERR, ER retention motif found in Kv1.1, Kv1.2, and Kv1.6; FTS, forward trafficking signal in Kv1.4; PDZ BD, PDZ-binding motif in all Kv1a subunits; Y132, tyrosine residue critical for tyrosine kinasedependent suppression of Kv1.2; S440/S441, serine residues critical for phosphorylationdependent trafficking of Kv1.2. (b) Crystal structure of the Kv1.2/Kvb2 complex (Protein Data Bank accession number 2A79). (See text for full caption.)

FIGURE 5.3 Subcellular localization of homotetrameric Kv1a subunits in COS-1 cells. Recombinant Kv1 expression monkey kidney fibroblast COS-1 cells. Intact cells were stained with ectodomain-directed antibodies (red), and then detergent permeabilized and stained with cytoplasmic domain-directed antibodies (green). (a) Kv1.1, (b) Kv1.4, (c and d) Kv1.2.

FIGURE 8.2 A large number of proteins and molecules act in concert to regulate alternative pre-mRNA splicing. In this hypothetical model of mutually exclusive splicing, all neurons express a number of regulatory proteins that either repress or enhance inclusion of one of the exon pair. (See text for full caption.)

FIGURE 8.3 Sites of alternatively spliced exons mapped on CaV2.1 and CaV2.2 proteins and the CaV2.2 gene. (a) and (b) illustrate the tetrameric structure and the twodimensional transmembrane topology of CaV2.1 and CaV2.2 proteins, respectively. Approximate locations of alternatively spliced exons are highlighted with blue and green triangles and are numbered. The four main structural domains (I, II, III, and IV) are labeled and the constitutively expressed exons encoding each domain shown with black lines. (c) illustrates the human CaV2.2 gene. Exons are denoted as solid boxes and introns as lines. (See text for full caption.)

FIGURE 8.4 Proteins that interact with the Cav2.2 subunit mapped onto a two-dimensional model of the channel. Several proteins interact with Cav2.2 channels, most notably in the II–III intracellular linker and C-terminus. A number of proteins associated with the synaptic machinery, and with synaptic transmission in general, interact with the II–III linker of Cav2.2, including CSP (cysteine string protein) (Miller et al., 2003), SNAP-25 (Catterall, 1999), synaptotagmin 1 (Sheng et al., 1997), syntaxin 1A (Sheng et al., 1994), huntingtin (Swayne et al., 2005), RGS12 (Richman and Diverse-Pierluissi, 2004), and b-arrestin (Puckerin et al., 2006). The asterisk (*) denotes proteins whose binding is either known or expected to be modified by alternative splicing. (See text for full caption.)

CaV2.2e[37b]

GPCR

CaV2.2e[37a]

GPCR

AP

ICa

FIGURE 8.5 Hypothesized physiological impact of alternative pre-mRNA splicing at the e37a/e37b site in CaV2.2. (See text for full caption.)

FIGURE 11.1 M-current in sympathetic neurons. (a) Biophysical properties of heterologously expressed M-channels. Kv7.2/7.3 heteromultimers were expressed in CHO cells and recorded from under whole-cell voltage-clamp. Currents were evoked by a family of 800 ms square voltage pulses from a holding potential of 70 mV to a range of potentials between 80 and þ40 mV (10 mV increments). The lower panel shows the activation curve from the current traces shown above. Plotted are the tail current amplitudes versus the test-pulse voltage. The tail-current amplitudes reflect the fractional activation of the channels at the end of the preceding test pulses. (b) Superimposed are the current-clamp record from an SCG neuron (black) and the voltage-clamp trace of Kv7.2/7.3 current activation from a CHO cell expressing the channels (red). Both traces are synchronized for timescale. (See text for full caption.)

FIGURE 11.2 Gq/11-coupled signaling in mammalian cells. Depicted is the canonical pathway triggered by the activation of Gq- or G11-coupled receptors in neurons: activation of phospholipase C (PLC) results in hydrolysis of membrane phosphatidylinositol 4,5- bisphosphate (PIP2, brown shapes) and release of inositol triphosphate (IP3, brown octagons) and diacylglycerol (DAG, green rectangle). The former may trigger release of Ca2þ (green spheres) from IP3-sensitive stores, whereas the latter can activate protein kinase C (PKC, ochre shape) or be further degraded to phosphatidic (PA, pink ovals)or arachidonic (AA, green ovals)acids.Each of these intermediates is capable of triggering diverse downstream signaling pathways.

FIGURE 11.4 Purinergic stimulation provokes PIP2 hydrolysis, but does not depress ICa. (a) Plotted are normalized Ca2þ current amplitudes recorded from a superior cervical ganglion (SCG) neuron under perforated patch-voltage-clamp. (See text for full caption.)

Slo1 (KCNMA1) gene (a)

* **

5'-UTR

1

2

*

3 4 5

6 7

8

* *

*

9 10 11 12 13 14 15 16 17 18 19 20 21

22 23 24 25

* 26

27 3'-UTR TGA

ATG

400 bp

M1 M2 M3 M4 mSlo1 MANGGGGGGGSSGGGGGGGGGSGLRMSSNIHANHLSLDASSSSSSSSSSSSSSSSSSSSSVHEPKMDALIIPVTMEVP hSlo1 MANGGGGGGGSSGGGGGGGG-SSLRMSSNIHANHLSLDASSSSSSSSSSSSSSSSSSSSSSVHEPKMDALIIPVTMEVP

(b)

*

N

3,4

+

+ S5 S1 S2 S3 S4

S0

+

2,3

* * 1,2

SSQA(V)DG RCK1

S6 DRD(G)DV

9,10

*

18,19

*

*

16,17

S7

S8

S10

S9

C RCK2 23,24

*

* “Ca2+ bowl”

FIGURE 12.1 Gene map and protein topology of Slo1 (KCNMA1) gene. (a) Gene map showing constitutive exons (gray boxes, at scale) and introns (broken lines, not to scale) of human Slo1. Known splice sites in various species are marked with an arrow and a star. Enlarged region shows probable translation start sites (methionine, M1–M4) in human and mouse and high degree of homology between species, which applies for the rest of the protein (not shown). Most of studies have used clones beginning at M3 (red). (b) Slo1 protein topology. Seven transmembrane domains are marked as S0–S6. Cytoplasmic hydrophobic domains are marked as S7–S10. Regulator of conductance of Kþ domains, RCK1 (blue) and RCK2 (pink) positions (T. Yusifov and R. Olcese, personal communication). Ca2þ bowl, calcium sensing domain (green). Arrowheads mark junctions of translated constitutive exons; numbers of corresponding exons are only given for known alternative splice sites (*) (e.g., 1,2 marks where the translated protein of exon 1 joins the translated protein of exon 2). Red boxes and sequences mark human mutations linked to generalized epilepsy and paroxysmal movement disorder (D/G), and autism (A/V).

FIGURE 12.3 A simplified scheme of Slo1 channel transcriptional regulation by estrogen, channel maturation, and degradation. Estrogen binds to the estrogen receptor a after crossing the plasma membrane, causing dimerization of the receptor. Dimerized receptor enters the nucleus and binds to estrogen response elements (ERE) present in Slo1 gene enhancing transcription. mRNA migrates to the cytoplasm and interacts with the translational machinery to form polyribosomes. Slo1 protein is inserted to endoplasmic reticulum (ER) membrane where it assembles into tetramers and associates or not associates with b subunits (depending on the cell type). Tetrameric channels are transferred to the cell membrane. Splice variant 1 insert (SV1) retains insertless Slo1 and b1 subunit in the ER. Surface expression of the channel can be regulated by b subunits via endocytosis. Direct binding of estrogen to Slo1 induces channel degradation by the proteasome. Dotted arrows indicate probable events: phosphorylation favoring forward traffic and association with partners in the cytosol during traffic to the membrane.

FIGURE 13.2 The overall architecture of the KChIP1–Kv4.3N complex. All three panels have the same color codes with some secondary structural elements labeled specifically. (a) One KChIP1 molecule in gold interacts simultaneously with two Kv4.3Ns in blue. (b) The 4:4 complex of KChIP1–Kv4.3N shown on this panel is generated from the complex on panel A through symmetric operations. (c) KChIP1–Kv4.3N complex in 4:4 showing the clamping effect of KChIP1 molecule on the tetramer of Kv4.3. (See text for full caption.)

FIGURE 13.3 Significant structural rearrangements occur to KChIP1 upon Kv4.3 N-terminal binding. Surface representations are shown to an isolated KChIP1. (See text for full caption.)

FIGURE 13.4 The N-terminal inactivation peptide of Kv4.3 is completely sequestered in a hydrophobic groove on the surface of KChIP1. (a) The N-terminal hydrophobic peptide of Kv4.3 binds to an elongated hydrophobic groove on the surface of KChIP1. KChIP1 is shown in the surface representation, and the N-terminal inactivation peptide of Kv4.3 is shown in ribbon. The Kv4.3 N-terminal residues involved in hydrophobic contacts with KChIP1 are colored in magenta. (b) An SDS-PAGE gel showing the pull-down results for the point mutants of both KChIP1 and the N-terminal peptide of Kv4.3. The single mutation of KChIP1 Y134E and the triple mutation W8E, P10E, and A15E in Kv4.3 completely abolish their interactions with their respective wild-type partners. GST-pull-down assay is used to detect the interaction between WT Kv4.3N and KChIP1 mutants (left panel), whereas various His-tagged Kv.3 mutants are immobilized on Ni-resin and WT KChIP1 is allowed to flow through Ni-resin (right panel). In both cases, the resin is visualized on SDS-PAGE followed by coomassie staining. (c) Sequence alignments of the N-terminal peptide of Kv4-family proteins among different species. (d) Effects of KChIP mutation within the first interface on Kv4.3. The representative traces recorded from oocytes injected with cRNAs of WT Kv4.3 alone, WT Kv4.3 þ WT KChIP1, and WT Kv4.3 þ KChIP Y134E. The left side panel depicts currents recorded from oocytes held at 80 mV by a family of pulses from 60 to 40 mV in 10 mV increments for 1 s, and the right panel shows recovery from inactivation for varying lengths of time at step from 80 to þ40 mV. (e) The representative traces of Kv4.3 triple mutant (W8E-P10E-A15E) alone and the triple mutant þ WT KChIP1 recorded from oocytes under the same protocol as in (d).

FIGURE 13.5 The second contact interface between KChIP1 and Kv4.3 T1 domain. (a) The interaction within the second interface is mediated by both hydrophobic contacts and salt bridges. The KChIP1 molecule and the Kv4.3 peptide (residues 70–78) are shown in surface representation and dark green ribbon, respectively. The residues of Kv4.3 colored in magenta are involved in hydrophobic interaction with KChIP1. The blue, red, and white represent the positive, negative, and hydrophobic surface of Kv4.3, respectively. (b) A close-up view of the interaction within the second interface between KChIP1 and Kv4.3N. The KChIP1 molecule is shown in light green ribbon and colored in green, and the residues involved in the interaction with Kv4.3N are highlighted in yellow. The O and N atoms are shown as red and blue, respectively. The salt bridges are indicated by black dashed dots. (c) Sequence alignment of Kv4.3 peptide (70–78) with corresponding sequences of other Kv proteins. The residues F73 and F74 of Kv4-family labeled in magenta square on the top form hydrophobic contacts with KChIP1 molecule and those with blue squares make salt bridges with KChIP1. (d) The representative traces are recorded from oocytes injected with cRNAs of Kv4.3 double mutant (E70A, F73E) that disrupts the second interface, in the presence or absence of WT KChIP1 or the KChIP1 the triple mutant (L39E, Y57A, K61A). The left panel depicts currents from oocytes held at 80 mV by a family of pulses from 60 to 40 mV in 10 mV increments for 1 s, and the right panel shows recovery from inactivation for varying lengths of time at step from 80 to þ40 mV every 8 s.

FIGURE 13.7 Comparison of the modeled Kv4.3–KChIP1 channel complex with Kv1.2– Kvb2. (a) Side views of Kv1.2/Kv4.3 T1-KChIP complex in which Kv4.3 T1 domain fused with transmembrane-spanning domains of Kv1.2 (left panel) and Kv1.2-Kvb2 complex (right panel). The tetrameric subunits of Kv1.2 channels are labeled with colors of cyan, yellow, pink, and green, respectively. KChIP1 and Kvb2 are labeled in blue and wheat, respectively. (b) Top views of panel A, showing KChIPs positioned between two adjacent T1 domains (left panel) and Kvb2 beneath the T1 domains (right panel).

PART I NEURONAL VOLTAGE-GATED ION CHANNEL FUNCTIONS

1 NEURONAL L-TYPE VOLTAGE-GATED CALCIUM CHANNELS ALEXANDER SCRIABINE1 AND DAVID J. TRIGGLE2 1

Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA 2 School of Pharmacy and Pharmaceutical Sciences, State University of New York, Buffalo, NY 14260, USA

1.1 STRUCTURE AND DISTRIBUTION Although the L-type calcium (Ca2þ) channel has long been associated with cardiovascular physiology and pharmacology and the pharmacological and therapeutic effects of a structurally diverse group of blockers, notably of the 1,4-dihydropyridine family (Fleckenstein, 1983; Goldmann and Stoltefuss, 1991; Triggle, 2004, 2006) (Fig. 1.1), it is also widely distributed in the peripheral and central nervous system where its roles are being increasingly examined. The application of established calcium channel antagonists, such as nifedipine, does not appear to have dramatic neuronal effects, but application of dihydropyridine activators, such as Bay K 8644, produces profound neuronal and behavioral disturbances, indicating the potential pathological and therapeutic importance of these channels (Lipscombe et al., 2004; Striessnig et al., 2006). The apparent general lack of the effect of 1,4-dihydropyridines on transmitter release suggests that L-type channels play an unimportant role in presynaptic calcium entry coupled to transmitter release, but rather are involved in longer term events such as neuronal plasticity and the control of gene expression.

Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

3

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NEURONAL L-TYPE VOLTAGE-GATED CALCIUM CHANNELS

FIGURE 1.1 The structural formula and receptor site organization for the three principal drug classes active at the L-type calcium channel. A separate receptor site exists for each of the drug classes. That for 1,4-dihydropyridine activator and antagonists has been best described and explored.

However, this may be an oversimplification based in part upon an excessive reliance on the prototypical pharmacology of 1,4-dihydropyridines to define channel properties and function. The neuronal L-type voltage-gated calcium channel (NLTCC) is a member of a family of voltage-gated calcium channels, which in turn belong to a superfamily of voltage-gated ion channels, including those for potassium (Kþ) and sodium (Naþ). It is likely that the potassium channel represents an ancestral member whose functions have been progressively modified by processes of gene duplication and mutation. A number of general reviews are available for this superfamily of ion channels (McDonough, 2004; Catterall, 2005; Lacinova, 2005; Zamponi, 2005; Triggle, 2006). The voltagegated calcium channel family is a heteromeric association of subunits as depicted in Fig. 1.2—a1, b, a2d, and g—and their biophysical and pharmacological properties as well as their expression are influenced significantly by the totality of the subunit interactions. Since there are several members of each subunit class with alternative splicing, the number of potential permutations with attendant variations in localization and biophysical and pharmacological properties is quite large. 1.1.1

Classification

Voltage-gated calcium channels are divided into two main classes, high voltage activated (HVA) and low voltage activated (LVA), and each of these classes is further subdivided (Catterall et al., 2005). The HVA channels are subdivided into five types (L, T, N, P/Q, and R) on the basis of their physiological and pharmacological properties. An interesting account of the history of the discovery of calcium channels has been provided by Tsien and Barrett (2005). The properties of L-type (for longlasting) channels are summarized in Table 1.1. Figure 1.3 depicts the overall sequence

STRUCTURE AND DISTRIBUTION

TABLE 1.1

5

Properties of L-Type Voltage-Gated Calcium Channels (Cav1.1–1.4)

Physiological properties Conductance (pS) Activation threshold Deactivation rate Inactivation rate Permeation Function

25 High Fast Slow Ba2þ > Ca2þ E–C coupling, CV system, smooth muscle, endocrine cells, neurotransmitter release (eye, ear)

Pharmacological properties 1,4-Dihydropyridines Phenylalkylamines Benzothiazepines Benzimidazoles v-Conotoxin GV1A v-Conotoxin MVIIC v-Agatoxin IVA v-Agatoxin IIIA Cd2þ block Ni2þ block Radioligands of choice Tissue expression Disease relevance

Sensitive Sensitive Sensitive Insensitive Insensitive Insensitive Insensitive Sensitive Potent Weak 3 ½H-cis-(þ)-Diltiazem, 3 ½H-desmethoxyverapamil, 3 ½H-isradipine Widespread: CV system, neurons, endocrine tissue, skeletal muscle, smooth muscle Hypertension, angina, malignant hyperthermia, hypokalemic periodic paralysis, night blindness

FIGURE 1.2 A schematic representation of the organization of the subunits of the voltagegated calcium channel.

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NEURONAL L-TYPE VOLTAGE-GATED CALCIUM CHANNELS

FIGURE 1.3 The organization of the voltage-gated calcium channel family.

homology between the several channel classes. The L-type voltage-gated channel, the principal focus of this chapter, belongs to the HVA class (although the potential dependence of activation and inactivation varies between subtypes) and has been particularly well investigated from structural, functional, and pharmacological perspectives. Drugs acting at this class of channel (Fig. 1.1) not only have found widespread use in the treatment of a number of cardiovascular disorders, but have also been widely explored for their potential application in a number of other disorders, including neuronal pathologies, from achalasia through depression to tinnitus and vertigo. 1.1.2

Structure

A schematic representation of the overall organization of the voltage-gated calcium channel a1 subunit is depicted in Fig. 1.1. The a1 subunit consists of four homologous domains, and the S4 segments contain regularly arrayed positively charged lysine and arginine residues, a feature associated with the voltage sensitivity and channel opening properties of these channels. This subunit makes up the pore-forming and voltagesensing components of the channel as well as containing the major drug binding sites. The S5–S6 linkers each contain a critical glutamic acid residue that comprises in total the selectivity filter of the channel. A summary of the sizes of individual cloned a1 subunits of the Cav1.1–1.4 (L-type) family is provided in Table 1.2. Interaction with other subunits is important for both expression and biophysical and pharmacological properties of the channel.

STRUCTURE AND DISTRIBUTION

TABLE 1.2

7

Sizes of Cloned Cav1 a1 Subunits

Subunit

Origin

MW (kDa)

No. of Residues

Cav1.1 Cav1.2 Cav1.2 Cav1.2 Cav1.3 Cav1.4

Rabbit skeletal muscle Rabbit heart Rabbit lung Rat aorta Human pancreas Human retina

212 242.8 242.5 243.6 247.6 219.5

1873 2171 2166 2169 2181 1966

Data from compilation of Lacinova (2005).

The cytosolic b subunit b1–4, coded by four genes (CACNB1–4) and with a number of splice variants, interacts through specific domains on that subunit and on the S1–S2 linker: the b interaction domain (BID) of approximately 30 residues and the a interaction domain of approximately 18 residues (Pragnell et al., 1994; Hofmann et al., 1999; Van Petergem et al., 2004; Cens et al., 2005; Doering and Zamponi, 2006). The b subunits are widely distributed in excitable tissues. The b subunit resembles a membrane-associated guanylate kinase, the GK domain of which provides a hydrophobic cleft for calcium channel binding (Takahashi et al., 2004). The AID–BID complex may also provide an additional site for drug interaction (Triggle, 2004, 2006). The a2d 1–4 subunit, coded by four genes CACNA2D1–D4 and with a number of splice variants, comprises two components linked by a disulfide bond with a membrane-spanning d component and an extracellular a2 component (De Jongh et al., 1990; Doering and Zamponi, 2006). These subunits are also widely distributed in excitable tissues. The a2d4 subunit also generates a drug binding site, characterized in particular for drugs such as gabapentin and pregabalin used for pain relief. The g 1–8 subunit is coded by eight genes, CACNG1–8, and is of particular importance in skeletal muscle (Cav1.1) and neuronal (Cav2 and Cav3) channels (Lacinova, 2005; Doering and Zamponi, 2006). The g subunits are integral membrane proteins with four transmembrane domains and intracellular C and N termini. Of particular importance to understanding both channel function and pharmacology is the question of the actual subunit combinations that make up the various channel types: it is likely that this is tissue and cell specific and the details remain largely unresolved. 1.1.3

Subtypes

Four principal classes of the L-type channel exist represented by the a subunit of Cav1.1–1.4 and associated with the b, a2d, and particularly for the Cav1.1 class, the g subunit. The specific permutations of association of these subunits and their splice variants remain to be fully established. However, the Cav1.1 channel, which mediates excitation–contraction coupling in skeletal muscle, is composed of the principal a1 subunit together with b1, a2d1, and g 1 subunits. The organization of the other three L channel types remains less certain, and it is possible that the subunit compositionvaries and is tissue dependent (Cens et al., 2005; Doering and Zamponi, 2006). In addition to the primary classification, other subtypes of the channel may exist through the

8

NEURONAL L-TYPE VOLTAGE-GATED CALCIUM CHANNELS

TABLE 1.3 Channel

Interaction of 1,4-Dihydropyridines with Splice Variants of the L-Type

Nifedipine Nisoldipine (þ)-Isradipine SDZ 2017-180

KD (nm), 100 mV, CaV1.2a (Cardiac)

KD (nm), 50 mV, CaV1.2b (Smooth Muscle)

47 2.1 15 91

10 0.56 2.1 100

Data from Morel et al. (1998).

existence of splice variants. Such subtypes may exhibit both tissue-dependent localization and tissue-selective pharmacology (Soldatov et al., 1986; Welling et al., 1993, 1997; Hu and Marban, 1998; Morel et al., 1998; Zhulke et al., 1998; Lacinova et al., 2000; Safat et al., 2001). Thus, several comparisons have been made of the properties and 1,4-dihydropyridine sensitivity of splice variants of the Cav1.2 (cardiac and smooth muscle) subunit. The a-splice variant (cardiac isoform) has a lower sensitivity to 1,4-dihydropyridines than the b-splice variant (smooth muscle). Representative data are shown in Table 1.3. Splice variants have been investigated in other tissues, including brain and neuroendocrine cells, but evidence for selective distribution and function is lacking (Safat et al., 2001). 1.1.4

Distribution

Although the dominant physiological and pharmacological expression of L-type channel activity is usually assumed to be localized to the cardiovascular system, these channels are in fact widely distributed throughout the body including the peripheral and central nervous systems (Bean, 1989; Hess, 1990; Miljanich and Ramachandran, 1995; Bech-Hansen et al., 1998; Strom et al., 1998; McRory et al., 2004; Herlitze and Mark, 2005; Lai and Jan, 2006). Within neurons, selective cellular localization occurs. Cav1.4 is present in the retina where loss-of-function mutations cause night blindness (Bech-Hansen et al., 1998; Strom et al., 1998; McRory et al., 2004). Cav1.2 and Cav1.3 are more widely distributed in nervous tissue, neuroendocrine cells, and hair cells (Herlitze and Mark, 2005). The role of Cav1.3 channels in hair cells in the cochlea is linked to the development of these cells and to the associated development of highconductance calcium-activated potassium BK channels (Nemzou et al., 2006). The widespread distribution of the Cav1.2 and Cav1a.3 channels within both the cardiovascular and nervous systems has made determination of their neuronal roles through pharmacological intervention difficult since the available L-type channel ligands have powerful cardiovascular properties that may overshadow or complicate any activities produced in neurons. However, selective elimination of 1,4-dihydropyridine sensitivity from Cav1.2a1 subunits permits the role of Cav1.3 channels to be examined through pharmacological dissection (Bourient et al., 2004). Cav1.3 stimulation was shown to selectively contribute to Fos expression, to neurotransmitter release in the ventral striatum, and to be associated with depression-like behavioral effects.

STRUCTURE AND DISTRIBUTION

9

Within neurons, L-type channels enjoy selective localization. In rat cortex and hippocampus, Cav1.2 and Cav1.3 channels are principally localized in cell bodies and proximal dendrites, with the Cav1.2 type being concentrated in clusters and the Cav1.3 more dispersed (Hell et al., 1993; Ludwig et al., 1997). In apparent contrast, L-type channels are associated in rat globus pallidus neurons predominantly with distal dendrites, where the authors suggest that the proximal localization reported in earlier studies may represent channels in the process of transport (Hanson and Smith, 2002). The issue of the subunit association of these a1 subunits remains unclear, but the work of Ludwig et al. (1997) on the rat brain suggests that this may be cell specific, thus providing further diversity of channel structure and function. NLTCCs do not have a major role in neurotransmitter release but are certainly involved in the control of gene transcription activity (Dolmetsch et al., 2001; Zhang et al., 2002; Deisseroth et al., 2003; Evans and Zamponi, 2006), and the subsequent changes in protein expression may be linked to changes in synaptic strength and the regulation of transmitter phenotype (Brosenitsch et al., 1998; Deisseroth et al., 2003). Proteolytic cleavage of the Cav1.2 channel generates a C-terminal fragment, calcium channel-associated transcription regulator (CCAT), that translocates to the nucleus where it interacts with an endogenous promoter to control the expression of a number of genes associated with signaling and excitability in neurons (Gomez-Ospina et al., 2006). Other voltage-gated calcium channels behave similarly, and this may be a general control mechanism (inter alia, Hell et al., 1993; Westenbroek et al., 1998; Kordasiewicz et al., 2006). However, the level of such activity may differ between L-type channel subtypes: in rat hippocampal neurons, Cav1.3 plays a more important role in pCREB signaling than does Cav1.2 (Zhang et al., 2005b, 2006). 1.1.5

Mechanisms of Action: Activation, Inactivation, and Drug Action

The Cav1.1–1.4 class of channels show overall similar structure and pharmacology but differ quantitatively in a number of important aspects. The traditional view of this channel class is that they are activated by strong depolarization, are relatively slowly activated, have large single-channel conductance, show calcium-dependent inactivation, and are very sensitive to the 1,4-dihydropyridine family of ligands (Lipscombe et al., 2004). However, at least some channels formally of the Cav1.2 and 1.3 classes, those most widely distributed in the nervous system, show distinct behavior, in particular activating at relatively polarized levels of membrane potential and showing differential sensitivity to 1,4-dihydropyridines (inter alia, Xu and Lipscombe, 2001; Lipscombe, 2002; Lipscombe et al., 2004; Helton et al., 2005). Recombinant neuronal Cav1.2 and Cav1.3 channels open rapidly over a wide range of membrane potentials and carry significant calcium current in response to single stimulus. However, 1,4-dihydropyridines do not block calcium entry in response to single action potential stimuli but are effective in blocking current in response to step depolarization or to long trains of action potentials. This reflects the significant state-dependent interactions of 1,4-dihydropyridines (Section 1.2.2). Furthermore, 1,4-dihydropyridines completely block Cav1.2 channels, but only partially block Cav1.3 channels (Helton et al., 2005). These observations indicate that the role of neuronal L-type channel activation in response to brief stimuli may have been underestimated because of an excessive

10

NEURONAL L-TYPE VOLTAGE-GATED CALCIUM CHANNELS

reliance on 1,4-dihydropyridines as pharmacological markers of L-type channel activity. 1.1.6

Interaction with Other Cellular Components

In addition to interaction with b, g, and a2d subunits, the principal a1 subunit of the voltage-gated calcium channel interacts with a variety of other proteins that regulate its expression, trafficking, and activity (for reviews see Catterall et al., 2005; Herlitze and Mark, 2005; Lee and Catterall, 2005; Stanley and Chan, 2005; Evans and Zamponi, 2006). Channels of the Cav2.1 and 2.2 classes interact with the bg subunits of G proteins to mediate differential inhibition and with the SNARE protein complex that mediates exocytosis of transmitters. Cav1.1 channels interact with ryanodine receptors to mediate excitation contraction coupling in skeletal muscle and also with A kinase anchoring proteins (AKAPs) that anchor cAMP-dependent protein kinase. Cav1-type channels undergo both Ca2þ-dependent inactivation and facilitation, best established for Cav1.2 channels, mediated predominantly through calmodulin binding to the IQ-domain of the C-terminal portion of the a1 subunit (reviewed in Lee and Catterall, 2005).

1.2 CLASSES OF DRUGS Of the three structural classes of drugs depicted in Fig. 1.1—the benzothiazepinones, the phenylalkylamines, and the 1,4-dihydropyridines—the latter represent the largest class studied both clinically and experimentally. They are also quantitatively the most active, exhibiting pharmacology in the nanomolar concentration range, and they also include both antagonist and activator species. However, drugs active at L-type channels are not confined to these three structural scaffolds, and many diverse agents exhibit antagonist properties. The structure–function relationships of drugs active at L-type channels have been extensively reviewed over many years (inter alia, Janis and Triggle, 1983, 1984a, 1984b; Janis et al., 1987; Triggle et al., 1989; Goldmann and Stoltefuss, 1991; Rampe and Triggle, 1993; Triggle, 2003, 2004, 2006; Budriesi et al., 2007). Hence, only a very brief overview primarily of the 1,4-dihydropyridines, the principal therapeutic and molecular tools active at the L-type channel, will be presented here. 1.2.1

Structural Requirements

The basic structural requirements for antagonism and activation are depicted in Fig. 1.4, and the structural formulas of clinically available 1,4-dihydropyridines are depicted in Fig. 1.5. The 1,4-dihydropyridines are of particular interest for several reasons. First, they are extremely potent ligands for the L-type channel. Second, they exhibit both activator and antagonist properties. Third, they exhibit considerable stereoselectivity of action. Fourth, they show substantial state-dependent mode of interaction. Fifth, the 4-aryl-1,4-dihydropyridine nucleus is a “privileged structure’’ capable, when decorated with the appropriate substituents, of interacting with a variety of receptors and ion channels (Triggle, 2003).

CLASSES OF DRUGS

11

FIGURE 1.4 The structural features that determine activity of activator and antagonist in the 1,4-dihydropyridine family of drugs active at the L-type calcium channel.

Bay K 8644 serves as the prototypical activator and such compounds show highly differential pharmacology whereby one enantiomer is an activator and the other an antagonist (Fig. 1.6). Stereoselectivity is quite generally observed with all 1,4-dihydropyridines, but the extent of chirality depends on the substituents around the 1,4-dihydropyridine ring. Thus, nitrendipine has a modest stereoselectivity factor of approximately 5–10, whereas amlodipine has a (+) factor of approximately 1000.

FIGURE 1.5

The clinically available 1,4-dihydropyridines.

12

NEURONAL L-TYPE VOLTAGE-GATED CALCIUM CHANNELS

FIGURE 1.6 Stereochemical requirements for activator and antagonist activity in 1,4dihydropyridines. The S-enantiomers are activators and the R-enantiomers are antagonists.

1.2.2

State-Dependent Actions

The 1,4-dihydropyridines (as well as verapamil and diltiazem) are characterized by their state-dependent mode of interaction at the L-type channel. These interactions arise because drugs may exhibit differential affinity and/or access to their binding sites in the resting, open, or inactivated states of the channel (Hille, 1977; Hondeghem and Katzung, 1985; Triggle, 1989; McDonough and Bean, 2006). Transitions between these states are determined by changes in chemical or electrical potential and by the kinetics of channel opening and closing. Drugs that interact preferentially with the open or inactivated state of the channel will show an increase in apparent affinity under those physiological or pathological conditions that increase the availability of those channel states. In addition, the structure of the drug and overall physicochemical properties—charged/uncharged and hydrophilic/hydrophobic characteristics—can control the access of the drug to the receptor site. Charged and polar drugs may access their binding sites through a polar and hydrophilic pathway, including the channel pore, whereas nonpolar drugs can access binding sites through membranedelineated pathways. Both diltiazem and verapamil exhibit frequency-dependent interactions and hence their use, particularly of verapamil, in certain tachyarrhythmias, whereas nifedipine and other 1,4-dihydropyridines exhibit voltage-dependent interactions consistent with a preferential interaction with the inactivated states of the channel, a property that underscores their general vascular selectivity (Bean, 1984; Sanguinetti and Kass, 1984; Wei et al., 1986; Triggle, 1989; Zhen et al., 1992; Sun and Triggle, 1995).

FUNCTION

1.2.3

13

Privileged Structures

Finally, the 4-aryl-1,4-dihydropyridine nucleus is a privileged structure and with appropriate ring substituents can access a diverse set of channels and receptors (Triggle, 2003). Certain dihydropyridines also interact with T-, N-, and P/Q-type calcium channels (Cohen et al., 1992; Kumar et al., 2002; Zhou et al., 2002; Yamamoto et al., 2006). Agents such as amlodipine, cilnidipine, barnidipine, benidipine, and nicardipine that can also block N-type channels may have some therapeutic cardiovascular advantage as well as interact at the N-type channels in neuronal tissues.

1.3 FUNCTION 1.3.1

Role of Subunits and Isoforms

The role of NLTCCs in the neuronal function is determined by the type and location of neurons as well as by the composition of channels, their subunits, and isoforms. Each channel complex consists of the pore-forming a1 subunit and three regulatory subunits (a2, b, and g). Three isoforms of the a1 subunit have been identified in the central nervous system: Cav1.2a1, Cav1.3a1, and Cav1.4a1. They have distinctly different neurological functions. The first two isoforms often occur together, expressed in the same cells, while Cav1.4a1 is found mainly in the retinal neurons (Baumann et al., 2004). The regulatory subunits also contribute to heterogeneity of NLTCCs. The a2d1 subunit has been intensively studied and identified as a molecular target for the analgesic action of pregabalin and gabapentin (Field et al., 2006; Joshi and Taylor, 2006). The techniques available to study physiological functions of NLTCCs are either pharmacologic or genetic. Pharmacologic methodology calls for the use of L-type Ca2þ channel activators or antagonists. Unfortunately, specific ligands for isoforms of the a1 subunit of NLTCCs have not yet been discovered, and the currently available ligands for L-type channels are not isoform specific. The genetic methodology utilizes “knockout’’ mice lacking speciﬁc isoforms of NLTCCs (Schulla et al., 2003; Sinnegger-Brauns et al., 2004). The neurological phenotype of Cav1.3a1/ mice exhibits inner hair cell dysfunction and cochlear sensory cell degeneration but appears neurologically normal. It has been suggested (Clark et al., 2003) that certain Ca2þ channel isoforms may support distinct behavioral functions. New insights into the role of NLTCCs and the Cav1.3a1 isoform in neuronal function were provided by the discovery of a link between these channels and macromolecular signaling complex formed by Shank and other modular adapter proteins as well as a link with G-proteincoupled receptors (Olson et al., 2005; Zhang et al., 2005). Using a mouse model without dihydropyridine (DHP)-sensitive Cav1.2a1 subunits (Cav1.2 DHP/ mice), Sinnegger-Brauns et al. (2004) found that in the ventral striatum of these mice agonistinduced glutamate and 5-HT release was abolished, while dopamine and norepinephrine release remained intact. This observation demonstrated differences in the functions

14

NEURONAL L-TYPE VOLTAGE-GATED CALCIUM CHANNELS

of Cav1.2a1 and Cav1.3a1 isoforms, although both isoforms appear to control emotional behavior in mice. On the basis of experiments suitable for the dissection of function of the two isoforms, Striessnig et al. (2006) concluded that selective inhibitors of channels containing Cav1.3a1 isoforms can be expected to have antidepressant and anxiolytic properties. 1.3.2

Synaptic Plasticity and Memory

A brief period of high-frequency electrical activity applied artificially to a neuronal pathway can enhance the strength of synapses for various periods of time. This phenomenon is called long-term potentiation (LTP). It can be induced in the cerebral cortex, hippocampus, and other brain areas. Many features of LTP resemble those involved in memory storage, and LTP is widely used in memory research in attempts to elucidate molecular mechanisms of memory (Kandel, 2001). In rat hippocampus, there are at least three types of LTP (short lasting or LTP1, of intermediate duration or LTP2, and long lasting or LTP3). LTP3 is selectively dependent on NLTCCs (Raymond and Redman, 2006). In rat basolateral amygdala, LTP is induced at least in part due to influx of Ca2þ through channels containing Cav1.2 isoforms (Pinard et al., 2005). In the superior cervical ganglion of the rat, however, ganglionic transmission is mediated primarily by P/Q- and N-type channels with only 14% contribution by L-type calcium channels (Cifuentes et al., 2004). Antagonists of NLTCCs, for example, nimodipine, reduce the extent of LTP in rat hippocampal neurons and abolish the induction of long-term depression (LTD), induced by postsynaptic spiking prior to presynaptic activation (Bi and Poo, 1998). The role of hippocampal NLTCCs containing Cav1.2 channels in synaptic plasticity and spatial memory was studied in Cav1.2HCKO mice in which the CACNAIC (Cav1.2) gene was inactivated (Moosmang et al., 2005b). These investigators found that the late phase of long-term potentiation (L-LTP) is lost in the hippocampus and neocortex of these animals and hippocampus-dependent spatial memory is severely impaired. A decreased activation of mitogen-activated protein kinase (MAPK) pathway and a reduced cAMP response element-dependent transcription were found in CA1 pyramidal neurons of Cav1.2HCKO mice. Phosphorylation of cAMP response element binding protein (CREB) at Ser133 is considered to be an important step in the induction of gene expression critical for memory (Moosmang et al., 2005a); it is impaired in Cav1.2HCKO mice. These observations suggest that selective inhibitors of Cav1.2 subtypes of NLTCCs could impair spatial memory. On the contrary, there is evidence that calcineurin, the only Ca2þ-activated protein phosphatase in the brain, negatively modulates learning, memory, and neuronal plasticity (Mansuy, 2003). Calcineurin has been identified as the key signal in the extinction of fear memory (Lin et al., 2003). It also impairs spatial memory in mice (Mansuy et al., 1998). Ca2þ needed for activation of calcineurin enters neurons at least partially through NLTCCs and is likely to enhance calcineurin-induced negative modulation of learning and memory, so that inhibitors of NLTCCs could be expected to improve learning and spatial memory. It is currently unknown whether Ca2þ entry through channels containing Cav1.2 or Cav1.3 isoforms is linked to the activation

FUNCTION

15

of calcineurin. If it is, a selective inhibitor of these isoforms could improve learning and spatial memory. The idea that antagonists of NLTCCs could improve memory and learning and be useful in the treatment of Alzheimer’s disease was originally based on the calcium hypothesis of Alzheimer’s disease and aging (Landfield, 1987; Disterhoft et al., 1994; Khachaturian, 1995). One of the key elements of this hypothesis involved breakdown of [Ca2þ] homeostasis and elevation of intraneuronal calcium as a factor contributing to neuronal degeneration and death. An antagonist of NLTCCs, nimodipine, has been shown to facilitate learned behavior in rats with neocortical injury (LeVere and Sandin, 1989). More recently, nimodipine has been shown to ameliorate age-related memory decline in aged rats. This effect was associated with the decline in abnormally high level of expression of channels containing Cav1.3 subunit in the hippocampus of these animals (Veng et al., 2003). Some clinical studies suggested that nimodipine activates cognition in patients with vascular or primary degenerative dementia (Tobares et al., 1989; Fischhof et al., 1993) and another antagonist nitrendipine (Forette et al., 1998) reduces the incidence of dementia in the elderly hypertensive population. High cytosolic calcium concentrations in neurons inhibit asecretase cleavage of amyloid precursor protein (APP) and increase intraneuronal levels of b-amyloid peptide (Ab1–42) (Pierrot et al., 2004). NLTCCs antagonists can be expected to reduce cytosolic calcium in neurons. Morich et al. (1996) reported clinical experience with nimodipine in patients with probable Alzheimer’s disease (AD). There was no convincing evidence of effectiveness of nimodipine in AD, but this drug improved performance of patients in Buschke’s Selective Reminding test, which is considered to be an index of memory storage. This finding suggests that Ca2þ entry through NLTCCs may modulate storage of memory in humans. The failure to demonstrate convincingly cognition activation with antagonists of NLTCCs in humans may be explained by the dual role of intraneuronal Ca2þ. It improves some aspects of memory but facilitates neurodegeneration and activates calcineurin, which impairs cognition. Antagonists of NLTCCs may also have effects other than those mediated by calcium channels, and these effects may oppose the consequences of calcium channel blockade. Nimodipine, for example, has been reported to stimulate Ab1–42 secretion in neuroblastoma cell cultures, an effect apparently not involving calcium channels (Facchinetti et al., 2006). This effect, if it occurs in vivo, would tend to oppose the putative cognition enhancing effect of this drug. 1.3.3

Pain

During the last two decades, substantial evidence has accumulated that voltage-gated calcium channels (VGCCs) are involved in the perception of pain (Cao, 2006; Yaksh, 2006). Pain behavior appears to be altered primarily by the Cav2.2 subunits of N-type channels, located in the presynaptic terminals where they seem to control neurotransmitter release (e.g., glutamate or substance P). The involvement of calcium channels in the control of pain was supported by the discovery of the analgesic activity of a2d subunit ligands, gabapentin, pregabalin, and L-phenylglycine, in neuropathic pain (Taylor, 2004; Frampton and Foster, 2005; Lynch et al., 2006), although not all ligands of this subunit attenuated neuropathic pain in rat spinal cord ligation model

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NEURONAL L-TYPE VOLTAGE-GATED CALCIUM CHANNELS

(Lynch et al., 2006). The a2d protein is encoded by four genes, a2d1, a2d2, a3d 3, and a2d4, and replacement of a single amino acid (arginine in a position 217) in a2d1 subunit prevented gabapentin or pregabalin binding and nearly abolished their analgesic activity in mutant mice (Field et al., 2006). This finding strongly supports the importance of the a2d 1 subunit for the analgesic action of these two compounds. The involvement of NLTCCs in pain perception is likely to depend on the nature of the a2d1 subunit in these channels. Some of the early pharmacologic studies with L-type calcium channel antagonists, for example, 1,4-dihydropyridines, demonstrated their antinociceptive activity and interactions with opiates (Hoffmeister and Tettenborn, 1986; Del Pozo et al., 1987). The activity was dependent on the test, doses of the drugs, and routes of their administration, and some of the findings were contradictory. Nimodipine and nifedipine antagonized acetic acid-induced writhing following their intracerebroventricular (i.c.v.) administration to mice (Miranda et al., 1993). In the tail withdrawal test, the same drugs had antinociceptive activity in rats by chronic subcutaneous (s.c.) administration (Martin et al., 1996). In hot plate test, however, nimodipine, but not nifedipine, verapamil, or diltiazem, had analgesic effect (Miranda et al., 1992). It has been suggested that 1,4-dihydropyridines may affect pain perception at the spinal level (Martin et al., 1996). On the basis of the available evidence, L-type calcium currents appear to be only marginally involved in pain perception. The possibility that antagonists of NLTCCs could be useful as adjuncts to analgesics has been extensively studied. Many experimental studies explored the interaction of 1,4-dihydropyridines with opiates. In the rat tail-flick test, nimodipine, at 1 mg/kg i.p., or lercanidipine, at 0.3 mg/kg i.p., potentiated analgesia caused by k-opioid receptor agonists and prevented the development of tolerance to the opiates (Gullapalli and Ramarao, 2002a). Chronic administration of nimodipine to rats at 1 mg/kg/day i.p. for 10 days increased morphine (2 mg/kg i.p.) induced analgesia. The effect was additive to that of naloxone (Gullapalli and Ramarao, 2002b). Zhang et al. (2003) demonstrated that NLTCC antagonists inhibit morphine sensitization in mice and proposed that NLTCCs are involved in the development of morphine-induced neural and behavioral plasticity. In diabetic rats, nimodipine at 0.3–3.0 mg/kg i.p. potentiated the antinociceptve effects of morphine (Gullapalli et al., 2002). The route of administration and the duration of treatment appear to determine the ability of nimodipine to potentiate morphine. Lee and Yoburn (2000) found that nimodipine when administered s.c. to mice by a minipump at 100 mg/kg/day over 7 days, but not by single s.c. administration at 100 mg/kg, potentiated morphine-induced analgesia. This effect of nimodipine is not specific for opioids. Nociception induced by 5-HTP in mice was potentiated by nimodipine, nifedipine, or verapamil (Liang et al., 2004), and nimodipine was found to enhance the antihyperalgesic effects of diclofenac in formalin pain model in rats (Sukriti and Pandhi, 2004). In an attempt to explain the mechanism of interaction of calcium channel antagonists with morphine, Shimizu et al. (2004) pretreated mice with high doses (40–80 mg/kg i.p.) of diltiazem, nimodipine, or verapamil prior to morphine (4 mg/kg s.c.) and found that these drugs potentiate analgesic effects and increase serum levels of morphine.

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By acute administration to healthy volunteers, diltiazem, nimodipine, or verapamil did not enhance the analgesic effects of morphine (Hasegawa and Zacny, 1997). In patients with cancer pain, nimodipine did not enhance morphine-induced analgesia (Roca et al., 1996). Also, in patients undergoing colorectal surgery, neither oral nifedipine nor intravenous nimodipine increased the analgesic potency of morphine (Zarauza et al., 2000). In patients undergoing knee replacement surgery, oral nimodipine increased morphine consumption without enhancing its analgesic effect (Casey et al., 2006). There is currently no definitive explanation for the apparent discrepancy between animal and human studies in respect to the ability of NLTCC antagonists to potentiate the analgesic effects of morphine. To better understand the nature of the discrepancy, the optimal blood levels of NLTCCs antagonists required for the enhancement of morphine analgesia in animals should be determined, so that the same blood levels can be achieved in clinical studies. It is conceivable that sustained release formulations of NLTCC antagonists administered for at least a week would enhance the analgesic effects of morphine. 1.3.4

Epilepsy

Epileptogenic activity in neurons is thought to be activated by an inward Ca2þ current. After entering neurons at least partially through NLTCCs, calcium ions are thought to regulate various aspects of synaptic activity, including epileptogenesis (De Lorenzo, 1986). The specific molecular mechanisms involved in the epileptogenesis are poorly understood, and the role of various subunits and isoforms of NLTCCs in the epileptogenic activity is not yet known. It has been suggested that antagonists of T-type calcium channels are effective in treatment of absence seizures, while NLTCCs may control partial seizures (Kułak et al., 2004). The NLTCC activator BAY K 8644 has proconvulsant activity in animals. Most antagonists of NLTCCs have been shown to have anticonvulsant activity at least in some animal models of epilepsy. Clinical data are contradictory. Anticonvulsant activity of nifedipine and nimodipine has been demonstrated in small open studies, but controlled studies with either nifedipine (Larkin et al., 1992) or nimodipine (Larkin et al., 1991; Meyer et al., 1995) were disappointing. Nimodipine has also been used in the prevention of eclampsia, but appears to be less effective than magnesium sulfate (Belfort et al., 2003). 1.3.5

Drug and Ethanol Dependence

NLTCCs appear to play a role in drug addiction and alcohol dependence, but the effectiveness of the antagonists in the treatment of drug addiction remains controversial. Nimodipine, 5–20 mg/kg s.c., or isradipine, 1–3 mg/kg s.c., inhibited selfadministration of morphine or cocaine in drug-na€ıve mice (Kuzmin et al., 1992). At 20 mg/kg s.c., nimodipine decreased the sensitivity of rodents to the reinforcing effects of cocaine (Kuzmin et al., 1996). Cocaine-induced elevation of plasma catecholamines was prevented by nimodipine in squirrel monkeys (Trouve et al., 1990). Nitrendipine, flunarizine, or diltiazem protected rats from convulsions and death caused by a large dose of cocaine (60 mg/kg i.p.) (Trouve and Nahas, 1990).

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Withdrawal signs in morphine-dependent rats were effectively antagonized by NLTCCs (Ramkumar and El-Fakahany, 1988). Chronic exposure to cocaine facilitated the function of L-type calcium channels in pyramidal neurons of medial prefrontal cortex of rats (Nasif et al., 2005). It appears that antagonists of NLTCCs may have a place in the treatment of cocaine toxicity or of withdrawal symptoms, but in cocaine-dependent patients craving for cocaine was not affected by nimodipine (Rosse et al., 1994). Dihydropyridine-type antagonists of NLTCCs were found to interact with ethanol in animals, reversing seizures and other symptoms associated with ethanol withdrawal (Little et al., 1986; Littleton et al., 1990), blocking self-administration of 5% (but not higher concentrations) of ethanol (Smith et al., 1999), and blocking tolerance to the antinociceptive effects of ethanol during withdrawal (Gatch, 2006). Physical dependence on alcohol is associated with an increased number of dihydropyridine-sensitive calcium channels in the rat brain (Dolin et al., 1987). More recently, Katsura et al. (2005, 2006) demonstrated that physical dependence on ethanol in mice is accompanied by increased expression (and possibly function) of Cav1.2 and Cav1.3 isoforms and a2/d1 subunits of NLTCCs in mouse brain. Selective antagonists of channels containing these isoforms may reduce or abolish the dependence on ethanol and possibly on other habit-forming substances. Antagonists of NLTCCs, nimodipine, verapamil, or diltiazem, attenuated nicotineinduced locomotor sensitization and place preference in mice (Biala, 2003). The same drugs attenuated the antinociceptive effects of nicotine as well as cross-tolerance to the antinociceptive actions of nicotine and morphine (Biala and Weglinska, 2006). These findings suggest that a common mechanism involving L-type calcium channels may be responsible for the development of tolerance to the antinociceptive effects of morphine and nicotine. Chronic administration of nicotine leads to upregulation of Cav1.2, Cav1.3, Cav1.4, and a2d 1 subunits in mouse brain (Hayashida et al., 2005). Anxiogenic effects of nicotine in mice as well as the development of tolerance to this effect were attenuated by nimodipine, flunarizine, verapamil, or diltiazem (Biala and Budzynska, 2006). Attenuation of nicotine effects by NLTCCs antagonists may not, however, be mediated solely by calcium channels. It has been recently shown that nimodipine and nifedipine can also block nicotinic acetylcholine receptors (nAChRs) directly (Wheeler et al., 2006). 1.3.6

Hearing

NLTCCs and specifically Cav1.3 isoform are essential for synaptic transmission in cochlear inner hair cells and hair cell development in mice (Brandt et al., 2003, 2005; Nemzou et al., 2006). Cav1.3/ mice are deaf and show outer hair cell loss at the apical cochlea, while heterozygous (Cav1.3þ/) mice have increased hearing threshold for low-frequency sounds (Dou et al., 2004). These findings indicate the importance of the Cav1.3 subtype of NLTCCs for normal hearing. Antagonists of NLTCCs could, therefore, be expected to impair hearing. However, nimodipine improved hearing in patients with sudden hearing loss (Handrock, 1985; Theopold, 1985). In rats, it improved cochlear microphonics (Jastreboff and Brennan, 1988) and prevented

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neuronal degeneration in the cochlear nerve (Sekiya et al., 2002), but did not protect gerbils from noise-induced hearing loss (Boettcher et al., 1998). 1.3.7

Vision

Light-dependent Ca2þ influx into photoreceptors in the retina is controlled by Cav1.4 and Cav1.3 subtypes of L-type calcium channels. These channels are located presynaptically in retinal synapses and control neurotransmitter release, primarily glutamate, but also GABA, nitric oxide, and dopamine (Barnes and Kelly, 2002). Cav1.3 is consistently expressed in AII amacrine cells, retinal neurons that have a critical role in night vision (Habermann et al., 2003). Mutations of the gene that encodes retinal Cav1.4-type channels are linked to the night blindness type 2 (CSNB2) (Hoda et al., 2006). No effect of NLTCCs on night vision has been described in the literature, but nimodipine was found to improve visual field and color vision in patients with normal tension glaucoma (Piltz et al., 1998). At 90 mg/day, nimodipine in combination with aspirin, 100 mg/day, improved visual field and hearing dysfunction in a patient with Susac’s syndrome (Wildemann et al., 1996). The pathogenesis of this syndrome is unknown, but its symptomatology includes memory loss, impaired hearing, and vision loss and may conceivably involve NLTCCs. 1.3.8

Gene Transcription

The most important and critical function of NLTCCs is probably the coupling of neuronal activity to gene transcription. Nuclear transcription factors (i.e., pCREB and NFATc44) are activated by Ca2þ influx via postsynaptic L-type calcium channels (Bito et al., 1996; Dolmetsch et al., 2001). In hippocampal neurons, at low levels of stimulation nuclear pCREB signaling is preferentially mediated by the Cav1.3 subtype of NLTCCs (Zhang et al., 2006). The mechanism linking calcium channels to genes involves calcium channel-associated transcription regulator (CCAT). It binds to nuclear proteins and regulates the expression of endogenous genes controlling neuronal signaling and excitability (Gomez-Ospina et al., 2006). CCAT increases dendritic length and promotes contacts between neurons and extracellular matrix. 1.3.9

Cell Differentiation

Ca2þ influx through NLTCCs also affects expression of genes involved in cell proliferation, programmed cell death, and differentiation of neurons. According to D’Ascenzo et al. (2006), differentiation of neural stem/progenitor cells (NSCs) isolated from brain cortex of newborn mice depends on the Ca2þ influx through NLTCCs containing Cav1 isoforms. Immature GABAergic neurons are particularly sensitive to low Ca2þ levels, and Ca2þ influx through L- and T-type channels protects immature neurons from apoptosis (Pardo and Honegger, 1999). Also, in cerebellar Purkinje neurons, Ca2þ influx through L-type channels appears to be more important in the early rather than in the late stages of their development (Gruol et al., 2006).

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1.3.10

Interactions of NLTCCs with Neurotransmitter Systems

The function of NLTCCs should not be viewed in isolation from other neurotransmitter systems. Ca2þ entry through NLTCCs is capable of modulating signaling of most neurotransmitter receptors, and many neurotransmitters can modulate the function of NLTCCs. Dopaminergic signaling plays a key role in the physiology and pathology of the central nervous system. D1 receptor-mediated CREB phosphorylation depends on NLTCCs. D1 receptor stimulation in striatal neurons reverses the effects of NLTCC antagonists on CREB phosphorylation, so that antagonists promote rather than block Ca2þ entry into the neurons (Eaton et al., 2004). Interdependence between dopaminergic and NMDA receptors and NLTCCs has been demonstrated (Cepeda and Levine, 1998). Cannabinoid receptor CB1 was described to inhibit calcium influx through NLTCCs in neonatal rat nucleus solitarius (Endoh, 2006), and opioids were found to modulate NLTCCs through orphan opioid receptor activation (Hurle et al., 1999). The extent of the interplay between receptors and neurotransmitters is not yet fully appreciated and the molecular mechanisms of these interactions are not yet completely understood, but modulation of other neurotransmitter systems is likely to be an important function of not only NLTCCs but also all ion channels.

1.4 CHANNELOPATHIES AND INHERITED DISORDERS Over the past two decades, an increasing number of mutations in voltage-gated calcium channels have been mapped and linked to a number of inherited disorders. A number of comprehensive reviews are available (inter alia, Lorenzo and Beam, 2000, 2005; Muth et al., 2001; Pietrobon, 2002; Striessnig et al., 2004; Pietrobon, 2005; Biduad et al., 2006; Bracey and Wray, 2006; Cannon, 2006). However, the majority of these mutations and inherited disorders are associated with non-L-type channel genes (Cav2 and Cav3), L-type channels, or associated proteins involved in muscle (skeletal, smooth, and cardiac) function. Associated with Cav2 and Cav3 genes are hemiplegic migraine, several ataxias, juvenile myocolonic epilepsy, and idiopathic generalized epilepsies, including childhood absence epilepsy. Associated with skeletal muscle are (1) hypokalemic periodic paralysis (hypoPP), a periodic muscle weakness associated with reduced serum Kþ levels and linked to missense mutations in Cav1.1 (a1S); (2) malignant hyperthermia (MH) linked to multiple mutations in the ryanodine receptor associated with the voltage-sensing dihydropyridine receptor and linked to life-threatening body temperature increases during a number of pharmacological interventions, including general anesthesia and skeletal muscle relaxants; and (3) central core disease, also associated with defects in ryanodine receptors. Two cardiac muscle disorders, arrhythmogenic right ventricular cardiomyopathy and familial polymorphic ventricular tachycardia, are also linked to defects in the ryanodine receptor. Defects in L-type channels associated with neuronal disorders have been described for Cav1.2 and particularly for Cav1.4. A mutation in the Cav1.2 gene is associated with childhood disorder termed “Timothy syndrome’’ associated with multiple

CLINICAL IMPLICATIONS AND FUTURE PERSPECTIVES

21

electrophysiological defects and sudden death caused by cardiac arrhythmias and contributed by gain of function mutations with reduced channel inactivation (Splawski et al., 2004, 2005) and aberrant phosphorylation of Ser49 in the S6 helix of domain 1 (Erxleben et al., 2006). These individuals have webbed ﬁngers and toes (syndactyly), cognitive abnormalities, and autism. The latter pathology is of particular interest although the generalized defect produced by this mutation makes attribution to a speciﬁc neuronal pathway difﬁcult. By contrast, mutations in the Cav.4 channel, almost totally distributed in and linked to retinal function (Bech-Hansen et al., 1998; Strom et al., 1998), have been well described as linked to incomplete congenital stationary night blindness (CSNB2) and characterized by varying levels of night blindness and a reduced visual acuity. Some 60 mutations have been described: approximately 50% are missense mutations leading to nonfunctional proteins, while the remainder lead to expressed channels with varying levels of dysfunction (McRory et al., 2004; Hoda et al., 2006). It is likely that more mutations will be discovered in Cav1 channels linked to neuronal dysfunction, although difficulties exist because this class of channels is so widely expressed (Gargus, 2006). However, knockout studies will provide valuable leads (Muth et al., 2001). Thus, mice lacking Cav1.3 channels have both sinoatrial node dysfunction and congenital deafness, the latter being associated with degeneration of hair cells (Platzer et al., 2000; Nemzou et al., 2006). Mice lacking the Cav1.2 gene have impaired glucose tolerance and insulin secretion, the latter reflecting the absence of an exocytotic component of fusion of secretory granule attached to this channel (Schulla et al., 2003). In addition, it is highly plausible that since the multiple cellular calcium signaling and regulatory mechanisms are closely linked, changes in one may lead to compensating changes in others. Thus, a missense mutation in the Cav2.1 channel underlies the behavior of tottering mice that have ataxia, paroxysmal dystonia, and spontaneous behavioral arrest (Pietrobon, 2002). Since the inducible dystonia component of this mutation can be blocked by L-type antagonists, including diltiazem, nifedipine, and verapamil, and since Cav1.2 channels are significantly upregulated in Purkinje and cerebellar cells, it appears that Cav2.1 dysfunction has produced a compensating upregulation of Cav1.2 channels (Campbell and Hess, 1999). Similarly, Cav1.3 knockout mice have impaired pancreatic islet cell function but have a compensatory overexpression of the Cav1.2 gene (Namkung et al., 2001).

1.5 CLINICAL IMPLICATIONS AND FUTURE PERSPECTIVES In spite of important advances in the physiology and molecular biology of calcium channels during the past 20 years, very little progress has been made in the development of calcium channel ligands as drugs for the treatment of central nervous system diseases. No clinical use for the available activators of NLTCCs has been found. BAY K 8644 and similar activators of NLTCCs produce dystonia, self-injurious behavior, and convulsions in rodents (Jinnah et al., 2000; Kasim and Jinnah, 2003) and are unlikely to be tried in humans.

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The antagonists, while widely used in cardiovascular diseases, have found only limited use in the treatment of CNS diseases (Scriabine et al., 1989, 1991; Scriabine, 2002). As noted in a recent review (Triggle, 2007), in the CNS a variety of calcium channel types contribute to neuronal activity and brain damage is likely to involve multiple pathways, so that blockade of a single channel type may not be the right strategy. Also, the failure of nimodipine in pivotal trials in either Alzheimer’s disease or stroke (Mohr, 1991; Morich et al., 1996) discouraged further trials with antagonists of NLTCCs in these conditions. Subarachnoidal hemorrhage is currently the only FDA-approved indication for nimodipine in the United States. This drug is, however, prescribed “off-label’’ in the prevention of migraine attacks and cluster headache in the United States and is approved for the treatment of “organic brain syndrome’’ in Germany and some of the other European countries. Further clinical trials with NLTCC antagonists in migraine as well as in drug and ethanol addiction should be considered. Such studies will probably be conducted with novel compounds, selective for subunits or isoforms of NLTCCs, or compounds with multiple sites of action. There are no selective NLTCCs yet, but Kiewert et al. (2006) described the pharmacology of NGP1-01, a polycyclic amine, which blocks NLTCCs as well as NMDA channels. Its neuroprotective potency is similar to that of nimodipine and it is structurally related to memantine, so the authors suggested its possible usefulness in stroke as well as in the treatment of neurodegenerative diseases. There is an obvious need for more basic and translational research on the function of NLTCC isoforms and their ligands. The observed interactions of NLTCCs with neurotransmitters or their receptors suggest the use of NLTCCs as adjuncts in the therapy of CNS diseases.

1.6 SUMMARY L-type calcium channels are widely distributed in the central nervous system. Like in other tissues, the channels consist of the pore-forming a1 and three regulatory subunits (a2, b, and g). Three isoforms of a1 subunit have been identified in the central nervous system: Cav1.2, Cav1.3, and Cav1.4. Channels with different isoforms of the a1 subunit differ in some of their functions. Cav1.2-containing channels appear to be involved in cognition and memory. Cav1.3-containing channels have many functions similar to those of Cav1.2-containing channels, but are essential for the synaptic transmission in cochlear inner hair cells and control hearing. Cav1.4-containing channels control Ca2þ influx into photoreceptors of the retina and are involved in the control of vision. L-type calcium channels are also involved in pain perception, neuronal excitability, gene transcription, and cell differentiation. They interact with other transmitter and receptor systems. Activators of L-type calcium channels are proconvulsant and neurotoxic. Antagonists are neuroprotective but have thus far found only limited use in the treatment of CNS diseases. In the United States, only nimodipine has been approved for the prevention of neurological deficits following subarachnoidal hemorrhage. L-type calcium channel antagonists, including nimodipine, appear to be effective in

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the prevention of migraine and cluster headache and can conceivably find new applications in the treatment of dementia, in drug and alcohol dependence, or as adjuncts in the treatment of epilepsy. Specific inhibitors of channels with different isoforms should be developed. Cav1.3a1 antagonists can be expected to have antidepressant and/or anxiolytic properties.

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Zhang Q, Li J-X, Zheng J-W, Liu R-K, Liang J-H, 2003. L-type Ca2þ channel blockers inhibit the development but not the expression of sensitization to morphine in mice. Eur J Pharmacol 467: 145–150. Zhang H, Maximov A, Fu Y, Xu F, Tang T-S, Tkatch T, Surmeier D J, Bezprozvanny I, 2005a. Association of Cav1.3 L-type calcium channels with Shank. J Neurosci 25: 1037–1049. Zhang X, Odom DT, Koo SH, Conkright MD, Canettieri G, Best J, Chen H, Jenner R, Herbolsheimer E, Jacobsen E, et al., 2005b. Genome-wide analysis of cAMP response element binding protein occupancy, phosphorylation and target gene activation in human tissues. Proc Natl Acad Sci USA 102: 4459–4464. Zhang H, Fu Y, Altier C, Platzer J, Surmeier J, Bezprozvanny I, 2006. Cav1.2 and Cav1.3 neuronal calcium channels: differential targeting and signaling to pCREB. Eur J Neurosci 23: 2297–2310. Zhen W, Stoltefuss J, Goldmann S, Triggle DJ, 1992. Pharmacological and radioligand binding studies of 1,4-dihydropyridines in rat cardiac and vascular preparations: stereoselectivity and voltage-dependence of antagonist and activator interactions. Mol Pharmacol 41: 535– 541. Zhou X, Ono H, Ono Y, Frohlic E, 2002. N- and L-type calcium channel antagonist improves glomerular dynamics, reverses nephrosclerosis, and inhibits apoptosis and proliferation in an LNAME/SHR model. J Hypertens 20: 993–1000. Zhulke RD, Bouron A, Soldatov NM, Reuter H, 1998. Ca2þ channel sensitivity towards the blocker isradipine is affected by alternative splicing of the human a1C subunit gene. FEBS Lett 42: 220–224.

2 VOLTAGE-GATED N-TYPE AND T-TYPE CALCIUM CHANNELS AND EXCITABILITY DISORDERS ELIZABETH TRINGHAM1 1 2

AND

TERRANCE P. SNUTCH1,2

Neuromed Pharmaceuticals, Rm 301, 2389 Health Sciences Mall, Vancouver, BC, Canada Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada

2.1 INTRODUCTION Calcium (Ca2þ) is a ubiquitous signaling molecule involved in a diverse array of cellular processes ranging from control of membrane excitability to gene transcription, all of which require that intracellular levels be tightly regulated. In particular, voltage-gated Ca2þ channels (Cav) regulate transmembrane fluxes of calcium in response to membrane depolarization. Ca2þ channel currents can generally be grouped into two major classes of Cav channels: high voltage activated (HVA) and low voltage activated (LVA or “T-type”). Based on their pharmacological and biophysical properties, the HVA class can be further subdivided into L-, N-, P/Q-, and R-type channels. The main pore-forming a subunits of the Cav channels are encoded by 10 different genes, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1I, and CACNA1S, with a greater variety of subtypes for each class arising from a substantial degree of alternative splicing. Associated with the pore-forming subunit of HVA channels are several accessory subunits, a2d (four genes in mammals), b (four genes in mammals), and g (up to eight genes in mammals), although the precise complement is not well determined biochemically for all of the HVA channel types. Further, HVA channel Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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FIGURE 2.1 Biophysical properties of high-voltage-activated Cav channels and T-type channels (LVA). (a) Voltage dependence of activation and inactivation of T-type channels is hyperpolarized compared to HVA channels. T-type channels inactivate faster than HVA channels (b), but deactivate (close) more slowly upon repolarization of the membrane potential (c). Adapted from Snutch, 2008.

complexity occurs as a result of the interaction between the main pore-forming HVA Cav subunits and various alternatively spliced forms of the accessory subunits. This complexity in the molecular makeup underlying the Cav channel composition creates a daunting assortment of functional diversity in the physiological roles of Cav channels. As progress has been made in assigning physiological roles to particular Cav channel complexes, it has also unveiled their contributions to numerous pathophysiological conditions beyond the classically defined role of L-type channels in hypertension, angina, and cardiac arrhythmias. More recent interest has focused on understanding the roles of N- and T-type channels, which despite having quite distinct biophysical and pharmacological properties (Fig. 2.1) appear to contribute to the pathophysiology of disorders that are characterized by changes in neuronal excitability. Biophysically, Ntype channels activate at higher potentials than T-type channels, exhibit slower kinetics of activation and inactivation, and recover more slowly from inactivation but deactivate more quickly. Three different genes, CACNA1G, CACNA1H, and CACNA1I, encode the pore-forming subunits of Cav3.1, Cav3.2, and Cav3.3 T-type channels, respectively, whereas N-type (Cav2.2) channels are encoded by a single gene, CACNA1B. Pharmacologically, to date there are no reported subtype-selective T-type channel blockers, which has limited the characterization of these channels in vivo. In contrast, selective peptide blockers with nanomolar affinity to N-type channels are available, providing insight into their roles in neuronal physiology. Despite some pharmacological limitations, the combination of available agents together with molecular genetic animal model and limited clinical data also provide valuable insight into the pathophysiological roles in which both N- and T-type channels are involved.

2.2 PATHOPHYSIOLOGY OF PAIN Major advances in the field of pain have expanded our current understanding of the mechanisms underlying different pain states as well as some of the complexities of the signaling pathways involved in the processing of painful stimuli. One concept that has emerged as a common theme among different pain states, whether arising from neuropathic or inflammatory pain, is the spontaneous and persistent repetitive activation of primary afferent neurons (Kajander and Bennett, 1992; Kajander et al., 1992;

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McLachlan et al., 1993; Devor et al., 1994; Petersen et al., 1996; Ramer et al., 1997; Song et al., 1999; Zhang et al., 1999). These changes in excitability result in exaggerated transmitter release and enhanced postsynaptic excitability of neurons in the dorsal horn, which in turn leads to central sensitization or “windup.” Whether pain arises from damage of tissue as a result of sensitization of sensory terminals by peripheral chemical mediators or nerve damage as in neuropathic pain, both lead to maladaptive changes in central neuronal excitability and hence to behavioral phenotypes such as hyperalgesia and allodynia. Although a number of transmitters are released by primary afferent neurons, at the core of central sensitization is the excitatory neurotransmitter glutamate, the release of which from C and Ad fibers is controlled by Cav channels (Fig. 2.2). During inflammation and neuropathic pain states, both somatic and presynaptic Ca2þ channel activities are elevated leading to an increase in ectopic discharges and

FIGURE 2.2 Primary afferent nociceptive pathway. Following stimulation of primary afferent Ad and C fibers, second-order neurons in the spinal dorsal horn are activated and sensitized by neurotransmitters released into the presynaptic cleft. The second-order neurons ascend as part of the spinothalamic tract where they synapse onto third-order neurons in the thalamus that radiate to the somatosensory cortex where pain is perceived. A subset of neurons transmits proprioceptive information via the dorsal column. N-type calcium channels at primary afferent terminals in the spinal dorsal horn trigger the release of excitatory (glutamate) and modulatory (substance P and CGRP) transmitters onto second-order neurons. In the primary afferent pathway, T-type calcium channels are concentrated at both free nerve endings and DRG cell bodies. Both N-type and T-type calcium channels are also found in second-order spinal neurons as well as in higher brain regions such as the thalamus and cortex. Adapted from Hildebrand and Snutch, 2006.

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glutamate release. It is this increase that results in the activation of glutamate receptors, specifically NMDA receptors through voltage-dependent Mg2þ unblock, which then prolongs neurotransmission and leads to “windup” in spinal nociceptive neurons of the dorsal horn. Further, as Cav channels are also known to fine-tune neuronal Ca2þ dynamics and hence shape action potential waveforms and control firing frequency, they present an interesting opportunity concerning nociceptive therapies. 2.2.1

Role of N-Type Channels in the Pathophysiology of Pain

Since native N-type channels were first identified more than 20 years ago in sensory chick dorsal root ganglion (DRG) using electrophysiology techniques (Nowycky et al., 1985), RNA expression studies have revealed that they are exclusively expressed in neurons of both the central and peripheral nervous systems and in neurally derived cells such as neuroendocrine cells (Dubel et al., 1992). Immunohistochemical studies show that N-type channels are distributed at the subcellular level along dendrites and cell bodies of many neurons and also at a subset of nerve terminals (Westenbroek et al., 1992). Their pivotal presynaptic location suggests that they are likely to be directly involved in synaptic transmission by controlling transmitter release and hence processing of sensory information Koyano et al., 1987. Indeed, the release of neuropeptides from the spinal cord, such as calcitonin gene-related peptide and substance P, is coupled to N-type channels (Holz et al., 1988; Maggi et al., 1990; Santicioli et al., 1992; Evans et al., 1996; Smith et al., 2002). Further, utilizing the peptide N-type Cav blocker w-conotoxin GVIA (w-CTx-GVIA, see below), the direct involvement of N-type channels in regulation of glutamatergic synaptic transmission, a major transmitter in afferent A and C fibers, has been demonstrated between primary afferent neurons and the spinal cord (Gruner and Silva, 1994). Following nerve injury, some inconsistencies have been noted with regard to changes in the expression of N-type channels. For example, N-type immunoreactivity increases in both the small dorsal root ganglion and lamina II of the spinal cord following chronic nerve compression injury (Cizkova et al., 2002), while no change in Cav2.2 mRNA levels is apparent in L5/L6 in a nerve ligation model of neuropathic pain (Luo et al., 2001). In other studies, whole-cell electrophysiological recordings of DRGs following nerve ligation or fura-2 microfluorometry of DRGs from axotomized neurons both demonstrate a reduction in the w-CTx-GVIA-sensitive N-type current (Baccei and Kocsis, 2000; Fuchs et al., 2007). While biochemical evidence implicates N-type channels in the pathophysiology of pain, the precise sensory modalities that N-type channels regulate have been elucidated by peptidic subtype-selective N-type channel blockers. Toxins derived from the venoms of cone snails and hunting spiders have been shown to be potent pain relievers in various animal models of neuropathic and inflammatory pain. One such toxin, w-CTx-GVIA, is derived from the venom of the cone snail, Conus geographus. At nanomolar concentrations, w-CTx-GVIA is a selective and irreversible blocker of N-type channels that acts by occluding the pore of the channel (Olivera et al., 1984, 1987; Wagner et al., 1988; Boland et al., 1994; Feng et al., 2003). Early evidence for the involvement of N-type channels in nociceptive

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responses was demonstrated by autoradiographic studies showing that radiolabeled w-CTx-GVIA binds in a highly localized manner in both the soma and presynaptic terminals of both myelinated and unmyelinated nociceptive primary afferent neurons at the level of superficial laminae I and II of the dorsal spinal cord (Kerr et al., 1988; Gohil et al., 1994). Intrathecal application of w-CTx-GVIA attenuates tactile allodynia and thermal hyperalgesia in the chronic constriction injury, partial sciatic nerve injury, and vincristine models of sensory peripheral neuropathy (Xiao and Bennett, 1995; Yamamoto and Sakashita, 1998; Scott et al., 2002; Fukuizumi et al., 2003a). In the models of inflammatory pain in rats, w-CTx-GVIA reduces hyperalgesia induced by intraplantar injection of carrageenan (Yokoyama et al., 2003) and attenuates the algogenic response of bradykinin and a,b-methylene ATP when injected intraplantarly into mice (Kato et al., 2002). Antinociceptive effects have also been reported with w-CTx-GVIA in the formalin (Murakami et al., 2001, 2004) and capsaicin inflammatory pain models (Sluka, 1997), both of which activate unmyelinated C fibers (Dickenson et al., 1987; Winter et al., 1995; McCall et al., 1996). In a postoperative rat pain model, intrathecal administration of w-CTxGVIA also dose-dependently attenuates incision-induced allodynia (Cheng et al., 2006). In contrast, responses to intrathecal administration of w-CTx-GVIA have provided mixed results in the acute thermal tail-flick antinociception test, with some groups reporting no change in the basal tail-flick latencies (Suh et al., 1997; Scott et al., 2002) while others finding dose-dependent increases in tail-flick latencies in rats and mice (Omote et al., 1996; Lia and Prado, 1999; Fukuizumi et al., 2003a). Spinal nociceptive responses to acute peripheral mechanical stimulation have also implicated N-type channels in increasing threshold responses in the tail and paw pressure tests (Omote et al., 1996; Fukuizumi et al., 2003a). Considering the electrophysiological properties of the neurons involved in sensory signaling, it is interesting to note the commonality among different pain states such as neuropathic and inflammatory pain, namely, that both lead to spontaneous activity in primary afferent fibers (Wall and Devor, 1983; Babbedge et al., 1996; Boucher et al., 2000; Liu et al., 2000; Wu et al., 2001; Djouhri et al., 2006; Rojas-Piloni et al., 2007). This spontaneous activity originating in the peripheral afferents is thought to mediate maladaptive changes in the central nervous system (CNS) and is implicated in sensory abnormalities, including hyperalgesia and allodynia. This peripheral afferent signal is transmitted with a high degree of fidelity to the CNS and studies have shown that Ca2þ influx through N-type channels is critical to both sensitization and plasticity. Extracellular electrophysiological recordings within dorsal horn neurons following spinal nerve ligation show that increased neuronal excitability elicited by application of mechanical, thermal, or electrical stimulation in rats is dose-dependently reduced by application of w-CTx-GVIA to the spinal cord (Matthews and Dickenson, 2001b). In spinal cord neurons that have become excitable following intraarticular injection of either mustard oil or kaolin and carrageenan, w-CTx-GVIA reduces neuronal excitability in both models suggesting a prominent role of N-type channels in afferent C-fiber-mediated hyperexcitability of spinal neurons (Neugebauer et al., 1996; Nebe et al., 1998). Intradermal application of formalin induces a biphasic

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(phase I and phase II) increase in activity in C fibers that corresponds to the biphasic behavioral response. In agreement with its ability to suppress nociceptive behavior, w-CTx-GVIA reduces the spontaneous action potential firing rate in both phases (Dickenson and Sullivan, 1987; McCall et al., 1996; Diaz and Dickenson, 1997). Antinociceptive responses have also been reported for two other N-type channel preferring toxins, w-conotoxin CVID (w-CTx-CVID) and w-conotoxin MVIIA (w-CTx-MVIIA), both of which act by physically occluding the pore of Cav2.2 channels at nanomolar concentrations. w-CTx-MVIIA, derived from Conus magus (Olivera et al., 1985, 1987), reduces the neuropathy-induced allodynia following spinal nerve ligation (Bowersox et al., 1996; Urban et al., 2005) as well as thermal hyperalgesia in both a chronic constriction injury and a partial sciatic nerve injury model (Yamamoto et al., 1998). Mechanical hyperalgesia, assessed following inflammation induced by intraplantar injection of Freund’s complete adjuvant, is also dose-dependently reduced following intrathecal administration of w-CTx-MVIIA (Bowersox et al., 1996; Wang et al., 1998). Interestingly, in the formalin test, both phase I (acute phase) and phase II (tonic phase) are suppressed by w-CTx-MVIIA at doses that are 1000-fold more potent than morphine (Malmberg and Yaksh, 1994, 1995; Bowersox et al., 1996). Similarly, w-CTx-CVID, derived from Conus catus (Lewis et al., 2000; Nielsen et al., 2000), reduces mechanical and tactile allodynia in a spinal nerve ligation model (Scott et al., 2002; Blake et al., 2005) as well as being antinociceptive in the rat Freund’s complete adjuvant model (Smith et al., 2002). Acute nociceptive responses in the rat tail-flick model appear unaffected by either wCTx-CVID or w-CTx-MVIIA (Scott et al., 2002; Blake et al., 2005). Following continuous intrathecal infusion with w-CTx-MVIIA, however, a significant reduction in the response to the hot plate is observed (Malmberg and Yaksh, 1995). Of interest is the observation that although the three subtype-selective w-conotoxins have similar affinity for the N-type channel, they are different in their rank order of potency in attenuating neuropathic pain (Scott et al., 2002). w-CTx-GVIA is the most potent with a three- to fourfold higher potency than w-CTx-MVIIA and w-CTx-CVID, but conversely w-CTx-CVID provides the greatest therapeutic margin, as animals display fewer adverse effects such as serpentine tail movements and body shaking. While this may possibly be explained by actions at off-target sites in the CNS other than N-type channels, as these effects are not noted in the Cav2.2 knockout mice, they support the notion that conotoxins have preferential affinities for different variants of N-type channels. For example, Adams et al. (2003) found that while transmitter release at preganglionic nerve terminals is regulated by N-type channels, w-CTx-CVID but not w-CTx-MVIIA blocks transmitter release. To address chronic pain conditions in patients refractory to current therapies, including morphine, the administration of a synthetic version of w-CTx-MVIIA, currently marketed as Prialt, has recently become available. Prialt was approved by the Food and Drug Administration (FDA) in 2005 to treat severe chronic cancer or AIDS pain and in some cases has resulted in complete or near-complete pain relief in patients whose symptoms were previously unmanageable even by intrathecal morphine (Brose et al., 1997; Mathur, 2000; Staats et al., 2004; Wallace, 2006). These results indicate that the animal models of pain used to evaluate the role of N-type

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channels in somatosensory processing of pain were highly predictive of human clinical outcome. The lack of tolerance of Prialt in humans was also predicted in animal models of pain as continuous intrathecal infusion of w-CTx-MVIIA in rats minimally developed tolerance after 7 days (Malmberg and Yaksh, 1995). Side effects with Prialt clinical use have been reported and can be present in the form of hallucinations, cognitive impairment, and alterations in mood and consciousness, thereby providing the opportunity for improved N-type channel blockers that more specifically target those channels resident in sensory pathways (Penn and Paice, 2000; Snutch, 2008). In support of in vivo behavioral tests using pharmacological tools to assign sensory modalities regulated by N-type channels, mice strains in which the Cav2.2 N-type has been genetically removed have for the most part substantiated the results using w-conotoxins. The data from three knockout strains from different laboratories clearly show the involvement of the N-type channels in the development of allodynia and hyperalgesia associated with neuropathic and inflammatory pain (Kim et al., 2001; Saegusa et al., 2001, 2002). However, for acute pain, the role of N-type channels is somewhat ambiguous as results are inconsistent among the labs. Interestingly, the three independent Cav2.2-deficient mouse strains exhibit surprisingly few deleterious effects despite the wide CNS distribution and known role of N-type channels in synaptic transmission. The most marked behavioral alteration related to CNS function is decreased anxiety, whereas in the autonomic nervous system, sympathetic nerve dysfunction is observed as a reduction in the baroreflex and elevated heart rate and blood pressure (Ino et al., 2001). As the assortment of splice variants encoding Cav channels is being unraveled, the precise alternatively spliced isoform of Cav2.2 expressed in sensory neurons has recently unfolded (see Lipscombe et al., Chapter 3 in this volume). A Cav2.2 splice isoform containing exon 37a is preferentially expressed in capsaicin receptorexpressing nociceptive neurons whereas the exon 37b variant is ubiquitously expressed (Bell et al., 2004). Of particular interest are the recent findings by Altier and colleagues showing that when splice isoform-specific small interfering RNA (siRNA) against exon 37a Cav2.2 channels is injected intrathecally, this results in a reduction in both thermal and mechanical hyperalgesia in inflammatory and neuropathic pain models, as well as in basal thermal nociception. Tactile neuropathic allodynia is, however, equally mediated by exon 37a- and 37b-containing neurons (Altier et al., 2007). In addition to these in vivo findings is the identification of a distinct mode of G-protein-mediated voltage-independent inhibition unique to Cav2.2e[37a] (Raingo et al., 2007). This is in contrast to the well-defined voltage-dependent G-protein inhibition of Cav2.2 channels promoted by the liberation of Gbg from Gi/o heteromers following agonist–receptor binding (i.e., morphine) (Herlitze et al., 1996; Ikeda, 1996). Although both exon 37a- and 37b-containing Cav2.2 splice isoforms undergo varying degrees of voltage-dependent inhibition, voltage-independent inhibition is only present in the Cav2.2 channels containing the 37a variant. The signaling pathway coupling G-protein-coupled receptors (GPCRs) to voltage-independent inhibition of Cav2.2e[37a] is mediated by the Ga subunit with downstream activation of a tyrosine kinase, which phosphorylates a single tyrosine residue (Y1747) in exon 37a. The

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physiological significance that has been speculated of this bifurcating pathway is that it maximizes the efficiency of inhibition by using both Ga and Gbg subunits from Gi/o heteromers, while simultaneously providing adaptive changes to Ca2þ influx depending on the level of neuronal activity (Ikeda and Dunlap, 2007). This will no doubt stimulate interest in generating a Cav2.2e[37a]-selective blocker that might have an increased therapeutic index while narrowing the risk of unwanted side effects. 2.2.2

Role of T-Type Channels in the Pathophysiology of Pain

Despite being identified in rat and chick dorsal root ganglion over 20 years ago (Carbone and Lux, 1984a, 1984b; Nowycky et al., 1985), a prominent role of T-type channels in the pathophysiology of pain has only recently emerged. This has been attributed in part to the discovery of three distinct subunit genes, CACNA1G (Cav3.1), CACNA1H (Cav3.2), and CACNA1I (Cav3.3), that encode T-type channels, as well as the existence of several classes of nonselective T-type channel blockers that have been used to tease out physiological and pathophysiological roles. In comparison to N-type channels, T-type calcium channels are much more widely expressed. T-type calcium channel currents are found in many tissues and cell types, including both central and peripheral neurons, heart, adrenal glands, kidney, smooth muscle, embryonic skeletal muscle, pituitary, pancreas, retina, and testes (PerezReyes, 2003). In the somatosensory pathway, the expression of T-type channels is localized to the dorsal root ganglion, dorsal horn spinal neurons, and the thalamus. In the dorsal root ganglion neurons, characterization of these primary afferents using electrophysiological techniques determined that T-type channels are exclusively expressed in small- and medium-sized DRG neurons but are essentially devoid of expression in the large DRG neurons (Schroeder et al., 1990; Scroggs and Fox, 1992; Shin et al., 2003). More recently, a novel subset of small capsaicin-sensitive DRG neurons called “T-rich” neurons has been identified that express only T-type channels with little contribution of the calcium conductance attributed to HVA channels (Nelson et al., 2005). Generally, the nociceptive C and Ad fibers are considerably smaller than Aa and Ab fibers that conduct primarily proprioceptive and tactile information (Harper and Lawson, 1985a, 1985b), which supports the contribution of T-type channels in the pathophysiology of pain. Of the three genes known to encode T-type channels, in situ hybridization and reverse-transcription PCR studies have identified the Cav3.2 subtype as the most prominent subtype in DRG neurons, whereas Cav3.3 expression is lower and Cav3.1 is virtually undetected in small to medium DRGs (Talley et al., 1999; Bourinet et al., 2005). In another subset of medium-sized DRG neurons, the Cav3.2 T-type channel is found to be present in D-hair cell mechanoreceptors (Shin et al., 2003). At the level of the spinal cord, in situ hybridization studies have demonstrated that the dorsal horn neurons in the superficial lamina also express Cav3.2 (Talley et al., 1999). In contrast to the predominant role of the Cav3.2 subtype of T-type channel in the primary and secondary order neurons of the somatosensory pathway, the thalamus expresses all three subtypes, albeit differentially expressed in subsets of thalamic neurons. Cav3.1 is predominantly expressed in

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the thalamocortical (TC) neurons, which relay information from the periphery to the cortex, and Cav3.2 and Cav3.3 are found in the reticular thalamic neurons that provide inhibitory input to TC neurons (Talley et al., 1999). In the absence of subtype-selective blockers for Cav3.1-, Cav3.2-, or Cav3.3containing T-type channels, knockdown and knockout studies have provided the greatest insight into the sensory modalities conveyed by the individual isoforms. Intrathecal injection of antisense oligonucleotides against each subtype identified Cav3.2 T-type channel as having a major pronociceptive role, whereas antisense directed at Cav3.1 and Cav3.3 did not significantly affect the nociceptive behavioral responses in rats (Bourinet et al., 2005). Cav3.2 T-type channels have effects on both acute and chronic pain as knockdown of Cav3.2 results in antiallodynic and antihyperalgesic effects in rats with a chronic constriction nerve injury and is antinociceptive in na€ıve rats when a noxious pressure is applied to the paw or a noxious thermal stimulus is applied to the tail. Concomitantly, whole-cell recordings reveal that T-type currents are reduced by 70–90% in small- to medium-sized DRGs, following a 50% reduction in Cav3.2 mRNA levels. In contrast, in mice lacking the Cav3.2 subtype of T-type Ca2þ channel, thermal hyperalgesia and mechanical allodynia are not reduced following spinal nerve ligation (Choi et al., 2007). Responses to acute pain, such as noxious mechanical (tail clip) and thermal (tail flick and hot plate) stimuli in na€ıve mice, are, however, reduced in Cav3.2deficient mice as are responses to intradermal capsaicin and formalin and visceral injections of acetic acid and MgSO4, both of which induce abdominal writhing. Although the role for Cav3.1 channels in processing of sensory inputs is predicted to be minimal as expression levels in the DRGs are extremely low or undetected, Cav3.1 knockout mice are unexpectedly found to have increased visceral pain response when induced by intraperitoneal injections of acetic acid or MgSO4 (Kim et al., 2003). In addition to increased visceral pain responses in the mutant mice, there are also changes in the firing patterns in the ventroposterolateral (VPL) thalamocortical neurons. Administration of acetic acid in wild-type mice increases both single spikes and bursts of spike activity in VPL neurons, but in the Cav3.1 knockout mice, only the frequency of the single spikes increases. However, the burst activity is not observed either prior to or following intraperitoneal administration of acetic acid in Cav3.1 knockout mice. This change in pattern of firing is likely to decouple the TC neurons from efficiently activating neurons in the nRT, which in turn provide the inhibitory feedback to the VPL neurons to induce burst firing. By preventing burst firing, the “sensory gate” is removed and sensory signals are able to reach the somatosensory cortex. This antinociceptive role of the thalamus is not thought to be effective at controlling acute pain responses, which are mediated by local reflexes in the periphery. Supporting this notion that Cav3.1 does not contribute to acute pain pathways is a lack of any difference in pain responses to thermal (tail flick and paw withdrawal) or mechanical stimuli (von Frey) in both Cav3.1 mutant and wild-type littermates. However, in the complete Freund’s adjuvant model of chronic inflammation, a model that involves central sensitization, hyperalgesia scores also do not differ in wild-type and mutant mice following intraplantar injection.

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Although there are no high-affinity subtype-selective T-type channel blockers available to support in vivo knockout and knockdown studies, there are nonselective compounds known to inhibit these channels, which have provided insight into the role of T-type channels in processing nociceptive signals. Ethosuximide, a known antiepileptic drug, inhibits cloned Cav3.1, Cav3.2, and Cav3.3 (0.3–1 mM) at therapeutic plasma levels (0.3–0.7 mM; Gomora et al., 2001) and also dosedependently suppresses both thermal hyperalgesia and mechanical allodynia following L5/L6 spinal nerve ligation when administered i.p. (Dogrul et al., 2003). This is in contrast with the lack of effect seen when ethosuximide is injected intrathecally, suggesting a peripheral mechanism of action. Similarly, no effect is seen when injected intraplantarly into the injured paw, suggesting that ethosuximide likely acts in the periphery at the level of the DRGs and not at sensory nerve endings. In other neuropathy models, paclitaxel- and vincristine-induced models of neuropathy, ethosuximide injected i.p. produces significant reversal of mechanical allodynia and hyperalgesia (Flatters and Bennett, 2004). Barton et al. 2005 also report antinociceptive effects of ethosuximide in both the early and late stages of the formalin test and in the acute tail-flick test as well as reversal of capsaicin-induced mechanical allodynia following i.p. injection. In contrast, intrathecal administration of ethosuximide fails to affect the nociceptive responses in the rat formalin test (Cheng et al., 2007). In addition to the behavioral studies, during in vivo recordings from single neurons in the dorsal horn in which ethosuximide is directly applied to the spinal cord, the responses of the dorsal horn neurons to mechanical and thermal stimuli were suppressed in sham-operated and spinal nerve-ligated animals (Matthews and Dickenson, 2001a). Two other antiepileptic drugs that are structurally diverse from ethosuximide and for which T-type inhibition has been reported are zonisamide and phenytoin. In cultured neurons, 50 mM zonisamide inhibits 40% of the T-type current in neuroblastoma cells (Kito et al., 1996) and 60% at 500 mM in cerebral cortex neurons (Suzuki et al., 1992). Interestingly, this compound suppresses neuropathy-induced thermal hyperalgesia, but not mechanical allodynia, in the Bennett chronic constriction rat model (Bennett and Xie, 1988; Hord et al., 2003) and is reported to provide relief in humans with refractory neuropathic pain (Guay, 2003; Takahashi et al., 2004) as well as prophylaxis for migraine patients (Drake et al., 2004). Phenytoin, which is used clinically in humans to treat neuropathic pain (McCleane, 1999; Finnerup et al., 2005), blocks Cav3.1 and Cav3.2 (140 and 8.3 mM, respectively; Todorovic et al., 2000) expressed in human embryonic kidney (HEK) cells, as well as native T-type channels in DRG neurons (IC50 ¼ 8.3 mM) and N1E-115 neuroblastoma (3–100 mM; Matsuki et al., 1984; Twombly et al., 1988). Though not studied in wide variety of animal models, phenytoin dose-dependently reduces bradykinin-induced pain when applied subcutaneously (Foong and Satoh, 1983); however, in acute models, i.p. administration of phenytoin is superior at reducing thermal pain in pawwithdrawal test than mechanical pain as measured in the tail-pressure test (Sakaue et al., 2004). Some of the most potent T-type channel blockers are comprised of neuroleptics used to treat a variety of psychiatric disorders, including schizophrenia, Tourette’s

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disorder, and obsessive-compulsive disorder. In particular, pimozide inhibits in a nonpreferential manner Cav3.1, Cav3.2, and Cav3.3 with IC50 values of 50 nM (Santi et al., 2002). Although not extensively tested in a variety of pain models, pimozide when administered at low doses in the mouse formalin test is not highly efficacious (Saddi and Abbott, 2000), yet in humans it suppresses the pain associated with trigeminal neuralgia (Lechin et al., 1989; Green and Selman, 1991). Another compound well known for its inhibition of the T-type channels as well as HVA channels (P/Q-, R-, and L-types) is mibefradil, which inhibits cloned and native T-type channels with IC50 values ranging from 0.1 to 4.7 mM depending on the assay conditions and cell type (Jimenez et al., 2000; Martin et al., 2000). In contrast to ethosuximide, when mibefradil is administered both intraperitoneally and injected directly into the injured limb of rats, it effectively reduces the tactile allodynia induced by spinal nerve ligation of L5/L6 (Dogrul et al., 2003). A reduction in thermal hyperalgesia is also reported following i.p. administration. Of relevance is the fact that mibefradil does not cross the blood–brain barrier when administered systemically and also does not suppress neuropathic behaviors upon direct i.t. administration, again suggesting a peripheral mechanism of action (Ertel et al., 1997). In the capsaicin assay, the results were mixed in that systemic (i.p) administration has no effect on allodynia, yet when injected intracisternally (i.c.), capsaicin-induced mechanical allodynia is dose-dependently reduced (Barton et al., 2005). In a postoperative pain model involving an incision into the paw, no antiallodynic action is observed following i.t. administration of mibefradil (Cheng et al., 2006). However, mibefradil inhibits both phases of the formalin response when injected i.p. or i.t., but is ineffective in suppressing acute nociceptive responses in the tail-flick reflex when administered i.p. (Barton et al., 2005; Cheng et al., 2007). Also noted is a lack of effect on an acute nociceptive stimulus (tail-flick test) when mibefradil is administered following i.t. administration (Dogrul et al., 2001; Barton et al., 2005). Another class of compounds that inhibit native T-type channels in DRG neurons is 5a-reduced neuroactive steroids, a class of compounds also known to potentiate GABAA ligand-gated channels. Of the various 5a-reduced neuroactive steroids examined, (3b,5a,17b)-17-hydroxyestrane-3-carbonitrile (ECN) was identified as inhibiting T-type channels in DRGs (IC50 300 nM) but being devoid of effects on GABAA receptors (Todorovic et al., 1998). To assess the peripheral role of T-type channels in nociceptive processing of acute noxious thermal stimulus to the paw, ECN injected intradermally dose-dependently produces analgesia as seen by the increase in latency to paw withdrawal (Pathirathna et al., 2005a). In rats with neuropathic pain (Bennett and Xie, 1988), intradermal injection of ECN alleviates both thermal and mechanical hyperalgesia in ligated rats and thermal and mechanical nociception in sham-operated rats (Pathirathna et al., 2005b). This antinociceptive effect on the thermal stimulus in ligated and sham-operated rats is not reversed by bicuculline, a GABAA antagonist, suggesting that the effect is most likely due to T-type channel inhibition. A novel role of peripheral T-type channels in amplifying nociceptive signals was unveiled when the effects of redox agents were examined on cloned and native T-type

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channels. Todorovic et al. (2001) found that the endogenous reducing agent, L-cysteine, augments T-type currents by 130% in small DRG neurons and in recombinant Cav3.2 channels by a similar amount and is reversed by the oxidizing agent 5,50 -dithio-bis-(2-nitrobenzoic acid) (DTNB). The effects are mimicked by the reducing agent dithiothreitol (DTT). Conversely, the oxidizing agent DTNB, when applied alone, inhibits the peak T-type calcium channel current in small DRG neurons and Cav3.2 channels expressed in HEK cells, both by about 50%. When applied intradermally into the ventral side of the paw, the reducing agents decrease pawwithdrawal latencies (PWLs), while the oxidizing agent prolongs PWLs. Cysteine also produces a hyperalgesic response to noxious mechanical stimulus, while DTNB is analgesic and reverses the effect of L-cysteine. In providing further evidence that T-type channels boost nociceptive signals, mibefradil reverses the thermal hyperalgesic response of L-cysteine and DTT and enhances the analgesic effect of DTNB. Similarly, reducing cysteine analogues, L-cysteine, D-cysteine, and D,L-homocysteine, induce potent dose- and time-dependent hyperalgesia and conversely endogenous oxidizing cysteine analogues, L-cysteine, D-cysteine, and D,L-homocysteine, induce potent dose- and time-dependent analgesia in an acute model of thermal peripheral nociception in intact rats (Pathirathna et al., 2006). In a neuropathic pain model induced by chronic constriction of the sciatic nerve, both L-cysteine and DTT increase the thermal hyperalgesic response in both nerve-injured and sham-operated rats, while DTNB reduces the neuropathy-induced thermal hyperalgesia and produces analgesia in sham-operated rats (Todorovic et al., 2004). Both DTNB and mibefradil are able to abolish the L-cysteine-induced increase in thermal hyperalgesia rats with neuropathic pain and in sham-operated rats. At the cellular the level, the mechanisms underlying changes in neuronal excitability that lead to the development of allodynia and hyperalgesia are not well understood (Kajander and Bennett, 1992; Kajander et al., 1992; McLachlan et al., 1993; Devor et al., 1994; Petersen et al., 1996; Ramer et al., 1997; Song et al., 1999; Zhang et al., 1999.). Recent molecular and behavioral pharmacology data have provided a strong linkage for the contribution of T-type channels to the pathological perceptions of pain, but the biophysical properties of T-type channels themselves provide even further evidence that they contribute to hyperexcitability observed in DRGs after peripheral trauma or inflammation. Under normal physiological conditions, T-type channels are capable of regulating the pacemaker activity of neurons, contributing to rebound burst firing and oscillatory behavior and hence making them likely contributors to the hyperexcitability of primary afferent neurons in neuropathic and inflammatory pain states. One of the first studies investigating the involvement of T-type channels in processing sensory information was by White et al. (1989) who reported that Ca2þ influx by LVA channels produces an afterhyperpolarizing potential in dorsal root ganglion that triggers a burst of action potentials, which is abolished by 100 mM Ni2þ. Supporting evidence using the “Trich” subset of DRGs that express a high density of T-type channels showed that the endogenous redox agent, L-cysteine, which promotes an increase in the current amplitude and shifts the gating properties, also lowers the nociceptor excitability threshold and induces burst firing (Nelson et al., 2005). Also in the D-hair

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mechanoreceptor neurons, a role of Cav3.2 T-type channels in inducing a slow depolarization has been identified, which leads to a lowering of the voltage threshold for the generation of action potentials and hence an increase in neuronal excitability (Dubreuil et al., 2004). In a streptozotocin model of diabetic neuropathy, the T-type currents were shown to have increased by twofold in medium-sized DRGs with a concomitant depolarizing shift in the steady-state inactivation, which results in a greater “window current,” suggesting that T-type channels can activate more readily at physiological membrane potentials (Jagodic et al., 2007). Interestingly, Jagodic et al. also identified a newly expressed T-type channel that only partially inactivates at a membrane potential of 40 mV, a voltage that completely inactivates the T-type channels in DRGs from control rats, and likely contributes to the reduced threshold for burst firing in DRGs neurons from diabetic rats. In contrast, T-type channel currents in DRGs were reduced after neuropathic injury was induced by chronic constriction of the sciatic nerve or axotomy of L5 neurons in rats (McCallum et al., 2003; Hogan, 2007). Also observed in these DRGs following the induction of neuropathic pain is an increase in the rate of deactivation, which also supports the loss of T-type channels that deactivate slowly in a voltagedependent manner. As the ability of neurons to fire repetitively requires that there is sufficient influx of Ca2þ to activate Ca2þ-activated Kþ channels that in turn hyperpolarize the cell, a reduction in Ca2þ at the entry during the slow deactivation mode at the end of an action potential may contribute to increased excitability of the DRGs from injured rats. Ultimately it will be interesting to determine whether the ability of T-type channel blockers to provide pain relief is dependent solely on their affinity of inhibiting Cav3.2 versus Cav3.1 and Cav3.3 channels or some combination thereof. Although the available evidence is suggestive of a peripheral mechanism of action, whether the efficacy is enhanced by the CNS-penetrant compounds also needs to be determined. Given the critical ability of Cav3.1 to alter the sensory gate in the thalamus, the effects of modulators on these central channels, which remain to be fully elucidated, may be quite important.

2.3 CONTRIBUTIONS OF T-TYPE CHANNELS TO THE PATHOPHYSIOLOGY OF EPILEPSY Epilepsy is a complex disorder of spontaneous recurrent seizures characterized by neuronal hyperexcitability leading to hypersynchronization of neural networks. Phenotypically, seizures present themselves in different forms depending on the site of origin and subsequent recruitment of additional CNS structures. Although the cellular substrate(s) underlying the genesis of seizure activity are largely unknown, genetic, pharmacological, and physiological evidence all implicate an involvement of T-type calcium channels. The involvement of T-type channels in seizure disorders is primarily implicated in idiopathic generalized epilepsies (IGEs) with evidence arising from studies using transgenic and mutant animals as well as population analysis studying linkages to genetic mutations in humans.

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Of the many IGEs, T-type channels have been shown to be critical for seizure activity in absence epilepsy. Absence epilepsy is characterized by a brief and sudden loss of consciousness, which is temporally correlated with 3 Hz bilateral synchronous spike-wave discharges (SWDs) involving the thalamocortical circuitry (Williams, 1950; Niedermeyer and Primary, 1996). Specifically, rebound burst firing in neocortical cells, thalamic reticular neurons, and thalamocortical relay neurons is known to be evoked by low-threshold Ca2þ potentials, which are thought to give rise to SWDs (Llinas and Jahnsen, 1982; Deschênes et al., 1984; Jahnsen and Llinas, 1984; Coulter et al., 1989a; Huguenard and Prince, 1992; De la Pena and Geijo-Barrientos, 1996; Destexhe and Sejnowski, 2002). In rats, the mRNAs of all three T-type isoforms are expressed in the thalamocortical pathway, though differentially in thalamus and neocortex (Talley et al., 1999). Cav3.2 and Cav3.3 are expressed in the thalamic reticular nucleus, whereas Cav3.1 is dominantly expressed in the thalamocortical neurons. More recently, splice variants of Cav3.1 have been differentially localized in the thalamic circuitry, which may account for the divergent burst ﬁring in TC and GABAergic interneurons (Broicher et al., 2007a). In the neocortex, Cav3.1 and Cav3.3 mRNA levels are diffusely distributed through most layers of the cortex. Conversely, Cav3.2 mRNA is regionally restricted to layer 5 cortical pyramidal neurons, with little expression in other cortical areas. Of interest is that although the neural networks involved in SWDs all express one or more T-type channel isoform, the initiation and generalization has only recently been demonstrated as arising from the cortex. Meeren et al. (2002) have demonstrated through nonlinear association analysis that SWDs originate from the perioral region of the somatosensory cortex of Wistar Albino Glaxo rats from Rijswijk (WAG/Rij), a genetic model of absence epilepsy. Multisite ﬁeld potential recordings from other cortical areas consistently lag behind the perioral region, with the cortical focus leading the thalamus during the ﬁrst 500 ms. More in-depth electrophysiological studies using in vivo intracellular recordings from genetic absence epilepsy rats from Strasbourg (GAERS) demonstrate that the neural substrate involved in the initiation and intracortical propagation of ictal activity leading to generalization of related thalamic nuclei is found in layer 5/6 of the perioral somatosensory cortex (Polack et al., 2007). These neurons display pronounced hyperactivity relative to more superficial cortical neurons in that their membrane potentials are more depolarized and show distinctive interictal and preictal 9–11 Hz oscillations as well as enhanced bursting activity. It is postulated that these changes in layer 5/6 neurons create local oscillations (Silva et al., 1991) that lead to the generation of SWDs that propagate secondarily and intra- and interhemispherically throughout the somatosensory cortex and other cortical regions and the thalamic nuclei. Synchronization of SWDs in the thalamocortical and corticothalamic neurons sets into motion a unified oscillatory network that is driven by the ictogenic properties of the cortical neurons while the thalamocortical neurons provide resonant circuitry to sustain the activity. In support of the “cortical focus theory” of absence epilepsy, infusion of ethosuximide (a first choice antiabsence drug) into the perioral region of GAERS animals but not into the thalamus immediately reduces SWDs (Sherwin,

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1989; Richards et al., 2003; Manning et al., 2004). Actions of ethosuximide on native T-type currents from isolated thalamocortical neurons and neurons from the thalamic reticular nucleus have, however, been mixed, with some groups demonstrating up to a 40% reduction in current amplitude at therapeutic concentrations (Coulter et al., 1989; Huguenard and Prince, 1994; Huguenard, 2002) while other studies observing no inhibition (Pfrieger et al., 1992; Leresche et al., 1998). More recently, a study on cloned channels confirms that ethosuximide inhibits cloned Cav3.1, Cav3.2, and Cav3.3 channels (IC50s 0.3–1 mM) at therapeutic plasma levels (0.3–0.7 mM; Gomora et al., 2001). Further evidence providing insight into the role of T-type channels in the generation of SWDs and absence-like seizures is from Cav3.1 knockout mice (Kim et al., 2001). Not only are Cav3.1 KO mice resistant to baclofen-induced 3–5 Hz spike-wave discharges as measured by EEG, intracellular recordings from thalamocortical neurons show an absence of rebound burst firing action potentials in thalamic slices when injected with negative current. In freely moving mice, field potential recordings from the ventroposteromedial and ventroposterolateral nuclei also show that responses to baclofen-mediated intrathalamic oscillations, which are required for SWD discharges, are also diminished. On the other hand, low doses of bicuculline, which evokes SWDs originating from the cortex (Steriade and Contreras, 1998), when injected into Cav3.1 KO mice are capable of generating SWDs in both the thalamus and cortex. Similarly, Cav3.1 KO mice are not resistant to 4-aminopyridine (4-AP)induced tonic–clonic seizures. While these experiments highlight a critical role of Cav3.1 in generating absence-like seizures, the lack of protection in other models may be indicative of the dominant involvement of either Cav3.2 or Cav3.3 in the cortex for bicuculline-induced SWDs and mechanistically different pathways for 4-AP-induced seizures. Cross-breeding Cav3.1 KO mice with other models of absence seizures, such as lethargic (b4lh/lh), tottering (a1Atg/tg), or stargazer (g2stg/stg) mutant mice models or mice harboring a null mutation for the pore-forming Cav2.1 subunit of P/Q-type channels, results in strongly or completely suppressed cortical SWD paroxysmal activities (Song et al., 2004). Interestingly, in all these murine models, T-type channel currents are elevated in thalamocortical neurons by up to 50%, although no alteration in mRNA levels are apparent (Zhang et al., 2002; Nahm et al., 2005). In GAERS animals, elevated increases in reticular thalamic T-type channel currents (55%) have also been reported with a concomitant increase in Cav3.1 and Cav3.2 mRNA levels in the ventral posterior thalamic relay nuclei and thalamic reticular nucleus, respectively (Tsakiridou et al., 1995; Talley et al., 2000). More recently, a homozygous, missense, single nucleotide (G–C) mutation has been identified in the Cav3.2 gene from GAERS, though the functional effects are unknown (Kyi et al., 2006). However, in Cav2.1// Cav3.1/þ mice, T-type channel currents are decreased by 25%, yet animals are still capable of generating SWDs indicating that baseline levels, but not necessarily augmented levels, of T-type channel currents are sufficient for the genesis of spontaneous SWD activity. As protection against absence seizures was afforded by deletion of Cav3.1, it will be interesting to determine whether complete and selective pharmacological inhibition of Cav3.1 channels provides complete

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suppression of SWDs in these genetic models and pharmacological models of absence seizures. In humans, the role of T-type channels as a susceptibility gene leading to the pathophysiology of absence epilepsy has been speculated due to the known function of T-type channels in the thalamic and cortical physiology. All three subtypes, Cav3.1, Cav3.2, and Cav3.3, have been examined in patients with childhood absence epilepsy (CAE) and other forms of IGEs. Population analyses studying linkages to genetic mutations in humans have identified numerous missense mutations in the Cav3.2 gene. Chen et al. (2003a) reported 12 mutations in 14 patients of Chinese Han ethnicity that were not observed in 230 control patients. In some patients, two or more mutations were identified and numerous polymorphisms were reported in both patients and seizure-free individuals. In an expanded study, which included patients with CAE, juvenile myoclonic epilepsy, and febrile convulsions, three new missense mutations have been identified, though they were also found in the control group (Heron et al., 2004). More recently, 28 new variants have been identified in the Cav3.2 gene from the Chinese Han population of which only some were found in CAE patients (Liang et al., 2006). In a study attempting to investigate whether common polymorphisms in the Cav3.2 gene are associated with CAE in the Chinese Han population, carriers with three different polymorphisms were identified as at a higher risk of developing CAE than noncarriers (Liang et al., 2007). In a predominantly Caucasian population, over 100 variants of the Cav3.2 gene have been found in patients with IGEs as well as temporal lobe epilepsy (Heron et al., 2007). In contrast, in an evaluation of Caucasian European patients with CAE, linkage analysis was unable to detect any of the Chinese variants in 220 patients (Chioza et al., 2006). The human Cav3.2 mutations are located mainly in exons 6–12 encoding the domain I–II linker region. Exogenous expression of the mutations introduced into rat and human Cav3.2 channels reveals that in some instances channel biophysical properties are altered while other changes are “biophysically silent” (Khosravani et al., 2004, 2005; Vitko et al., 2005; Peloquin et al., 2006). Alterations in the biophysical properties mainly result in gain-of-function phenotypes as seen by a more depolarized steady-state inactivation and hyperpolarized voltage dependence of activation. Further insight into the possible roles of the “biophysically silent” mutations and SNPs in loop I–II was revealed when deletion of this region was found to increase Cav3.2 plasma membrane expression with a predicted overactivity to occur in neurons (Vitko et al., 2007). Therefore, the effect of the gain of function as a result of changes in biophysical properties on the Cav3.2 channels as well as biophysically silent mutations that may serve to promote increased plasma membrane expression both result in increased T-type channel activity. A study investigating whether Cav3.1 channels are involved in the etiology of IGEs has proposed a linkage to the CACNA1G gene (Singh et al., 2007). However, while 13 variants were identified in 123 Japanese and Hispanic patients with IGEs, many were found in both patients and control individuals. In the Han Chinese population, no Cav3.1 mutations were identified in patients with CAE, but six single nucleotide polymorphisms (SNPs) were found (Chen et al., 2003b). Overall, the distribution of SNPs was not significantly different in control and CAE patients in the Chinese Han

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population and therefore the SNPs are not considered important for susceptibility to nonconvulsive seizures (Wang et al., 2006). Though the pathophysiological role of T-type channels has primarily focused on IGEs, they are also likely to be involved in other seizure types as RNA expression studies have shown that Cav3.1 and Cav3.3 are prominently expressed in the human CNS, including structures known to generate seizure activity, such as the thalamus, cortex, hippocampus, and amygdala. In situ hybridization studies in rat brain have also revealed diffuse mRNA expression of Cav3.1, Cav3.2, and Cav3.3 in all brain structures (Craig et al., 1999; Talley et al., 1999). Interestingly, at the subcellular level subtypespecific polyclonal antibodies against Cav3 channel proteins indicate that the three T-channel subtypes are differentially localized in the soma and dendrites (McKay et al., 2006). Cav3.1 immunolabeling is prominent in the soma and proximal region of the dendrites, while somatic and proximal middendritic regions largely express Cav3.2. Distribution of Cav3.3 is further distinct in that expression is found in the soma as well as extended throughout the arborization of the dendrites in selective neurons. In concordance with this immunohistochemical study are previous electrophysiological studies that have identified T-type currents in both the somatic and dendritic compartments of central neurons (Karst et al., 1993; Markram and Sakmann, 1994; Magee and Johnston, 1995; Mouginot et al., 1997; Jung et al., 2001; Isope and Murphy, 2005). Two particular animal models of seizures have substantiated the potential role of T-type channels in seizure types other than IGEs. In the pilocarpine-induced status epilepticus rat model, a model of human complex partial seizures with secondary generalization, a threefold increase in T-type tail current density is observed in hippocampal neurons with a concomitant 54% increase in the number of neurons with intrinsic burst firing, which normally fire in a regular mode; this was inhibited by Ni2þ with an IC50 of 27 mM (Su et al., 2002). In a focal model of epilepsy induced by kindling of the rat hippocampus, patch-clamp recordings from in situ slices show an 80% increase in T-type channel currents in hippocampal neurons compared to recordings from control rats (Faas et al., 1996). These elevated T-type channel currents are still present 6 weeks after the last kindling stimulation and support a role of T-type channels in epileptogenesis. Definitive pharmacological proof-of-concept studies confirming that T-type calcium channels represent important drug targets for the treatment of different epilepsies have been hampered by the lack of high-affinity subtype-selective blockers. Besides ethosuximide, which is used to treat patients with absence epilepsy and inhibits both native (0.2–24 mM) and cloned T-type channels (Cav3.1, Cav3.2, and Cav3.3; 0.3– 1 mM) at therapeutically relevant concentrations of 0.3–0.7 mM (Coulter et al., 1989b; Huguenard and Prince, 1994; Gomora et al., 2001; Huguenard, 2002), only a handful of other AEDs with activity at T-type Ca2þ channels have been identified. Valproate, a broad spectrum antiepileptic drug used in the treatment of patients with IGEs, is thought to primarily provide efficacy by inhibiting GABA transaminase to promote GABAergic transmission and through inhibition of sodium channels; this compound is also known to block T-type channels (reviewed in Czapinski et al., 2005). Initial studies report the partial inhibition of T-type channel currents at

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high concentrations in rat nodose ganglion neurons relative to the therapeutic plasma concentration (Kelly et al., 1990; Todorovic and Lingle, 1998). However, even at cloned Cav3.1 channels, 10 mM valproate only modestly inhibited the Ca2þ channel current by 10% (Lacinova et al., 2000). More recently, the antagonistic action of valproate and ethosuximide on LVA channels of thalamic relay neurons in WAG/Rij rats has been systematically compared to nonepileptic control rats (Broicher et al., 2007b). Both ethosuximide (0.25–0.75 mM) and valproate (1 mM) inhibit T-type Ca2þ channel currents with higher affinity in acutely isolated thalamocortical relay neurons from WAG/Rij rats than in comparable neurons from nonepileptic rats. In addition, ethosuximide delays the onset of the low-threshold spike and increases the tonic action potentials in thalamocortical neurons from WAG/Rij rats, an effect that is different to that observed in control rats. A role for T-type channels in temporal lobe seizures has been suggested as fluoxetine, a reuptake inhibitor of serotonin used to treat depression, not only inhibits LVA (IC50 ¼ 6.8 mM) and HVA (1–2 mM) calcium channels in cultured hippocampal pyramidal cells, but also reduces Kþ-induced seizure-like activity in hippocampal slices (Wong et al., 1995; Deak et al., 2000). In addition, in rodents, fluoxetine dose-dependently protects animals from limbic seizures evoked by bicuculline applied into the deep prepiriform cortex (Prendiville and Gale, 1993) as well as by increasing the after-discharge threshold of hippocampal seizures induced by electrical stimulation (Wada et al., 1995). In genetically epilepsy-prone rats (GEPR), fluoxetine in a dose-dependent manner reduces convulsions induced by sound stimulus (Dailey et al., 1996). Interestingly, in humans when fluoxetine is coadministered with valproate, carbamazepine, or phenobarbital or in mice with phenytoin, carbamazepine, and ameltolide, the effects of the AEDS are enhanced (Leander, 1992; Favale et al., 1995). Other studies examining roles of T-type channels in partial and generalized seizures have proposed that the therapeutic action of phenytoin and zonisamide may be in part due to T-type inhibition (Suzuki et al., 1992; Todorovic et al., 2000). Phenytoin is used clinically to treat partial and generalized seizures and is known to primarily inhibit sodium channels. It also inhibits recombinant T-type channels at concentrations close to the maximal therapeutic concentration (Cav3.1 IC50 ¼ 124 mM; Cav3.2 IC50 ¼ 8.3 and 192 mM). Zonisamide, an adjunctive therapy in adults with partial onset seizures with multiple sites of action, shows T-type current blockade in cultured neurons isolated from rat cerebral cortex of 38% at 50 mM (Suzuki et al., 1992). In addition, flunarizine, a potent blocker of recombinant Cav3.1 (IC50 ¼ 0.53 mM), Cav3.2 (IC50 ¼ 0.36 mM), and Cav3.3 (IC50 ¼ 0.84 mM) channels, also exhibits anticonvulsant properties in both animals and humans (Santi et al., 2002). In humans, flunarizine administered as an adjuvant therapy is efficacious in patients with complex partial seizures with or without secondarily generalized seizures as well as reflex seizures (Starreveld et al., 1989; Durrheim et al., 1992; Pledger et al., 1994). In various chemoconvulsant and electroconvulsant animal models, flunarizine monotherapy or coadministration results in protection (Desmedt et al., 1975; De Sarro et al., 1986, 1992; Drago et al., 1986; Pohl and Mares, 1987; Mack and Gilbert, 1992; Rodger and Pleuvry, 1993; Becker and Grecksch, 1995; Joseph et al., 1998a, 1998b), though in some models no protection is

REFERENCES

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observed for cocaine-induced seizures, cortical stimulation, amygdala kindling, or pentylenetetrazol seizures (Trommer and Pasternak, 1989; Gasior et al., 1996, 1999).

2.4 CONCLUSIONS In summary, there is considerable pharmacological, genetic, and physiological evidence implicating N-type and T-type calcium channels in a number of pathophysiological conditions centered around neuronal hyperexcitability. Although there exist clinical agents that nonselectively block T-type calcium channels, there are no subtype-specific drugs yet available; thus, there remains both significant clinical challenge and opportunity. In the case of N-type channels and intractable pain, while Prialt provides excellent clinical proof of concept, its peptidic nature requiring intrathecal administration together with its narrow therapeutic window provides the opportunity for developing equally efficacious but safer and orally available N-type channel blockers.

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3 VOLTAGE-GATED SODIUM CHANNELS: MULTIPLE ROLES IN THE PATHOPHYSIOLOGY OF PAIN SULAYMAN D. DIB-HAJJ1,2, BRYAN C. HAINS1,2, JOEL A. BLACK1,2, 1,2 AND STEPHEN G. WAXMAN 1

Department of Neurology and Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New Haven, CT 06510, USA 2 Rehabilitation Research Center, VA Connecticut Healthcare System, West Haven, CT 06516, USA

3.1 INTRODUCTION Voltage-gated sodium channels underlie the generation and conduction of action potentials (AP) in excitable cells. Sodium channels in the plasma membrane are closed at rest but undergo structural changes in response to depolarization of the cell membrane, leading to cycling of the channel through closed, open, inactive, and repriming states (Hille, 2001). Channel opening is transient, allowing the flow of sodium ions down their concentration gradient, thus generating an inward current, and most channels rapidly inactivate, within milliseconds of opening, and then undergo conformational changes to recover (reprime) from inactivation. The different sodium channel isoforms cycle through these states with different kinetics and voltagedependent properties so that it is possible to identify individual currents produced by some channels in the presence of other members of the family. Sodium channels are heteromultimers of a large a subunit and smaller auxiliary b subunits (Catterall, 2000). The a subunit alone is necessary and sufficient to produce a Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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functional channel, whereas b subunits and an array of other cytosolic channel partners regulate trafficking and anchoring of the channels at the cell membrane. Still other channel partners modulate biophysical properties of the channels either by direct binding or by inducing posttranslational modifications, for example, phosphorylation (Wood et al., 2004; Abriel and Kass, 2005; Liu et al., 2005; Cantrell, 2001). The a subunits of sodium channels are large polypeptides (size range: 1700–2000 amino acids) that are organized into four domains (DI–DIV), each consisting of six transmembrane segments that are connected by intra- and extracellular linkers (Fig. 3.1) (Catterall, 2000). The b subunits, however, are type I membrane proteins, each with a single transmembrane segment and a larger extracellular domain that has an immunoglobulin fold (Catterall, 2000). Nine a subunits (Nav1.1–Nav1.9) and several of their cognates have been identified in mammals and lower vertebrates (Goldin et al., 2000; Goldin, 2002; Catterall et al., 2005). Many of these channels produce sodium currents with distinct biophysical and pharmacological properties (Rush et al., 2007). Expression of sodium channels is regulated in spatial and temporal patterns, which will be discussed in the coming sections. Because neuropathic pain has peripheral and central components, sodium channel dysregulation in both PNS and CNS may play a role in establishment and maintenance of this pain state. The following sections will review our current knowledge of neuronal sodium channels Nav1.1–1.3, Nav1.6–1.9, with a major focus on those expressed within dorsal root ganglion (DRG) and higher order neurons along pain signaling pathways, and their demonstrable or putative roles in neuropathic pain.

FIGURE 3.1 Schematic of the pore-forming a subunit of voltage-gated sodium channel. The pore-forming subunit of sodium channels is a long polypeptide with 24 transmembrane segments that are organized into four homologous domains (DI–DIV). The N- and C-termini of the channel, and loops 1–3 (L1–L3) that joins the four domains, are cytosolic and have been shown to house sequence motifs for channel partner binding and for phosphorylation of the channel. The binding of different classes of cytosolic proteins and phosphorylation of the channels have been shown to regulate channel trafficking and polarized distribution within neuronal compartments and/or biophysical properties of the channel. The S4 transmembrane segment in each of the domains is a voltage sensor, and the gray sphere in L3 designates the tetrapeptide IFMT, which acts as the fast-inactivating particle of the channel. The extracellular linkers may be sites of N-glycosylation of channels.

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3.2 TISSUE DISTRIBUTION AND SUBCELLULAR LOCALIZATION OF SODIUM CHANNELS Sodium channel expression is tissue and developmentally regulated across the different species where it has been investigated. Neuronal sodium channels, Nav1.1–1.3 and Nav1.6–1.9, can be found within CNS and PNS neurons and within glia, whereas Nav1.4 and Nav1.5 are expressed primarily in skeletal and cardiac myocytes, respectively, (Catterall et al., 2005) and will not be discussed further in this chapter. Most neurons express more than one sodium channel isoform, and the specific channel complement influences the electrogenic properties of these neurons. For example, neurons in adult rodent DRG can express up to five sodium channel isoforms (Fig. 3.2A), and the current profile and voltage-dependent gating properties of the two channels that are resistant to micromolar concentrations of tetrodotoxin can be distinguished even in the presence of other sodium currents (Fig. 3.2B). Dysregulation of sodium channel expression under pathological conditions can lead to neuronal hyper- or hypoexcitability, which contributes to the disease phenotype. The genes for sodium channels Nav1.1, 1.2, 1.3, and 1.7 are clustered on human and mouse chromosome 2 and are thus thought to have a common lineage (Goldin, 2002). Despite their common ancestry and chromosomal location, the expression of these channels is differentially regulated in CNS and PNS tissues and during development. Nav1.1 is expressed in both CNS (Beckh et al., 1989) and DRG neurons (Black et al., 1996) and in cardiac muscle (Maier et al., 2002). Nav1.1 and Nav1.2 are expressed at low levels during embryogenesis and reach adult levels by P14–P21 in the rat CNS (Beckh et al., 1989; Westenbroek et al., 1989; Felts et al., 1997). Nav1.2 is expressed at low levels within DRG neurons during embryogenesis and its expression is significantly attenuated by P0 (Waxman et al., 1994; Black et al., 1996; Schafer et al., 2006). In rodents, Nav1.3 is the predominant isoform during embryogenesis in CNS and DRG neurons; its expression is significantly attenuated after birth (Beckh et al., 1989; Waxman et al., 1994), but adult human CNS neurons continue to produce higher levels of Nav1.3 compared to their rodent counterparts (Whitaker et al., 2001). Nav1.3 transcripts, however, continue to be detected in sympathetic ganglia in adult rodents, at levels comparable to those of other channels (Rush et al., 2006). Nav1.1 and Nav1.3 appear to be predominantly localized to somatodendritic compartments of myelinated neurons (Westenbroek et al., 1989; Whitaker et al., 2001). Recently, Nav1.1 was detected at the initial segment of axons in the inner plexiform layer of the retina (Van Wart et al., 2005); however, Nav1.1 is not detectable in optic nerve axons (Craner et al., 2003). The subcellular distribution of Nav1.1 in adult rat DRG neurons is not well defined. In contrast, Nav1.2 is present along premyelinated axons and is targeted to immature nodes of Ranvier during myelination of CNS axons (Boiko et al., 2001; Kaplan et al., 2001) but is then restricted to the somatodendritic compartment, stretches of axons that lack myelin, and totally nonmyelinated axons (Westenbroek et al., 1989, 1992; Boiko et al., 2001). Considering the very low levels of Nav1.2 in rodent DRG neurons after birth (Waxman et al., 1994; Black et al., 1996), it is not surprising that this channel is not detected at nodes of Ranvier in peripheral myelinated axons (Schafer et al., 2006).

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FIGURE 3.2 Multiple sodium channels and currents in adult DRG neurons. (A) Sodium channel a subunit mRNAs (left panels) and protein (right panels) visualized by subtype-specific riboprobes and antibodies. Transcripts and protein for five different sodium channels (Nav1.1, Nav1.6, Nav1.7, Nav1.8, and Nav1.9) are present at moderate-to-high levels in DRG neurons. Nav1.2 and Nav1.3 are not detectable in adult DRG neurons. Scale bar: 50 mm. (B) Voltagegated sodium currents recorded by whole-cell patch clamp in adult DRG neurons. (a) Only fast, TTX-sensitive sodium current (presumably composed of Nav1.1, Nav1.6, and Nav1.7) is observed in a muscle afferent DRG neuron, which exhibits little overlap between activation (filled circles) and steady-state inactivation (unfilled circles). (b) A small DRG neuron displays only slow, TTX-resistant sodium current (Nav1.8); activation and steady-state inactivation curves are depolarized compared to fast, TTX-sensitive current. (c) Persistent, TTX-resistant sodium current (Nav1.9) recorded from a small DRG neuron from Nav1.8-null mouse. Activation (unfilled circles) and steady-state inactivation (filled circles) show significant overlap (window currents). (Modified and reproduced with permission from Black et al. (1996), Black et al. (2004), Cummins et al. (1999), Sleeper et al. (2000), and Waxman et al. (1999).)

Nav1.6 is located on human chromosome 12 and is expressed in CNS neurons and glia (Burgess et al., 1995; Schaller et al., 1995) and in peripheral sensory neurons (Black et al., 1996; Dietrich et al., 1998) and sympathetic ganglia (Rush et al., 2006). The expression of Nav1.6 in CNS neurons increases after birth, reaching adult levels by

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P14 (Schaller and Caldwell, 2000), whereas it is detected within DRG neurons at E17 and becomes robust by P0 (Felts et al., 1997; Chung, ). Nav1.6 channels have also been detected in immune cells, for example, microglia and macrophages (Craner et al., 2005). The distribution of Nav1.6 in human CNS tissue is in close agreement with that in rodents (Whitaker et al., 1999). Nav1.6 is the major sodium channel at mature nodes of Ranvier in myelinated axons in the CNS, where it replaces Nav1.2 (Boiko et al., 2001; Kaplan et al., 2001) upon the formation of compact myelin and node maturation, and in the PNS (Schafer et al., 2006). In contrast, Nav1.6 is present in the somata and diffusely along substantial lengths of unmyelinated fibers arising from small DRG neurons (Black and Waxman, 2002; Rush et al., 2005). The focal distribution of Nav1.6 at nodes is disrupted in demyelinated lesions in patients with multiple sclerosis (MS) and in mice with experimental autoimmune encephalomyelitis (EAE) (Waxman et al., 2004). The remyelination of experimental lesions with Schwann cells (Black et al., 2006) or olfactory ensheathing cells (Sasaki et al., 2006) leads to the restoration of Nav1.6 immunolabeling at nodes of Ranvier. Nav1.7 is expressed only in sensory and sympathetic neurons in the PNS (Black et al., 1996; Felts et al., 1997; Sangameswaran et al., 1997; Toledo-Aral et al., 1997; Rush et al., 2006) even though it is encoded by the gene SCN9A, which is clustered at the same chromosomal region with SCN1A–SCN3A, the genes that encode Nav1.1–1.3. More important, Nav1.7 is produced by the majority of functionally identified nociceptive neurons (Djouhri et al., 2003). An earlier report of Nav1.7 detection in brain samples (Sangameswaran et al., 1997) has not been confirmed by other studies (Black et al., 1996; Felts et al., 1997; Toledo-Aral et al., 1997). Nav1.7 has also been detected in a variety of other cell types including neuroendocrine cells (Klugbauer et al., 1995; Toledo-Aral et al., 1997), smooth muscle cells (Holm et al., 2002; Jo et al., 2004; Saleh et al., 2005), and in metastatic cancer cells (Diss et al., 2005; Fraser et al., 2005). Nav1.7 is uniformly distributed in the somata and along the unmyelinated Cfibers within the sciatic nerve (Rush et al., 2005). This channel accumulates within neurite tips in DRG neurons in culture (Toledo-Aral et al., 1997), which suggests an analogous accumulation at nerve terminals in vivo. The presence of Nav1.7 at nerve endings would be consistent with the role of this channel in amplifying generation potentials due to its enabling biophysical properties (Cummins et al., 1998). Nav1.8 is a sensory neuron-specific channel that is normally expressed within DRG and trigeminal ganglia but not in CNS neurons (Akopian et al., 1996; Sangameswaran et al., 1996). Nav1.8 is present in most peptidergic and nonpeptidergic small DRG neurons (Fjell et al., 1999; Amaya et al., 2000; Sleeper et al., 2000; Djouhri et al., 2003a; Fang et al., 2005; Rush et al., 2005). Nav1.8 immunostaining signal is abundant in the somata of small DRG neurons (Amaya et al., 2000; Sleeper et al., 2000) and in unmyelinated fibers in the sciatic nerve (Rush et al., 2005). Nav1.8 is also expressed in trigeminal neurons (Bongenhielm et al., 2000; Eriksson et al., 2005) and has been detected in unmyelinated fibers innervating cornea (Black and Waxman, 2002). Nav1.8 is also present within a subpopulation of medium-sized DRG neurons that produce Ab fibers (Rizzo et al., 1994, 1995; Amaya et al., 2000; Djouhri et al., 2003a) and has been reported at 20% of nodes of Ranvier in myelinated axons that innervate tooth pulp (Henry et al., 2005). The reporting of Nav1.8 at nodes of Ranvier in spinal cord tracts

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(Arroyo et al., 2002) is inconsistent with the lack of Nav1.8 message in normal CNS tissue, although a small number of myelinated axons that emanate from DRG neurons and project to the spinal cord might express it. The slowly inactivating Nav1.8 current is absent from proprioceptive, muscle afferent DRG neurons (Rizzo et al., 1994) that have continuous central projections within spinal cord tracts; thus, these cells could not be the source of the putative Nav1.8 signal in that tissue. Nav1.9 is the other sensory neuron-specific sodium channel within DRG and trigeminal ganglia (Dib-Hajj et al., 1998; Tate et al., 1998), and within myenteric neurons (Rugiero et al., 2003). Within DRG neurons, Nav1.9 expression has been reported in small-diameter nonpeptidergic IB4þ neurons (Fjell et al., 1999a; Amaya et al., 2000; Fang et al., 2002; Rush et al., 2005). Low levels of Nav1.9 transcripts were detected by reverse transcription-polymerase chain reaction (RT-PCR) assays in cerebral hemispheres and retina but not in cerebellum or spinal cord, or in satellite or Schwann cells within DRG (Dib-Hajj et al., 1998). Reports of wider distribution of Nav1.9 within CNS tissue (Jeong et al., 2000) have not been confirmed by other studies and are inconsistent with the absence of a persistent TTX-R current, which is attributable to Nav1.9. A report contending that BDNF could activate Nav1.9 channels in CNS neurons (Blum et al., 2002) has yet to be independently reproduced, casting doubt on the validity of the initial observations. Nav1.9 is abundant in the somata of the majority of small nonpeptidergic DRG neurons (Amaya et al., 2000; Sleeper et al., 2000) and along unmyelinated fibers and nerve endings in the cornea (Black and Waxman, 2002) and in the skin (Dib-Hajj et al., 2002; Rush et al., 2005). Nav1.9 is rare in Ab neurons (Dib-Hajj et al., 1998b; Tate et al., 1998b; Fjell et al., 1999a; Amaya et al., 2000; Fjell et al., 2000) but, when present, is restricted to nociceptive neurons (Fang et al., 2002). The presence of Nav1.9 at nerve endings is consistent with its predicted role of amplifying subthreshold stimuli (Herzog et al., 2001).

3.3 ROLE OF SODIUM CHANNELS IN TRAUMA-INDUCED PAINFUL NEUROPATHIES There is substantial evidence for a critical role of sodium channels in acquired and inherited painful neuropathies. A better understanding of the role of individual channels in chronic pain syndromes has been obtained from studies of animal models and human patients. The following sections will discuss our current knowledge about dysregulation and modulation of sodium channels in painful neuropathies. 3.3.1

Nerve Transection

Three of the most commonly used animal model for studying dysregulation of voltagegated sodium channels following nerve injury are sciatic nerve transection that entails cutting and tying off axons within the sciatic nerve at the midthigh level, spinal nerve ligation (SNL) that is caused by tight ligature of the spinal nerve originating from an individual DRG (Kim and Chung, 1992), and spared nerve injury (SNI) where two of the branches of the sciatic nerve, the tibial and the common peroneal, are cut sparing

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the third branch (sural) (Decosterd and Woolf, 2000). The main advantages of these models are that the injury is highly reproducible, permits easier application of exogenous factors, for example, neurotrophic factors or transformed cells that secrete desired factors, to the proximal nerve stump, and is amenable to assessment of behavioral pain responses. The application of exogenous factors has allowed investigations of the contribution of specific growth factors to the regulation of expression of voltage-gated sodium channels within DRG neurons (Dib-Hajj et al., 1998a; Boucher et al., 2000; Leffler et al., 2002). These injury models are similar in that nerve regeneration is prevented so that the severed axons cannot regrow to reach target tissues, either by threading the proximal end of the nerve into a silicon cuff (Waxman et al., 1994), or by effecting transection through a tight and permanent ligature of the spinal nerve (Kim et al., 2001, 2002) or the branches of the sciatic nerve (Decosterd and Woolf, 2000). The absence of axonal regeneration to peripheral tissues may not provide an exact parallel to clinical cases of trauma to human patients. However, each of these injury models results in the intermingling of injured and intact axons, which may lead to injury-mediated effects on the spared axons as will be discussed later. Neuropathic pain caused by transection of axons both in animal models and in human patients has a structural component at the site of injury and molecular and cellular components as well, which originate within the cell bodies of DRG neurons. The two components of the injury response are not mutually independent and reflect the interplay of neurons and various cell types, for example, immune cells, glia, and peripheral tissue cells such as keratinocytes. These cellular components constitute an integrated information unit that produces and transmits injury signals from the periphery to higher order centers within the CNS. For simplicity, we will separately discuss cellular, molecular, and structural components of response to injury. The physical transection of axons prevents the flow of electrical and chemical information between the neuronal cell bodies and their peripheral targets. At the molecular level, the physical transection of axons activates an injury response gene expression program that leads to an altered profile of ion channels, among other changes within DRG neurons (Costigan et al., 2002; Xiao et al., 2002), and secondorder neurons in the dorsal horn of the spinal cord (Yang et al., 2004a). The changes in gene expression underlie a new postinjury state of hyperexcitability of injured primary afferents and higher order neurons, which is manifested in neuropathic pain symptoms, for example, tactile allodynia and phantom pain (pain from an amputated limb). Although a multitude of gene products are dysregulated following trauma, studies from animal models and empirical data from human patients point to a key role of sodium channels in producing this neuronal hyperexcitability and the neuropathic pain state. Dynamic regulation of sodium channel expression after injury with some channels “turned off ” and others “turned on” is now a well-established contributor to hyperexcitability of DRG neurons (Fig. 3.3) (Waxman, 1999, 2001). Transcripts and protein levels of the Nav1.3 channel, which are detected only at exceedingly low levels in adult rat neurons, are upregulated following axotomy (Waxman et al., 1994; Dib-Hajj et al., 1996; Black et al., 1999). In contrast, transcripts and protein levels of Nav1.8 and Nav1.9 are significantly reduced in injured neurons (Dib-Hajj et al., 1996; Dib-Hajj

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FIGURE 3.3 Alterations in expression of Nav1.3, Nav1.8, and Nav1.9 in DRG neurons following peripheral transection of sciatic nerve. (A) RT-PCR analyses of control (C) and peripherally axotomized (A) DRG demonstrates upregulation of Nav1.3 and downregulation of Nav1.8 and Nav1.9 at 7–12 days following axotomy. (B) Contralateral (contra) and ipsilateral (ipsi) DRG reacted with isoform-specific antibodies for Nav1.3, Nav1.8, and Nav1.9 display an upregulation of Nav1.3 signal and a downregulation of immunofluorescent signal for Nav1.8 and Nav1.9 within DRG neurons. Scale bar: 50 mm. (C) Whole-cell patch-clamp recordings of control and axotomized small DRG neurons. (a) The time course for recovery from inactivation at 80 mV is faster in axotomized (open circles) than in control (ﬁlled circles) neurons. (b) Time constants for recovery from activation plotted as function of voltage. Time constants for axotomized (open circles) are smaller than that for control (ﬁlled circles) neurons. (c) and (d) Slowly inactivating (Nav1.8) and persistent (Nav1.9) TTX-resistant currents are reduced in small DRG neurons following peripheral axotomy. Scale bar: 100 mm. (Modiﬁed and reproduced with permission from Black et al. (1999), Dib-Hajj et al. (1996), Dib-Hajj et al. (1998a), and Sleeper et al. (2000).)

et al., 1998b; Sleeper et al., 2000; Decosterd et al., 2002), and those for Nav1.1, Nav1.6 and Nav1.7, the other voltage-gated sodium channels that are expressed in DRG neurons (Black et al., 1996), are reduced but to a lesser extent (Kim et al., 2001, 2002). Dysregulation of Nav1.3 following axotomy is not a generalized response to injury or a recapitulation of an embryonic expression profile. For example, transection of the

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sciatic nerve injures the peripheral axons of both sensory neurons of DRGs and primary motor neurons within the spinal cord; however, Nav1.3 is upregulated only in DRG neurons (Waxman et al., 1994; Dib-Hajj et al., 1996; Black et al., 1999) and not in motor neurons (Black and Waxman, unpublished observations). In addition, axotomy does not upregulate Nav1.2, another channel that is expressed within DRG neurons during embryogenesis (Waxman et al., 1994). Finally, transection of the central projections of DRG neurons (dorsal rhizotomy) does not cause an upregulation of Nav1.3 (Sleeper et al., 2000). The reduction of transcripts and protein for the Nav1.8 and Nav1.9 channels in axotomized DRG neurons is paralleled by the loss of the total TTX-R current in these neurons (Rizzo et al., 1995; Cummins and Waxman, 1997). The identification of the specific current profiles of Nav1.8 (Akopian et al., 1996; Sangameswaran et al., 1996; Akopian et al., 1999) and Nav1.9 (Cummins et al., 1999) permitted the unequivocal demonstration that both these channels and the sodium currents that they produce are downregulated after sciatic nerve transection (Cummins et al., 2000) and SNL (Gold et al., 2003) but not rhizotomy (Sleeper et al., 2000). The loss of Nav1.8 and Nav1.9 within injured DRG neurons has been confirmed in human patients suffering from traumatic injury to their ganglia (Coward et al., 2000). While the loss of these channels is likely to influence the electrogenic properties of injured DRG neurons, it is not clear whether their loss might contribute to hyperexcitability or increased spontaneous firing of these neurons (however, see discussion below on innervation of tissues by regenerating nerves). In contrast to the loss of the TTX-R currents in axotomized DRG neurons, the major effect on the TTX-S currents is a change in the repriming properties but with little effect on the current density. Axotomy of DRG neurons causes the loss of slowly repriming and the emergence of rapidly repriming TTX-S sodium current (Cummins and Waxman, 1997). Nav1.7 channels produce a slow-repriming TTX-S channels when expressed in native DRG neurons (Herzog et al., 2003) or in a heterologous expression system (Cummins et al., 1998). Thus, the loss of the slow-repriming TTX-S current in injured neurons (Cummins and Waxman, 1997) has been attributed to the reduction in Nav1.7 transcripts in DRG neurons from rats with SNL injury (Kim et al., 2002). The data from this animal study are consistent with studies on human patients with traumatic injury to their peripheral nerves, which shows that Nav1.7 immunostaining is significantly reduced in injured DRG neurons compared to control (Coward et al., 2001). The contribution of Nav1.6, the other TTX-S sodium channel that is known to be present in small DRG neurons (Black et al., 1996, 2002), to the sodium current profile in control and injured DRG neurons is not clear. Nav1.6 has been shown to rapidly reprime when expressed in DRG neurons, similar to its behavior in a heterologous expression system (Herzog et al., 2003). SNL injury reduces Nav1.6 transcript levels in rat DRG neurons (Kim et al., 2002), but the absence of a specific blocker or an intrinsic biophysical property that is not cell dependent hinders the determination of the effect of this injury on the Nav1.6 current. Thus, the contribution of Nav1.6 to DRG neuronal hyperexcitability after nerve injury remains less well understood than that of other channels.

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The emergence of a rapidly repriming TTX-S current in DRG neurons has been attributed, at least partially, to the upregulation of Nav1.3 following axotomy. Nav1.3 is virtually undetectable in normal adult DRG but is significantly upregulated following axotomy (Waxman et al., 1994; Black et al., 1996, 1999; Dib-Hajj et al., 1996) and in adult DRG neurons that have been dissociated and kept in culture for several days (Black et al., 1997). More important, Nav1.3 produces a rapidly repriming TTX-S current in DRG neurons and in a heterologous expression system (Cummins et al., 2001) with similar properties to those observed within injured DRG neurons (Cummins and Waxman, 1997). Injury-induced upregulation of Nav1.3 with its rapidly repriming properties and the ability to produce a significant current in response to the application of small, slow depolarizations (ramp stimulus) (Cummins et al., 2001) could support a lower threshold for single action potential firing and sustained highfrequency firing, which are hallmarks of neuronal hyperexcitability in injured neurons. The regenerative response of peripheral axons can produce a neuroma, which is a collection of de- and dysmyelinated axon sprouts and connective tissue at the proximal end of the transected axons (Fried et al., 1991). Neuromas have been shown to be sites of ectopic impulse generation and spontaneous firing of DRG neurons, which can lead to sensitization (for recent reviews see Black, ; Amir et al., 2006; Devor, 2006). For patients, painful neuromas can be worse than motor deficits associated with the injury. Interestingly, neuromas may not be painful, and in these cases, the accumulation of ankyrin G, which is normally clustered at nodes of Ranvier and initial segments, is reduced compared to that of painful neuromas or neurofibroma (Kretschmer et al., 2002, 2004). Treatment generally involves surgeries to resect the neuroma and implant the nerve end in different tissues (Vernadakis et al., 2003; Lewin-Kowalik et al., 2006). Early studies demonstrated accumulations of sodium channels at the distal tips of transected axons (Devor et al., 1989; England et al., 1996a). Importantly, Nav1.3 was specifically shown to accumulate within axon tips in the neuroma in rats (Black et al., 1999) in addition to its upregulation within the cell bodies of peripherally axotomized DRG neurons. The intensity of Nav1.3 immunoreactivity is highest at the distal region of the transected nerve, with only background levels of immunofluorescence at distances greater than 500–1000 mm proximal to this region. The accumulation of Nav1.3 within the neuroma suggests a molecular basis for the generation of ectopic discharges, which are known to emanate from this region (Scadding, 1981; Burchiel, 1984a, 1984b; Matzner and Devor, 1994). Consistent with a significant role of TTX-S sodium channel(s) in this process, application of 20 nM TTX to the neuroma significantly attenuates ectopic firing (Liu et al., 1999, 2001). The accumulation of Nav1.3 at the neuroma is coupled with the coaccumulation of the cell adhesion molecule contactin/F3 (Shah et al., 2004) (Fig. 3.4). Contactin has been shown to associate with several sodium channels and increase their current density both in heterologous expression systems (Kazarinova-Noyes et al., 2001; Shah et al., 2004) and in native DRG neurons (Rush et al., 2005). Previously, it was shown that contactin levels at the plasma membrane of hypothalamic neurons are regulated in an activity-dependent manner (Pierre et al., 2001). Therefore, a positive feedback loop could exist, leading to an increased contactin-mediated Nav1.3 surface expression at

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FIGURE 3.4 Nav1.3 and contactin are upregulated and colocalized in sciatic nerve neuroma— Images of uninjured control (contralateral) and transected (ipsilateral) sciatic nerve show that contactin and Nav1.3 accumulate within axon-like profiles within the neuroma that forms at the ligation site (arrow), with less immunostaining in proximal regions of the transected nerve. Colored images in the original publication (Shah et al., 2004) show that contactin and Nav1.3 exhibit extensive colocalization within the neuroma (not shown). (Modified and reproduced with permission from Shah et al. (2004).)

the neuroma, which could exacerbate neuropathic pain following nerve injury (Shah et al., 2004). In a limited number of human cases, Nav1.7 and Nav1.8 have been reported to accumulate within neuromas of trunk nerves (Coward et al., 2000; Kretschmer et al., 2002). Recently, Nav1.7 accumulation has been reported in painful neuromas of the lingual nerve but not in nonpainful neuromas of the same nerve (Bird et al., 2007). The accumulation of Nav1.8 at neuromas in human patients is in stark contrast to the findings from animal studies where sciatic nerve was studied. Similarly, the accumulation of Nav1.7 at sciatic nerve neuromas in animal models of injury has not been reported in the literature. Consistent with the downregulation of Nav1.8 and Nav1.9 mRNA and protein in DRG neurons following peripheral axotomy, these channels do not accumulate within the neuroma at 9–14 days following sciatic nerve transection (Black, ). The absence of Nav1.8 and Nav1.9 from neuromas is consistent with the blocking of ectopic firing at the neuroma by the application of 20 nM TTX (Liu et al., 1999, 2001), a TTX concentration that is several orders of magnitude lower than the Kd of these two TTX-R channels (Akopian et al., 1996; Cummins et al., 1999). Studies on human patients share the common weakness of small numbers of samples and a paucity of control tissue, two deficiencies that stress the need for caution in interpreting their outcomes.

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Chronic Constriction Injury

A chronic constriction injury (CCI) of the sciatic nerve is produced by placing loose ligatures around the nerve at midthigh level, which results in the comingling of injured and intact fibers for a long distance within the nerve and produces behavioral signs of spontaneous pain and cutaneous hyperalgesia in rodents (Bennett and Xie, 1988). DRG neurons subjected to CCI develop abnormal spontaneous activity and adrenergic sensitivity in the intact ganglion in vivo (Kajander et al., 1992; Xie et al., 1995) and in vitro (Zhang et al., 1997) or in dissociated DRG neurons (Petersen et al., 1996; Study and Kral, 1996). A linkage between Wallerian degeneration and hyperalgesia in CCI was confirmed by the demonstration that the onset of, and recovery from, hyperalgesia is delayed, with parallel delays in recruitment of macrophages to the site of injury, cytokine production, and degeneration of nerve fibers in the C57Bl/wld mouse strain compared to C57BL/6 (Myers et al., 1996; Sommer and Schafers, 1998). Most myelinated and unmyelinated axons (60–80%) undergo Wallerian degeneration following CCI (Basbaum et al., 1991; Carlton et al., 1991), an observation that has been verified functionally by the reduction in the anterograde axonal transport of acetyl cholinesterase (Filliatreau et al., 1994). CCI appears to produce effects that are a combination of axotomy and inflammation of nerve fibers. CCI triggers changes in sodium channel expression in both primary (DRG) and higher order sensory neurons, which may contribute to DRG neuron hyperexcitablity (Dib-Hajj et al., 1999; Hains et al., 2004a). The pattern of sodium channel dysregulation in DRG neurons at 14 days following CCI (Fig. 3.5) was similar but less extensive compared to that following sciatic nerve transection (Dib-Hajj et al., 1999), consistent with the observations that most of the sciatic nerve fibers are transected following this injury (Basbaum et al., 1991; Carlton et al., 1991). A reduction in transcripts and currents attributed to the TTX-R sodium channels Nav1.8 and Nav1.9 has been observed (Dib-Hajj et al., 1999; Decosterd et al., 2002), and an increase in the level of the TTX-S Nav1.3 channels is thought to contribute to the shift in the repriming properties of the TTX-S current in these neurons (Dib-Hajj et al., 1999). A similar reduction in the TTX-R current but with a hyperpolarizing shift in the voltagedependence of activation was independently reported (Kral et al., 1999). The difference might be attributable to recording of the currents in acutely dissociated DRG neurons from injured animals (Kral et al., 1999), compared to neurons in culture for 24 h (Dib-Hajj et al., 1999). At this point, however, it remains to be determined if contactin and Nav1.3 accumulate in injured axons proximal to the loose ligatures in the CCI model, similar to their accumulation at neuromas (Shah et al., 2004). The effect of CCI on the dysregulation of Nav1.8 and the contribution of this channel to DRG neuronal hyperexcitability, however, are a subject of controversy. Another study using CCI as an injury model reported no change in Nav1.8 transcripts and slowly inactivating TTX-R current density but a reduction in NaV1.8-specific immunolabeling within cell bodies and an accumulation of Nav1.8 in the sciatic nerve (Novakovic et al., 1998). An explanation that might reconcile the apparent discrepancy between this study and those discussed above would be to invoke an upregulation of Nav1.8 in intact nerves that comingle with degenerating axons of the sciatic nerve leading to an increased Nav1.8 immunostaining in these fibers. None of the reports on CCI measured levels of Nav1.8 in identified, uninjured DRG neurons following injury.

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FIGURE 3.5 Chronic constriction injury (CCI) of sciatic nerve upregulates Nav1.3 and downregulates Nav1.8 and Nav1.9 in DRG neurons. (a) RT-PCR products from control (C) and CCI DRG 14 days postsurgery exhibit a significant downregulation of Nav1.8 and Nav1.9 mRNA (Nav1.8: 512 bp; Nav1.9: 392 bp). Nav1.3 transcripts are upregulated in CCI neurons (Nav1.3: 412 bp). (b) In situ hybridization of small neurons from control and CCI DRG shows that hybridization signals for Nav1.8 and Nav1.9 are significantly reduced in CCI neurons, while hybridization signal for Nav1.3 is upregulated. Scale bar: 20 mm. (c) Recovery from inactivation is accelerated after CCI; repriming kinetics of TTX-sensitive current is signiﬁcantly increased in CCI neurons compared to control neurons. Patch-clamp recording demonstrates a signiﬁcant reduction in TTX-resistant currents in CCI neurons compared to control neurons. (Modiﬁed and reproduced with permission from Dib-Hajj et al. (1999).)

An upregulation of Nav1.8 in spared DRG neurons is in agreement with the finding that levels of substance P are significantly upregulated following CCI in DRG neurons with spared axons, possibly due to the exposure to inflammatory cytokines, for example, NGF, during Wallerian degeneration of sciatic nerve fibers (Ma and Bisby, 1998). NGF is known to increase levels of Nav1.8 within DRG neurons in vitro and in vivo (Dib-Hajj et al., 1998; Leffler et al., 2002). An elevation of Nav1.8 in spared neurons, whose axons comingle with damaged neighbors, would lead to neuronal hyperexcitability and might explain the lower incidence of ectopic firing following saphenous nerve transection in Nav1.8-null neurons (Roza et al., 2003) and the amelioration of neuropathic pain behavior in CCI animal injury models by knockdown of Nav1.8 using antisense oligonucleotides (Lai et al., 2002; Gold et al., 2003; Joshi et al., 2006). 3.3.3

Radicular Injury

Compression injuries either to the nerve root or to the DRG itself cause hyperexcitability that is manifested by periods of prolonged rapid firing and radicular pain (Howe et al., 1977). Radicular pain is a common clinical diagnosis that could have both mechanical and inflammatory components underlying its pathogenesis (Van Zundert et al., 2006). However, the molecular pathophysiology leading to neuronal

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hyperexcitability in compressed DRG neurons is not as well understood as that following SNL or axotomy. An animal model has been developed to investigate the molecular basis for DRG neuron hyperexcitability following chronic compression injury of DRG (CCD) (Hu and Xing, 1998). Compression of DRG neurons by placement of a metal rod in the intervertebral foramen, producing foramen stenosis and chronic compression of the ganglion, causes hyperexcitability of dissociated neurons or those in intact ganglion (Hu and Xing, 1998; Zhang et al., 1999; Ma and LaMotte, 2005). Spontaneous activity of DRG neurons following CCD is regulated by PKA (Hu et al., 2001), and these neurons demonstrate an enhanced response to inflammatory mediators (Song et al., 2003; Sun et al., 2006), both features that are consistent with neuronal hyperexcitability. CCD leads to dysregulation of sodium channel Nav1.9, albeit with less marked changes than SNL or axotomy (Abe et al., 2002). This injury, however, does not cause an increase in the levels of Nav1.3 transcripts or a significant reduction in the Nav1.8 channel transcript (Abe et al., 2002) unlike the effects of SNL and axotomy (see discussion above). However, recordings from identified cutaneous afferents revealed an 8 mV hyperpolarizing shift in voltage dependence of activation of TTX-S channels and an increase in the peak amplitude of the slowly inactivating TTX-R current, and a reduction in voltage-gated potassium current (Tan et al., 2006). The lack of correlation between electrophysiological changes and changes in channel transcript levels (Abe et al., 2002) suggests a posttranscriptional or posttranslational modulation of channels in the CCD injury model. The totality of the evidence strongly suggests that plasticity of expression, and possibly modulation, of sodium channels in compressed DRG neuron contribute to neuronal hyperexcitability in radicular pain.

3.4 ROLE OF SODIUM CHANNELS IN INFLAMMATION-INDUCED PAINFUL NEUROPATHIES Inflammation-induced pain behavior shows the same hallmark responses as those of neuropathic pain, including spontaneous activity of nociceptors, lowered threshold for action potential generation, and exaggerated evoked response to stimuli. The inflammation-induced pain symptoms are attenuated by treatment with sodium channel blockers (for a recent review see Amir et al., 2006), indicating a key role of these channels in neuronal hyperexcitability following tissue or nerve inflammation. The empirical data from human patients have been supported by animal studies, which demonstrate plasticity in the expression of sodium channels within nociceptors following inflammation of skin and muscle (Okuse et al., 1997; Tanaka et al., 1998; Tate et al., 1998; Gould et al., 1999; Black et al., 2004; Nassar et al., 2004) or viscera (Yoshimura et al., 2001; Bielefeldt et al., 2002; Beyak et al., 2004; Malykhina et al., 2004). Studies using knockout or knockdown models of specific sodium channels have identified Nav1.7, Nav1.8, and Nav1.9 as significant contributors to inflammation-induced pain. Inflammation of hindpaw in the rat causes an upregulation of Nav1.7 in DRG neurons that project to the inflamed area (Fig. 3.6) (Black et al., 2004; Gould

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FIGURE 3.6 Sodium channel protein expression and sodium currents in DRG neurons from saline-injected naive and carrageenan-injected rats. (a) Sections from saline and carrageenan-injected DRG were reacted with isoform-specific antibodies to Nav1.7 and Nav1.8. Sodium channels Nav1.7 and Nav1.8 exhibit greater immunolabeling in carrageenan neurons compared to saline-injected neurons. Microdensitometry quantitative analysis demonstrates that the immunofluorescent signals for Nav1.7 and Nav1.8 are significantly (*p 5 0.05) upregulated in small (5 25 mm) DRG neurons from carrageenan-injected DRG compared to saline-injected contralateral to carrageenan-injected (contra carrageenan) and saline-injected naive rat (saline) DRG. Scale bar: 50 mm. (b) Representative families of voltage-activated TTX-S (upper) and TTX-R (lower) sodium current traces for small neurons cultured from saline (left) and carrageenan (right) DRG are shown. Both TTX-S and TTX-R sodium current densities are signiﬁcantly greater in carrageenan- than saline-injected small DRG neurons. (Modiﬁed and reproduced with permission from Tanaka et al. (1998) and Black et al. (2004).)

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et al., 2004). Nav1.7 shows the most robust increase in transcript and protein levels following carrageenan-induced inflammation of the hindpaw in rats, and this is accompanied by a significant increase in the amplitude of TTX-S current in DRG neurons (Black et al., 2004). Inflammation-induced increase in the levels of Nav1.7 transcript and protein might be regulated by the increased levels of NGF, which is known to upregulate Nav1.7 expression in cell lines (Toledo-Aral et al., 1995, 1997) and in DRG neurons (Gould et al., 2000). In the absence of specific channel blockers, it is difficult to definitively attribute the increase in the TTX-S current solely to increased Nav1.7 protein levels. However, knockdown of Nav1.7 in primary afferents has been shown to prevent inflammation-induced thermal hyperalgesia in mice injected with complete Freund’s adjuvant in their hindpaw (Yeomans et al., 2005). A direct role of Nav1.7 in inflammatory pain has been confirmed in studies using Nav1.7 knockout mice. Mice that lack Nav1.7 in DRG neurons show almost complete absence of inflammation-induced pain (Nassar et al., 2004). Additional evidence has come from patients with the painful neuropathy inherited erythromelalgia (which will be discussed in a separate section) who show swelling, erythema, and local increase in skin temperature (Novella et al., 2007), symptoms that are common to inflammatory conditions, as a result of gain-of-function mutations of Nav1.7. Thus, Nav1.7 appears to play an important role in inflammation-induced pain. Chronic inflammation causes an increase in the level of Nav1.8 channels in affected DRG neurons, which may contribute to neuronal excitability leading to pain. Carrageenan-induced inflammation of rat hindpaw causes an increase in the Nav1.8 current 4 days postinjection (Tanaka et al., 1998), which is paralleled by an increase in the intensity of immunoreactivity of DRG neurons to Nav1.8-specific antibody (Black et al., 2004). CFA injection into rat hindpaw does not increase levels of Nav1.8 transcripts or protein within DRG neurons after 48 h (Okuse et al., 1997) but appears to induce an increase in the translocation of the channel protein to a larger number of myelinated and unmyelinated axons in sciatic nerve (Coggeshall et al., 2004). Colitis, which is induced 10 days after injection of trinitro genzone sulfonic acid causes an increase in the Nav1.8 current density 10 days postinjection (Beyak et al., 2004). Reducing the levels of the channel in DRG neurons by the administration of anti-Nav1.8 antisense oligonucleotides ameliorates inflammatory pain induced by PGE2 (Khasar et al., 1998) or CFA (Porreca et al., 1999; Joshi et al., 2006). Similarly, acetic acid-induced urinary bladder pain is attenuated using anti-Nav1.8 antisense oligonucleotide treatment (Yoshimura et al., 2001). Thus, an increase in Nav1.8 levels in DRG neurons can contribute to the development of inflammatory pain. Chronic or acute application of inflammatory mediators, injected subcutaneously in vivo or applied directly to DRG neurons in culture, causes an increase in the amplitude of the TTX-R slowly inactivating Nav1.8 current. NGF, which functions as an inflammatory cytokine in addition to being a trophic factor (Hefti et al., 2006), contributes to the chronic upregulation of Nav1.8 levels within DRG neurons under inflammatory conditions. NGF treatment triggers an increase in Nav1.8 expression in DRG neurons both in vivo (Dib-Hajj et al., 1998a; Fjell et al., 1999b; Leffler et al., 2002) and in vitro (Fjell et al., 1999b; Cummins et al., 2000). Acute application

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of inflammatory mediators PGE2 (England et al., 1996; Gold et al., 1996), NGF and its second messenger ceramide (Zhang et al., 2002), and endothelin-1 (Zhou et al., 2002) have been shown to increase the TTX-R Nav1.8 current density, and the increase is accompanied by a hyperpolarizing shift in voltage dependence of activation of the channel. However, activation of p38 MAP kinase in DRG neurons, either by treatment with TNF-a (Jin and Gereau, 2006), a major inflammatory cytokine, or by anisomycin (Hudmon, 2008) to induce a stress response, increases the amplitude of the TTX-R current but without a change in the gating properties of the channel. The increase in Nav1.8 current density by activated p38 MAPK is mediated by the direct phosphorylation of two residues in loop 1 of Nav1.8 (Hudmon, 2008). Inflammation of visceral organs has been associated with an increase in the current density of the slowly inactivating TTX-R current but without a hyperpolarizing shift in the voltage dependence of activation (Yoshimura et al., 2001; Bielefeldt et al., 2002; Malykhina et al., 2004). These observations suggest that diverse inflammatory modalities may regulate the Nav1.8 current in different ways. Alternatively, altered gating properties of Nav1.8 in some studies may reflect the use of different recording conditions. Studies using Nav1.8-null mice confirm the important role of this channel in painful neuropathies. Mice that lack Nav1.8 demonstrate a delayed development of carrageenan-evoked (Akopian et al., 1999), an attenuated NGF-evoked (Kerr et al., 2001), thermal hyperalgesia, and reduced visceral pain responses to inflammatory mediators and referred hyperalgesia (Laird et al., 2002). Nav1.8-null mice also show deficits in visceral pain following parasite-induced chronic inflammation (Hillsley et al., 2006), which is consistent with the presence of Nav1.8 in sensory DRG neurons innervating the colon (Gold et al., 2002). These findings are best explained by the role of this channel in rendering neurons hyperexcitable (Rush et al., 2006). The contribution of Nav1.9 to inflammatory pain, while not as well understood as that of Nav1.7 and Nav1.8, has begun to be clarified by studies of mice that lack this channel, in which the persistent Nav1.9 TTX-R current is absent. An increase in the level of Nav1.9 transcripts was reported 7 days after CFA injection into the hindpaw of rats (Tate et al., 1998). PGE2, which acts on this channel via a G-protein-coupled mechanism, increases the current density of Nav1.9 (Rush and Waxman, 2004). The increase in the current density of Nav1.9 via G-proteins has been linked to a reduction in the threshold for action potential firing and an increase in spontaneous firing (Baker et al., 2003). This effect of Nav1.9 on neuronal excitability is consistent with predictions from computer simulations that this channel contributes to setting resting membrane potential and amplifying subthreshold stimuli (Herzog et al., 2001). In agreement with a role of Nav1.9 in inflammatory pain, heterozygotes and Nav1.9-null mice show deficits in the late phase of formalin-induced pain, and Nav1.9null mice show a reduction in the duration of thermal hyperalgesia following carrageenan or CFA injection into the hindpaw (Priest et al., 2005). Recently, similar findings in another Nav1.9-null mouse were reported in another study, which extended the analysis to involve several inflammatory mediators that act through distinct second

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messenger systems (Amaya et al., 2006). However, there was no difference in pain behavior between wild-type and Nav1.9-null mice when PGE2 was injected into the subarachnoid space of spinal cord, suggesting that Nav1.9 does not contribute to direct central sensitization (Amaya et al., 2006), consistent with the restriction of the channel’s expression to peripheral sensory neurons (Dib-Hajj et al., 1998b; Tate et al., 1998). Furthermore, NGF-mediated effects were not different between wild-type and Nav1.9-null mice (Amaya et al., 2006), consistent with the expression of Nav1.9 in DRG neurons that respond to glial-derived neurotrophic factor but not NGF (Cummins et al., 2000; Fjell et al., 1999a). However, unlike Nav1.8-null mice, yet another Nav1.9-null mouse model showed no deficits in nematode-induced inflammatory visceral pain (Hillsley et al., 2006). It is noteworthy that Nav1.9 has been detected in myenteric sensory neurons (Rugiero et al., 2003) but not in afferents innervating the bladder (Black et al., 2003). Taken together, these data support an important role of Nav1.9 in some (but not all) inflammatory pain conditions.

3.5 ROLE OF SODIUM CHANNELS IN PAINFUL DIABETIC NEUROPATHY Sodium channel dysregulation has been investigated in diabetic neuropathy, which was evoked in rats by treatment with streptozotocin (STZ) that resulted in tactile allodynia 6 weeks following the injection of the drug (Craner et al., 2002). Dysregulated expression of sodium channels (Fig. 3.7) was evident in diabetic rats 8 weeks after the onset of allodynia with significant increases in transcript levels in small DRG neurons (525 mm) of Nav1.3 and Nav1.6, no change in Nav1.1, Nav1.7 and Nav1.9, and a reduction in Nav1.8 (Craner et al., 2002). Protein levels generally mirrored transcript levels in the diabetic neurons, except for a small increase in Nav1.9 protein (Craner et al., 2002). Notably, the expression of Nav1.9 was shown to increase substantially in large DRG neurons. Nav1.7 was reported to increase as judged by immunoblot assay in another study (Hong et al., 2004). Dysregulation of sodium channel transcript and protein levels in diabetic neurons is reflected in changes to the TTX-S and TTX-R sodium currents in these neurons (Hong et al., 2004). Electrophysiological studies showed an increase in the TTX-S sodium current, consistent with the upregulation of Nav1.3, Nav1.6, and Nav1.7 channels (as discussed above). Importantly, there was a significant increase in the current in response to a small, slow depolarization (ramp stimulus) of the diabetic neuron (Hong et al., 2004). The increase in ramp current is consistent with an upregulation of Nav1.3 and/or Nav1.7 channels since both the channels have been shown to produce a robust ramp current both in DRG neurons and in heterologous expression systems (Cummins et al., 1998; Cummins et al., 2001; Herzog et al., 2003). The increase in ramp currents in diabetic neurons may contribute to a lowered threshold for firing in these neurons. The electrophysiological studies, however, have also shown that STZ-induced diabetes increases the slowly inactivating TTX-R current and shifts the voltage

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FIGURE 3.7 Sodium channel expression in control, 1-week, and 8-week allodynic DRG neurons in the rat streptozotocin model of diabetes. Nav1.3 and Nav1.6 immunoreactivity is increased in both 25 and 425 mm diameter diabetic DRG neurons at 1- and 8-week allodynia compared to control neurons. Nav1.8 immunolabeling in both neuron size classes at 1-week allodynia is similar to that exhibited by control neurons; in contrast, 425 mm DRG neurons display decreased Nav1.8 immunoreactivity at 8-week allodynia. Scale bar: 25 mm. (Modiﬁed and reproduced with permission from Craner et al. (2002).)

dependence of activation and steady-state inactivation in a hyperpolarizing direction (Hong et al., 2004), in disagreement with the reduction in transcript and protein levels of this channel (Craner et al., 2002; Hong et al., 2004). In an attempt to reconcile this paradoxical finding, Hong et al. (2004) show an elevated level of serine/threonine phosphorylation of Nav1.8 in diabetic neurons. Phosphorylation of Nav1.8 at serine residues in L1 causes an increase in the peak current of this channel in a PKA- and PKC-dependent manner (Fitzgerald et al., 1999; Vijayaragavan et al., 2004) and by stress activated mitogen-activated protein kinase (MAPK) p38 (Hudmon, 2008). Thus, the reduction of the total protein of Nav1.8 in DRG neurons appears to be overcompensated by the modulation of the channel by phosphorylation, which may enhance the stability of the channel at the cell surface and/or alter its gating to increase single-channel properties. The increased levels of Nav1.8 current are predicted to enhance neuronal excitability. The functional consequences of diabetic-induced changes to the persistent TTX-R Nav1.9 current (Cummins et al., 1999), however, remain to be examined.

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FIGURE 3.8 Nav1.3 expression and pain-related phenomena after spinal cord injury (SCI). In the lumbar dorsal horn of intact animals (a), Nav1.3 protein expression is very low. In contrast, 28 days following T9 SCI, Nav1.3 expression is upregulated in lumbar dorsal horn. At

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3.6 SODIUM CHANNEL DYSREGULATION AND CENTRAL SENSITIZATION AND CENTRAL PAIN Peripheral injury leads to sensory neuronal hyperexcitability that propagates postsynaptically to second- and third-order nociceptive neurones in the CNS; this process has been termed as central sensitization, and it shares many aspects of LTP (Ji, 2004; Woolf, 2004; Campbell and Meyer, 2006). The release of neurotransmitters from primary afferent terminals in the superficial lamina of the dorsal horn is traditionally thought to be a major determinant of central sensitization (Hunt and Mantyh, 2001). The LTP model of inflammatory hyperalgesia is supported by recent findings showing that low-level activity of presynaptic nociceptive fibers, similar to the activity during inflammation of peripheral targets, can lead to central sensitization, suggesting the presence of a synaptic amplifier in the dorsal horn (Ikeda et al., 2006). In this chapter, we will concentrate on the role of sodium channel dysregulation, which also appears to contribute to central sensitization and central pain. Sodium channel dysregulation in second- and third-order nociceptive neurons has been shown to contribute to central sensitization following peripheral nerve injury (Fig. 3.8). Specifically, CCI of sciatic nerve causes the upregulation of sodium channel Nav1.3 in primary sensory neurons, and this expression plasticity is extended postsynaptically into higher order neurons (Hains et al., 2003). In additional, contusion spinal cord injury (SCI) results in the upregulation of Nav1.3 in neurons along the spinothalamic tract, which are located caudal to the site of injury (Hains et al., 2003). The available evidence suggests that expression of Nav1.3 may contribute to hyperexcitability of these neurons, enlargement of peripheral receptive fields, and central pain (Waxman and Hains, 2006). The dysregulated expression of Nav1.3 in spinal cord neurons following peripheral injury or spinal cord injury suggests that sodium channel blockers may be effective therapeutic agents after SCI. Indeed, phenytoin treatment provides neuroprotection and improves functional outcome in an experimental model of 3

this timepoint, antisense (AS) oligodeoxynucleotides generated against Nav1.3 or its mismatch (MM) sequence were topically administered to the lumbar spinal cord. Extracellular unit recordings of multireceptive neurons revealed hyperresponsiveness to application of brush (BR), press (PR), pinch (PI), increasing strength von Frey filaments (0.39 g, 1.01 g, 20.8 g), and a noxious thermal stimulus (47 C) in MM-treated animals after SCI (c). In AS-receiving animals, evoked hyperresponsiveness of dorsal horn neurons after SCI was reduced. Nav1.3 expression was similarly low in the ventral posterolateral (VPL) nucleus of the thalamus in intact animals (e). In contrast, 4 weeks after SCI, Nav1.3 expression was upregulated in the VPL (f), but not adjacent structures. Unit recordings of VPL neurons following SCI and MM administration revealed no changes in hyperresponsiveness following stimulation of hindpaw receptive fields (g), however, AS administration resulted in reduced evoked responsiveness of VPL neurons (h). Lumbar intrathecal administration of AS, but not MM, resulted in a partial restoration of paw withdrawal thresholds to mechanical stimulation of the hindpaw (i), as well as paw withdrawal latencies to noxious thermal stimulation (j). Dotted line represents mechanical and thermal thresholds in intact animals. *p 5 0.05 SCI versus SCI þ AS. (Modified and reproduced with permission from Hains et al. (2004a, 2005).)

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contusion SCI (Hains et al., 2004). In addition, several studies have shown that the knockdown of Nav1.3 in central nocieptive neurons following peripheral (CCI injury model) or contusion SCI reduces hyperexcitability of these neurons and attenuates neuropathic pain behavior (Hains et al., 2003, 2004a, 2006). Two articles, however, have presented contradicting findings to the prevailing view of a key role of Nav1.3 in neuropathic pain (Lindia et al., 2005; Nassar et al., 2006). Mice with global or DRG-specific knockout of Nav1.3 develop normally, are fertile, and, surprisingly, have been reported to demonstrate normal neuropathic pain behavior after nerve injury (Nassar et al., 2006). It is possible though that either global or DRG-specific deletion of Nav1.3 leads to unexpected gene expression adjustments that might influence the behavior of the mice. This view is consistent with the modest effect of knocking out Nav1.8 on neuropathic pain behavior (Akopian et al., 1999) despite the fact that Nav1.8 contributes most of the current underlying the depolarizing phase of action potential in the neurons where it is expressed (Renganathan et al., 2001; Blair and Bean, 2002). Alternatively, the role of Nav1.3 in rendering primary and higher order neurones hyperexcitable might be redundant, and it may be replaced by another channel in the knockout mice. Lindia et al. (2005) have confirmed that SNL causes an upregulation of Nav1.3 within DRG neurons and have reported that the administration of specific antisense oligonucleotides intrathecally reduces the Nav1.3 immunoreactivity by 50% but does not ameliorate the mechanical or cold allodynia typical of this animal model (Lindia et al., 2005). In contrast, studies by Hains et al. (2003, 2004a, 2006) have reported near-complete reversal of injury-induced Nav1.3 upregulation in secondand third-order neurons following intrathecal administration of Nav1.3 antisense oligonucleotides. It is possible that a 50% reduction in Nav1.3 is not enough to attenuate neuropathic pain behavior. Alternatively, differences in antisense chemistry, synthesis, purification, dose, or delivery methods could be significant factors that affect the outcome of these studies. In addition, because none of these studies have investigated global gene expression following the antisense administration, it is possible that off-target effects, either pro- or antinociceptive, might have lead to the different outcomes. A direct test of the contribution of overexpression of Nav1.3 on excitability of DRG neurons could shed light on the potential role of this channel in painful neuropathies.

3.7 SODIUM CHANNELS AND INHERITED PAINFUL NEUROPATHIES Inherited painful peripheral neuropathies may have diverse etiologies. Mutations in sodium channel Nav1.7 have been identified in patients suffering from inherited erythromelalgia (IEM) (Waxman and Dib-Hajj, 2005; Dib-Hajj, 2006) and paroxysmal extreme pain disorder (PEPD) (Fertleman et al., 2006). Recently, individuals with congenital and complete loss of Nav1.7 have been reported to be “indifferent” to pain while showing no deficits in other sensory modalities (Cox et al., 2006). Thus, similar to acquired sodium channelopathies following trauma or inflammation, some inherited painful disorders are sodium channelopathies.

SODIUM CHANNELS AND INHERITED PAINFUL NEUROPATHIES

3.7.1

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Inherited Erythromelalgia

Genetic studies in human patients with early-onset inherited erythromelalgia, a heritable disorder characterized by severe painful symptoms of the extremities that are accompanied by reddening of skin, have linked it to mutations in Nav1.7 (Waxman and Dib-Hajj, 2005; Dib-Hajj, 2006). Early-onset primary erythromelalgia (also known as erythermalgia) is a lifelong, autosomal dominant disorder that has been linked to missense mutations in Nav1.7 (Yang et al., 2004b; Dib-Hajj et al., 2005; Drenth et al., 2005; Michiels et al., 2005; Han et al., 2006; Harty et al., 2006). All these mutations have been shown to lower the threshold for channel activation, and most cause an increase in the amplitude of ramp currents and slowed deactivation kinetics (Cummins et al., 2004; Dib-Hajj et al., 2005; Choi et al., 2006; Han et al., 2006; Harty et al., 2006; Lampert et al., 2006). The introduction of these mutant channels into DRG neurons, such as Nav1.7/A863P, renders these neurons hyperexcitable by lowering the threshold for single action potential firing and increases the number of action potentials in response to a graded stimulus (Fig. 3.9) (Dib-Hajj et al., 2005; Harty et al., 2006; Rush et al., 2006). The expression of some mutant Nav1.7 channels in DRG neurons has been associated with a depolarization of the resting membrane potential of these neurons (Harty et al., 2006; Rush et al., 2006). This effect of mutant Nav1.7 channels brings the resting membrane potential of DRG neurons closer to the activation threshold voltage of Nav1.8 (Akopian et al., 1996; Sangameswaran et al., 1996). Depolarization of neuronal resting membrane potential in neurons that do not normally produce Nav1.8 channels is predicted to cause these neurons to become hypoexcitable because of resting inactivation of the TTX-S channels in these cells, which have hyperpolarized voltage dependence of activation and inactivation compared to Nav1.8 (Rush et al., 2007). Indeed, Nav1.7 mutant-mediated neuronal hyperexcitability appears to be dependent upon the presence of Nav1.8 because expression of L858H mutant Nav1.7 in superior cervical ganglion (SCG) neurons, which do not express endogenous Nav1.8, renders these neurons hypoexcitable (Rush et al., 2006). The coexpression of L858H Nav1.7 mutant and Nav1.8 in SCG neurons restores excitability of these neurons to near-normal levels although the L858H mutation depolarizes these cells (Rush et al., 2006), showing that the response to depolarization is conditioned by the absence (SCG neurons) or presence (DRG neurons) of Nav1.8. However, depolarization of the resting membrane potential does not explain all aspects of the behavior of DRG neurons housing Nav1.7 mutant channels (Harty et al., 2006), suggesting that other changes in the properties of mutant Nav1.7 channels may contribute to the hyperexcitability of DRG neurons in patients with inherited erythromelalgia. 3.7.2

Paroxysmal Extreme Pain Disorder

Recently, another inherited painful syndrome, paroxysmal extreme pain disorder (PEPD), previously known as familial rectal pain (Bednarek et al., 2005; Fertleman and Ferrie, 2006), has been linked to a different set of mutations in Nav1.7 (Fertleman et al., 2006). PEPD is manifested as severe pain and flushing in the lower body in infants during bowel movement, transforming with age to ocular and mandibular

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FIGURE 3.9 Nav1.7/A863P mutation decreases action potential threshold and increases frequency of firing in small, current-clamped DRG neurons. (a) Responses of a current-clamped DRG neuron transfected with WT Nav1.7 DNA to a series of subthreshold and suprathreshold depolarizing current steps. Starting at subthreshold stimulus intensity, the current amplitude was increased in 5 pA increments to intensity well beyond threshold. RMP for this cell was 55 mV and threshold was 310 pA. A DRG neuron expressing WT channels responds to a 1 s depolarizing current step, that is one, two, and three times the current threshold for action potential generation by the firing of up to two spikes (at 3 threshold). (b) The same threshold protocol applied to a DRG neuron transfected with the A863P mutant DNA elicits AP with a smaller current injection. RMP for this cell was 45 mVand threshold was 95 pA. Arrows with numbers indicate the current step amplitude used to elicit the labeled response. A DRG neuron expressing A863P mutant channels responds to a 1 s depolarizing current step, that is, one, two, and three times the current threshold for action potential generation by the firing of up to 11 spikes (at 3 threshold). (Modified and reproduced with permission from Harty et al. (2006).)

pain (Fertleman and Ferrie, 2006). Three mutations were characterized by whole-cell patch clamp analysis and were shown to impair fast inactivation of the mutant channels but with no effect on channel activation (Fertleman et al., 2006). Impaired fast inactivation allows more sodium current to flow through the mutant channel and is

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predicted to increase DRG neuron hyperexcitability. Interestingly, patients with PEPD tend to respond favorably to treatment with the anticonvulsant drug carbamazepine (Fertleman et al., 2006), unlike patients with inherited erythromelalgia who are refractory to long-term treatment with nonselective sodium channel blockers (Novella et al., 2007). 3.7.3

Congenital Insensitivity to Pain

Congenital insensitivity to experience pain is a very rare clinical phenotype. Recently, this trait has been shown to be inherited in an autosomal recessive manner in three consanguineous families from Pakistan, and nonsense mutations in Nav1.7 have been genetically linked to this phenotype (Cox et al., 2006). Patients missing functional Nav1.7 channels do not experience pain from inserting sharp objects in their hands or walking on hot surfaces (burning coal) or after bone fracture and tongue biting, but without deficits in other sensory modalities, for example, sensation of warmth or touch (Cox et al., 2006). Parents are asymptomatic, suggesting that SCN9A does not cause haploinsufficiency. The truncated channel protein produces no sodium current when expressed in the mammalian cell line HEK 293, suggesting a molecular pathophysiological cause for the phenotype (Cox et al., 2006). These data are in agreement with the results from animal studies using mice that lack Nav1.7 (Nassar et al., 2004), although the human and mouse cases differ in that the neuropathic component of pain in the animal model is intact (Nassar et al., 2005) whereas it appears to be missing in the patients (Cox et al., 2006).

3.8 CONCLUSIONS Sodium channelopathies underlie acquired and inherited neuropathies, some forms of epilepsy, and diseases of skeletal and cardiac muscles (Waxman, 2001; Lai et al., 2004; Viswanathan and Balser, 2004; Wood et al., 2004; George, 2005; Meisler and Kearney, 2005; Moss and Kass, 2005; Waxman and Dib-Hajj, 2005; Cannon, 2006; Venance et al., 2006; Waxman and Hains, 2006). Dysregulated expression of several sodium channels under a variety of pathological conditions induced by trauma or inflammation, and metabolic disorders, such as diabetes, has been linked to chronic pain, and blocking of sodium channels has been shown to ameliorate, or at least partially ameliorate, pain symptoms in some clinical studies of neuropathic pain in humans (Waxman, 2001; Lai et al., 2004; Wood et al., 2004; Waxman and Hains, 2006). Importantly, existing sodium channel blockers can be effective in treatment of some cases of neuropathic pain but suffer from undesirable side effects because they are not isoform selective (Rice and Hill, 2006). The contribution of individual channels to the pain state, however, is only now becoming better understood. As the roles of different channel isoforms under pathological conditions are elucidated, the identification of sodium channel isoforms that play dominant roles in pain should contribute to mechanism-based diagnosis and identify potential targets for treatment.

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Rush AM, Dib-Hajj SD, Liu S, Cummins TR, Black JA, Waxman SG, 2006. A single sodium channel mutation produces hyper- or hypoexcitability in different types of neurons. Proc Natl Acad Sci USA 103: 8245–8250. Rush AM, Waxman SG, 2004. PGE2 increases the tetrodotoxin-resistant Nav1.9 sodium current in mouse DRG neurons via G-proteins. Brain Res 1023: 264–271. Saleh S, Yeung SY, Prestwich S, Pucovsky V, Greenwood I, 2005. Electrophysiological and molecular identification of voltage-gated sodium channels in murine vascular myocytes. J Physiol Lond 568: 155–169. Sangameswaran L, Delgado SG, Fish LM, Koch BD, Jakeman LB, Stewart GR, Sze P, Hunter JC, Eglen RM, Herman RC, 1996. Structure and function of a novel voltage-gated, tetrodotoxinresistant sodium channel specific to sensory neurons. J Biol Chem 271: 5953–5956. Sangameswaran L, Fish LM, Koch BD, Rabert DK, Delgado SG, Ilnicka M, Jakeman LB, Novakovic S, Wong K, Sze P, Tzoumaka E, Stewart GR, Herman RC, Chan H, Eglen RM, Hunter JC, 1997. A novel tetrodotoxin-sensitive, voltage-gated sodium channel expressed in rat and human dorsal root ganglia. J Biol Chem 272: 14805–14809. Sasaki M, Black JA, Lankford KL, Tokuno HA, Waxman SG, Kocsis JD, 2006. Molecular reconstruction of nodes of Ranvier after remyelination by transplanted olfactory ensheathing cells in the demyelinated spinal cord. J Neurosci 26: 1803–1812. Scadding JW, 1981. Development of ongoing activity, mechanosensitivity, and adrenaline sensitivity in severed peripheral nerve axons. Exp Neurol 73: 345–364. Schafer DP, Custer AW, Shrager P, Rasband MN, 2006. Early events in node of Ranvier formation during myelination and remyelination in the PNS. Neuron Glia Biol 2: 69–79. Schaller KL, Caldwell JH, 2000. Developmental and regional expression of sodium channel isoform NaCh6 in the rat central nervous system. J Comp Neurol 420: 84–97. Schaller KL, Krzemien DM, Yarowsky PJ, Krueger BK, Caldwell JH, 1995. A novel, abundant sodium channel expressed in neurons and glia. J Neurosci 15: 3231–3242. Shah BS, Rush AM, Liu S, Tyrrell L, Black JA, Dib-Hajj SD, Waxman SG, 2004. Contactin associates with sodium channel Nav1.3 in native tissues and increases channel density at the cell surface. J Neurosci 24: 7387–7399. Sleeper AA, Cummins TR, Dib-Hajj SD, Hormuzdiar W, Tyrrell L, Waxman SG, Black JA, 2000. Changes in expression of two tetrodotoxin-resistant sodium channels and their currents in dorsal root ganglion neurons after sciatic nerve injury but not rhizotomy. J Neurosci 20: 7279–7289. Sommer C, Schafers M, 1998. Painful mononeuropathy in C57BL/Wld mice with delayed wallerian degeneration: differential effects of cytokine production and nerve regeneration on thermal and mechanical hypersensitivity. Brain Res 784: 154–162. Song XJ, Zhang JM, Hu SJ, LaMotte RH, 2003. Somata of nerve-injured sensory neurons exhibit enhanced responses to inflammatory mediators. Pain 104: 701–709. Study RE, Kral MG, 1996. Spontaneous action potential activity in isolated dorsal root ganglion neurons from rats with a painful neuropathy. Pain 65: 235–242. Sun JH, Yang B, Donnelly DF, Ma C, LaMotte RH, 2006. MCP-1 enhances excitability of nociceptive neurons in chronically compressed dorsal root ganglia. J Neurophysiol 96: 2189–2199. Tan ZY, Donnelly DF, LaMotte RH, 2006. Effects of a chronic compression of the dorsal root ganglion on voltage-gated Naþ and Kþ currents in cutaneous afferent neurons. J Neurophysiol 95: 1115–1123.

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4 THE ROLE OF ION CHANNELS IN THE ETIOLOGY AND DEVELOPMENT OF GLIOMAS Amy K. Weaver and Harald Sontheimer Department of Neurobiology and Center for Glial Biology in Medicine, The University of Alabama, Birmingham, AL 35294, USA

4.1 INTRODUCTION Over the last decades, several studies have characterized ion channel expression in glial cells (Sontheimer and Waxman, 1993; Ransom and Sontheimer, 1995; Sontheimer et al., 1996; Bordey and Sontheimer, 1997, 1998b, 2000; MacFarlane and Sontheimer, 1997, 2000a; Parkerson and Sontheimer, 2004; Olsen et al., 2006). By and large, these studies agree on the notion that these channels do not participate in any form of electrical signaling but instead engage in extracellular ion homeostasis or support fundamental aspects of cell biology. An excellent example for a homeostatic role of ion channels is the glial inwardly rectifying Kþ channel, Kir4.1, which has been shown to constitute the primary uptake pathway for neuronally released Kþ from the extracellular space (Sontheimer et al., 1996; Higashi et al., 2001; Olsen et al., 2006, 2007). Its elimination results in a complete breakdown of extracellular Kþ homeostasis (Neusch et al., 2006; Kucheryavykh et al., 2007). Numerous studies have demonstrated a role for delayed rectifying Kþ channels of the Kv1.x family in cell cycle progression. In astrocytes, Kv1.5 is reversibly phosphorylated by Src kinase as cells enter S-phase leading to a marked cell depolarization. Suppression of Kv1.5 activity or inhibition of Src kinases causes reversible cell cycle arrest (MacFarlane and Sontheimer, 2000b). Importantly, acute Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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injury causes gliosis and a similar activation of Kv1.5 in many glial cell types, including retinal glial cells (Bordey et al., 2001). As the participation of ion channels in cell proliferation became more compelling, numerous investigations examined tumor cells that show aberrant, uncontrolled cell proliferation. These studies propose that certain channels, the ether-a-go-go (EAG) channel family being a prominent example, may be so strongly connected to the oncogenic phenotype of these cancers that these channels could actually be considered oncogenes (Meyer et al., 1999; Kunzelmann, 2005; Camacho, 2006). This chapter will focus exclusively on a subset of primary central nervous system (CNS) tumors, gliomas, to illustrate how ion channel function promotes the unique biology of these cancers. Most of the primary brain tumors originate from glial cells or their progenitor cells and are collectively called gliomas. These rapidly proliferating tumors include astrocytomas, glioblastomas, and oligodendrogliomas and share an unusual propensity to invade the brain. This feature is particularly pronounced in the highest tumor grade, glioblastoma multiforme, which is the most frequent adult glioma. By invading the normal brain parenchyma, they escape complete surgical resection and make effective radiation therapy challenging. Consequently, gliomas carry an unusually high mortality rate with most patients succumbing to the disease within 12–18 months of diagnosis, typically due to regrowth of the tumor (Kleihues et al., 1995; Ohgaki and Kleihues, 2005). Glioma migration/invasion requires complex and well-arranged interactions of molecular motors with the cytoskeleton and adhesion sites of cells interacting with other cells or the extracellular matrix surrounding them (for reviews see Giese et al., 1994; Gladson, 1999; Uhm et al., 1999; Tonn and Goldbrunner, 2003; Visted et al., 2003; Demuth and Berens, 2004; Zamecnik, 2005). It also involves the production and release of matrix-degrading enzymes (Ohnishi et al., 1993; Maidment et al., 1996; Deryugina et al., 1997; Treasurywala and Berens, 1998; Belien et al., 1999; Gladson, 1999; Nakada et al., 2003) and chemotactic interactions with neighboring cells (Merzak et al., 1994; Giese et al., 1995, 1998; Chicoine and Silbergeld, 1997; Maidment et al., 1997; Manning et al., 2000; Ritch et al., 2003; Farin et al., 2006). Many of these features are shared with other cancers. However, in stark contrast to other cancers, glioma cells spread by active cell migration rather than spreading passively via hematogenous routes. As a result, gliomas rarely ever metastasize outside the CNS, they readily invade and metastasize within the brain and spinal cord. This is somewhat surprising since the extracellular space in the mature brain is very small. Cells that navigate the narrow and tortuous extracellular space will need to be able to adjust their shape and volume to fit through these narrow spaces. Indeed, as illustrated for two examples in Fig. 4.1, glioma cells that penetrate through the extracellular space are elongated, wedge-shaped with slender cell processes (Soroceanu et al., 1999). This morphology is consistent with an overall shrinkage of the cell body and an elongation of invadipodia, the leading processes of cells actively engaged in invasion. Cell shrinkage requires the secretion of cytoplasm, principally KCl and water from the cell. As further discussed below, and illustrated in Fig. 4.6a, this is accomplished by the coordinated activity of defined Kþ and Cl channels, which are the principal diffusional release pathways for these ions.

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FIGURE 4.1 (a) Semithin section through a coculture of tumor spheroids and fetal rat brain aggregates, stained with toluidine blue. Glioma cells are seen advancing between two normal rat brain cells (arrows). Scale bar, 20 mm. (b) Area of detail is of the same preparation as in (a), analyzed by transmission electron microcopy. Glioma cells are easily recognized because of the abundance of ribosomes and other organelles that incorporate lead citrate and give a darker appearance. Arrows indicate area of contact between an elongated tumor cell and two other membranes, presumably of the fetal rat brain. Scale bar, 1 mm. (Adapted from Soroceanu et al., 1999).

4.2 Kþ CHANNELS IN GLIOMAS Potassium channels are essential to establish and maintain a negative resting potential in many cells including nonexcitable cells. At least 40 different Kþ channels can now be identified molecularly and many cells express more than one channel type. In the earliest biophysical studies of glial cells (Kuffler, 1967), it was noted that the glial cell membrane appeared to be almost exclusively permeable to Kþ ions. Not surprisingly, later studies showed that glial cells express Kþ channels at a very high density. Through the use of genetic tools, it has now become evident that the resting potential of astrocytes and retinal glial cells is mainly established by the activity of Kir4.1, a voltage-dependent inwardly rectifying Kþ channel (Chvatal et al., 1995; Li et al., 2001; Kofuji et al., 2002). Kir4.1 channel expression arising upon differentiation of progenitor cells into mature postmitotic cells is developmentally regulated in astrocytes and in oligodendrocytes (Ransom and Sontheimer, 1995). The malignant transformation of astrocytes, that is, formation of gliomas, correlates with a depolarization of the resting membrane potential and, indeed, this seems to be due to the loss of functional Kir4.1 channels (Olsen and Sontheimer, 2004). Surprisingly however, Kir4.1 channels are still expressed, but are mislocalized to a perinuclear region, presumably the ER indicating an inability to correctly traffic the channel to the membrane. Recombinant expression studies suggest that insertion of Kir4.1 into glioma membranes reestablishes a negative resting potential and is sufficient to cause growth inhibition (Higashimori and Sontheimer, 2007). This study also demonstrates that a downregulation of Kir4.1 channels is required to allow glial and glioma cells to

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proliferate and from a mechanistic point of view, a depolarizing shift in resting membrane potential is essential for a cell’s progression through S-phase. An involvement of Kþ channels in growth control of cancer cells is now widely accepted (for review see Wonderlin and Strobl, 1996; Kunzelmann, 2005), suggesting that Kþ channels may be excellent therapeutic cancer targets. Many studies have implicated, in particular, two Kþ channel families in growth control. These are the large conductance, calcium-activated Kþ (BK) channels and the human ether-agogo-like (hERG) channels. While very little is known about hERG channels in the glioma, BK channels are highly expressed and are probably the most well-studied ion channel in the glioma field. A decade ago we noted the presence of large outward currents in cells from patient biopsies that were activated by membrane depolarizations larger than þ50 mV (Ullrich et al., 1998). Subsequent studies identified these currents as Kþ selective, sensitive to micromolar concentrations of Ca2þ, and inhibited by Iberiotoxin (IbTX), tetraethylammonium(TEA) chloride, and tetrandrine (Ransom and Sontheimer, 2001), all indicative of BK channel expression and representative examples are shown in Fig. 4.2a. The BK channels are a member of the family of calcium-activated Kþ channels and their names derive from their very large single-channel conductances (>200 pS in glioma cells). These channels are unique among Kþ channels in that their activity can be modulated by changes in intracellular calcium concentration in addition to changes in membrane voltage. Interestingly, the BK channel expressed in glioma cells is a novel splice variant with a 36 amino acid insert that confers an increased sensitivity to calcium when compared to other known BK channel splice variants (Ransom et al., 2002; Liu et al., 2002) (Fig. 4.2b). Because of this, these channels are well suited to respond to various mitogenic and motogenic signals that affect intracellular calcium concentration. This combined with the fact that BK currents account for most of the voltage-dependent currents in glioma cells has led investigators to examine the function of these channels in gliomas. Through the use of specific pharmacology, it has been discovered that BK channels indeed play a role in glioma cell proliferation, albeit a complicated one. Straightforward examination of glioma cell proliferation under normal culture conditions has revealed no effect of the specific BK channel inhibitors, IbTX and paxilline. However, when serum was removed from the culture media (Weaver et al., 2004) or when cells were stimulated to proliferate by raising extracellular Kþ concentration (Basrai et al., 2002), these drugs inhibited glioma cell proliferation. While this may seem counterintuitive, it actually may reveal a significant problem with the use of glioma cell lines in research. The brain does not exist in a state of unlimited nutrient and oxygen supply. In fact, gliomas proliferate at such a rate that they rapidly exhaust the nutrients and oxygen that are available to them, suggesting that current culture conditions containing fetal calf serum and 21 % oxygen do not accurately mimic the conditions in vivo. In agreement with this, gliomas are characterized by overexpression and/or constitutive activation of growth factor receptors (Collins, 1995; Di et al., 2000). One such receptor, the erbB2 growth factor receptor, is constitutively active in gliomas and appears to be capable of modulating BK channel activity through changes

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FIGURE 4.2 BK currents in glioma cells (a) whole-cell currents from a D54-MG cell with the indicated concentrations of iberiotoxin, a selective BK channel inhibitor. Inset: The time course of current inhibition in this cell by iberiotoxin (current measured at þ140 mV). Iberiotoxin inhibition was reversible. (b) Average activation curves with the indicated [Ca2þ]i. Data are from 4–17 outside-out patches from D54MG glioma cells. Error bars are omitted for clarity. Solid lines are drawn according to the Boltzmann equation. (Adapted from Liu et al., 2002; Ransom et al., 2002).

in [Ca2þ]i (Olsen et al., 2005) providing direct evidence that BK channels can be regulated by proliferative signaling. In addition to its role in proliferation, BK channel involvement in migration/ invasion has also been documented. The results of these studies, however, were also complex. In 2000, Bordey et al. (2000) showed that muscarinic activation of acetycholine (ACh) receptors activated BK channels as well as inhibited glioma cell migration in a two-dimensional motility assay. These results were corroborated 3 years later by Kraft et al. (2003) who noticed that activation of BK channels with phloretin and NS1619, a synthetic BK channel opener, also inhibited migration of another glioma cell line. Contrary to these findings, a later study by Weaver et al. (2006)

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examined the effect of BK channel inhibitors and NS1619 on glioma cell migration across a transwell barrier, a technique used to mimic the physical constraints encountered in vivo, and found that inhibition, not activation, of BK channels inhibited migration of yet another glioma cell. It remains to be seen whether these different effects are due to different assay systems or due to the use of different glioma cell lines. However, a recent study suggests that BK channels are found in lipid rafts on the lamellipodia, the invading edge, of glioma cells where they specifically respond to both mitogenic and motogenic signals (Weaver et al., 2007). Together these findings may indicate that BK channel activation can have different cellular consequences, depending on the upstream signal responsible for the calcium changes activating the channels. In light of the high expression of BK channels in gliomas in vitro and in vivo and their described role in growth control and cell invasion, one may wish to think of them as potential therapeutic targets. Indeed, BK channels have two specific blockers and several synthetic openers making it possible to target these channels without nonspecifically affecting other ion channels. Unfortunately, however, BK channels have fundamental roles in smooth muscle contraction/relaxation and help control bursting frequency in neurons. All of this must be taken into account when designing possible therapeutic strategies to target these channels. The human ether-a-go-go family of ion channels is made up of three subfamilies, the ether-a-go-go or EAG channels, the ether-a-go-go-related or ERK and the ethera-go-go-like or ELK channels. For the purposes of the chapter, we will collectively refer to these channels as the EAG channels. All EAG channels give rise to outwardly rectifying, slowly activating, noninactivating Kþ currents that activate in response to depolarizing voltages. This family of channels is encoded by eight genes, Kv10.1–10.2, Kv11.1–11.3, and Kv12.1–12.3, and many of these channels seem to be involved in cell cycle regulation (Pardo et al., 1998). In fact, the first discovery of their association with the cell cycle came from neuroblastoma. The whole-cell patch-clamp recording indicated that EAG current density varied widely from cell to cell (Meyer and Heinemann, 1998). However, if cells were synchronized in the G1 phase of the cell cycle, current density was the same in all cells. Furthermore, as cells moved through the cell cycle, current density changed with the largest density found in G1. This same phenomenon was later demonstrated for breast cancer cells as well (Ouadid-Ahidouch et al., 2001). Increasing the link between EAG cells and cancer means that EAG transcripts and/or currents can be found in a large number of cancers including neuroblastoma, breast, gastrointestinal, endometrial, and leukemic and colon cancers (Meyer et al., 1999; Smith et al., 2002; Suzuki and Takimoto, 2004; Ousingsawat et al., 2007). In the past 5 years, this list has grown to include gliomas (Patt et al., 2004). Specifically, both eag1 and erg1 transcripts have been discovered in patient biopsies of both low-and high-grade gliomas. Surprisingly, pharmacological inhibition of hERG currents appears to dramatically reduce secretion of vascular endothelial growth factor (VEGF) specifically as epidermal growth factor secretion was unaffected (Masi et al., 2005). Both growth factors are known to be secreted from gliomas and secretion of VEGF, in particular, has been of interest recently as a potential target to limit the growth of gliomas in vivo. Studies on EAG channels in gliomas are still in

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their infancy, but if the above results are substantiated, EAG channels may be the ideal Kþ channels to target therapeutically. Currently, specific inhibitors for these channels are unavailable, but given their intimate association with cancer, drug development to generate specific inhibitors is a worthy pursuit. 4.3 Cl CHANNELS IN GLIOMAS A signature feature of nonmalignant glia is a very negative resting membrane potential (Kuffler, 1967), established primarily by the activity of the Kir4.1 inwardly rectifying Kþ channels. As discussed in the previous section, glioma cells do not express functional Kir channels and as a consequence have a relatively depolarized resting membrane potential of approximately 30 to 40 mV (Olsen and Sontheimer, 2004). We have already alluded to the fact that the absence of these channels is essential for cells to go through mitosis (Higashimori and Sontheimer, 2007). Surprisingly, however, the negative resting potential is at least in part established by a contribution of a resting Cl conductance (Ransom et al., 2001). As illustrated in Fig. 4.3, when recorded with amphotericin patch clamp so as to not dialyze the cell, the application of the Cl channel inhibitor NPPB caused the resting conductance to decrease markedly and input resistance to increase, while TEA had little effect. These data suggest that glioma cells indeed have Cl channels that show significant open probability at rest and, secondly, that even at 40 mV, an outward-directed driving force exists for Cl ions to leave the cells. The latter is particularly surprising as in many cell types, neurons being a prime example, Cl conductance stabilizes the resting potential rather than depolarizes it, as is evident from the inhibitory action of GABA-gated Cl channels (Ebihara et al., 1995). Using the fluorescence Cl indicator SPQ, we have been able to demonstrate directly that intracellular Cl in glioma cells is maintained around 80–100 mM through the activity of the NKCC1 transporter (unpublished). This value is at least twice as high as in astrocytes (Kimelberg, 1981) and approximately 10-fold higher than in most neurons (Ebihara et al., 1995). This fact will become important as we discuss the participation of Cl channels in cell invasion, which requires an efflux of Cl from these cells to promote cell shrinkage. Over the past 8 years, we have made a concerted effort to further identify and characterize the underlying Cl channels in glioma cells. Cl channels cloned to date fall into four molecular superfamilies (Valverde, 1999): the CFTR channels, Ca2þ-activated Cl channels (Agnel et al., 1999), voltage-dependent anion-selective channels (VDACs) (Wunder and Colombini, 1991; Dermietzel et al., 1994; Dolder et al., 1999), and the ClC channels (Jentsch et al., 1999; Valverde, 1999). Of these, neither the CFTR nor Ca2þ-activated channels are present in gliomas but three members of the ClC family are consistently present in patient biopsies and isolated glioma cells (Olsen et al., 2003). These include ClC-2, ClC-3, and ClC-5. Biophysically, currents mediated by ClC-2 and ClC-3 channels could be identified. Unfortunately, specific inhibitors for Cl channels are not available and hence most investigators rely on a combination of differential biophysical and pharmacological properties

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FIGURE 4.3 The resting chloride conductance of glioma cells is present in nondialyzed cells (amphotericin-perforated patch clamp). (a) Double y-axis plot of current (at 40 mV) and input resistance (Rin) as a function of time in an amphotericin-perforated patch-clamped cell. NPPB (165 mM), but not TEA (1 mM), blocked an inward current at typical resting potentials (40 mV) and increased the input resistance. Inset shows current responses to a voltage step from 40 to 60 mV before and after NPPB application (used to calculate input resistance). The series resistance of this cell was 22 MW. (b) Summary of NPPB effects on input resistance in cells recorded from the perforated patch configuration. (c) Summary of NPPB effects on current at 40 mV in cells recorded from the perforated patch configuration. Data in (b and c) are displayed as mean SE (Ransom et al., 2001).

to identify the underlying channels. We have complemented such approaches with the use of antisense oligonucleotides specific for each of the ClC channels of interest to more unequivocally identify currents as being mediated by ClC-2 and ClC-3, respectively. As illustrated in Fig. 4.4a and b, ClC-2 channels give rise to inwardly rectifying currents that are insensitive to NPPB but blocked by Cd2þof 200 mM. Currents are enhanced if voltage steps are preceded by a short hyperpolarization to 120 mV. ClC-3 channels, by contrast, give rise to currents that are outwardly rectifying and show time-dependent inactivation at positive potentials. Currents are inhibited by DIDS and NPPB but not Cd2þ. Currents with this identical biophysical signature are also inhibited by chlorotoxin, a 36 amino acid peptide isolated from Leiurus quinquestriatus and suggested to be a Cl channel blocker (DeBin et al., 1993) (Fig. 4.4c). However, unlike NPPB, Cltx requires several minutes to achieve complete current inhibition (Ullrich et al., 1996). This discrepancy was recently explained by the

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FIGURE 4.4 Voltage-dependent inward Cl currents in human glioma cells. (a) Inward currents were evoked with voltage steps from 140 to þ20 mV from a holding potential of 40 mV. Cells were hyperpolarized to 120 mV for a minimum of 20 s to increase the activation of inward current. Representative traces of inward Cl current before (top) and after Cd2þ (200 mM) (middle) and the subtracted Cd2þ-sensitive current (bottom) are shown. (b) I–V plot of Cd2þ (200 mM)-sensitive Cl currents evoked from the same voltage step protocol. Currents returned after washout of Cd2þ (Olsen et al., 2003) (c) Whole-cell currents were recorded before and after the application of 1 mM Cltx. A near complete inhibition of ClC-3 currents is achieved with Cltx. Of note, unlike the block by NPPB, which develops within seconds, complete block by Cltx requires 10–15 min, and the example illustrated is recorded 15 min after the application of Cltx (Weaver et al., 2007).

identification of the actual receptor for Cltx as a protein complex consisting of matrix metalloproteinase 2 (MMP-2), its receptor MT1-MMP, and the ClC-3 Cl channel (Deshane et al., 2003; McFerrin and Sontheimer, 2005). Rather than blocking the channel directly, binding of Cltx to glioma cells causes the internalization of this molecular complex and hence the removal of functional ClC-3 channels from the plasma membrane into caveoli, a specialized vesicle associated with lipid rafts (McFerrin and Sontheimer, 2005). Although the underlying interactions and the signal that causes internalization of this complex remain elusive, the requirement for MMP-2 as target for Cltx has recently been confirmed in a related tumor model in vivo (Veiseh et al., 2007).

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In agreement with the hypothesized role of Cl channels in the glioma cell shape changes that facilitate cell invasion, all inhibitors of ClC-3 have been shown to also effectively inhibit glioma cell invasion. The simplest way to mimic the spatial constraints encountered by invading glioma cells is through the use of the so-called transwell chambers, which are modified Boyden chambers in which cells are allowed to migrate through pores of 5 mm from one chamber to another toward a chemoattractant molecule. Replacement of Cl in the medium with impermeant anions impeded the ability of cells to shrink sufficiently to transverse these barriers as did the inhibition of Cl channels with NPPB, DIDS, or Cltx (Ransom et al., 2001). The latter also inhibited cell invasion in situ using a spheroid confrontation assay. Together with animal studies in which it was demonstrated that Cltx selectively homes in on the tumor cells (Soroceanu et al., 1999), these studies paved the way for a clinical use of Cltx in patients with malignant gliomas as discussed at the end of this review.

4.4 VOLTAGE-GATED SODIUM CHANNELS Voltage-gated sodium (Naþ) channels are responsible for the depolarizing upstroke of the action potential in neurons and muscle cells. This family of channels consists of nine alpha subunits (Nav1.1–1.9) that combine with two beta subunits to form a heterotrimer. Each of the nine alpha subunits is expressed in the brain and gives rise to voltage-dependent currents that rapidly inactivate. These channels are characterized by their sensitivity to tetrodotoxin (TTX), a toxin isolated from the puffer fish. Because these channels are the principal substrates for excitability, it came as a great surprise when Ritchie and colleagues demonstrated them also in glial cells (Ritchie, 1992), albeit at much lower density. Subsequent studies suggested that unlike neurons, astrocytic Naþ channels function to maintain the intracellular Naþ concentration at a level sufficient to fuel Naþ-Kþ-ATPase activity and thus energy production for the cell (Sontheimer et al., 1994). Most of the cancer researches are performed in cell lines that derive from patient biopsies and have often been cultured over hundreds of passages in the lab. Electrophysiological recordings from glioma cell lines indicated a lack of expression of voltage-gated Naþ channels. Since astrocytes often lose ion channel expression in passages in vitro, this prompted investigators to examine glioma cells in human brain slices and/or cells acutely dissociated from patient biopsies. With this approach, Patt et al. (1996) recorded whole-cell TTX-sensitive Naþ currents from oligodendroglioma and oligoastrocytoma cells (Fig. 4.5a). Interestingly, oligodendrocytes, the presumed precursor to the tumors examined in this study, do not normally express Naþ channels leading one to speculate that the expression of these channels may be integral to the malignant transformation and propagation of these tumors. Bordey and Sontheimer (1998a) independently reported similar findings from astrocytomas. Specifically, the whole-cell patch recordings of low grade astrocytomas identified Naþ currents identical to those found in oligodendrogliomas. Possibly the most intriguing discovery of both of

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FIGURE 4.5 Astrocytoma cells express tetrodotoxin (TTX)-sensitive, voltage-dependent Naþ currents. (a) Whole-cell inward currents in response to 8 ms step depolarizations starting at 70 mV incremented by 10 mV from a holding potential of 110 mV. Currents before exposure to TTX (left) and after a 3-min exposure to 100 nM TTX (right). (b) Current–clamp traces from a glioma cell of a tumor biopsy after a short depolarizing current pulse. Spike amplitude was 52 mV, threshold was approximately 33 mV, rise time (beginning of pulse to peak value) was 15.1 ms, duration of spike from threshold was 11.3 ms, and corresponding ratio for the same recorded cell was 1.1 (Adapted from Bordey and Sontheimer, 1998a).

these studies is that upon injection of current, glioma cells are capable of generating spikes (Fig. 4.5b) akin to neuronal action potentials. However, the half-maximal voltage for inactivation of these channels in glioma cells is 47 mV, making it highly unlikely that these cells could ever generate an action potential in vivo since the typical resting membrane potential of a glioma cell is 40 mV. What makes this finding quite interesting is the fact that glioma cells express Naþ channels at a sufficient density to generate action potentials and hence express a much larger number of Naþ channels than that found in the nonmalignant glia. The reasons for this are entirely unclear. However, given the proliferative capacity of glioma cells, it has been speculated that these channels may serve a similar function in gliomas as in astrocytes, that is, supporting the activity of the cells Naþ/Kþ-ATPase. Thus,

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because glioma cells are metabolically quite active, a greater Naþ influx may be required to sustain their Naþ-Kþ-ATPase. Attempts have been made, albeit unsuccessful, to identify the subunit responsible for the Naþ currents in glioma cells. Glioma cell lines and biopsies, alike, seem to variably express each of the nine Naþ channel alpha subunits (Schrey et al., 2002). Moreover, functional studies are difficult since expression of these channels is lost in culture (Patt et al., 1996). A recent study was able to recover Naþ channel expression in glioma cell lines through a combination of removal of the fetal calf serum that is normally present in culture media and addition of nerve growth factor (NGF) (Kraft et al., 2001). In the future, this approach may not only make possible to assess the function of voltage-gated Naþ channels in gliomas, but may also allow to study pathways responsible for their regulation. However, regardless of the function of Naþ channels in gliomas, they make poor pharmacological targets due to their ubiquitous expression in the brain and their importance in all aspects of nervous system function.

4.5 VOLTAGE-GATED CALCIUM CHANNELS Arguably, the most ubiquitous ion channel expressed and found in almost all cell types and animal species are the voltage-gated calcium channels (VGCCs). These are comprised of a pore-forming a1 subunit along with three or four additional modulatory subunits. Five members of the VGCC family are encoded by 10 genes and thus there are 10 different a1 subunits: the L-type channels are encoded by Cav1.1–1.4, the N-, P/Q-, and R-type channels are encoded by Cav2.1–2.3, respectively, and the T-type channels are encoded by Cav3.1–3.3. The L-, N-, P/Q- and Rtype channels give rise to currents that are activated by large depolarizations and hence are collectively called the high-voltage activated (HVA) calcium channels. The L-type VGCCs can be blocked by a family of compounds termed dihydropyridines that include verapamil, nifedipine, and nicardipine. The remaining HVA channels each exhibit unique pharmacology; however, all HVA channels are sensitive to the heavy metal Cd2þ. In contrast, the T-type calcium channels are activated by very small depolarizations and give rise to small, transient currents. These channels are typically referred to as low-voltage activated (LVA) channels. In addition to being characterized by unique voltage dependence, LVA channels are not sensitive to typical VGCC antagonists and are instead inhibited by the heavy metal Ni2þ and the organic compound mibefradil. Much like Naþ channels, VGCCs can be found in all excitable cells where they have well-defined roles in action potential generation and neurotransmitter release. Indeed, therapeutically targeting VGCCs has proven extremely successful in the treatment of cardiac disorders such as hypertension and arrhythmia. In nonexcitable cells, these channels have been shown to be involved in both cell proliferation and cell migration. In the mid-1980s, it was discovered that coapplication of the L-type VGCC inhibitors verapamil, nifedipine, or nicardipine with various chemotherapeutic agents actually enhanced the cytotoxic effect of these drugs and in

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some cases overcame the adaptive resistance that gliomas can develop against chemotherapy drugs (Kaba et al., 1985; Merry et al., 1986; Bowles et al., 1990; Kondo et al., 1995). Interestingly, calcium imaging in the presence or absence of one of the L-type VGCC inhibitors, verapamil, indicated a lack of verapamil-sensitive calcium fluxes in glioma cells (Huet and Robert, 1988), leading researchers to conclude that calcium channel blockers exert their chemotherapeutic-enhancing effects by a mechanism independent of its inhibition of VGCCs. Subsequent research suggested that verapamil actually downregulated the expression of P-glycoprotein, a protein thought to be responsible for the multidrug-resistant phenotype of gliomas (Kiwit et al., 1994; Abe et al., 1995), although recent evidence has questioned whether this is actually the case (Huet et al., 1992; Rieger et al., 2000). Aside from their direct effect on tumor cells, the L-type VGCC inhibitors may act on other cell types to enhance drug delivery to the tumor site. Specifically, two studies from the late 1990s indicate that intravenous administration of nimodipine may increase blood–tumor barrier (BTB) selectively, leaving the BTB unaffected (Matsukado et al., 1994) and allowing higher concentrations of chemotherapeutic agents to reach the tumor specifically (Zenke et al., 1996). Regardless of the mechanism by which verapamil, minodipine, and nicardipine act to enhance cytotoxity of common anticancer drugs and despite the seeming lack of L-type VGCC expression in gliomas, the use of their inhibitors may offer a new twist on conventional therapies to enhance their outcome. Indeed, several animal studies indicate that combining the common chemotherapy treatment with the L-type VGCC inhibitors might be a successful approach to combating gliomas. Analysis of mRNA transcripts has revealed that gliomas express Cav3.1, a T-type VGCC (Latour et al., 2004). Furthermore, this channel appears to be a unique splice variant in that when exogenously expressed, it appears to have slower inactivation kinetics compared to the known Cav3.1 channels. Emerging research is indicating that of all the VGCCs, the T-type channel seems to be expressed in several cancers (Panner and Wurster, 2006). Indeed, a recent study has indicated that the T-type channels may be involved in the glioma proliferation as pharmacologic inhibition with mibefradil, the most widely used T-type channel inhibitor, and knockdown of the channel with antisense oligonucleotides reduces glioma proliferation by 50 % (Panner et al., 2005). The involvement of T-type calcium channels in the proliferation of cancer cells may not be surprising. Unlike the other VGCCs, the T-type channels activate in response to much smaller depolarizations that are reasonably achieved in nonexcitable cells. Furthermore, these channels have been reported to be regulated by calmodulin in addition to voltage (Barrett et al., 2000). Calmodulin is a common signaling molecule activated by growth factors known to be constitutively active in gliomas. The potential modulation of these ion channels by growth factors positions them as the primary calcium entry pathway in proliferative signaling cascades. Evidence for this already exists in gliomas. Specifically, platelet-derived growth factor–BB (PDGF–BB) induces a sustained Ca2þ increase in glioma cell lines that can be inhibited by the T-type channel inhibitor NDGA (Saqr et al., 1999). Increases in [Ca2þ]i are absolutely required for cell cycling. Despite a lack of electrophysiological evidence for the

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functional T-type VGCCs, molecular biology and pharmacology indicate that these channels may be intricately involved in these proliferative calcium signals and, therefore, might be potential therapeutic targets. Unfortunately, specific pharmacological inhibitors of the T-type channels do not currently exist. However, the implication of their involvement in multiple cancers as well as various other disorders such as chronic pain and epilepsy is driving a concerted effort to develop specific inhibitors as future therapeutic tools.

FIGURE 4.6 (a) Model representing glioma cell shape and volume-adaptive changes that occur during invasion in spatially restricted conditions. These changes, accompanied by water loss and cytoskeletal rearrangements, are mediated by ion fluxes through Cl and Kþ ion channels and other ion transport mechanisms. (b) Immunocytochemistry demonstrates a colocalization of both ClC3 channel protein and BK channel protein with the cholera toxin-B subunit as evidenced by yellow staining in the merged image. This subunit specifically binds to GM1 ganglioside, a well-characterized component of lipid rafts. (c) Biochemical separation of lipid raft proteins indicates that both ClC3 and BK channels can be found in the second detergent insoluble fraction of one such separation. This fraction contains common lipid raft proteins including Caveolin-1, a marker of a subset of lipid rafts. (Weaver et al., 2007). (See the color version of this figure in Color Plate section.)

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4.6 USE OF CHANNEL INHIBITORS FOR THE TREATMENT OF GLIOMAS It becomes quite evident from the above studies that several ion channels are prominently expressed in glioma cells and appear to be necessary for distinct aspects of glioma cell biology. BK channels appear to confer an advantage to cells to survive in conditions of growth factor starvation. The downregulation of Kir4.1 appears necessary for cells to maintain cycling. Similarly, it is clear that inhibition of certain channels, gBK channels or ClC-3 Cl channels, impedes the ability of cells to invade in vitro. While the mechanistic interdependence of channel function and the underlying biology are not always obvious, a good mechanistic model can be presented to explain how Kþ and Cl channels collaborate to support shape changes of invading cells. As illustrated in Fig. 4.6a, fluid dynamics is the mechanistic underpinning in that a coordinated flux of KCl and water is the requirement to attain a cell volume conducive to cell invasion. Not surprisingly therefore, all channels involved in fluid secretion colocalize to the invading aspects of cells where they are clustered in specialized lipid raft domains (Fig. 4.6b and c). Kþ and Cl channels may serve a similar role in other inexcitable cells including other cancer cells. This complex of channels, localized to lipid rafts, may provide an opportune therapeutic target. Unfortunately, blockers for BK channels lack glioma selectivity and useful aquaporin blockers do not exist. Fortunately, the putative Cl channel blocker Cltx causes functional inhibition of Cl currents by disrupting the membrane association of ClC-3 channels. Since Cltx binds almost exclusively to the cancer cells in the brain (Soroceanu et al., 1998) and lacks specific binding to normal human organ tissues, its clinical use was proposed for targeting experiments in animal models. Clinical grade Cltx has now been manufactured and introduced into a phase I clinical study that reported its findings recently (Mamelak et al., 2006). As illustrated in Fig. 4.7, Cltx shows remarkable glioma specificity in patients where the molecule is retained at the

FIGURE 4.7 Axial view of T1-Wc, (a) coregistered (b) and SPECT (c) (day 8) images. Longterm retention of 131I-TM-601 radioactivity at tumor site was observed in all patient scans (Hockaday et al., 2005). (See the color version of this figure in Color Plate section.)

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tumor site for up to 8 days. This retention indicates that Cltx is endocytosed and trapped inside the glioma cell in vivo analogous to our previous culture experiments. Importantly, the phase I clinical trial established drug safety and paved the way for a multicenter phase II clinical trial that is currently enrolling patients with malignant glioma. ACKNOWLEDGMENTS This work was supported by NIH grants RO1-NS031234, RO1-NS036692, and P50CA97247.

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Olsen ML, Schade S, Lyons SA, Amarillo MD, Sontheimer H, 2003. Expresssion of voltagegated chloride channels in human glioma cells. J Neurosci 23: 5572–5582. Olsen ML, Weaver AK, Ritch PS, Sontheimer H, 2005. Modulation of glioma BK channels via erbB2. J Neurosci Res 81: 179–189. Olsen ML, Higashimori H, Campbell SL, Hablitz JJ, Sontheimer H, 2006. Functional expression of K(ir)4.1 channels in spinal cord astrocytes. Glia 53: 516–528. Olsen ML, Campbell SL, Sontheimer H, 2007. Differential distribution of Kir4.1 in spinal cord astrocytes suggests regional differences in kþ homeostasis. J Neurophysiol 98: 786–793. Ouadid-Ahidouch H, Le BX, Roudbaraki M, Toillon RA, Delcourt P, Prevarskaya N, 2001. Changes in the Kþ current-density of MCF-7 cells during progression through the cell cycle: possible involvement of a h-ether-a-go-go Kþ channel. Receptors Channels 7: 345–356. Ousingsawat J, Spitzner M, Puntheeranurak S, Terracciano L, Tornillo L, Bubendorf L, Kunzelmann K, Schreiber R, 2007. Expression of voltage-gated potassium channels in human and mouse colonic carcinoma. Clin Cancer Res 13: 824–831. Panner A, Wurster RD, 2006. T-type calcium channels and tumor proliferation. Cell Calc 40: 253–259. Panner A, Cribbs LL, Zainelli GM, Origitano TC, Singh S, Wurster RD, 2005. Variation of T-type calcium channel protein expression affects cell division of cultured tumor cells. Cell Calc 37: 105–119. Pardo LA, Bruggemann A, Camacho J, Stuhmer W, 1998. Cell cycle-related changes in the conducting properties of r-eag Kþ channels. J Cell Biol 143: 767–775. Parkerson KA, Sontheimer H, 2004. Biophysical and pharmacological characterization of hypotonically activated chloride currents in cortical astrocytes. Glia 46: 419–436. Patt S, Labrakakis C, Bernstein M, Weydt P, Cervos-Navarro J, Nisch G, Kettenmann H, 1996. Neuron-like physiological properties of cells from human oligodendroglial tumors. Neuroscience 71: 601–611. Patt S, Preussat K, Beetz C, Kraft R, Schrey M, Kalff R, Schonherr K, Heinemann SH, 2004. Expression of ether a go-go potassium channels in human gliomas. Neurosci Lett 368: 249–253. Ransom CB, Sontheimer H, 1995. Biophysical and pharmacological characterization of inwardly rectifying Kþ currents in rat spinal cord astrocytes. J Neurophysiol 73: 333–345. Ransom CB, Sontheimer H, 2001. BK channels in human glioma cells. J Neurophysiol 85: 790– 803. Ransom CB, O’Neal JT, Sontheimer H, 2001. Volume-activated chloride currents contribute to the resting conductance and invasive migration of human glioma cells. J Neurosci 21: 7674– 7683. Ransom CB, Liu X, Sontheimer H, 2002. BK channels in human glioma cells have enhanced calcium sensitivity. Glia 38: 281–291. Rieger L, Rieger J, Winter S, Streffer J, Esser P, Dichgans J, Meyermann R, Weller M, 2000. Evidence for a constitutive, verapamil-sensitive, non-P-glycoprotein multidrug resistance phenotype in malignant glioma that is unaltered by radiochemotherapy in vivo. Acta Neuropathol (Berl) 99: 555–562. Ritch PA, Carroll SL, Sontheimer H, 2003. Neuregulin-1 enhances motility and migration of human astrocytic glioma cells. J Biol Chem 278: 20971–20978. Ritchie JM, 1992. Voltage-gated ion channels in Schwann cells and glia. TINS 15: 345–351. Saqr HE, Guan Z, Yates AJ, Stokes BT, 1999. Mechanisms through which PDGF alters intracellular calcium levels in U-1242 MG human glioma cells. Neurochem Int 35: 411–422.

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5 SHAKER FAMILY KV1 VOLTAGEGATED POTASSIUM CHANNELS IN MAMMALIAN BRAIN NEURONS HELENE VACHER1 AND JAMES S. TRIMMER1,2 1

Department of Neurobiology, Physiology, and Behavior, College of Biological Sciences, University of California, Davis, CA 95616, USA 2

Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis, CA 95616, USA

5.1 INTRODUCTION TO SHAKER FAMILY VOLTAGE-GATED POTASSIUM OR KV1 CHANNELS 5.1.1

KV1 Channels and Neuronal Excitability

Electrical excitability is a fundamental property of neurons. Diversity in intrinsic neuronal excitability and function is generated by the variable expression, subcellular localization, and activity of a complex repertoire of neuronal ion channels (Llinas, 1988). Dynamic regulation of intrinsic excitability can further alter the behavior of neurons and confer plasticity to neuronal signaling (Mohapatra et al., 2007). Moreover, aberrant expression, localization, and function of ion channels can result in channel-based pathophysiologies or channelopathies (Cannon, 2006). Thus, an understanding of how neurons regulate the expression and localization of ion channels is critical to understanding the complexity of normal neuronal function, its dynamic modulation to achieve plasticity, and defects that lead to neuronal dysfunction. Among the large complement of ion channels expressed in mammalian neurons are an especially wide variety of voltage-dependent potassium (Kv) channels. Among Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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these, members of the Kv1 subfamily of Kv channels regulate diverse neuronal electrical properties, including action potential amplitude and duration, the frequency of cell firing, the kinetics and amount of neurotransmitter release, and the cells resting membrane potential (Dodson and Forsythe, 2004). Kv1 channels are supramolecular protein complexes composed of four pore-forming and voltage-sensing principal, or a, subunits plus up to four associated auxiliary or Kvb subunits (Pongs, 1999). In heterologous cells, these Kv1a and Kvb subunits can heteromultimerize to yield biophysically and pharmacologically distinct a4b4 channel complexes (Pongs, 1999). Kv1 channels can also associate with various scaffolding proteins and enzymes, which can impact channel localization, turnover, and function (Schulte et al., 2006). As such, the functional characteristics, abundance, and subcellular localization of Kv channel complexes are determined by diverse protein–protein interactions, both between constituent Kv1a and Kvb subunits, and between these subunits and a wide variety of interacting proteins (Levitan, 2006). 5.1.2

Expression of Kv1 Channel Subunits

Expression of Kv1 channel genes is highly regulated, with specific promoter elements acting in concert with transcriptional machinery to achieve precise temporal and spatial cellular patterns of expression (Mandel and McKinnon, 1993). Although evidence for dynamic regulation of ion channel translation has not been provided, it is clear that multiple posttranslational processes, including protein–protein interactions mediating subunit assembly, folding, and ultimately export from the endoplasmic reticulum (ER), intracellular trafficking and appearance in the plasma membrane, and removal via endocytosis play a key role in regulating Kv1 channel expression. In addition, interactions with specific intracellular enzymes can lead to the covalent modification of Kv1a and Kvb subunit polypeptides, impacting diverse characteristics of Kv1 channels in neuronal membranes. Many Kv1 channel a subunits carry specific intracellular trafficking signals that regulate their exit from the rough ER, where they are synthesized and inserted in a topologically correct manner into the membrane, and their stepwise transit through the biosynthetic endomembrane system. Polarized sorting of Kv1 channels to axon- or dendrite-directed cargo vesicles in the trans-Golgi network, targeted insertion at discrete sites within axonal or dendritic plasma membrane subdomains, and active retention at these sites to maintain discrete patterns of channel localization in the face of membrane fluidity are also mediated by discrete signals on Kv1 channel a subunits and dictate which channels are acting at which sites in neurons. Kvb subunits can also profoundly influence these characteristics. Mechanisms that regulate channel assembly, folding, and ER export of Kv1 channels, as well as mechanisms used to establish their correct subcellular localization, are beginning to be understood in detail. The ER export competence of membrane proteins seems to be determined by two major mechanisms: the chaperone-based quality control machinery that senses and acts on the folding state of proteins while in the ER and trafficking control machinery recognizing specific trafficking determinants. Whether these systems operate independently to control exit of proteins from

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the ER or are simply different faces of the same system is not clear (Mancias and Goldberg, 2005). Specific mechanisms also exist to sort newly synthesized membrane proteins into axon- or dendrite-destined transport vesicles (Tang, 2001). It seems likely that, given the highly restricted localization of many Kv1 channels, mechanisms exist for insertion of channel transporting vesicles at specific sites (juxtaparanodal, axon initial segment, etc.). Once present in the plasma membrane, such restricted localization, as well as inclusion of Kv1 channels in high-density clusters, must be maintained in the face of the dynamic lateral mobility of the lipid bilayer (Hedstrom and Rasband, 2006). This chapter focuses on recent insights into the expression and localization of mammalian brain Kv1 channels, and the mechanisms mediating their intracellular trafficking and the generation and maintenance of their distinct subcellular localizations. This chapter focuses on Kv channels whose principal or a subunits are members of the classical Shaker-related mammalian Kv family (i.e., Kv1) and not other subfamilies of Kva subunits (i.e., Kv2–Kv11).

5.2 MOLECULAR PROPERTIES OF KV1 CHANNELS 5.2.1

Nomenclature of Kv1 Channel Subunits and Genes

Kv1 channels are members of a diverse gene family. The nomenclature system for Kv channel a subunits, originally proposed by Chandy and colleagues (Chandy, 1991; Gutman et al., 2005) and now widely accepted, is based primarily on the relatedness of amino acid sequences between the different Kva subunits. The principal physiologically permeant ion of these channels is denoted by the chemical symbol (K for potassium), followed by the abbreviation of the ligand, which in this case is voltage (V). The remainder of the nomenclature relates to the gene families within these ion channel groups. The prototypical Kv channels have been divided into four families (Kv1–Kv4) based on the relative similarity of their amino acid sequences and on their relatedness to their single-gene orthologues in Drosophila: Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw), and Kv4 (Shal). A parallel nomenclature for Kv channel a subunit genes has also been developed, with genes named KCN*, the four gene families assigned the letters A–D (i.e., Kv1–Kv4 ¼ KCNA–KCND), and the specific gene numbers following the Kv nomenclature (Kv1.1 ¼ KCNA1, Kv1.4 ¼ KCNA4, Kv2.1 ¼ KCNB1, etc.). This review focuses exclusively on mammalian Kv1a subunits encoded by paralogous KCNA genes that are the orthologues of the Drosophila Shaker gene (Salkoff et al., 1992). In mammals, there are seven different Kv1a subunits (Kv1.1–Kv1.7) that are the products of the KCNA1–KCNA7 genes, respectively. Only Kv1.1–Kv1.6 are expressed in mammalian brain (Kalman et al., 1998; Gutman et al., 2005). None of the Kv1a subunit genes generates messenger RNAs that are subject to alternative splicing (Chandy and Gutman, 1995). Kvb auxiliary subunits that coassemble with Kv1a subunits to form native mammalian brain Kv1 channels are also members of a family of related genes. There exist three genes encoding these Kv1-associated Kvb subunits: KCNAB1

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(Kvb1), KCNAB2 (Kvb2), and KCNAB3 (Kvb3). Alternative splicing of transcripts from the KCNAB1 gene yields three different Kvb1 subunits: Kvb1.1, Kvb1.2, and Kvb1.3. There are two alternative forms of Kvb2 (Kvb2.1 and Kvb2.2) generated by alternative splicing of transcripts from the KCNAB2 gene (Akhtar et al., 1999). These paralogous genes are the orthologues of the Drosophila Hyperkinetic gene (Chouinard et al., 1995). 5.2.2

Kv1a and Kvb Subunit Expression in Mammalian Brain

In mammalian brain, the expression of Kv1a and Kvb subunits is primarily restricted to neurons, although glial cells may also express a subset of the neuronal repertoire (Vacher et al., 2006). In neurons, Kv1 channels are found predominantly on axons and nerve terminals, although dendritic expression is also found in certain neurons (Trimmer and Rhodes, 2004). Kv1 family members exhibit extensive coassembly to generate heteromeric channels with distinct characteristics (Pongs, 1999). In addition, assembly with auxiliary subunits can dramatically impact expression, localization, and function of the resultant channel complexes (Li et al., 2006). For example, inclusion of the Kvb1.1 subunit in Kv1 channel complexes containing Kv1.1 or Kv1.2 dramatically alters channel gating properties, converting the channels from sustained, or delayed rectifier type, to rapidly inactivating, or A-type (Rettig et al., 1994). The specific subunit composition of native complexes can also dramatically impact the expression level, localization, and function of Kv1 channels in mammalian neurons (Vacher et al., 2006). The predominant Kv1 cellular staining pattern throughout the brain is neuronal and subcellularly axonal. The three most abundant Kv1 subunits expressed in mammalian brain, Kv1.1, Kv1.2, and Kv1.4, are found predominantly localized to axons and nerve terminals (Trimmer and Rhodes, 2004). In many cases, these subunits are components of heteromeric channel complexes, as Kv1.1, Kv1.2, and Kv1.4 exhibit precise patterns of colocalization (Rhodes et al., 1997) and extensive association as shown by copurification (Rhodes et al., 1997; Shamotienko et al., 1997; Coleman et al., 1999). However, the subunit composition of channels containing these subunits varies across brain regions (Scott et al., 1994a). The Kv1.1a subunit in mammalian brain appears to be segregated into two major subpopulations: one associated with Kv1.2 and the other associated with Kv1.4. Kv1.1 and Kv1.2 are found in the absence of Kv1.4 in cerebellar basket cell terminals (Fig. 5.1a) (McNamara et al., 1993; Wang et al., 1993, 1994; Rhodes et al., 1995, 1997; Laube et al., 1996; McNamara et al., 1996), in the juxtaparanodal membrane adjacent to axonal nodes of Ranvier (Fig. 5.1b) (Wang et al., 1993, 1994; Rhodes et al., 1995, 1997; Rasband et al., 1998, 1999; Vabnick et al., 1999; Rasband et al., 2002, Rasband, 2004), and in the terminal segments of axons (Dodson et al., 2003). Kv1.1 and Kv1.2 are also present at axon initial segments (Dodson et al., 2002; Inda et al., 2006; Van Wart et al., 2007), sometimes in association with Kv1.4 (Fig. 5.1c), where they control axonal action potential waveform and synaptic efficacy (Kole et al., 2007). Kv1.1/Kv1.2 channels also play a role in m-opioid receptor-mediated modulation of GABAergic inputs into basolateral amygdala neurons (Finnegan et al., 2006) and in serotonin-modulated glutamate release from thalamocortical nerve

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FIGURE 5.1 Immunofluorescence staining of Kv1a and Kvb subunits in mammalian brain. (a) Staining for Kv1.1 (blue), Kv1.2 (green), and Kvb2 (red) in adult rat cerebellar cortex. Note overlap of all three fluors yielding white signal in basket cell terminals (arrowheads). (b) Staining for Kv1.2 (blue) in adult rat optic nerve (green ¼ Nav channels, red ¼ Caspr). Image courtesy of Dr. Matthew Rasband. (c) Staining for Kv1.4 (green) in the axon initial segment of a cultured hippocampal neuron (blue ¼ MAP2). Image courtesy of Drs. Yasuhiro Ogawa and Matthew Rasband. (d) Kv1.2 (red) in adult rat hippocampus (green ¼ Kv2.1). Arrowheads point to prominent Kv1.2 staining in perforant path presynaptic terminals in the middle molecular layer of the dentate gyrus. (See the color version of this figure in Color Plate section.)

terminals (Lambe and Aghajanian, 2001). Low-threshold, slowly inactivating axonal Kv1.2-containing channels, presumably containing either Kv1.4a or Kvb1 subunits to confer inactivation, are involved in the flexible properties of intracortical axons of layer 5 pyramidal neurons and may contribute significantly to intracortical processing (Shu et al., 2007). Kv1-containing channels are also important in setting the firing rate of layer II/III pyramidal neurons (Guan et al., 2007). Kv1.1 and Kv1.4 are found robustly expressed in the relative absence of Kv1.2 within the striatal efferents in globus pallidus and pars reticulata of substantia nigra (Sheng et al., 1992; Rhodes et al., 1997). Within the excitatory circuitry of the hippocampus, a number of patterns for expression of these three Kv1a subunits emerge, providing a striking example of the complex heterogeneity of subunit association (Rhodes et al., 1997). Kv1.1, Kv1.2, and Kv1.4 are highly expressed in the middle third of the molecular layer of the dentate gyrus (Fig. 5.1d), where they are associated with axons and terminals of the medial perforant path (Sheng et al., 1992, 1994; Wang et al., 1993, 1994; Rhodes et al., 1995, 1997; Veh et al., 1995; Monaghan et al., 2001). Kv1.1, Kv1.2, and Kv1.4 are also found in Schaffer collateral axons, while Kv1.1 and Kv1.4 colocalize, in the absence of Kv1.2, in mossy fiber axons (Sheng et al., 1992, 1994; Wang et al., 1993, 1994; Cooper et al., 1998), where they regulate Ca2þ influx and transmitter release (Geiger and Jonas, 2000).

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However, in spite of their colocalization, it is not clear that heteromeric channel complexes containing coassociated Kv1.1, Kv1.2, and Kv1.4 are present on perforant path and Schaffer collateral axons. Lesions in entorhinal cortex have distinct effects on the distribution of Kv1.2 and Kv1.4 in the middle third of the dentate molecular layer, suggesting that while these subunits may colocalize at the light microscope level, they may be expressed, along with Kv1.1, on different components of the perforant path (Monaghan et al., 2001). Similar results are obtained with lesions placed in other subfields. For example, predominant Kv1 channels appear to be composed of Kv1.1 with Kv1.4 in CA3, while in the Schaffer collateral pathway Kv1.1 is likely to be associated with Kv1.2 and Kv1.4 (Monaghan et al., 2001). Electron microscopic immunohistochemical studies have demonstrated that Kv1.1, Kv1.2, and Kv1.4 are concentrated in the axonal membrane immediately preceding or within axon terminals (Wang et al., 1993, 1994; Cooper et al., 1998). The immunoreactivity for Kv1.1 and Kv1.2 has been localized to the preterminal axonal membrane in stratum radiatum (Wang et al., 1993, 1994), while immunoreactivity for Kv1.4 has been localized to the preterminal extensions of mossy fiber axons (Cooper et al., 1998). Activation of Kv1 channels at these sites can play a critical role in regulating nerve terminal excitability and thereby regulate neurotransmitter release, as shown by pharmacological and genetic knockdown of Kv1 function (Dodson and Forsythe, 2004). The other Kv1a subunits appear to be expressed at lower levels in mammalian brain. Kv1.6 is found predominantly in interneurons, although some dendritic staining is seen throughout the brain on principal cell dendrites, which also express Kv1.1 and Kv1.2 (Rhodes et al., 1997) and presumably underlie the “D” current (Storm, 2000; Yuan and Chen, 2006). Kv1.3 is highly expressed in the cerebellar cortex. The bulk of this expression is in the parallel fiber axons of cerebellar granule cells, as strong in situ hybridization signal is present in the granule cell layer (Kues and Wunder, 1992), while strong immunostaining (Veh et al., 1995) and 125Imargatoxin binding (specific for Kv1.2 and Kv1.3) (Koch et al., 1997) are found in the molecular layer. The molecular layer also contains high levels of staining for Kv1.1 (Fig. 5.1a) (Veh et al., 1995; Rhodes et al., 1997), suggesting that Kv1.1 and Kv1.3 have the opportunity to form heteromeric channels on parallel fibers. The expression of Kv1.5 in the brain is overall quite low (Felix et al., 1999). What Kv1.5 expression there is may be restricted to nonneuronal cells. For example, Kv1.5 and Kv1.3 are components of delayed rectifier currents in glia (Khanna et al., 2001b; Chittajallu et al., 2002; Pannasch et al., 2006) and endothelial cells (Millar et al., 2007). In mammals, Kv1.7 is expressed in skeletal muscle, heart, and pancreatic islets, but not brain (Kalman et al., 1998). In situ hybridization, immunoprecipitation, and immunohistochemical analyses have also localized sites of expression of Kvb1 and Kvb2 in mammalian brain (Rettig et al., 1994; Rhodes et al., 1995, 1996, 1997). Kvb2 appears to be a component of many, if not all, Kv1-containing channel complexes in mammalian brain, and immunoreactivity for Kvb2 is present in each and every location where immunoreactivity for Kv1-family a subunits is observed (Rhodes et al., 1996, 1997). The Kvb1 subunit, which exerts dramatic effects on the inactivation kinetics of Kv1

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channels, appears to be included more selectively. Interestingly, the pattern of immunoreactivity for Kvb1 closely matches the expression pattern for Kv1.1 and Kv1.4, in that Kvb1 is found to colocalize with Kv1.4 in the medial perforant path, in the mossy fiber pathway, and in striatal efferents to the globus pallidus (Rhodes et al., 1996, 1997). Kvb2 is found in the absence of Kvb1 at many sites that exhibit colocalized Kv1.1 and Kv1.2, for example, in cerebellar basket cell terminals (Fig. 5.1a) and juxtaparanodes of nodes of Ranvier (Rhodes et al., 1997). As such there exist distinct pairings of Kv1a and Kvb subunits in heteromeric channel complexes in mammalian brain. 5.2.3

Structure of Kv1 Subunits

Detailed information on the primary, secondary, tertiary, and quaternary structures of mammalian Kv1a subunits is now available. Kv1a subunits exhibit extensive amino acid sequence identity within the transmembrane and pore-forming domains. The Shaker Kv channel from Drosophila melanogaster was the first Kv channel to be characterized (Tempel et al., 1987) and remains one of the best-studied Kv channels. Mammalian Kv1a subunits exhibit 70% amino acid identity with Shaker, and only 50% identity with a subunits from other mammalian Kv channel subfamilies Kv2– Kv4 (Salkoff et al., 1992). Shaker channels (MacKinnon, 1991) and mammalian Kv1 channels (Long et al., 2005) are tetramers of a subunits, and each a subunit consists of six transmembrane segments S1–S6 (Fig. 5.2a). The fourth transmembrane segment, or S4, of each subunit acts as the main component of the “voltage sensor” module, which detects the electrical potential across the membrane and controls the voltage-dependent gating of the channel (Papazian et al., 1991; Yellen, 1998; Swartz, 2004). The ionic conductance pathway or pore, which is responsible for rapid and selective potassium ion flux, is formed by the close association of the last two transmembrane segments (S5 and S6) from each of the four a subunits around a central water-filled cavity (Doyle et al., 1998; Long et al., 2005). Kvb subunits exhibit weak overall sequence similarity (McCormack and McCormack, 1994) but striking structural similarity (Gulbis et al., 1999) to aldo-keto reductase enzymes, and enzymatic activity against artificial substrates has recently been demonstrated (Weng et al., 2006). The structure (Fig. 5.2b) of a mammalian Kv1 channel complex formed by four Kv1.2a and four Kvb2 subunits reveals striking structural details of a Kv1 channel complex (Long et al., 2005). The portion of the Kv1.2a subunit between transmembrane segments S1–S4 comprises the voltage-sensing module, which is a distinct structure from the pore-forming module comprising the portion of the Kv1.2a subunit between transmembrane segments S5–S6 (Long et al., 2005). The modular structure of these domains and the relatively independent nature of the voltage sensor module are consistent with recent functional studies (Soler-Llavina et al., 2006). However, the specific conformation of the voltage sensor and pore modules in their resting and active states, and how the voltage-dependent movement of one is allosterically coupled to opening of the pore, remains unresolved and a focus of much recent research (Swartz, 2004).

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FIGURE 5.2 Structure of Kv1a and Kvb subunits. (a) Cartoon of the predicted topology of Kv1a subunits. The predicted transmembrane topology of a Kv1a subunit and sites defined as important determinants of intracellular trafficking and polarized expression are shown schematically. T1, tetramerization domain important for Kvb-mediated intracellular and polarized trafficking of Kv1a subunits; NLG, N-linked glycosylation site of Kv1.1–Kv1.5; ERR, ER retention motif found in Kv1.1, Kv1.2, and Kv1.6; FTS, forward trafficking signal in Kv1.4; PDZ BD, PDZ-binding motif in all Kv1a subunits; Y132, tyrosine residue critical for tyrosine kinasedependent suppression of Kv1.2; S440/S441, serine residues critical for phosphorylationdependent trafficking of Kv1.2. (b) Crystal structure of the Kv1.2/Kvb2 complex (Protein Data Bank accession number 2A79). Only two Kv1.2a and Kvb2 subunits are shown for clarity. TM, transmembrane domain; T1, tetramerization domain; Kvb, Kvb2 subunit. (c) Higher resolution view of the pore region of Kv1.2 with residues critical to the function of the ERR motif highlighted. Views of the Kv1.2 pore helices (S5 and S6) in blue with the side chains of the four critical residues depicted. The top panel shows wild-type Kv1.2 and the bottom panel the predicted structure of Kv1.2 with the four residues critical to the ERR function of Kv1.1 substituted at the equivalent positions. Note the change in the nature of the side chains forming the external face of the channel. Figures generated with Swiss PDB viewer. (See the color version of this figure in Color Plate section.)

5.3 DETERMINANTS OF INTRACELLULAR TRAFFICKING OF KV CHANNELS 5.3.1

Trafficking Signals Regulating Retention of Kv1 Channels in the ER

Neurons use diverse mechanisms to control the cell surface expression levels of Kv1 channels, which can profoundly affect neuronal excitability and signaling (Hille, 2001). Different levels of expression of Kv1a subunits lead, by mass action, to their proportional incorporation into channel complexes and the formation of tetrameric channels with different stoichiometries of Kv1a subunits. The precise stoichiometry of Kv1a subunits within a tetramer influences channel trafficking, localization, and function. Kv1 homomeric channels formed by different a subunits exhibit distinct protein stabilities and internal organelle retention characteristics. These patterns are altered upon coassembly to heteromeric complexes, such that the precise composition of Kv1a and Kvb subunits could affect both the overall level of the protein and its

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trafficking characteristics. As such, subunit composition can affect Kv1 channel cell surface expression levels as well as function. The overall steady-state cell surface expression levels and subunit composition appear to be regulated by a hierarchical system of regulatory steps, many of which operate at the level of the ER. The ER is a highly versatile protein factory that is equipped with chaperones and folding enzymes essential not only for protein folding and exit but also for retention and degradation of misfolded or aberrantly assembled protein complexes. Different Kv1a subunits exhibit high amino acid sequence identity (Stuhmer et al., 1989) but show striking differences in trafficking and functioning (Papazian, 1999; Manganas and Trimmer, 2000; Trimmer and Rhodes, 2004). The primary determinant for regulating trafficking of Kv1a subunits appears to be a potent ER retention (ERR) signal consisting of residues in the turret region (Fig. 5.2a) at the external face of the channel pore domain (Manganas et al., 2001b; Zhu et al., 2001). Cell surface expression of Kv1 subunits can also be influenced by a cytoplasmic C-terminal VXXSL motif (Fig. 5.2a) that acts as a forward trafficking signal (FTS) (Li et al., 2000) and by auxiliary Kvb subunits (Shi et al., 1996). In heterologous cells, these motifs dictate the steady-state distribution of Kv1 channels, such that homotetrameric Kv1.1 (and Kv1.6) channels are essentially localized to the ER (Fig. 5.3a), Kv1.4 channels mainly to the cell surface (Fig. 5.3b), and Kv1.2 channels to both the ER (Fig. 5.3c) and

FIGURE 5.3 Subcellular localization of homotetrameric Kv1a subunits in COS-1 cells. Recombinant Kv1 expression in monkey kidney fibroblast COS-1 cells. Intact cells were stained with ectodomain-directed antibodies (red), and then detergent permeabilized and stained with cytoplasmic domain-directed antibodies (green). (a) Kv1.1, (b) Kv1.4, (c and d) Kv1.2. (See the color version of this figure in Color Plate section.)

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the cell surface (Fig. 5.3d) (Manganas and Trimmer, 2000; Tiffany et al., 2000; Zhu et al., 2003). Kv1.1 homomeric channels in the ER appeared to be properly folded and assembled as tetramers with no evidence of aggregation or gross misfolding (Manganas et al., 2001a; Zhu et al., 2003). The generation of chimeras between Kv1.1 and Kv1.4 revealed that any Kv1.4a subunit containing the Kv1.1 pore region (P-loop), including the turret domain, was ER retained (Manganas et al., 2001b). Conversely, any Kv1.1a subunit containing the Kv1.4 P-loop was efficiently exported from the ER. An alignment of the Kv1.1 and Kv1.4 P-loop sequences revealed three key positions, all in the turret domain, which differed between Kv1.1 and Kv1.4 (Fig. 5.2a). Mutation of those in Kv1.1 to those in Kv1.4 yielded functional Kv1 channels that have Kv1.4-like trafficking patterns, and vice versa (Manganas et al., 2001b). Studies of these determinants for Kv1 channel surface expression have been primarily conducted in heterologous expression systems, although critical experiments have been reproduced in mammalian neurons, where the Kv1.1 ERR signal was also shown to function in cultured hippocampal neurons (Manganas et al., 2001b). Analyses of a large number of Kv1.1/Kv1.4 chimeras and truncation, deletion, and point mutants suggest that this luminal ERR motif is dominant over the cytoplasmic Cterminal FTS and also dominant over any effects of Kvb subunit coexpression on Kv1a subunit trafficking (Manganas et al., 2001b). Although small but measurable increases of Kv1.1 surface expression have been observed in cells coexpressing Kvb2 subunits (Shi et al., 1996), the effects are small compared to the effects of Kvb auxiliary subunits on homotetrameric Kv1.2 channels (Shi et al., 1996; Campomanes et al., 2002). Three amino acid residues (A352, E353, and Y379) within the highly conserved pore region of Kv1.1 are critical to functioning of the ERR (Manganas et al., 2001b). Subsequent studies yielded similar results and also revealed an additional role for S369 in the Kv1.1 ERR (Zhu et al., 2003, 2005). The function of the Kv1.1 ERR appears to be dictated only by these four pore regions of amino acid residues (Fig. 5.2c), in that point mutations in other Kv1 subunits at equivalent positions control the cell surface trafficking efficiency of these a subunits (Manganas et al., 2001b; Zhu et al., 2005). However, regulation of trafficking via these positions in the pore region is not transferable to Kv2, Kv3, and Kv4 subfamily members and seems to be unique to the Kv1 subfamily (Zhu et al., 2005). The location of the pore-localized ERR signal in Kv1.1 and the equivalent positions that regulate trafficking of other Kv1a subunits are unusual among membrane proteins in their ER luminal localization. One intriguing and surprising aspect of these findings is that three of the four P-loop residues (A352, E353, and Y379 in Kv1.1) that dictate Kv1 family trafficking also determine high-affinity binding to the mamba snake neurotoxin a-dendrotoxin or a-DTX (Hurst et al., 1991; Tytgat et al., 1995; Imredy and MacKinnon, 2000). Moreover, each of the Kv1 family members that binds a-DTX (Kv1.1, Kv1.2, and Kv1.6) exhibit a strong degree of ERR relative to the Kv1 family members (Kv1.3, Kv1.4, and Kv1.5) that lack the critical binding residues and do not bind a-DTX (Dolly and Parcej, 1996; Manganas and Trimmer, 2000; Tiffany et al., 2000; Manganas et al., 2001b). This allows for speculation that the ERR function of Kv1.1 (and of Kv1.2 and Kv1.6) may be mediated by a resident ERR receptor protein that binds to the turret domain of Kv1.1 in a fashion similar to the binding of a-DTX. Such a model is

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supported by the fact that exogenous expression of soluble DTX in the lumen of the ER leads to release of Kv1.1 from ER retention and efficient expression of Kv1.1 homotetrameric channels on the cell surface (Vacher et al., 2007). This mechanism is attractive in that should the resident ERR receptor resembles a-DTX, it may block the pore of ER-retained Kv1 channels, preventing any deleterious effects of ERlocalized Kv1 channels on the ionic integrity of the ER. This hypothetical ERR receptor for Kv1.1 and other a-DTX-sensitive Kv1a subunits remains to be identified. 5.3.2

Trafficking Signals Responsible for ER Export

Intrinsic sequences and/or interaction with auxiliary subunits can regulate ER export. Kv1.4 homotetrameric channels expressed in a wide variety of heterologous expression systems (COS-1, HEK293, CHO, MDCK cell lines), and in cultured hippocampal neurons, are efficiently expressed on the cell surface (Bekele-Arcuri et al., 1996; Shi and Trimmer, 1999; Li et al., 2000; Manganas and Trimmer, 2000). A large proportion of the steady-state Kv1.4 cellular pool (80–90%) is present on the cell surface (Fig. 5.3b). This is due to the lack of the critical amino acids in the turret region that mediate ER retention in Kv1.1 (Manganas et al., 2001b), combined with the presence of a unique ER export or FTS in the cytoplasmic carboxyl-terminal region (Li et al., 2000). This cytoplasmic FTS appears to be recessive to the turret domain ERR signal (which would be luminal in the ER), as Kv1.4 chimeras with active ERR signals from Kv1.1 but that are otherwise composed of Kv1.4 (including an intact FTS) are efficiently retained in the ER. However, simple deletion of the FTS decreases the level of Kv1.4 surface expression (Li et al., 2000; Zhu et al., 2003). Kvb subunits do not normally affect the trafficking of wild-type Kv1.4a subunits. This may be simply due to the fact that the inherent trafficking properties of these proteins may be near the maximum efficiency possible. However, Kv1.4 mutants lacking the FTS exhibit robust responses to Kvb coexpression (Li et al., 2000). Kvb1 and Kvb2 coexpression promotes the stability, N-linked glycosylation, and surface expression of homomeric Kv1.2 channels (Shi et al., 1996; Campomanes et al., 2002). The effects of Kvb subunits such as Kvb2 are mainly to increase the overall levels of those subunit combinations that already exhibit intermediate trafficking efficiencies, in that Kvb2 effects are obvious for Kv1.2, heteromeric channels containing Kv1.1, Kv1.2, and Kv1.4, and FTS-deficient Kv1.4 mutants, but are not detectable for Kv1.1 or Kv1.4 homotetramers. One possible explanation is that the main consequence of Kvb2 association is the stabilization of Kv1a subunits in the ER, as observed for Kv1.2 homotetramers (Shi et al., 1996). This stabilization could indirectly impact surface abundance by increasing the number of chances that a Kv1 channel complex would have to achieve ER export competence, through chaperone-assisted folding and/or saturation of retention receptor sites, by simply increasing the half-life of the channel complex in the ER. The fact that Kvb subunits such as Kvb2 are able to similarly promote plasma membrane abundance of different Kv1 channels regardless of precise a subunit composition implies that these cytoplasmic subunits are not directly involved in masking retention signals, the mechanism used by KChIP cytoplasmic subunits to facilitate trafficking of Kv4

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channels (Shibata et al., 2003; Scannevin et al., 2004), but mediate their effects indirectly via other mechanisms, such as stabilization or folding. 5.3.3

Posttranslational Regulation of Kv1 Channel Expression Levels

Among the enormous variety of posttranslational modifications, the only known modifications of Kv1a subunits are asparagine N-linked glycosylation (NLG) (Shi and Trimmer, 1999) and phosphorylation (Yang et al., 2007), and for Kvb subunits only phosphorylation (Gong et al., 1999). Posttranslational modification of proteins can play an important role in the proper folding, assembly, and trafficking, as well as in dynamic regulation of function. In this section, we will discuss the role of glycosylation and phosphorylation of Kv channels in channel trafficking. NLGs of membrane and secreted proteins have been shown to promote proper protein folding in the ER as well as to alter protein transport and targeting (Helenius and Aebi, 2001). In general, NLGs of Kv1 channels are not required for efficient trafficking to the cell surface but do appear to increase their surface expression by decreasing channel turnover rates and increasing channel stability. For example, blocking NLG of Shaker-type Kv channels dramatically decreases channel stability and steady-state cell surface expression levels but does not affect the folding (Santacruz-Toloza et al., 1994) and assembly of functional channels, or their transport to the cell surface (Khanna et al., 2001a). However, overexpression of the lectin-like protein chaperone calnexin (Bergeron et al., 1994; Hammond and Helenius, 1994) acts to promote Kv1.2 trafficking and increased cell surface expression, presumably through an interaction with NLGs (Manganas and Trimmer, 2004). Kv1.1–Kv1.5, but not Kv1.6, a subunits contain a single consensus site for NLG, which is located in the extracellular linker segment between transmembrane segments S1 and S2 (Fig. 5.2a). In native mammalian brain Kv1.1, Kv1.2, and Kv1.4a subunits, this NLG site carries a sialic acid-bearing oligosaccharide chain, conferring a strong negative charge to the extracellular glycan chain (Sheng et al., 1993; Thornhill et al., 1996; Shi and Trimmer, 1999; Manganas and Trimmer, 2000). Absence of these negative charges affects channel function (Thornhill et al., 1996; Watanabe et al., 2003). Important to many of the published studies of Kv1 trafficking, the NLG of these Kv1a subunits, and the spatial segregation of oligosaccharide-processing enzymes within the secretory pathway, allows tracking of the biosynthetic trafficking of Kv1a subunits by straightforward biochemical analyses of the glycan chain (Trimmer, 1998). Kv1 channel a subunits are also extensively modified by covalent phosphorylation. This posttranslational modification, governed by a diverse array of protein kinases and phosphatases, can modulate the expression and the function of supramolecular complexes formed from Kv1a subunits, Kvb auxiliary subunits, and interacting proteins. One important question is whether phosphorylation can dynamically regulate Kv1 trafficking. Steady-state Kv1.1 current density increases in transfected cells upon stimulation of specific protein kinases (Winklhofer et al., 2003). However, whether this is mediated through effects on biosynthetic trafficking, endocytosis/ turnover, or through changes in biophysical properties of active surface channels has not been directly assessed. Muscarinic stimulation of M1 receptors, or treatment with

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phorbol esters, tyrosine phosphatase inhibitors, and Ca2þ ionophore, yields enhanced Kv1.2 tyrosine phosphorylation and acute suppression of Kv1.2 functional expression (Huang et al., 1993; Tsai et al., 1997). Mutating a specific Tyr (Y132) in the Kv1.2 N-terminal linker connecting the T1 domain to the first S1 transmembrane segment (Fig. 5.2a) reduces the extent of stimulus-induced suppression by 50% (Huang et al., 1993). The bulk of the suppression of Kv1.2 functional expression in response to these treatments appears to be due to stimulus-induced increases in Kv1.2 endocytosis (Nesti et al., 2004). Kv1.2 interaction with the actin binding protein cortactin, which can also influence levels of Kv1.2 surface channels, is also regulated through tyrosine phosphorylation of Kv1.2 (Hattan et al., 2002). Expression of the Kv1.2a subunit appears to be especially sensitive to regulation by phosphorylation. Recent application of mass spectrometry-based identification of in vivo phosphorylation sites on ion channels (Park et al., 2008) has allowed for identification of novel serine phosphorylation sites on the C-terminus of Kv1.2a subunits purified from mammalian brain (Yang et al., 2007). Interestingly, analyses of Kv1.2 purified from rat, mouse, and human brain led to identification of the same set of serine phosphorylation sites, suggesting a conserved and fundamental function for phosphorylation at these C-terminal sites. As detailed above, Kv1.2a subunits are unique among Kv1a subunits in that they lack strong ERR (Manganas et al., 2001b) and FTS (Li et al., 2000) trafficking signals, such that homotetrameric Kv1.2 channels exhibit highly variable trafficking characteristics heavily influenced by other coexpressed Kv1a (Manganas and Trimmer, 2000) and auxiliary Kvb (Shi et al., 1996; Tiffany et al., 2000; Campomanes et al., 2002) subunits. Kv1.2 trafficking also differs between different cells in a population (Fig. 5.3c and d) (Shi et al., 1996; Manganas and Trimmer, 2000; Tiffany et al., 2000), and between different cell types (Manganas and Trimmer, 2000). This suggests conditional regulation of Kv1.2 trafficking, as can occur upon changes in protein phosphorylation state as recently shown for the KCNK3 potassium channel (O’Kelly et al., 2002). Kv1.2 phosphorylation at a subset of these sites (S440 and S441) identified in vivo occurs during intracellular trafficking of Kv1.2 (Fig. 5.2a), such that cell surface Kv1.2 channels are phosphorylated at these sites, but ER Kv1.2 pools are not. Mutation of these sites suppresses intracellular trafficking and leads to accumulation of Kv1.2 in the ER (Yang et al., 2007). Importantly, inclusion of Kv1.2 with altered phosphorylation state into heteromeric Kv1.2/Kv1.4 complexes also suppresses their trafficking, suggesting that incorporation of Kv1.2 into heteromeric Kv1 channel complexes such as predominate in brain may allow for dynamic regulation of their trafficking through stimulus-induced changes in phosphorylation state (Yang et al., 2007). The amplitude of Kv1.2 currents in heterologous cells is enhanced upon PKA activation (Huang et al., 1994). This involves phosphorylation at an N-terminal residue (T46), which leads to increased channel open probability and larger Kv1.2 currents (Huang et al., 1994). A recent report suggests that regulation of Kv1.2 by cAMP-based signaling is complex, with PKA-dependent and independent events (Connors et al., 2008). Such regulation involves not only T46 but also two of the C-terminal Ser sites identified as being phosphorylated in vivo in the studies detailed above (Yang et al., 2007). Changes in Kv1.2 phosphorylation appear to regulate cell surface expression

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levels and activity of Kv1.2 channels through effects on trafficking, endocytosis, and gating and may provide a crucial link between cell signaling pathways and electrical excitability. 5.3.4

Auxiliary Subunits of Kv1 Channels and Intracellular Trafficking

The best characterized of ion channel auxiliary subunits are the cytoplasmic Kvb subunits associated with Kv1 family members (Pongs et al., 1999). The bulk of Kv1 channel complexes in mammalian brain have associated Kvb subunits (Rhodes et al., 1996). Kvb subunits lack putative transmembrane domains and potential glycosylation sites, suggesting that they are cytoplasmic proteins (Scott et al., 1994b). Moreover, cryo-electron microscopy studies place their localization on the cytoplasmic face of the channel complex (Orlova et al., 2003; Sokolova et al., 2003). The Kvb subunits are each approximately 300 amino acids in length and share a common conserved core (over 85% amino acid identity), with the highest degree of variability in the amino terminus. The Kvb subunits are tetramers of oxidoreductase-like proteins arranged with fourfold symmetry similar to that of the integral membrane a subunits (Gulbis et al., 1999). Each oxidoreductase-like Kvb subunit contains an intact oxidoreductase active site with critical catalytic residues, and a bound NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate) cofactor, but the specific substrate for any enzymatic activity is unknown, and the biological function of the oxidoreductase-like structure of Kvb subunits remains a mystery (Gulbis et al., 1999). Activity against artificial substrates has recently been demonstrated (Weng et al., 2006), a crucial first step toward identifying in vivo substrates. Studies of Kv1 channel biosynthesis have shown that Kv1a and Kvb subunits coassemble in the ER and remain together as a permanent complex (Shi et al., 1996; Nagaya and Papazian, 1997). Kvb subunits attach to a Kv1 channel through an interaction with the N-terminal cytoplasmic T1 domain (Fig. 5.2a and b) (Sewing et al., 1996; Yu et al., 1996; Gulbis et al., 2000; Long et al., 2005). Differences in the functional effects of Kvb subunits can often be ascribed to the variations in the amino terminal variable domain. Arguably, the most dramatic functional effect conferred by the association of Kvb subunits with Kv1 channels is an increase in the kinetics of channel inactivation gating (Heinemann et al., 1995). In a striking example, Kvb1.1 subunits can normally convert noninactivating delayed rectifier Kv channels to a rapidly inactivating channel (Rettig et al., 1994; Leicher et al., 1998). Kvb1 and Kvb2 have also been shown to modulate voltage dependence of activation of Kv1 channels in heterologous expression systems (England et al., 1995). A number of additional roles have been proposed for the function of Kvb/Kv1a subunit interactions. Each of the Kvb subunits can promote the cell surface expression of coexpressed Kv1.2a subunits (Shi et al., 1996; Campomanes et al., 2002). In these cases, Kvb subunits may aid in proper protein folding and/or a subunit assembly and thus enhance transport from ER to Golgi to cell surface. Effects of Kvb subunits on inactivation (Tipparaju et al., 2007), but not trafficking (Campomanes et al., 2002), can be regulated by intrinsic oxidoreductase activity. Recent studies showed that calnexin, the resident ER trafficking machinery, can also regulate Kv1 channel trafficking (Manganas and Trimmer, 2004). Calnexin is a

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type I integral membrane protein found in the ER that functions as a molecular chaperone to facilitate the folding and assembly of newly synthesized membrane proteins (Bergeron et al., 1994; Hammond and Helenius, 1994). Calnexin has been defined as a lectin chaperone that binds to membrane glycoproteins. The interaction occurs between the N-terminal ER luminal domain of the calnexin and the luminal NLGs (Ou et al., 1993). Homotetrameric Kv1.2 channels, possessing a weak porelocalized ERR (Manganas et al., 2001b) and FTS (Li et al., 2000) signals responded to calnexin coexpression with increased intracellular trafficking and cell surface expression (Manganas and Trimmer, 2004). This suggests that calnexin may be acting in its classical role to assist in efficient folding and/or increased stability of Kv1.2a subunit glycoprotein in the ER. Moreover, the interaction of the Kv1.2 N-terminus with Kvb2 subunits increases the efficiency of addition of the N-linked oligosaccharide chain of Kv1.2a subunits (Shi and Trimmer, 1999) through an unknown mechanism but that presumably occurs through an allosteric conformational change at or near the site of addition of the N-linked oligosaccharide chain that is induced by Kvb binding. This in itself may lead to a higher proportion of Kv1.2a subunits that are competent for interaction with calnexin and a subsequent increase in the number of channels that would be correctly folded and thus ER export competent. Kvb2 subunits and calnexin coexpression are epistatic, suggesting that they share a common pathway for promoting Kv1.2 channel surface expression (Manganas and Trimmer, 2004). In contrast, the trafficking of homotetrameric Kv1.1 channels, which contain strong ERR motifs (and no FTR export motifs), could not be influenced by calnexin coexpression (Manganas and Trimmer, 2004). These studies together demonstrate that a diverse set of intrinsic trafficking motifs, covalent modifications, and interacting proteins are crucial to intracellular trafficking of Kv1 channels.

5.4 DETERMINANTS OF SUBCELLULAR COMPARTMENTALIZATION OF KV1 CHANNELS IN THE PLASMA MEMBRANE Polarized localization of ion channels is essential for neurons to generate and maintain local and specialized signaling domains such as that found at synapses and the nodes of Ranvier. As discussed above, different Kv1 channels show distinct patterns of subcellular localization in neurons (Fig. 5.1). In this section, we discuss possible mechanisms underlying the polarized localization of Kv1 channels observed in neurons and its dynamic regulation. There are several hurdles to study intracellular trafficking of Kv channels compared to other ion channels, such as glutamate receptors: (1) Kv1 channels form complex heteromultimeric channels in neurons, (2) both the N- and C-termini are in the cytoplasm, and extracellular domains are limited to relatively short segments between transmembrane segments, which complicates analyses of surface pool of proteins by using epitope tags, biotinylation, or ectodomain-directed antibodies, and (3) expression levels of Kv1 channels are relatively low. Despite these complications, there are several compelling studies describing trafficking determinants of Kv1 channels that provide insights into molecular mechanisms determining polarized localization of Kv1 channels in central neurons.

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SHAKER FAMILY KV1 VOLTAGE-GATED POTASSIUM CHANNELS

Determinants of Axonal Localization of Kv1 Channels

The precise determinants for the polarized expression and local clustering of most Kv1 channels are not well characterized. One of the problems has been recapitulating the heteromeric channels found in native neurons in model cell systems (e.g., polarized epithelial cells), compounded by the inefficient intracellular trafficking inherent to many of the Kv1a subunits. Moreover, for reasons that are not well understood, efficiently expressed Kv1a subunits, such as Kv1.4, when expressed in such model systems do not “behave”; for example, axonal Kv1.4 is found in the basolateral and not apical membranes of polarized MDCK epithelial cells (Le Maout et al., 1996). One possible explanation for such discrepancies is that recent studies have provided compelling data that cytoplasmic Kvb subunits are crucial to polarized trafficking of Kv1a subunits. While Kv1.2a subunits overexpressed in cultured hippocampal neurons exhibit somatodendritic localization, cotransfection of the Kvb2 subunit yields a pronounced axonal localization of Kv1.2 (Campomanes et al., 2002). Subsequent analyses of Kv1.2 deletion mutants and chimeric channels revealed that the amino terminal T1 domain, which comprises the Kvb subunit binding site (Sewing et al., 1996; Yu et al., 1996; Gulbis et al., 2000), is essential for axonal expression of Kv1.2 channels (Gu et al., 2003). Specific mutations to disrupt Kvb2 binding also disrupted axonal expression of Kv1a subunits in transfected neurons, further suggesting that endogenous Kvb subunits were contributing to the axonal Kv1 localization (Gu et al., 2003). Remarkably, the wild-type Kv1.2 T1 domain, but not mutants with altered Kvb binding, was able to direct the axonal localization of single-pass transmembrane reporter proteins (Gu et al., 2003). Thus, cytoplasmic Kvb subunits may affect not only early biosynthetic processing events and ER export (Shi et al., 1996), but also axonal localization (Campomanes et al., 2002; Gu et al., 2003) of Kv1 channels. Recent studies have shown that axonal targeting of Kv1 channels is dependent on the direct interaction of Kvb2 with the microtubule plus-end-tracking protein EB1 (Gu et al., 2006). Analysis of Kvb2 deletion mutants revealed that the disruption of its association with EB1 dramatically decreases Kv1.2 axonal targeting (Gu et al., 2006). Suppression of endogenous EB1 expression using siRNA also impairs axonal targeting of endogenous Kv1 channels but not voltage-gated sodium channels (Gu et al., 2006), which appear to target to axons by distinct mechanisms and motifs (Garrido et al., 2003). Another recent study shows that Kv1 channel axonal targeting is dependent not only on EB1 and Kvb2 but also on the interaction of the Kv1a subunit T1 domain with the kinesin KIF5B (Rivera et al., 2007). The role of Kvb2, which also binds at the T1 domain, was not addressed in this study, although endogenous Kvb2 was presumably expressed in the neurons in the cultured cortical slices used in these studies (Rivera et al., 2007). As such, the T1 domain of Kv1a subunits mediates multiple protein–protein interactions, including tetrameric assembly of Kv1a subunits, association with Kvb subunits (and their EB1 binding partner), and interaction with KIF5B. The important role of Kvb2 is underscored by the finding that deletion of the human Kvb2 gene in chromosome 1p36 deletion syndrome is closely linked to epilepsy that is present in a subset of these patients (Heilstedt et al., 2001) and that Kvb2 null mice also exhibit seizures and cold swim-induced tremors similar to that

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observed in Kv1.1-null mice (McCormack et al., 2002). However, the discrepancy that Kv1.2 localization is minimally affected in the cerebellar basket cell synaptic terminals in Kvb2 null mice remains to be addressed (McCormack et al., 2002), although Kvb1 can substitute for Kvb2 in effects on Kv1 trafficking (Shi et al., 1996; Campomanes et al., 2002), and Kvb1/Kvb2 double knockout mice have more severe defects than either single knockout (Connor et al., 2005). 5.4.2

Determinants of Kv1 Channel Clustering

Membrane-associated guanylate kinases (MAGUKs) such as PSD-95, Chapsyn-110, SAP102, and SAP97 function as scaffolding molecules to promote coclustering of membrane receptors and ion channels (Sheng and Sala, 2001). Mutational and structural analyses showed that the PDZ domains of PSD-95 and other MAGUKs bind to the PDZ-binding motif (S/TxV) in the carboxyl-termini of all Kv1a subunits (Fig. 5.2a) (Kim et al., 1995; Doyle et al., 1996; Kim and Sheng, 1996; Tiffany et al., 2000). In most mammalian central neurons, MAGUKs are found at synapses, most prominently in the postsynaptic density (Sheng and Sala, 2001) and Kv1 channels on axons (Trimmer and Rhodes, 2004). However, MAGUKs are found as prominent components of Kv1 complexes purified from mammalian brain (Schulte et al., 2006). Examples where Kv1 channels and MAGUKs, in this case PSD-95, exhibit robust colocalization in neurons are in the colocalization of Kv1.1, Kv1.2, and Kvb2 with PSD-95 in cerebellar basket cells terminals (Kim et al., 1995; Laube et al., 1996) and at the juxtaparanodal regions of nodes of Ranvier (Rasband and Trimmer, 2001). Although mutation of the Drosophila discs large gene, a homologue of PSD-95, results in failure to localize Shaker potassium channels to the neuromuscular junction of flies, mutation of PSD-95 in mice does not lead to altered Kv1 channel localization (Rasband et al., 2002). At the level of the light microscope, the localization of Kv1.1 and Kv1.2 in basket cell terminals and juxtaparanodes is normal in transgenic mice expressing a truncated form of PSD-95 that is no longer expressed at these sites. While MAGUKs remain as attractive candidates for the interacting proteins that cluster Kv1 channels at discrete plasma membrane subdomains in axons and near nerve terminals, the specific binding partners mediating the clustering and their regulation remain to be elucidated.

5.5 PATHOLOGICAL ALTERATIONS IN KV1 CHANNEL TRAFFICKING AND DISTRIBUTION Kv1 channels are major regulators of the membrane excitability of neuronal nerve terminals (Dodson and Forsythe, 2004). Failures in controlling Kv1 channel expression, localization, and function may result in severe pathological hyperexcitability, leading to epilepsy and/or ataxia. As detailed above, neurons display wellorganized patterns of Kv1 channel expression and localization that is tightly regulated through diverse cellular mechanisms. Aberrations in these molecular events could affect neuronal excitability and lead to neuronal dysfunction as discussed below.

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SHAKER FAMILY KV1 VOLTAGE-GATED POTASSIUM CHANNELS

Episodic Ataxia

Episodic ataxia type-1 (EA-1) is a neurological syndrome that is characterized by continuous myokymia and cerebellar ataxia (Kullmann, 2002). EA-1 is caused by mutations in the Kv1.1 subunit that recessively affect individuals (Kullmann, 2002). Genetic linkage studies in EA-1 patients have identified at least 17 mutations in the Kv1.1 gene (Cannon, 2006; Jen et al., 2007). Most of the EA-1 mutations are missense mutations that lead to Kv1.1 channels with altered biophysical characteristics (Zerr et al., 1998; Maylie et al., 2002). However, the single EA-1 nonsense mutation that results in the truncation of the last 79 amino acid residues in the C-terminus (Eunson et al., 2000) causes loss of expression, misfolding, and resultant intracellular aggregation of Kv1 channels (Manganas et al., 2001a). Interestingly, the expression of this mutant also leads to intracellular aggregation and retention of coassembled wild-type Kv1a and Kvb subunits (Manganas et al., 2001a), suggesting effects beyond those on Kv1.1a subunits. These dominant negative properties of the EA-1 mutant Kv1.1 resemble in many aspects to those of the mutant CFTR protein encoded by the predominant mutation in cystic fibrosis patients. 5.5.2

Epilepsy

Epilepsy is a common neurological disorder and, in some cases, can be linked to channel dysfunction. To date, single-gene ion channel mutations are the predominant cause of rare Mendelian idiopathic epilepsy syndromes (Mulley et al., 2003; Cannon, 2006), of which two encode Kv channels (Kv7.2 and Kv7.3) outside of the family on which this review focuses. Experimental knockout of Kv1.1 channels in mice results in early postnatal epilepsy (Smart et al., 1998), and knockout of Kv1.2 also yields mice with epilepsy and a shortened life span (Brew et al., 2007). This suggests significant roles of Kv1 channels in the development of epilepsy. Kv1.1 knockout mice have an enlarged hippocampus (Persson et al., 2007) and exhibit hippocampal damage and reorganization (Wenzel et al., 2007). However, expression levels and localization of other Kv1 family members, auxiliary subunits, and interacting proteins are remarkably unaffected (Wenzel et al., 2007), in sharp contrast to findings in Kv4.2 knockout mice (Menegola and Trimmer, 2006). A spontaneous mutation in mouse Kv1.1 was identified in the megencephaly mouse (Petersson et al., 2003). The mutation leads to expression of a severely truncated form of Kv1.1 that is phenotypically similar to the EA-1 truncation mutant described above (Manganas et al., 2001a), in that it has trafficking deficiencies that can be transferred to heteromeric Kv1 channel complexes (Persson et al., 2005). Remarkably, a recent report showed that the phenotypes of mice with a voltage-gated calcium channel mutation that leads to neuronal hypoexcitability and absence epilepsy, and the hyperexcitable seizure phenotype of Kv1.1 knockout mice, can be eliminated by simply crossing the two mice (Glasscock et al., 2007). This highlights the role of precise regulation of neuronal excitability in maintaining normal brain function and the molecular complexity of such regulation.

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5.6 CONCLUDING REMARKS Given the crucial role of Kv1 channels in regulating the excitability of mammalian presynaptic nerve terminal, somata, and dendrites, a better understanding of the molecular and cellular mechanisms of their intracellular trafficking subcellular targeting is of great biological and biomedical significance. Such information will not only provide insights into mechanisms that generate the observed patterns of expression and discrete localization of Kv1 channels in precise functional domains of the axonal and somatodendritic membrane, but could also provide information crucial to development of highly selective drugs that target this important class of ion channels and modulate neuronal function. Moreover, it is likely to provide important information as to the etiology and pathophysiology of diseases of neuronal dysfunction such as ataxia and epilepsy.

ACKNOWLEDGMENT This work was supported by National Institutes of Health Grant NS34383 (J. S. T.).

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Tytgat J, Debont T, Carmeliet E, Daenens P, 1995. The alpha-dendrotoxin footprint on a mammalian potassium channel. J Biol Chem 270: 24776–24781. Vabnick I, Trimmer JS, Schwarz TL, Levinson SR, Risal D, Shrager P, 1999. Dynamic potassium channel distributions during axonal development prevent aberrant firing patterns. J Neurosci 19: 747–758. Vacher H, Misonou H, Trimmer JS, 2006. Determinants of voltage-gated potassium channel distribution in mammalian neurons. In: Bean AJ (Ed.), Protein Trafficking in Neurons. Elsevier Academic Press, London, pp. 244–270. Vacher H, Mohapatra DP, Misonou H, Trimmer JS, 2007. Regulation of Kv1 channel trafficking by the mamba snake neurotoxin dendrotoxin K. FASEB J 21: 906–914. Van Wart A, Trimmer JS, Matthews G, 2007. Polarized distribution of ion channels within microdomains of the axon initial segment. J Comp Neurol 500: 339–352. Veh RW, Lichtinghagen R, Sewing S, Wunder F, Grumbach IM, Pongs O, 1995. Immunohistochemical localization of five members of the Kv1 channel subunits: contrasting subcellular locations and neuron-specific co- localizations in rat brain. Eur J Neurosci 7: 2189– 2205. Wang H, Kunkel DD, Martin TM, Schwartzkroin PA, Tempel BL, 1993. Heteromultimeric Kþ channels in terminal and juxtaparanodal regions of neurons. Nature 365: 75–79. Wang H, Kunkel DD, Schwartzkroin PA, Tempel BL, 1994. Localization of Kv1.1 and Kv1.2, two K channel proteins, to synaptic terminals, somata, and dendrites in the mouse brain. J Neurosci 14: 4588–4599. Watanabe I, Wang HG, Sutachan JJ, Zhu J, Recio-Pinto E, Thornhill WB, 2003. Glycosylation affects rat Kv1.1 potassium channel gating by a combined surface potential and cooperative subunit interaction mechanism. J Physiol 550: 51–66. Weng J, Cao Y, Moss N, Zhou M, 2006. Modulation of voltage-dependent Shaker family potassium channels by an aldo-keto reductase. J Biol Chem 281: 15194–15200. Wenzel HJ, Vacher H, Clark E, Trimmer JS, Lee AL, Sapolsky RM, Tempel BL, Schwartzkroin PA, 2007. Structural consequences of Kcna1 gene deletion and transfer in the mouse hippocampus. Epilepsia 48: 2023–2046. Winklhofer M, Matthias K, Seifert G, Stocker M, Sewing S, Herget T, Steinhauser C, SaalerReinhardt S, 2003. Analysis of phosphorylation-dependent modulation of Kv1.1 potassium channels. Neuropharmacology 44: 829–842. Yang JW, Vacher H, Park KS, Clark E, Trimmer JS, 2007. Trafficking-dependent phosphorylation of Kv1. 2 regulates voltage-gated potassium channel cell surface expression. Proc Natl Acad Sci USA 104: 20055–20060. Yellen G, 1998. The moving parts of voltage-gated ion channels. Q Rev Biophys 31: 239–295. Yu W, Xu J, Li M, 1996. NAB domain is essential for the subunit assembly of both alpha-alpha and alpha-beta complexes of shaker-like potassium channels. Neuron 16: 441–453. Yuan LL, Chen X, 2006. Diversity of potassium channels in neuronal dendrites. Prog Neurobiol 78: 374–389. Zerr P, Adelman JP, Maylie J, 1998. Episodic ataxia mutations in Kv1. 1 alter potassium channel function by dominant negative effects or haploinsufficiency. J Neurosci 18: 2842–2848. Zhu J, Watanabe I, Gomez B, Thornhill WB, 2001. Determinants involved in Kv1 potassium channel folding in the endoplasmic reticulum, glycosylation in the Golgi, and cell surface expression. J Biol Chem 276: 39419–39427.

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Zhu J, Watanabe I, Gomez B, Thornhill WB, 2003. Heteromeric Kv1 potassium channel expression: amino acid determinants involved in processing and trafficking to the cell surface. J Biol Chem 278: 25558–25567. Zhu J, Gomez B, Watanabe I, Thornhill WB, 2005. Amino acids in the pore region of Kv1 potassium channels dictate cell-surface protein levels: a possible trafficking code in the Kv1 subfamily. Biochem J 388: 355–362.

6 UNIQUE MITOCHONDRIAL ION CHANNELS: ROLES IN SYNAPTIC TRANSMISSION AND PROGRAMMED CELL DEATH Elizabeth A. Jonas Department of Internal Medicine, Yale University, New Haven, CT 06520, USA

6.1 INTRODUCTION Mitochondria produce ATP for a myriad of cellular processes including the highly energy-dependent events of synaptic transmission. Mitochondria exist at the center of a web of signaling within the cell that regulates cell fate. They contribute to a spectrum of cell death types from necrosis to apoptosis. In many forms of programmed cell death, mitochondria play a crucial role in regulating oxidative phosphorylation to produce or conserve precious energy supplies. They also manage cytosolic levels of calcium and zinc, key ions implicated in excitotoxic neuronal death. This type of death has been implicated in cellular metabolic decline, neurodegeneration, and aging. Mitochondria contain two membranes, an inner and an outer membrane. In canonical apoptotic death, mitochondria regulate the release of proapoptotic factors such as cytochrome c from the mitochondrial intermembrane space across the outer membrane. Cytochrome c and other proapoptotic molecules activate downstream enzymes, including caspases that destroy the cell contents and its nuclear material. Outer membrane mitochondrial voltage-gated ion channels are intimately involved in these cellular processes. These ion channels regulate the Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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release of ATP and the reuptake of ADP through mitochondrial membranes, the influx and efflux of calcium, sodium, potassium, and zinc, which help determine the membrane potential of the inner membrane and regulate matrix volume. In addition, a family of proteins known as the BCL-2 family, which reside in the outer membrane and inhibit the release of proapoptotic factors from mitochondria, have properties of ion channels. Knowledge of their roles in the regulation of cellular functions has been expanding. In particular, we now know that BCL-2 family proteins perform physiological functions as well as participate in cell death, including, for example, regulation of the strength and pattern of synaptic transmission. Mitochondria also use channels to import proteins from the cytosol. These channels may themselves be regulated by changes in mitochondrial membrane potential. Although little is known about how the outer membrane voltage is determined, it has become clear that its permeability is tightly controlled, in part by the voltage dependence of outer membrane conductances. The control of outer membrane permeability compartmentalizes the intermembrane space with implications for metabolic regulation and cell fate determination. Until late in the last century, investigators mainly concentrated on understanding the inner mitochondrial membrane and matrix mitochondria efficiently produce ATP by oxidative phosphorylation. Studies led to a consensus that the energy produced by the reduction of NAD(P) to NAD(P)H and FAD to FADH in the tricarboxylic acid cycle was used (by reoxidizing these species in the oxidation part of the reaction) for the transport of electrons from high to low energy electron acceptors and to pump Hþ ions out across the inner membrane, thereby producing a large electrochemical (voltage) gradient. The movement of Hþ ions down the gradient across the ATP synthase phosphorylates ADP, producing ATP (the phosphorylation part of the reaction). The inner mitochondrial membrane is a barrier similar to the plasma membrane that allows the separation of ion species to produce the electrochemical gradient. The potential energy produced by charge separation across the inner membrane can be used to produce work and for signaling through the actions of its ion channels. These channels regulate the ATP-producing activity of the mitochondria and the enzymatic activity of the matrix. They control cytosolic concentrations of several ions and they alter the concentration of signaling molecules. The chapter is divided into three parts, addressing the unique aspects of mitochondrial ion channel function in the nervous system. The first part will summarize the current knowledge of the functions of the unique mitochondrial channels in the outer membrane, including the voltage-dependent anion channel (VDAC) and the channels of the BCL-2 family. The second part will address the known unique mitochondrial inner membrane ion channels that regulate ion flux into and out of the matrix and will suggest that the outer and inner membrane channels exist in a protein complex that is highly regulated. The third part will address the combined the functions of the inner and outer membrane channels, including those of the BCL-2 family, and discuss how regulation of such channels may alter the physiological behavior of the neuronal synapse.

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6.2 OUTER MITOCHONDRIAL MEMBRANE CHANNELS 6.2.1

Role of VDAC in Mitochondrial Function

Perhaps the most prevalent ion channel in the mitochondrial outer membrane is the voltage-dependent anion channel (mitochondrial porin, VDAC), and no studies of mitochondria, metabolism, or cell death can ignore the biophysical characteristics or physiological behavior of this important molecule (Colombini et al., 1996; ShoshanBarmatz et al., 2006). The main function of VDAC appears to be its ability to conduct metabolites such as ATP, ADP, NADH, and pyruvate, in addition to metabolites whose molecular weight can reach almost 1000. VDAC, therefore, has different biophysical characteristics than other voltage-gated ion channels (Hodge and Colombini, 1997). Historically, it was thought that VDAC was always open, making the mitochondrial outer membrane like a leaky sieve, but many recent studies have contributed to the present notion that the opening and closing of VDAC are highly regulated (Kinnally and Tedeschi, 1994; Jonas et al., 1999). The 30 kDa VDAC protein is relatively small compared to the plasma membrane voltage-gated ion channels such as Naþ or Caþ channels, whose molecular weights are approximately 300 kDa. VDAC is highly conserved in tertiary structure from yeast to man and it is also clearly present in plants. Interestingly, different VDACs differ widely in their amino acid sequences even though their channel gating and selectivity properties are conserved, suggesting that different interacting partners may bind to alternative sequences to regulate channel behavior (Colombini et al., 1996). 6.2.1.1 Biophysical Characteristics of VDAC In all the VDAC sequences analyzed, the N terminus contains a sequence that can form an amphiphilic alpha helix. All the downstream sequences have 12 or more alternating hydrophobic/ hydrophilic segments, which form the beta sheet that makes up the wall of a cylinder or barrel (Blachly-Dyson et al., 1990). In this configuration, the hydrophilic inner surface through which water and solutes travel faces the pore and the hydrophobic outer surface is buried in the membrane (Blachly-Dyson et al., 1989). Site-directed mutagenesis confirmed that residues in the alpha helix and the beta sheet strands are necessary to define selectivity, whereas some residues that do not affect selectivity are assigned positions outside the transmembrane region (Colombini et al., 1996). All VDACs studied have a conserved set of biophysical features (Colombini, 1989; Colombini et al., 1996). The channels have a very large single-channel conductance of 1–4 nS when the purified protein is reconstituted into artificial lipid membranes. The open state of the channel prefers anions to cations 2:1 and also strongly prefers metabolically relevant anions such as ATP. The channels open at voltages close to 0 mV, either in the positive or negative direction, but undergo rapid transitions to several closed states at potentials less than 50 or more than þ50 (Fig. 6.1). These “closed” states are permeable to cations but not to anions such as metabolites. Some closed states are more stable than others, so that the channel tends to reopen from some of the states more frequently than from others. Since the selectivity of the channel

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Membrane conductance (pS)

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4 Amount pos. charge in pore: low Amount pos. charge in pore: high 3 2

+ + +

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FIGURE 6.1 Voltage dependence of membrane conductance of the voltage-dependent anion channel (VDAC). (a) The artist’s rendition simulates data recorded when the channel protein is reconstituted in artificial membranes. Increasing the number of charged residues in the pore increases the steepness of the voltage dependence. (b) Dependence of the open and closed states of VDAC on position of the charged selectivity moiety inside or outside of the pore.

changes dramatically upon gating, the electrostatic forces within the ion conducting pathway must also change. Certain residues therefore affect the channel behavior in the open state but not in the closed state. This finding suggests that a positively charged moiety moves out of the pore during gating (Peng et al., 1992) (Fig. 6.1). The removal of positive charges may lead to a decrease in the propensity to conduct anions (such as metabolites) in the closed state. The change should also result in a reduction in the volume of water in the pore and a reduction in the pore radius. The change in selectivity with gating also predicts that the positive charges that move during gating may comprise the voltage sensor (Colombini et al., 1996). Therefore, increasing the positive charge within the pore will increase the steepness of voltage dependence and decreasing the positive charge will decrease the steepness (Fig. 6.1) (Doring and Colombini, 1985). Interestingly, moving the sensor against a high salt gradient requires more energy than moving it down the salt gradient; therefore, it appears that kinetic energy from the movement of the salt may help move the sensor out of the channel (Zizi et al., 1998). Regulators of VDAC include a variety of polyanions, all of which have similar effects (Colombini, 1989). When they are added to one side of the membrane, they appear to favor the closure of the channel, possibly by drawing out the positively charged gate region (Mangan and Colombini, 1987). Konig’s polyanion (a VDAC inhibitor that is a copolymer of styrene, maleic acid, and methacrylic acid) increases the probability of the channel closing even in the absence of a membrane potential, suggesting that its hydrophobic regions allow it to bind tightly to the membrane as well as to the voltage sensor (Colombini et al., 1987). It induces a progressive decrease in conductance of the channel over time as the channel enters several closed states. In addition to exogenously applied inhibitors, a large (100,000 MW) endogenous soluble protein modulator of VDAC, most likely residing within the intermembrane space,

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also increases the voltage-dependence of the channel and favors the closed state (Holden and Colombini, 1988). Of the nucleotides that penetrate the open channel, only NADH and NADPH significantly affect channel gating. NADH doubles the voltage-dependence of VDAC (Zizi et al., 1994). The addition of NADH and NADPH to mitochondria with intact outer membranes results in a six-fold reduction of the permeability of the outer membrane to ADP (Lee et al., 1994). VDAC may favor the closed state under normal conditions due to the presence of negative anions and protein modulators in the intermembrane space. Immobile molecules in the intermembrane space may exert osmotic pressure, drawing water through the pore and decreasing hydrostatic pressure within the pore (Holden and Colombini, 1993). The decline in hydrostatic pressure decreases pore volume and finally favors channel closure (Zimmerberg and Parsegian, 1986). 6.2.1.2 Control of Metabolism by VDAC Hexokinase is the first enzyme in the glycolytic pathway (Lemasters and Holmuhamedov, 2006) and it requires ATP. It apparently binds to the outer surface of the outer membrane via its association with VDAC, giving it preferential access to the ATP that is leaving the mitochondrion and also giving the mitochondrion preferential access to ADP produced by hexokinase. Hexokinase thereby uses ATP that is made in mitochondria over exogenous ATP. The relationship between VDAC and hexokinase suggests that VDAC is the central regulator of the interaction between glycolysis and mitochondrial respiration (Golshani-Hebroni and Bessman, 1997). Contact sites between the two membranes may contain the colocalized adenine nucleotide transporter (ANT, transporter of ADP and ATP through the inner membrane), VDAC, and hexokinase (Hashimoto and Wilson, 2000). Increasing the proximity of VDAC to the inner membrane proteins provides ADP readily to the ANT, thereby possibly increasing the efficiency of

FIGURE 6.2 Regulation of mitochondrial metabolism by interaction of hexokinase with VDAC. On the left is shown the case where hexokinase is not bound to VDAC. Inefficient transfer of ATP and ADP across the two membranes leads to the loss of crosstalk between glycolysis and mitochondrial respiration in the matrix. On the right is shown the case where hexokinase is bound to VDAC. Efficient exchange of metabolites occurs, increasing crosstalk between glycolysis and mitochondrial respiration.

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production of ATP (Fig. 6.2). An increase in metabolic rate, by activating glycolysis and hexokinase, increases the likelihood that the two membranes will come into close contact (Hackenbrock, 1972a; Brdiczka et al., 1986). The ATP generated by mitochondrial respiration then further stimulates the enzymatic activity of hexokinase. In certain tumor cells, hexokinase is very highly expressed. In these cells, the unusually high amount of hexokinase may cause VDAC to move closer to the inner membrane, increasing the association between glycolysis and mitochondrial metabolism (GolshaniHebroni and Bessman, 1997). Some tumor cells may lack this type of modulation. In these cells, glucose inhibits rather than activates oxidative phosphorylation in a reaction known as the Crabtree effect. High hexokinase levels cause hexokinase to bind tightly to VDAC, inhibiting the conductance of metabolites (Colombini et al., 1996; Penso and Beitner, 1998). In these defective cells, the ANT is not present at the hexokinase sites and contact sites are absent (Denis-Pouxviel et al., 1987). However, the cells can be converted to normal metabolism by lowering glucose levels, thereby enhancing mitochondrial metabolism, which causes contact sites and VDAC/ANT connections to re-form. Hexokinase, therefore, can help the cell switch between glycolysis and mitochondrial metabolism. When hexokinase inhibits VDAC, presumably no mitochondrial ATP can exit the mitochondrion nor can ADP enter; therefore, the cell must switch from mitochondrial metabolism to glycolysis. However, the homeostatic mechanisms regulating VDAC closure are quite complex since glucose-6-phosphate, the product of the enzymatic activity of hexokinase, antagonizes the binding of hexokinase to VDAC and tends to favor the open state. Therefore, if glycolysis becomes inhibited downstream of glucose-6-phosphate, glucose-6-phosphate will accumulate and then VDAC may open again. 6.2.1.3 VDAC Structural Studies EM studies suggest that monomeric VDAC channels form a crystalline array on the outer membrane (Rostovtseva et al., 2005). A pore of 2.5–3 nm was suggested by EM studies and confirmed by the inability of nonelectrolytes to penetrate through the narrowest portion of the pore (Rostovtseva et al., 2005). When the channel is in its closed state, a rigid spheroid molecule of 1.9 nm (gamma cyclodextrin) can barely permeate. Studies of these nonpermeating macromolecules, which favor closure by producing tension within the channel, show that the change in channel volume upon closure is 20–40 nm3. This result supports a model whereby a global change in the pore structure occurs, rather than a local constriction at one point within the pore. 6.2.2

BCL-2 Family Ion Channels: Role in Programmed Cell Death in Neurons

Programmed cell death or apoptosis is the genetic predisposition of cells to die (Adams and Cory, 2007). In this fashion, cells are eliminated developmentally and throughout the life of an organism to remove old and damaged cells (Kroemer and Reed, 2000). Failure of the death program can lead to the unchecked growth of cancer cells, while untimely onset of cell death leads to degenerative changes. In the nervous system, premature cell death produces diseases such as Alzheimer’s disease and amyotrophic lateral sclerosis (Yuan and Yankner, 2000). In addition, during pathological brain insults such as ischemia, infection, or trauma, some brain cells die immediately while

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others die a delayed death long after the insult, by turning on programmed death pathways (Banasiak et al., 2000). A highly evolutionarily conserved form of cell death in vertebrate cells is organized by mitochondria, which appear to receive a signal to undergo permeabilization of their outer membranes (Green and Kroemer, 2004; Adams and Cory, 2007). The opening of the outer mitochondrial membrane is also sometimes referred to as the activation of the apoptosis channel (Dejean et al., 2005). This event occurs suddenly, leading to the release of several intermembrane space proteins such as cytochrome c (Green and Kroemer, 2004; Martinez-Caballero et al., 2005). The release of cytochrome c produces two events. On the one hand, the loss of cytochrome c compromises the ability of mitochondria to produce ATP and eventually to maintain the mitochondrial membrane potential. On the other hand, cytochrome c and the other factors released from the mitochondria activate downstream caspases that chew up hundreds of cellular proteins (Youle and Strasser, 2008). Exactly how mitochondrial membrane permeabilization occurs is still unclear. Nevertheless, it appears that the BCL-2 family proteins play a major role in either regulating or, through their ion channel function, by producing permeabilization of the outer membrane (Fig. 6.3). BCL-2 family proteins were first described when the gene encoding BCL-2 was identified in B-cell lymphomas (Tsujimoto et al., 1985). BCL-2 family proteins regulate the protection of cells against prodeath signals, including growth factor

FIGURE 6.3 Interaction of inner and outer mitochondrial membrane ion channels in different scenarios. (From left to right): (1) The release of cytochrome c from the intermembrane space during cell death through an outer membrane channel formed by Bax or a VDAC/D N BCL-xL complex. (2) During physiological functioning of mitochondria such as during cell growth or during inhibition of cell death, VDAC may open to release metabolites, assisted by interaction with BCL-xL. (3) Formation of a two-membrane-spanning complex of channel proteins. If the inner membrane channel is activated by the binding of cyclophilin D in the presence of calcium, an outer membrane channel such as VDAC may open in response to activation of a messenger within the intermembrane space, or (4) by formation of a channel complex spanning the two membranes. If the mPTP is activated, swelling of the mitochondrial matrix followed by bursting of the outer membrane may cause release of cytochrome c from the intermembrane space.

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deprivation, ultraviolet and gamma radiation, heat shock, tumor necrosis factor, viral infections, and free radical formation (Adams and Cory, 2007). In addition, BCL-2 family proteins regulate death stimuli that had previously been relegated to the realm of necrosis, such as ischemic cell death in the heart and brain (Bonanni et al., 2006). 6.2.2.1 BCL-2 Family Proteins Function as Ion Channels BCL-2 family proteins exhibit two important features: They produce ion channel activity in intracellular organelle membranes, and they are regulated by interactions with binding partners, including BCL-2 family members and other intracellular membrane components. As a result, the BCL-2 family proteins control the release of death-inducing mitochondrial factors such as cytochrome c, which is normally present in the intermembrane space, into the cytosol (Fig. 6.3). Certain BCL-2 family proteins may also enhance the release of metabolites from mitochondria by interaction with VDAC (Fig. 6.3) (Gottlieb et al., 2002) and interact with the members of the growth and survival signaling pathway (Zha et al., 1996). In some cases, antiapoptotic proteins can be rapidly converted into proapoptotic molecules by proteolytic cleavage after a cell death stimulus, such as the transformation of BCL-xL to DN BCL-xL, and this event dramatically increases the permeability of the mitochondrial outer membrane by increasing the single-channel conductance of the pore-forming protein (Fig. 6.3) (Cheng et al., 1997; Clem et al., 1998; Jonas et al., 2004). In neurons, the size of the mitochondrial channel conductance and its binding partners may determine whether a mitochondrial channel promotes cell survival or death. Both pro- and antiapoptotic BCL-2 family proteins produce ion channel activity when inserted into artificial lipid membranes (Schlesinger et al., 1997; Schendel et al., 1998). Many BCL-2 family proteins are localized to the outer mitochondrial membrane or translocated into the outer membrane upon a death stimulus (Wolter et al., 1997; Kaufmann et al., 2003). Three categories of BCL-2 proteins contribute to the regulation of cell death (Fig. 6.4). These are the antiapoptotic members (such as

FIGURE 6.4 BCL-2 family proteins. Canonical antiapoptotic proteins contain the BH4 domain that is lacking in proapoptotic BCL-2 family members such as cleaved BCL-xL (DN BCL-xL) and Bax. BH3-only molecules are traditionally thought to be proapoptotic, but they may also play physiological roles.

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BCL-xL, BCL-2, and MCL-1), the proapoptotic members such as Bax and Bak, and a large group of BH3-only proteins such as BID, BAD, PUMA, and NOXA (Galonek and Hardwick, 2006). The antiapoptotic members of the group are similar in structure and sequence to proapoptotic Bax and Bak, and in addition to the BH1–3 domains, contain a BH4 domain that is important for the antiapoptotic features of the molecules (Tsujimoto and Shimizu, 2000; Sugioka et al., 2003). Despite the presence of the BH4 domain in BCL-xL, at first glance, the ion channel activities of Bax and BCL-xL seem quite similar (Schlesinger et al., 1997). The three-dimensional structure of BCL-xL is comprised of seven alpha helices (Muchmore et al., 1996; Schendel et al., 1998). Two outer layers of amphipathic helices serve to screen the long hydrophobic alpha helices from the aqueous domain. A long proline-rich loop found between the first and second helices is absent in the proapoptotic members of the family. The loop may be vulnerable to protease digestion and contains phosphorylation sites. The BH1–3 domains of BCL-xL fold together to give a hydrophobic region involved in homo- and heterodimerization and contain domains important for interaction with proteins that contain only a BH3 domain. The structure of BCL-xL is strikingly similar to that of the diphtheria toxin membrane translocation domain and the pore-forming domains of bacterial colicins that kill sensitive cells via the formation of a highly conductive ion channel in the target cell’s plasma membrane. The overall organization of the colicin-like channels is that of a hydrophobic region containing the pore, shielded by amphipathic helices that keep the molecule soluble in the cytoplasm until insertion into a membrane activates the pore function. Two helices of Bax and BCLxL are insufficient to form a pore (Schendel et al., 1998) but their ability to homo- and heterodimerize may provide for interactions critical for their pore-forming ability. It is possible that channel activity observed in artificial lipid membranes is not the same as activity in vivo. Both BCL-xL and Bax have demonstrated avid pore-forming capability in lipid bilayers if lipid composition of the bilayer is carefully regulated (Minn et al., 1997; Schlesinger et al., 1997; Schendel et al., 1998; Antonsson et al., 2000). The BCL-xLchannel inlipidbilayers isa nonselectivechannel that favors the conductanceof cations over anions and displays multiple conductances, with the most common conductance value of about 276 pS (Minn et al., 1997). Both Bax and BCL-xL display similar channel activity with multiple conductances, but BCL-xL has a linear conductancewhereasBaxappears to be more rectified toward positive potentials, is more anionselective than BCL-xL, and has larger peak conductances (Schlesinger et al., 1997). Channel Activity of BCL-2 Proteins Recorded in Vivo The similar channel activities in these anti- and proapoptotic proteins raised several questions, the most important of which was whether the channel activity of the anti- and proapoptotic molecules was important at all for their anti- and proapoptotic functions, and if so,why were the channel activities so similar and could they prove more dissimilar in vivo? To examine these issues, the giant synapse of the squid stellate ganglion has been used as a model system to study the activity of the recombinant proteins in mitochondrial membranes inside a living neuron (Jonas et al., 2003, 2004). In this method, a concentric electrode arrangement, a clean patch pipette tip of small internal diameter is exposed to mitochondria inside the neuronal presynaptic ending, and channel activity

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FIGURE 6.5 Channel recordings performed on mitochondrial membranes within a living neuronal synapse. (a) A control recording and recordings of patches exposed to recombinant FL BCL-xL or Bax. (b) The panel shows recordings of patches exposed to Bax or DN BCL-xL, demonstrating the large conductance activity of the membrane in the presence of these proteins. (c) A mitochondrial recording within a hypoxic synapse and a recording of a patch exposed to a VDAC inhibitor (NADH) within a hypoxic synapse.

is recorded from a patch of outer mitochondrial membrane exposed to the inside of the pipette. When such patches are exposed to recombinant antiapoptotic full-length BCL-xL protein (FL BCL-xL), mitochondrial channel activity that is distinctly different from that of controls appears (Fig. 6.5). Application of FL BCL-xL produces characteristic activity with multiple conductances in mitochondrial patches within 5 min of the start of the recording. Unitary openings correspond to conductances between 100 and 760 pS. Channel activity switches rapidly between different conductance levels. Holding the voltage at different potentials reveals a current voltage relationship that is linear or very slightly outwardly rectifying (Jonas et al., 2003). Channel activity of recombinant Bax recorded in presynaptic mitochondrial membranes shares some features with that of recombinant FL BCL-xL. To form channels readily in vitro, however, it must be activated by treatment with detergent, which causes the protein to oligomerize (Hsu and Youle, 1998; Antonsson et al., 2000; Green and Kroemer, 2004). In isolated mitochondria or isolated outer mitochondrial membranes, this activation of Bax is mediated by endogenous tBID (N-terminally cleaved/truncated BID) (Roucou et al., 2002) or another BH3-only protein (Polster et al., 2001), which may also be present in squid (Jonas et al., 2005). The findings suggest that activation of

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Bax channel activity requires a mitochondrial membrane component (Roucou et al., 2002). When purified recombinant Bax protein lacking the C terminus (BaxDC) or full-length Bax protein is placed inside the patch pipette used to record from mitochondria in the squid presynaptic terminal, it evokes channel activity in the patches ranging between 100 and 750 pS and demonstrates outward rectification (Fig. 6.5). In 5–10% of the channels observed on mitochondria inside the neurons with recombinant Bax, a number of large openings with conductances >750 pS are detected in addition to the smaller conductances, similar to the large conductances reported for purified Bax in artificial lipid membranes (Dejean et al., 2005). In contrast to the intermediate conductance openings, the current–voltage relations for the large openings are linear (Fig. 6.5). Thus, it appears that including Bax in the patch pipette induces channel activity with two distinct properties, a large conductance state and a less conductive state that shares some properties with that induced by full-length BCL-xL. The findings suggest that the smaller conductance channel activity of Bax could represent the activity of Bax in a form prior to its exposure to a death stimulus or to activator BH3 peptides. However, this “inactive” form of Bax could have additional functions besides waiting to bring on cell death in response to a stimulus. Bax may alter synaptic function in the healthy cell. The evidence that Bax and Bak can act as prosurvival factors in neurons exists (Lewis et al., 1999; Middleton and Davies, 2001; Fannjiang et al., 2003). Both in vitro and in vivo data support the hypothesis that the functional activities of Bax depend on the specific cell in which Bax resides and the specific pathological stress that the cell is undergoing. It is possible that the protective effects of Bax and Bak in such models may result indirectly from their actions on synaptic activity (Fannjiang et al., 2003; Jonas et al., 2005). If the smaller conductance activity represents the activity of Bax in healthy cells, then the infrequently detected large conductance activity could be important for its death-promoting actions. Indeed, proteolytic cleavage of Bax accelerates the onset of its prodeath function (Wood and Newcomb, 2000) and it is conceivable that the observed spontaneous large activity could have been recorded in synapses in which a death stimulus had been activated by previous damage. 6.2.2.2 Endogenous Death Channels Produced by Bax-Containing Protein Complexes The first patch-clamp recordings of endogenous death channel activity were performed on mitochondrial outer membranes isolated from cells undergoing apoptosis (Pavlov et al., 2001). Pavlov et al. were able to detect an ion channel, mitochondrial apoptosis-induced channel (MAC), whose pore diameter was estimated to be of sufficient size (4 nm) to allow the passage of cytochrome c and larger proteins. The channel displays multiple conductances, the largest of which is 2.5 nS. The channel activity is expressed in mitochondrial outer membranes, inhibited in cells overexpressing BCL-2, and is similar to the activity of pure Bax expressed in artificial lipid membranes. The timing of cytochrome c release in apoptotic cells correlates well with the onset of MAC activity and with the translocation of Bax to mitochondrial membranes, further suggesting that such channel complexes include Bax protein. Moreover, MAC activity can be immunodepleted from mitochondrial membranes treated with anti-Bax antibodies.

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Inactivator BH3 protein

Activator BH3 protein

BAD

BID

BCL BCL-xL L

B Bax activation ti ti

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Cell death

FIGURE 6.6 Schematic of an example of one possible hierarchical model of the control of cell death by BCL-2 family proteins. From left to right, after a cell death signal, BAD (the inactivator BH3 protein) sequesters BCL-xL, freeing BID (the activator BH3 protein) to activate Bax. Bax oligomerizes and permeabilizes mitochondrial outer membranes, releasing cytochrome c to activate downstream caspase pathways.

6.2.2.3 Actions of BCL-2 Family Proteins are Regulated by Binding Partners The mechanisms by which antiapoptotic proteins such as BCL-2 and BCL-xL prevent cell death are poorly understood. Recent studies have focused on the ability of antiapoptotic proteins such as BCL-xL to bind to and sequester proapoptotic members of the BCL-2 family (Kim et al., 2006). BCL-2 and BCL-xL bind to Bax and to BH3 peptides, preventing the proapoptotic actions of these proteins (Galonek and Hardwick, 2006; Kim et al., 2006). Under resting conditions, BCL-xL and BCL-2 are bound to activator BH3 molecules, keeping them in a quiescent state. Data suggest that activation of inactivator BH3-only proteins such as BAD is the first step in the initiation of cell death. These inactivator BH3 proteins then bind to the antiapoptotic BCL-2 proteins BCL-xL and BCL-2, preventing them from binding to Bax or to activator BH3 peptides such as BID. The activator BH3 peptides can then bind to and activate Bax or bind to inhibitors of Bak, such as VDAC2 (Cheng et al., 2003). The end result is that Bax or Bak are free to homo-oligomerize and thereby permeabilize outer mitochondrial membranes. This hierarchy model explains the high killing potency of a subset of BH3-only proteins that is known to bind all antiapoptotic proteins (Fig. 6.6) (Galonek and Hardwick, 2006; Kim et al., 2006). Bax and BCL-xL share high structural homology in the BH1–BH3 domains (Schlesinger et al., 1997; Schendel et al., 1998). It is clear from the binding pair studies that both Bax and BCL-xL interact in complexes with the BH3-only proteins. When other proteins or binding partners compete for their binding, they both may get released from these complexes and can perform other functions. 6.2.3

Interaction of VDAC with BCL-2 Family Proteins

Because of the importance of VDAC in mitochondrial function, a role for VDAC in cell death has been postulated. The possibility that VDAC is a component of the permeability transition pore, which can in the absence of other outer membrane

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proteins release cytochrome c during programmed cell death, remains controversial. Against the idea that it is the sole member of the apoptotic channel is the finding that programmed cell death can occur in the absence of VDAC (Baines et al., 2007) and that the functional unit of VDAC is most likely a monomer (Rostovtseva et al., 2005) whose pore size is too small to release proapoptotic factors such as cytochrome c. However, several reports support the tendency of VDAC to self-assemble into dimers, trimers, and tetramers (Zalk et al., 2005; Malia and Wagner, 2007).Whether these structures could actually form in mitochondrial outer membranes during cell death is not known. VDAC dimerization is produced by As2O3 (Zheng et al., 2004) during the process of release of cytochrome c, and it has been suggested that VDAC oligomerization could be dependent on the presence of cytochrome c (Zalk et al., 2005). A further argument against the release of cytochrome c by VDAC is that VDAC only passes certain negatively charged metabolites, most not much larger than ATP. Large, positively charged cytochrome c is unlikely to pass through VDAC since permeation by cations is reduced in the open conformation. VDAC may interact with molecular regulators of apoptosis and metabolism such as hexokinase on the cytosolic side and creatine kinase in the intermembrane space. The precise mechanism by which association with these molecules regulates the permeability of VDAC is unknown (Wicker et al., 1993; Brdiczka et al., 1994). A description of the studies in which VDAC has been found to interact with BCL-2 family proteins follows. 6.2.3.1 Models of VDAC -BCL-2 Family Interaction Although the diameter of VDAC at its largest is not compatible with the release of cytochrome c, it is possible that VDAC-interacting proteins could regulate its pore diameter or interact in multimeric structures with VDAC, forming a pore large enough to release proapoptotic factors from the mitochondrion. One controversial model proposes that Bax interacts with VDAC to provide a large pore through which cytochrome c could permeate (Shimizu et al., 1999). Studies by this group suggest that the single-channel conductance of Bax, when combined with VDAC in planar lipid bilayers, is increased by a factor of 4–10 over VDAC and Bax channels alone, and the group also finds that liposomes containing these proteins are permeable to cytochrome c (Shimizu et al., 2000). The authors also found that the antiapoptotic BH4 domain of BCL-2 closed VDAC and inhibits cell death (Shimizu et al., 2000). An equally controversial model suggests that BCL-2 family proteins regulate mitochondrial membrane permeability by closing VDAC during apoptosis. This closure leads to a lack of metabolite exchange across the outer membrane eventually accompanied by inner membrane swelling and rupture of the outer membrane, thereby releasing cytochrome c (Vander Heiden et al., 2000). This process is prevented by the antiapoptotic protein BCL-xL, which by interacting with VDAC after a cell death stimulus maintains metabolic exchange across the outer mitochondrial membrane and prevents cell death (Gottlieb et al., 2002). Proapoptotic tBID has also been proposed as a VDAC interacting partner. In a study of VDAC reconstituted into artificial lipid membranes, the activated proapoptotic protein BID induced VDAC channel closure (Rostovtseva et al., 2004).

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6.2.3.2 Interactions of VDAC with BCL-xL A recent structural study of BCL-xL and VDAC illuminated potential sites of interaction between the two molecules in an artificial lipid environment. Recombinantly expressed VDAC (Malia and Wagner, 2007) was dissolved in a detergent buffer that mimicked the state of VDAC within the outer mitochondrial membrane. The structural spectrum resulting from the analysis of this solution reveals a folded protein of high beta sheet content, as described previously. Titration of VDAC with a large excess of metabolites results in chemical shifts indicative of low affinity binding of the metabolites to VDAC (especially NADH, NAD, and ATP), a result predicted by the requirement of low affinity binding for efficient transport. Exposing the dissolved VDAC solution to BCL-xL also causes a marked shift of the chemical spectrum of VDAC, indicating significant interaction between VDAC and BCL-xL. In complementary experiments, VDAC in turn causes a marked shift in the spectrum of BCL-xL. These results, and those of additional studies, demonstrate that VDAC appears to bind to BCL-xL in two areas. The first is the helical hairpin region of BCL-xL near the C-terminus that is the putative site of interaction of BCL-xL with membranes. The other is a region near the N-terminus that may form part of the BH4 domain. Previous studies had suggested that the BH4 domain of BCL-xL was necessary for the inhibition of VDAC channel opening by BCL-xL (Shimizu et al., 2000). Thus these structural findings support the findings of the artificial lipid bilayer studies. The model based on these findings predicts that the two membrane proteins are oriented parallel to each other in the membrane and bind to each other along the entire length of the helical hairpin of BCLxL and one face of the beta barrel of VDAC. Complex formation between BCL-xL and VDAC is also found by western blotting after cross-linking and size exclusion chromatography. These studies suggest that VDAC usually exists as a trimer (although oligomerization can be detergent dependent (Pappert and Schubert, 1983) and that within the trimer, BCL-xL binds to one or two VDACs, so that BCL-xL displaces one VDAC from a trimeric complex to form a new trimeric structure with BCL-xL as the third member. The data suggest that BCL-xL may change the structural properties of VDAC and enhance metabolite or cytochrome c conductance in different contexts. 6.2.3.3 BCL-xL Interaction with VDAC in Mitochondria Isolated from Ischemic Brain To prove that an interaction between BCL-xL and VDAC forms in vivo, a model system was studied in which application of recombinant BCL-xL protein to mitochondrial membranes produced channel activity that was attenuated by inhibiting VDAC (Jonas et al., 2004). BCL-xL is known to exist in two forms, a full-length antiapoptotic form and an N-truncated version (DN BCL-xL) that appears after death stimuli and acts as a cell killer protein (Clem et al., 1998). In mitochondria of synapses exposed to the death stimulus hypoxia or mitochondria isolated from ischemic brain, large channel activity is recorded that has biophysical features of the activity produced by application of DN BCL-xL to control mitochondria (Clem et al., 1998; Jonas et al., 2004; Bonanni et al., 2006). (Figs 6.3 and 6.5) Both the mitochondrial channel activity produced during hypoxia and the channel activated by the application of DN BCL-xL are strongly inhibited by millimolar concentrations of NADH. NADH binds with low

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affinity to VDAC, but in large concentrations can block its channel activity (Wunder and Colombini, 1991; Lee et al., 1994). NADH is ineffective on DN BCL-xL-induced channel activity when VDAC is absent (Basanez et al., 2001, 2002; Jonas et al., 2004). Furthermore, yeast mitochondria lacking VDAC (Lohret and Kinnally, 1995) do not respond to the application of recombinant DN BCL-xL with any change in channel activity, and NADH fails to inhibit the endogenous channel activity of the mitochondria lacking VDAC. These studies suggest that the large channel activity of mitochondria recorded after a death stimulus (hypoxia) is produced by a channel made up of a protein complex of VDAC and BCL-xL. Whether BCL-xL and VDAC actually interact biochemically or merely biophysically in vivo is still in question. 6.2.3.4 VDAC2 Inhibits Apoptosis In mammals, three different VDAC genes encoding distinct isoforms have been reported (Shoshan-Barmatz et al., 2006). More complex multicellular organisms have all three isoforms, suggesting that the different isoforms have specialized functions. Yeasts have two isoforms. Yeast VDAC1 protein forms pores when reconstituted into artificial lipid membranes, but yeast VDAC2 does not. VDAC1 and 3 are the predominant pore-forming isomers of VDAC in mammalian cells, but a role for VDAC2 has recently been suggested (Cheng et al., 2003). Cells deficient in VDAC2, but not VDAC1, are more susceptible to Bak oligomerization and apoptotic cell death, and these events are prevented by overexpression of VDAC2. In a model created to explain these findings, the authors suggest that the proapoptotic molecules tBID, BIM, or BAD may displace VDAC2 from Bak, enabling homooligo merization of Bak and release of cytochrome c through the mitochondrial outer membrane. 6.2.3.5 VDAC BAD Regulation of Metabolism and Cell Death The potential interaction of VDAC with hexokinase, as already described, places VDAC in a position to interact directly or indirectly with BAD, a BH3-only protein (Fig. 6.7). BAD is necessary in pancreas for the formation of a mitochondrial outer membrane complex of glucokinase (pancreatic hexokinase), protein kinase A, protein phosphatase 1, and Wiskott-Aldrich protein family member WAVE-1, a known actin binding protein. The BAD knockout animals or those in which BAD has been constitutively dephosphorylated display profound deficits in glucose homeostasis and glucose tolerance (Danial et al., 2003; Danial et al., 2008). In normal cultured cells deprived of glucose and in brain exposed to transient ischemia, BAD becomes dephosphorylated, translocates to mitochondrial membranes, and contributes to cell death, possibly by contributing to the formation of a cytochrome c-releasing pore (Fig. 6.7). Such a pore could be formed by the activation of Bax after sequestration of BCL-xL by BAD, or possibly by the activation of proapoptotic BCL-xL, after cleavage by caspases or calpains to form DN BCL-xL (Fig. 6.7) (Miyawaki et al., 2008). 6.2.4

VDAC Regulation of Learning and Memory

Studies of VDAC1 and 3 knockout mice suggest that VDAC plays an important role in the normal process of learning and memory (Weeber et al., 2002). Although there

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FIGURE 6.7 Physiological and pathological roles of BAD. In a normal pancreatic insulinsecreting cell, phosphorylated BAD nucleates a complex of proteins at the mitochondrial membrane that regulates normal glucose homeostasis. In BAD knockout animals (BAD/), the complex is disrupted and glucose metabolism is disturbed perhaps by a decrease in efficiency of ATP production in response to glucose stimulation. During cell death, BAD becomes dephosphorylated and participates in cytochrome c release from mitochondria, possibly by binding to BCL-xL and contributing to the formation of a channel produced by proapoptotic DN BCL-xL.

appears to be no obvious neuronal structural defect in the VDAC knockout mice, they are deficient in contextual fear conditioning and in the ability to learn to find a hidden platform in the Morris Water Maze test, suggesting that the mice lack normal capabilities for establishing memory and learning. In addition, hippocampal slice recordings from VDAC knockout mice demonstrate a decrease in certain forms of short-term facilitation. The cellular model proposed to underlie hippocampal memory formation and learning is long-term potentiation (LTP) of synaptic transmission. LTP is also significantly impaired in the knockout mice. Interestingly, low-dose CSA, which inhibits mitochondrial inner membrane depolarization, attenuates LTP in normal mice, suggesting that inner membrane ion channel activity is also involved in the onset of learning.

6.3 MITOCHONDRIAL INNER MEMBRANE CHANNELS Many studies of inner membrane physiology over the last half century were concerned with the management of calcium fluxes, calcium buffering by the matrix, and catastrophic calcium-induced depolarization of mitochondria (Nicholls and

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Akerman, 1982; Gunter and Pfeiffer, 1990; Bernardi, 1999). The next section will attempt to outline the inner membrane ion channel conductances as they relate to the mechanisms of calcium movement between the mitochondrion and the cytosol and the ability of the mitochondrion to produce energy in the form of ATP. Ion channels that regulate ion and metabolite fluxes across the inner membrane may form complexes with channels in the outer membrane such that the complex of channels may regulate the flow of ions and metabolites directly across the two membranes between the matrix and the cytosol. Regulation of complex formation may thereby alter metabolism and neuronal function. 6.3.1

Energy Dependence of Mitochondrial Calcium Accumulation

Calcium normally cycles constantly between the mitochondrial matrix and the cytosol. Calcium enters the matrix over its electrochemical gradient via the uniporter, a calcium-selective channel of the inner membrane (Litsky and Pfeiffer, 1997; Kirichok et al., 2004), and can be exchanged with sodium (sodium in, calcium out, Naþ/Ca2þ exchanger) linked to a sodium/hydrogen (Naþ/Hþ, hydrogen in, sodium out) exchanger and Hþ efflux via respiratory complexes (Fig. 6.8). These exchange pathways are used when cytosolic calcium levels are relatively low and no net accumulation of calcium occurs in the mitochondrial matrix (Fig. 6.8). Nevertheless, when calcium rises more precipitously in the cytosol, mitochondria can accumulate calcium and therefore may serve as major buffers for cytoplasmic calcium (Nicholls and Akerman, 1982). In this setting, Hþ ions are pumped out in 1. Calcium ci uniporter

Ca2+

3. Na +/H+ antiporter

5. Permeability transition pore

Ca2+ Na+

Na+

H+

2. Na +/Ca2+ exchanger

H+

H+

4. Electron transport complexes

H+

Ca2+ ADP

7. H +/Pi- cotransporter

ATP Pi

6. ATP synthase

CaPi

FIGURE 6.8 Management of calcium homeostasis in cells by mitochondrial calcium buffering. When cytosolic calcium levels are low, calcium cycles into and out of the matrix using channels and transporters (1–4). When cytosolic calcium levels rise, mitochondria can buffer calcium by the net exchange of calcium for Hþ ions, compromising the synthesis of ATP (6). The buffering of calcium is aided by the formation of a calcium phosphate complex in the matrix (7). Calcium can be rereleased through the transporters (2–4) or through a calciumactivated channel that may be related to the mPTP (5).

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exchange for calcium, so that net accumulation of calcium will occur together with net efflux of Hþ ions (Nicholls and Akerman, 1982). Therefore, the net accumulation of calcium leads to matrix alkalinization, and increases the difference in pH across the inner membrane. Eventually, no more Hþ can be lost from the matrix without compromising the maximum value of the membrane potential. When this happens, the membrane begins to depolarize toward 0 mV. More extensive calcium uptake can occur in the presence of anions (such as Pi, acetate, HCO3) that permeate upon cotransport with Hþ ions (Fig. 6.7). The cotransport of Hþ and anions is stimulated by the previous alkalinization of the matrix. The resultant reestablishment of the normal concentration of Hþ ions in the matrix then allows the respiratory chain to reestablish the membrane potential by pumping out Hþ ions again, so that there may be only a transient membrane depolarization during calcium uptake. If phosphate is the permeating anion, then a calcium phosphate precipitate forms in the matrix, so that free calcium remains low. Under these conditions, there is theoretically possible a massive accumulation of precipitated calcium but very little accumulation of free matrix calcium. In the presence of phosphate, unlike with other anions such as acetate that do not form a precipitate, there is no change in osmolarity of the matrix. One Hþ accumulates in the cytosol for each calcium taken up, as the precipitate forms and Hþ is freed up from hydrogen phosphate. The increase in free Hþ in the matrix stimulates the respiratory complexes to pump Hþ back out by accelerating the respiratory chain to maintain the membrane potential, just as if ATP were being made. Hþ ions can also be pumped out by reversal of the synthase, requiring ATP hydrolysis. All of the above described processes take energy, as Hþ is pumped out but calcium flows in at the expense of ATP production. If no phosphate is available, then this reaction is prevented and further alkalinization of the matrix occurs with gradual depletion of the Hþ pool, finally resulting in depolarization of the membrane potential, preventing further calcium accumulation. 6.3.2

Voltage-Dependent Inner Membrane Channels: The Calcium Uniporter

Calcium enters the matrix via the uniporter, which appears to be a calcium selective, voltage-dependent channel (Kirichok et al., 2004). By recording in the whole mitoplast configuration, the problem of dissipation of the membrane potential by calcium influx can be prevented by voltage-clamp. In this mode, a calcium-selective current is increased by varying cytoplasmic calcium concentrations. The membrane is highly conductive to calcium under these circumstances, suggesting that the permeability pathway constitutes a channel rather than a transporter. At micromolar concentrations of calcium, the current density is comparable to that of voltage-gated plasma membrane calcium channels that are exposed to millimolar calcium. This is because of the large electrochemical potential gradient or driving force across the mitochondrial membrane and because the channel is usually in the open state. The calcium current is inwardly rectifying, so that it is larger at negative potentials, as one would expect for calcium uptake in energized mitochondria. The current is inactivated by time to a plateau level still carrying significant current. The total current is inhibited effectively by ruthenium

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red, a known inhibitor of calcium uniporter activity, but in single-channel recordings of inside-out patches, ruthenium is ineffective at positive potentials. In the absence of calcium, single-channel recordings demonstrate that the channel conducts sodium with a constant current but conducts calcium with a flickering current at positive potentials. The flickering in a calcium-containing medium suggests that the channel is partially blocked while calcium is passing through the channel. Although the molecular identity of the calcium uniporter is still not known, one candidate for calcium uptake in heart is the ryanodine receptor, which is activated by calcium and inhibited by Mg2þ and ruthenium red (Beutner et al., 2001). The ryanodine receptor may play an important role in the regulation of calcium uptake during cardiac ischemia. 6.3.3

Other Inner Membrane Conductances: Mitoplast Recording Technique

Patch clamping of isolated mitochondrial inner membranes in the “mitoplast” configuration has yielded data on several different conductances, but the most well studied is the large-conductance voltage-dependent channel of the inner membrane, variously named in the literature, but most likely serving as the underlying conductance of the rapid calcium efflux pathway related to the mitochondrial permeability transition (Crompton, 1999; Halestrap et al., 2000). The channel or pore that may underlie this conductance was termed mitochondrial permeability transition pore (mPTP). To the mPTPs have also been ascribed various functions in addition to calcium efflux, most notably a role in the formation of the inner membrane component of a two-membrane-spanning mitochondrial death channel complex that has been particularly implicated in ischemic cell death in heart and brain (Fig. 6.3) (Baines et al., 2005; Halestrap, 2005; Nakagawa et al., 2005). The first “mitoplast” recordings were obtained in the 1980s from isolated inner membranes from cuprizone-fed animals. Cuprizone feeding yields giant mitoplasts (5 mM or more in diameter) compared to those derived from control animals (1–3 mM in diameter), creating an organelle closer to the size of a small cell. This type of mitoplast is easier to patch clamp. The recordings (Sorgato et al., 1987, 1989) reveal a slightly anion-selective channel of 108 pS that is voltage dependent in that there is much more current, both whole “cell” or single channel, at positive potentials. 6.3.4 Channel Activity Correlated with Permeability Transition: The Mitochondrial Permeability Conductance Pore (mPTP) The permeability transition is characterized by a sudden loss of mitochondrial membrane potential induced by an increase in permeability of the inner membrane to a large number of unrelated solutes (Gunter and Pfeiffer, 1990). Physiologically relevant molecules and those for which there is no known influx pathway enter by diffusion down their concentration gradients, because after permeability transition, all energy-dependent processes of the inner membrane are halted by the dissipation of the potential gradient. The transition is regulated by calcium on the matrix side and therefore it serves as a calcium efflux pathway from the matrix to the cytosol, but whether this channel is used by mitochondria during physiological activity is still

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controversial. Swelling of the matrix also occurs after permeability transition because of the rapid influx of ions and small molecules followed by water. Swelling can be measured optically by the decrease in light scattering by the dissolved solutes. Therefore, permeability transition as measured with optical techniques can be correlated with channel activity as measured by patch-clamp recording of mitoplasts. The “MCC” has been characterized as a multiconductance, voltage-dependent channel recorded in mitoplast (inner membrane) preparations (Kinnally and Tedeschi, 1994) Most frequently, the activity was more prominent at negative potentials, with closings to lower conductances at positive potentials. The activity was found to be inhibited by amiodarone or cyclosporine A. These inhibitors caused the channel to step through various lower conductance levels and remain in the closed or small conductance open state for many minutes. The channel has biophysical characteristics in common with the permeability transition pore (mPTP) (Gunter and Pfeiffer, 1990) and is also known as mitochondrial megachannel (MMC) (Szabo and Zoratti, 1991, 1992; Bernardi et al., 1992). It is activated by calcium, inhibited by Mg2þ, and is voltage dependent (Kinnally et al., 1991; Bernardi, 1992; Bernardi et al., 1992; Scorrano et al., 1997). The similarities between the conductances recorded by different groups also includes inhibition by ADP and by alterations in pH. The MCC has the same response to various agents that inhibit the MMC, including propranolol, amiodarone, dibucaine, and benzodiazepines (Kinnally et al., 1992; Zorov et al., 1992). In another study performed at that time, a 1.3 nS conductance channel was recorded that also demonstrated subconductance states (Petronilli et al., 1989). The channel was voltage dependent. When the matrix was made positive, the channel was more likely to enter subconductance states. Unlike VDAC, at all negative potentials, the channel remained at a high conductance. It was hypothesized that these channels could participate in the transport of proteins across the inner membrane, but subsequent studies identified other more specific protein-conducting channels, therefore its complete function is still not fully understood. Further studies of the 1.3 nS channel revealed that it was activated by calcium on the matrix side and inhibited by competition of calcium with Mg2þ and Ba2þ, Sr2þ, and Mn2þ (Szabo et al., 1992). The calciumactivated 1.3 nS conductance was also inhibited by CSA, a known inhibitor of the prolyl isomerase cyclophilin, on the matrix side (Szabo and Zoratti, 1991). Cyclophilin had previously been known to inhibit permeability transition measured optically. The finding that channel activity was inhibited by the binding of CSA to cyclophilin established that cyclophilin was in a complex with the channel pore protein, whose molecular nature was and is still not completely known. Studies of cyclophilin D knockout mice by two independent groups (Baines et al., 2005; Nakagawa et al., 2005) subsequently established that cyclophilin D, and by inference the mPTP, was involved in ischemic necrosis, because mitochondria isolated from the knockout mice were resistant to calcium-induced and CSA-inhibited permeability transition. Correlated with the resistance to permeability transition was the resistance to necrotic death induced by ROS and calcium overload and to cardiac ischemia/reperfusion injury (Nakagawa et al., 2005). Tissues from animals overexpressing cyclophilin D had abnormal swollen mitochondria and a propensity toward spontaneous cell death (Baines et al., 2005). Despite these findings, developmental apoptotic death appeared

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normal. The mice underwent normal development, raising suspicions that this type of death did not require cyclophilin-regulated inner membrane channel activity. The prevalent hypothesis is that the mPTP may contain both inner and outer membrane components, such as the ANT that normally transports ATP out of the inner membrane, VDAC, and cyclophilin D (Fig. 6.3). The role of the ANT remains unproven, because in hepatocyte mitochondria isolated from knockout animals lacking both liver-expressed isoforms of ANT, calcium-induced permeability transition still occurs and is nonresponsive to inhibitors of ANT such as bongkrekic acid (Kokoszka et al., 2004). Interestingly, when isolated brain mitochondria are tested for their responses to calcium and CSA, full substrates allow full permeability transition in response to introduction of high external calcium but restricted substrates (such as succinate only) produce a partial depolarization that is sensitive to CSA but not sensitive to another mPTP inhibitor, bongkrekic acid, suggesting that ANT is not involved in this type of transition. Maximum respiratory stimulation is restricted by the low-level use of the electron transport chain in the setting of the restricted substrates, which enter the chain distal to Complex I, so that maximum Hþ pumping cannot be attained (Brustovetsky and Dubinsky, 2000). A regulation of substrate use by mitochondria may protect mitochondria from maximum respiratory stimulation and reactive oxygen species (ROS) production at times of calcium overload. Nevertheless, ATP production will still be limited by the partial depolarization and could be insufficient in times of stress (Brustovetsky and Dubinsky, 2000). 6.3.5 Physiological Function of the Two-Membrane-Spanning Channel Complex: Regulation of Contact Point Formation It is tempting to speculate that VDAC forms a molecular complex with an inner membrane channel, so that metabolites and ions may enter and exit the matrix directly from the cytosol (Fig. 6.3). Coimmunoprecipitation studies support this hypothesis suggesting that VDAC, ANT, and cyclophilin D are biochemically linked (Crompton et al., 1998). VDAC clusters at contact sites where inner membrane MCC-like activity is present by patch recording (Brdiczka et al., 1986; Sandri et al., 1988; Moran et al., 1990). There are estimated to be about 37 contact sites per square micron of mitochondrial membrane, making it likely that there are 7 contact sites in a recording of a membrane patch of 0.5 . Because of the importance of communication between the matrix and the cytosol, it is possible that a functional mitochondrial channel could require the presence of both inner and outer membrane components at contact sites in series (Halestrap, 2005). This would provide for streamlined flow of ions and metabolites from the matrix bypassing the intermembrane space and exiting directly into the cytosol. If this were to be the only type of conduit, however, there would be no way for intermembrane space components to be taken up or released through a channel. For example, cytochrome c, which resides in the intermembrane space, could not be released to the cytosol in the absence of outer membrane rupture (Brustovetsky et al., 2002; Green and Kroemer, 2004) Opposing the idea that channels of the two

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membranes exist in a fixed complex is the finding that recordings of MCC in yeast mitoplasts (with intact contact points) derived from cells lacking VDAC have channel activity that is similar to that of MCC recorded in mitoplasts from wild-type cells (Lohret and Kinnally, 1995), and that normal developmental apoptosis (with cytochrome c release) occurs in cyclophilin D knockout animals despite the lack of normal inner membrane channel regulation (Baines et al., 2005; Nakagawa et al., 2005). Nevertheless, contact point formation may be transient. For example, metabolic functions of contact sites have been proposed. A contact between the membranes may occur depending on the need to release ATP, to permit the entry of ADP into the matrix, or to support the entry of calcium. Contact sites may form when oxidative phosphorylation is taking place (Knoll and Brdiczka, 1983). In keeping with this idea, ADP, atractyloside, and succinate, a substrate for mitochondrial respiration, appear to induce contacts, whereas glycerol and uncouplers such as DNP or the electron transport inhibitor antimycin A decrease their formation (Hackenbrock, 1972; Bucheler et al., 1991). Contact sites isolated by differential centrifugation are distinct in densities, containing only outer or only inner membranes and may contain all the channel and regulatory proteins in the complex spanning the two membranes (Ohlendieck et al., 1986). Bonanni et al. 2006 have observed a decrease in electron density of contact sites after ischemia, suggesting that channel regulatory proteins may disperse from the complex, perhaps as a result of damage to the electron transport chain during the ischemic event. One possible explanation for the multiconductance state of many mitochondrial channels could be the ability to increase conductance depending on an increasing interaction between the two membranes (Kinnally and Tedeschi, 1994). Finally, it is possible that VDAC may transiently interact with members of the BCL2 family and hexokinase, either to regulate metabolism or during cell death (Figs 6.3 and 6.6), and this interaction may enhance or decrease the efficiency of mitochondrial metabolism, suggesting that complex formation may be regulated by events occurring outside of the outer membrane.

6.4 TRANSLOCATOR OF THE INNER MEMBRANE (TIM) AND TRANSLOCATOR OF THE OUTER MEMBRANE (TOM) The protein translocators of the outer and inner mitochondrial membranes are waterfilled channels that provide pathways for mitochondrial proteins translated in the cytosol in the form of preproteins encoded by nuclear genes. Eight different tranlocator proteins of the outer membrane have been identified that participate in preprotein translocation, recognition of preproteins, and insertion of resident outer membrane proteins (Kunkele et al., 1998) and these eight proteins function as receptors and/or pore-forming subunits. Tom40 and Tom22 form the major constituents of the Tom complex (Kunkele et al., 1998). Tom40 is an integral membrane protein with mainly beta sheet structure that forms a cation-selective high conductance in artificial lipid bilayer recordings. It binds to mitochondrial targeting sequences added to the cis side of the membrane. The whole Tom complex forms a single-ring structure of 75 Awith a pore size of 16–20 A. Tom40 forms channels that are mostly open below 100 mV,

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with occasional, flickering closures and prominent subconductance states at negative potentials. The main conductance of Tom40 in 250 mM KCl is 360 pS, with a subconductance state of 150 pS and a cation over anion selectivity of 8:1. The current–voltage relationship shows voltage dependence with the greatest probability of opening near 0 and symmetrically increased tendency toward closure as voltage is increased on both sides of 0 mV. The channel is transiently blocked by mitochondrial preproteins and mitochondrial signal peptides, but not by related peptides lacking the mitochondrial signal (Hill et al., 1998). Upon direct comparison of the single-channel properties of Tom and Tim, both channels were found to demonstrate high conductance, voltage dependence, and slight cation selectivity. The channels were similarly affected by signal sequence peptides (Muro et al., 2003).

6.5 MITOCHONDRIAL CHANNEL CONDUCTANCE: EFFECTS ON SYNAPTIC STRENGTH Mitochondria are the predominant organelle within many presynaptic terminals. During frequent synaptic events, they affect intracellular calcium homeostasis and provide the energy needed for synaptic vesicle recycling and for the continued operation of membrane ion pumps. Recent discoveries have altered our ideas about the role of mitochondria in the synapse. Mitochondrial localization, morphology, and docking at synaptic sites may indeed alter the kinetics of transmitter release and calcium homeostasis in the presynaptic terminal. In addition, mitochondrial ion channel activity of BCL-xL alters synaptic transmitter release and the recycling of pools of synaptic vesicles. BCL-xL, therefore, not only affects the life and death of the cell soma, but its actions in the synapse may underlie the regulation of basic synaptic processes that subtend learning, memory, and synaptic development. Mitochondrial ion channels participate in the management of cytosolic calcium levels and in the release of ATP and are therefore potentially extremely important for the regulation of synaptic transmission (Blaustein et al., 1978). Different types of neuronal synapses contain different numbers of mitochondria with slightly different properties, depending on whether the main function of the mitochondria is to provide energy or buffer calcium. In most synapses, oxidative metabolism by mitochondria is crucial to successful neurotransmission (Nguyen et al., 1997). Moreover, mitochondrial bioenergetics are altered acutely in synapses that have undergone preconditioning, providing for enhanced oxidative competence (Nguyen et al., 1997), suggesting that an interaction may exist between neuronal plasticity and mitochondrial plasticity (Nguyen and Atwood, 1994). 6.5.1

Mitochondria Alter Calcium Homeostasis During Synaptic Events

Synaptic transmission depends on mitochondria not only for energy production, but also for maintaining calcium homeostasis within the presynaptic terminal (Kaftan et al., 2000; Nicholls and Budd, 2000; Atwood and Karunanithi, 2002; Jonas, 2006). During synaptic events, calcium influx through voltage-gated channels and the

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release of calcium from intracellular stores including mitochondria produce elevations of cytosolic calcium that enhance synaptic vesicle fusion (Stevens, 2003). In the crayfish neuromuscular junction, modulation of fast synaptic transmission is dependent on persistently elevated calcium levels inside the presynaptic terminal produced by mitochondrial calcium rerelease (Tang and Zucker, 1997) Different synapses may have different degrees of potentiation or depression of release of neurotransmitter, depending on their ability to buffer and make calcium available at certain times (Wang and Kaczmarek, 1998; Billups and Forsythe, 2002; Stevens, 2003). The proximity of sites of calcium influx to sites of vesicle fusion also contributes to enhancing the probability of fusion events (Sakaba and Neher, 2001; Schneggenburger et al., 2002). In some synapses, reaccumulation of vesicles to different pools that have been depleted may be differentially dependent on ATP (Jonas, 2006). Release of calcium from mitochondria participates in shaping the time course and amplitude of neurotransmitter release from presynaptic nerve endings after the invasion of the endings by action potentials. In the example of the crayfish neuromuscular junction, eliminating the ability of mitochondria to sequester calcium not only leads to a higher rise in cytosolic calcium inside the presynaptic terminal during a tetanus, but also to prevention of the normal potentiation of neurotransmitter release after the tetanus (Tang and Zucker, 1997). 6.5.2 Mitochondrial Presence at Presynaptic Sites Regulates Intense Synaptic Activity Mitochondrial ATP release also appears to play a role in the management of vesicle pool size both at rest and during synaptic stimulation. In Drosophila melanogaster, mitochondrial targeting is necessary for normal synaptic transmission at the neuromuscular junction (Stowers et al., 2002; Guo et al., 2005; Verstreken et al., 2005). Animals lacking mitochondrial targeting proteins such as Milton (Stowers et al., 2002), GTPase dMiro (Guo et al., 2005), or syntabulin (Cai et al., 2005) cannot target mitochondria normally to the presynaptic terminal and have abnormal synaptic transmission. Animals lacking normal mitochondrial division, which regulates the targeting of mitochondria to developing synapses, also have abnormal synapses (Li et al., 2004; Verstreken et al., 2005). 6.5.3 Mitochondrial ATP Production Regulates Normal Functioning of Synaptic Vesicle Pools Distinct pools of vesicles have different probabilities of release (Rizzoli and Betz, 2005). The readily releasable pool is defined as the vesicles that are immediately available for release, or “docked” at the active zone. In hippocampal synapses, for example, there appear to be approximately 5–10 vesicles that are docked at each active zone, but a single brief stimulus (such as an action potential) may release only one vesicle. The recycling pool is defined as the pool of vesicles that continue to be released and reaccumulate during moderate or physiological stimulation. This pool contains

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FIGURE 6.9 Regulation of mitochondrial ATP release enhances synaptic transmission, in part by regulating the refilling of depleted neurotransmitter-containing vesicle pools.

5–20% of all vesicles, but these estimates vary in different synapses. The reserve pool is defined as those vesicles that only release upon extremely frequent stimulation. The reserve pool of vesicles makes up about 80–90% of the vesicles in most terminals. The temperature-sensitive Drosophila shibire mutant (Kuromi and Kidokoro, 2002) demonstrates that the reserve pool of vesicles is normally mobilized only after the recycling pool is depleted. This mutant exhibits defective endocytosis at high temperatures, leading to an inability of vesicles to reaccumulate after exocytosis. In conditions of mild or moderate stimulation, which would not usually mobilize the reserve pool in controls, the reserve pool is mobilized at high temperatures in the mutant because it cannot refill its recycling pool. The experiments suggest that the reserve pool will be used only after the recycling pool has been depleted. The recycling pool, therefore, may contain vesicles that are privileged for release, either by their interaction with specific cytoskeletal elements, or their location, or both (Rizzoli and Betz, 2005). Surprisingly, however, the recycling pool is not located adjacent to the active zone. Rather, the vesicles of the recycling pool are distributed widely throughout the vesicle cluster (Rizzoli and Betz, 2004). ATP is required for certain steps in synaptic vesicle mobilization, release, and recycling. Specific ATP-dependent steps in synaptic transmission include refilling single vesicles with neurotransmitter (Takamori et al., 2000), membrane fission during endocytosis (Heidelberger, 2001), and coated pit formation (Smythe et al., 1989; Faundez and Kelly, 2000). Recent evidence suggests that ATP is required for normal functioning of vesicle pools (Kuromi and Kidokoro, 2002; Verstreken et al., 2005). Studies of the Drp1 mutation in Drosophila suggest that mobilizing the reserve pool requires ATP. These synapses do not contain mitochondria. An ATP-sensitive motor, the mysosin light chain kinase, which moves vesicles from pool to pool in an energydependent manner, is affected by the lack of locally released ATP brought on by the absence of mitochondria at synaptic endings. It is clear that mitochondria need to be targeted to the synapse for synaptic transmission to function normally during intense stimulation. How does the release of ATP from mitochondria increase at the time it is needed during intense stimulation?

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As seen in electron micrographs, the brainstem auditory synapse of the medial nucleus of the trapezoid body (MNTB) (the Calyx of Held), which is specialized to release neurotransmitter at extremely high frequency and fidelity, contains a mitochondrial adherens complex. The complex is a collection of filaments that tether mitochondria very closely to the synapse in a regulated fashion, orienting the matrix cristae perpendicular to the active zone (Rowland et al., 2000). It is likely that the organization of mitochondria within this specialized synapse enables the mitochondria to carry out precisely timed ATP release and calcium buffering. In hippocampal neurons, which have considerably different synaptic organization than that observed in the Calyx of Held, it appears that mitochondria are mostly untethered and that they can be either mobile or stationary. When hippocampal neurons are stimulated by local application of growth factors to points on the axon, mitochondria move preferentially to the stimulated site, presumably mimicking the in vivo situation where mitochondria might be targeted rapidly during growth or plasticity (Chada and Hollenbeck, 2003, 2004; Malaiyandi et al., 2005). 6.5.4

Mitochondrial Ion Channel Regulation During Synaptic Transmission

Specific targeting of mitochondria is thus required for normal synaptic transmission at high frequencies. The regulated targeting of mitochondria to sites of high energy demand suggests that the mechanisms of ATP production and release by mitochondria could very well be regulated during frequent synaptic events. Mitochondria are suggested to release ATP via VDAC, as described above. It is predicted that during synaptic events (such as synaptic plasticity), regulation of the opening of VDAC in the outer mitochondrial membrane could occur. Another prediction is that there is likely to be a second messenger that signals the opening of VDAC during synaptic events. The first evidence that mitochondrial ion channel activity could be regulated during synaptic events came from studies of mitochondrial membrane conductance during synaptic transmission in an intact presynaptic terminal of the squid stellate ganglion. Through the use of a double-barreled patch pipette (Jonas et al., 1997), recordings were made both at rest and during and after intense synaptic stimulation (Jonas et al., 1999). In control recordings within the resting squid presynaptic terminal, the conductance of mitochondrial membranes is low. In contrast, during frequent electrical stimulation of the squid presynaptic nerve, there occurs up to a 60-fold increase in activity and conductance of mitochondrial membrane patches within the presynaptic terminal (Jonas et al., 1999), a change that lasts for approximately 1 min after the stimulus. The delay and persistence of the mitochondrial membrane activity after stimulation implies that the mitochondrial outer membrane channel activity is not simultaneous with the opening of plasma membrane channels and suggests that the increase depends on an intracellular second messenger. Such a messenger could be calcium, which remains elevated in the squid terminal for approximately 1 min after stimulation, just as in the crayfish neuromuscular junction (Jonas, 2006). In keeping with these reports, in a calcium-deficient bathing medium, there is no change in mitochondrial conductance in response to stimulation of the presynaptic terminal, demonstrating that the evoked

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mitochondrial membrane channel activity is dependent on calcium influx into the terminal (Jonas et al., 1999). In addition, the uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), which depolarizes mitochondria, prevents the channel activity. The acute changes in mitochondrial membrane activity are correlated with synaptic plasticity, because FCCP application eliminates the short-term potentiation of the synapse that follows nerve stimulation. The findings imply that a calcium-dependent channel such as the mPTP might be involved in the change in conductance. The calcium-dependent site of that protein complex is in the matrix, however, and the recordings were performed on the outer mitochondrial membrane, suggesting that the activity could represent opening of an inner membrane channel whose activity might be linked to the opening of VDAC or another conductance in the outer membrane (Halestrap, 2005). A channel spanning two membranes could, therefore, permit the efflux of calcium (as well as ATP and other ions and metabolites) from the matrix into the cytosol during synaptic potentiation. In addition to mPTP components such as VDAC and ANT, BCL-xL and other BCL2 family members might also contribute to the regulation of channel activity by the protein complex during synaptic transmission. As we have seen, BCL-xL resides in the outer membranes of mitochondria (Kaufmann et al., 2003) and the recombinant protein induces mitochondrial ion channel activity (Jonas et al., 2003). BCL-xL could influence the release of calcium, ATP, or other metabolites into the cytosol during synaptic responses. In support of this hypothesis, injection of recombinant BCL-xL protein into the presynaptic terminal enhances the rate of rise of postsynaptic responses, resulting in an earlier latency for evoked action potentials in the postsynaptic cell as compared to the latency recorded in control presynaptic termini (Jonas et al., 2003). Interestingly, the injected BCL-xL protein produces potentiation of synaptic transmitter release in both healthy synapses and in those in which transmission had run down. Under these conditions, injection of BCL-xL protein into the terminal enhances the amplitude of the postsynaptic potential, restoring suprathreshold responses, in effect, giving the synapse a “new life.” If BCL-xL regulates the flux of metabolites across the outer mitochondrial membrane (Vander Heiden et al., 2001; Gottlieb et al., 2002), then this property predicts that the regulation of levels of ATP may enhance neurotransmission in the physiological setting. The evidence to support this hypothesis comes from the studies of the effect of ATP injection into the synapse on the degree of synaptic responses (Jonas et al., 2003; Verstreken et al., 2005). Direct microinjection of ATP into the synapse produces a similar degree and time course of enhancement of synaptic transmission as the effects of BCL-xL injection (Jonas et al., 2003) and, in fact, occludes the effects of injection of BCL-xL, suggesting that the two agents acted via the same mechanism. The findings imply that the conductance change could involve a calcium-dependent increase in the activity of ATP release regulated by BCL-xL. Although the injection of recombinant BCL-xL protein enhances transmission for a prolonged period, endogenous BCL-xL might participate in short-term responses of the mitochondrial membrane after high intensity stimulation. Recent findings suggest that this is indeed the case. The activities of BCL-xL can be disrupted by the application of ABT-737, a mimetic of the BH3-only protein BAD, that binds to BCL-xL with high

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affinity within a pocket of the three-dimensional structure that usually binds proapoptotic BH3-only proteins (Oltersdorf et al., 2005). The structure of ABT-737, a thioethylamino-2-4-dimethylphenyl analogue, was designed to bind to the threedimensional structure of BCL-xL/BCL-2. It displaces a GFP-tagged BH3-only protein from BCL-xL at mitochondrial surfaces in intact tumor cells. In cancer cell lines, ABT737 alone effectively induces cell death possibly via its ability to displace the prebound proapoptotic proteins Bax and Bak from BCL-xL (Oltersdorf et al., 2005). When applied to mitochondria within the squid presynaptic terminal just before synaptic transmission, ABT-737 inhibits the channel activity of mitochondrial membranes induced by synaptic stimulation, suggesting that BCL-xL is necessary for this activity. Recovery of vesicle pools after synaptic depression is also regulated by BCL-xL. Stimulation of the synapse at 2 Hz produces synaptic depression as the readily releasable pool is rapidly depleted (Swandulla et al., 1991). After the depletion, the more reluctantly releasable pools are accessed. During the recovery phase following the administration of a tetanus given against the background of continuous 2 Hz stimulation, the vesicles do not repopulate the readily releasable pool (because of the continued 2 H3 stimulation) but do repopulate the reluctant pools. The time course of the recovery of the reluctant pools is rapid (Sakaba and Neher, 2001) and is not affected by the previous injection of BCL-xL. If recovery from tetanic stimulation is measured during infrequent basal stimulation, full recovery of all pools occurs after a tetanus, as evidenced by the ability of the synapse to release as fully after the tetanus as it does during the control period at the beginning of the experiment. Nevertheless, the time course of the recovery of synaptic responses following the tetanus is slower than it is at 2 Hz, suggesting that when all the pools are repopulated, the most readily releasable––the first pool to be released at the onset of stimulation––repopulates quite slowly (Sakaba and Neher, 2001; Sakaba et al., 2005). The amount of recovery of this pool is significantly enhanced by BCL-xL injection when compared to recovery measured in controls. Thus, a slow component of the time course of recovery of the total vesicle pool is sensitive to the actions of BCLxL, and the pool that is affected may be the most readily releasable pool. BCL-xL appears to enhance the ability of this subset of neurotransmitter-containing vesicles to become available for release. If endogenous BCL-xL is necessary for recovery of synaptic vesicle pools, then ABT-737 might affect the rate of recovery from tetanic stimulation. In control squid synapses, recovery of neurotransmitter release after a tetanus generally occurs in less than 2 min. In contrast, in synapses exposed to ABT-737 just before tetanic stimulation, the rate at which the synapse recovers from high frequency firing is decreased (Hickman et al., 2008). As we have seen, the enhancement of synaptic responses by FL BCL-xL could be related to a difference in function of its ion channel compared to that of proapoptotic molecules such as Bax or DNBCL-xL. A key characteristic of the ion channel activity of BCL-xL is that it can induce ATP exchange across mitochondrial membranes (Vander Heiden et al., 2000, 2001). In particular, it performs this function in mitochondria from cells that have been exposed to apoptotic stimuli such as growth factor deprivation. In this pathological setting, BCL-xL may protect cells from death by

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maintaining ATP levels in the cell and by keeping VDAC in its open configuration. The delicate balance between the pro- and antiapoptotic BCL-2 family proteins may thereby regulate mitochondrial metabolism at times of stress and may control the onset of the eventual release of proapoptotic mitochondrial factors into the cytosol (Plas and Thompson, 2002). These factors and the compromise of mitochondrial function caused by the release of these factors may together cause the decline in synaptic responses. A surprising dichotomy of the effects of antiapoptotic molecules is that they may enhance the release of ATP from mitochondria (Gottlieb et al., 2002), but prevent the release of cytochrome c (Antonsson et al., 1997; Kluck et al., 1997). Binding of BCLxL and BCL-2 to proapoptotic molecules may inhibit channel activity of the proapoptotic molecules. Therefore, the channel activity of FL BCL-xL as well as its ability to alter the channel activities of prodeath molecules may comprise the antiapoptotic functions of FL BCL-xL. In summary, although large conductance mitochondrial membrane activity causes cell death and synaptic dysfunction, smaller conductance activity may be a prerequisite for a normal and healthy neuronal life.

ACKNOWLEDGMENTS This work was supported by NIH RO1 NS045876 and an American Heart Established Investigator Award (EAJ).

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7 REGULATION OF NEURONAL EXCITABILITY BY THE SODIUM-ACTIVATED POTASSIUM CHANNELS SLICK (SLO2.1) AND SLACK (SLO2.2) VALENTIN K. GRIBKOFF1,2 AND LEONARD K. KACZMAREK2 1

Discovery Research, Knopp Neurosciences Inc., 2100 Wharton Street, Suite 615, Pittsburgh, PA 15203, USA 2 Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA

7.1 INTRODUCTION Various chapters in this book describe neuronal voltage-gated ion channels that are regulated by intracellular messengers, including other ions. This chapter describes a subfamily of potassium (Kþ) channels, the Slo2 channels Slick and Slack, which encode the sodium (Naþ)-activated Kþ channels (KNa). As the name implies, these Kþ channels are regulated by the levels of intracellular Naþ ([Naþ]in) as well as by other ions and second messengers. Like Ca2þ, Naþ enters cells by multiple pathways, including specific voltage-gated ion channels. Unlike Ca2þ, which is an important second messenger in many cell types, Naþ currents have important roles principally in excitable cells, and therefore rapid changes or large, long-lasting changes in [Naþ]in are most prevalent in these cells. KNa channels are, therefore, not as widely distributed outside of the nervous system and muscle cells as Ca2þ-activated Kþ channels

Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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(including Slo1, the BKCa channel; see Chapters 12 and 15), although with the cloning of KNa channels, the presence of KNa subunits can be more precisely determined and they are being localized to additional areas. In neurons, Naþ plays a pivotal role in the intercellular communication, and its intracellular concentration varies significantly as a function of cellular activity. Neuronal KNa channels have evolved to respond to activity-dependent increases in intracellular Naþ, and can act as a brake on the longer term effects of rapid or prolonged action potential discharge resulting from intracellular Naþ accumulation. 7.2 SODIUM-ACTIVATED POTASSIUM CURRENT (KNa) 7.2.1

KNa in Cardiac Myocytes

The first recordings made from KNa channels were not made in neurons, but rather in isolated guinea pig ventricular myocytes (Kameyama et al., 1984). The channels were of large conductance (>200 pS in 150 mM Kþ outside/49 mM Kþ inside, among the largest conductance cardiac ion channels), inwardly rectifying near the Kþ reversal potential but outwardly rectifying at more depolarized potentials, and activated by intracellular Naþ in excess of 20 mM with an EC50 in excess of 60 mM. The channels were not affected by varying ATP levels or pH within a physiological range, nor were they activated by intracellular Ca2þ. Some of these original findings, such as the pH sensitivity of the channels, have been challenged (Veldkamp et al., 1994), and such discrepancies may reflect the molecular makeup of different KNa channels. Importantly, the high threshold of activation of these channels by intracellular Naþ, in cells where the “normal” concentration is no greater than 10 mM, suggested that the current would primarily be activated under conditions in which Naþ extrusion from these myocytes was compromised. This could be due to failure of the Naþ-Kþ pump, a condition that could also result in deregulation of intracellular Ca2þ concentration when Naþ accumulated to the point of failure of the Naþ–Ca2þ exchange mechanism. A condition in which these criteria for Naþ-dependent opening of the channel would be met is cardiac hypoxia (Kameyama et al., 1984; Bertrand et al., 1989; Dryer, 1994; Veldkamp et al., 1994). In ventricular myocytes, this apparent low sensitivity of KNa to intracellular Naþ has been observed consistently, with activation occurring only at Naþ concentrations greater than 20–30 mM such as during block of the Naþ–Kþ-pump (Luk and Carmeliet, 1990; Rodrigo and Chapman, 1990; Niu and Meech, 2000). While some activation of KNa has been reported following physiological activation during which intracellular [Naþ]in is increased only by about 30% above the normal concentration of approximately 7 mM (Rodrigo, 1993), overall these data support the contention that the primary activation of this current in myocytes would occur during pathological conditions. In neurons, however, the sensitivity of KNa to intracellular Naþ may be higher and physiological increases in [Naþ]in are often much larger than in the myocytes. An early and consistent finding with native KNa channels from ventricular cells has been the existence of multiple, regularly spaced subconductance states (Sanguinetti, 1990; Wang et al., 1991; Mistry et al., 1996). Subconductance states, representing

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the fractional opening of a channel, can have a number of potential underlying mechanisms, although most evidence suggests that the structures of voltage-gated ion channels provide opportunities for multiple partial gating conformations (Fox, 1987; Dani and Fox, 1991; Bezanilla, 2005; Chapman and VanDongen, 2005). Subconductance states are observed in many ion channels, including ligandgated ion channels, particularly in the presence of compounds or experimental conditions that result in partial channel block or incomplete activation, or at membrane potentials where the probability of channel opening is small (Moss and Moczydlowski, 1996; Bezanilla, 2005; Chapman and VanDongen, 2005). Wang et al (1991) examined the conductance of KNa channels in ventricular myocytes and using variance-mean analysis (Patlak, 1993), found at least 10 major conductance sublevels. Interestingly, they found that these channels displayed inward rectification with outward current reduced by increasing intracellular Mg2þ, Naþ, or Ba2þ, but the appearance of subconductance states was apparently unaffected by the direction of the current (inward versus outward) or the presence of outward current inhibition by Mg2þ, Naþ, or Ba2þ. Subsequent analysis has shown that ventricular KNa channels may have 12 subconductance levels during longer, stable recordings, divided into transient and stable subconductances (Mistry et al., 1996). There were two stable levels, which were about 30 and 70% of the conductance of the fully open channel and which occurred spontaneously and for only relatively brief periods during prolonged recordings. The kinetics of the two observed stable subconductance levels, their open and closed times under identical conditions, were approximately equal to those of full conductance transitions when they were present (in other words, they look pretty much like smaller versions of the full open state when they occur, while the transient levels have very different kinetics with only brief open times). KNa channels in ventricular myocytes, therefore, activate in a highly complex manner, with at least 10 transient subconductance levels that likely contribute little in terms of physiological effect and at least 2 stable subconductance levels that occur spontaneously but that thus far largely defy experimental induction. These data strongly suggest that there are multiple stable channel configurations of native KNa channels. Additional characteristics of peripheral KNa channels have been described. These include pH sensitivity (block by Hþ, as mentioned above) (Veldkamp et al., 1994) and inhibition by Kþ (Niu and Meech, 2000). KNa currents and channels were recorded from neurons soon after their discovery in myocytes (Bertrand et al., 1989; Haimann and Bader, 1989; Martin and Dryer, 1989; Haimann et al., 1990; Kubota and Saito, 1991). In the following section we will briefly describe neuronal KNa channels and their functions, the identification of the genes encoding KNa channels, and the beginning of the pharmacological characterization of these channels. 7.2.2

KNa and the Regulation of Neuronal Excitability

Neurons successfully fulfill their numerous roles in sensation, information processing, secretion, and motor control in part because of their ability to change their response repertoire as a function of prior input and activity. Many neuronal constituents contribute to neuronal plasticity (used here in this broadest sense), including a number

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of ion channels. Both Ca2þ and Naþ are charge carriers and hence important mediators of neuronal function, each with their own set of ion channels that specifically control the flux of these cations across the plasma membrane (see Chapters 1 – 3). Ca2þ also serves as an important second messenger in many cells, and its intracellular concentration is tightly regulated. Since changes in the intracellular concentrations of both cations can have significant neuronal consequences, one or more corresponding Kþ channels have evolved to indirectly regulate their intracellular concentration and mitigate the effects that increases in their intracellular concentrations can have on neuronal excitability. There are two subfamilies of Kþ channels, the largeconductance Ca2þ-activated Kþ channels (BKCa, maxi-K, KCa1.1) encoded by the Slo1 gene (Butler et al., 1993; Dworetzky et al., 1996; Salkoff et al., 2006) and the small-and intermediate-conductance Ca2þ-activated Kþ channels KCa2.1–2.3 and KCa3.1 (Kohler et al., 1996; Ishii et al., 1997; Joiner et al., 1997; Bond et al., 1999) that respond to changes in [Ca2þ]in. Similarly, KNa channels have evolved in neurons to regulate excitability changes that result from intracellular Naþ accumulation (molecular identity to be discussed below). Importantly, unlike in cardiac myocytes, there is increasing evidence that KNa plays an important role in the normal regulation of neuronal activity, particularly in neurons where high-frequency action potential discharge activity is an important physiological feature. This has suggested that activation of KNa in neurons differs from that in myocytes in several possible ways, each of which have been or are currently being approached experimentally. For example, neuronal KNa channels may not require the high levels of [Naþ]in required by KNa in cardiac myocytes (where [Naþ]in only reaches activating levels during pathological conditions such as ischemia), or the levels of [Naþ]in in neurons may reach activating levels (>10 mM) during normal neuronal activity, at least in critical neuronal microenvironments. Both of these possibilities may be true at different times; KNa channels may be modulated by intracellular messengers, which may affect responsiveness to [Naþ]in, and quite high levels of [Naþ]in can be reached in select loci following prolonged neuronal activity, particularly after high-frequency discharge. In particular, the membrane hyperpolarizations following single and multiple action potentials contribute to the determination of action potential frequency and the adaptation of frequency (the reduction in firing observed during prolonged action potential trains) that are critical to the function of many neurons (Gean and ShinnickGallagher, 1989; Faber and Sah, 2005; Gu et al., 2007; Khawaja et al., 2007). These afterhyperpolarizations (AHPs) generally have multiple components. They vary significantly between different neurons in terms of underlying ionic mechanism, duration, and influence on cellular function, but Kþ currents initiated by action potentials or their sequelae (voltage changes or increased [Ca2þ]in or [Naþ]in) are wellcharacterized mediators of these events (Kim and McCormick, 1998; Wikstrom and El, 1998; Nedergaard, 2004; Pedarzani et al., 2005; Wallen et al., 2007). KNa has been shown to be an important contributor to AHPs, with increasing influence during prolonged and high-frequency neuronal activity. Finally, KNa may also serve a role during neuronal ischemia that is similar to that postulated for KNa in cardiac cells. Native KNa channels have been recorded from neurons in many areas in the central nervous system (CNS) and in the periphery. In the vertebrate CNS, KNa channels have

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been recorded from neocortical neurons, sensory neurons, hippocampal neurons, neurons within the auditory nuclei, spinal cord, and the olfactory system, among others (Bertrand et al., 1989; Haimann and Bader, 1989; Haimann et al., 1990, 1992; Egan et al., 1992a, 1992b; Dryer, 1994; Safronov and Vogel, 1996; Franceschetti et al., 2003; Liu and Stan Leung, 2004; Hess et al., 2007; Yang et al., 2007). In the olfactory bulb, KNa channels may be one of the most prominent and frequently recorded Kþ channels (Egan et al., 1992b). KNa channels represent one of the major mechanisms for responding to neuronal activity, particularly to prolonged neuronal activity at high frequencies, and the effect of activation of these channels is to produce a hyperpolarization and a decrease in membrane resistance. By the late 1980s/early 1990s, KNa channels exhibiting all of the hallmark characteristics of cardiac KNa channels, such as sensitivity to high [Naþ]in, inward rectification due to Naþ at highly positive membrane potentials, channel rundown in excised patches, and resistance to Liþ substitution for Naþ, had been recorded from central neurons. In several CNS areas, the role of KNa has been particularly well documented. As examples of this, we will briefly review some of the data concerning KNa functions in neurons of the neocortex and hippocampus and in neurons in the auditory system (specifically the medial nucleus of the trapezoid body (MNTB)). The purpose is not to exhaustively review this literature, but to introduce the function of KNa in neurons. A number of studies have examined the roles of these channels in regulating the excitability of neocortical and hippocampal neurons. In cat sensorimotor cortex, early studies demonstrated the existence and importance of KNa in regulating the responsiveness of large layer V neurons. When Ca2þ influx was blocked to remove the stimulus for Ca2þ-activated Kþ currents, a slow outward current resulted either from evoking prolonged discharges or by injecting subthreshold depolarizing current sufficient to activate persistent Naþ current (Schwindt et al., 1989). The outward current was blocked or greatly reduced in amplitude when persistent Naþ current was blocked using tetrodotoxin, and it produced spike frequency adaptation that could be reduced by certain G-protein-coupled receptors, such as norepinephrine (Schwindt et al., 1989). In visual cortical neurons, exposure to high-contrast visual stimuli results in neuronal adaptation. This is defined as a progressive loss of neuronal responsiveness to the continued presence of the stimulus, a phenomenon that is mediated at the cellular level (and revealed in intracellular current clamp recordings) by a long-lasting membrane hyperpolarization that is reflected in the degree of firing rate reduction in these cells (Sanchez-Vives et al., 2000a, 2000b). As mentioned above, similar spike frequency adaptation has been recorded in many types of neurons and it can have several underlying mechanisms. Sanchez-Vives and colleagues have shown, using ferret primary visual cortical slices in vitro, that prolonged depolarizing current injections or prolonged sinusoidal currents injected at a frequency of 2 Hz produced variable slow spike frequency adaptation over many seconds in these neurons. The discharge was followed by a prolonged AHP that lasted many seconds and was mediated by one or more Kþ currents. While the block of voltage-gated Ca2þ currents reduced the rate of spike frequency adaptation, the AHP was not affected. The reduction in [Naþ]out, the ultimate source of [Naþ]in during prolonged neuronal activity, however, further reduced the remaining spike frequency adaptation and also

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significantly reduced the AHP (Sanchez-Vives et al., 2000a). KNa currents therefore may be important, along with KCa currents, in tuning the responses of visual cortical neurons to persistent stimuli and play a critical role in such phenomena as contrast adaptation. A role for KNa can also be demonstrated for fast-spiking neurons of the visual cortex, cells that are capable of discharging at rates up to 1 kHz without adaptation and without significant changes in spike amplitude. In these neurons, the action potential duration is typically brief, the Naþ channels recover from inactivation rapidly, and the action potentials are short because these cells express voltagedependent Kþ currents (KV3 family) that activate rapidly and produce transient current. Together these factors allow the generation of high-frequency trains of action potentials. In these cells, brief periods of stimulation do not result in spike frequency adaptation, but a recent study demonstrated that longer trains of high-frequency action potentials did induce a slow AHP of many seconds duration, an effect that reduced the cells’ responses to afferents for much of this period (Descalzo et al., 2005). This slow AHP had all the characteristics of a KNa-mediated membrane hyperpolarization, although KNa channels were not definitively identified as underlying the AHP. The importance of KNa in neuronal regulation in cortical and hippocampal neurons can also be demonstrated in intrinsically bursting neurons. As opposed to neurons that discharge primarily or exclusively in response to supra-threshold extrinsic stimuli, the intrinsically bursting neurons in cortex and hippocampus have conductances that initiate and terminate bursts of action potentials in the absence of synaptic influences (Franceschetti et al., 1993; Istvan and Zarzecki, 1994; Metherate and Aramakis, 1999; Sipila et al., 2006). Identifying and understanding these properties has clear relevance to cortical function, as well as dysfunction, as exemplified by their involvement in epileptiform activity (Traub and Wong, 1982; Silva-Barrat et al., 1992; Sanabria et al., 2001). In sensorimotor cortical neurons in vitro, intrinsic bursts are followed by AHPs that are key determinants of burst length and rhythmic discharge. In these cells, partial substitution of Naþ with Liþ had little effect on action potentials (since voltagedependent Naþ channels will conduct Liþ current), but significantly increased burst duration and reduced AHP amplitude, an effect that was also observed on the Kþ conductance evoked following the Naþ entry through Naþ channels (Franceschetti et al., 2003). The effect of Liþ substitution was not observed when voltage-dependent Naþ channels were blocked with tetrodotoxin. Thus, KNa currents, which are reduced on substitution of Naþ by Liþ, appear to play a significant role in AHP generation and in the burst rhythmicity in neocortical neurons. The influence of KNa is not limited to reducing excitability during and following the prolonged trains of action potentials or bursts. In hippocampal pyramidal neurons, the depolarizing afterpotentials (DAPs) are important regulators of neuronal excitability and can be produced by single-antidromic or current-evoked action potentials. The replacement of [Naþ]out by Liþ enhanced DAP amplitudes, produced spike bursts where single-action potentials had been evoked, and caused a positive shift in the reversal potential of the DAP (Liu and Stan Leung, 2004). These data suggest that even after a single-action potential, KNa can influence the subsequent excitability of central neurons. In the auditory system, the binaural pathway in the brainstem permits the localization of the source of sounds by comparing the time and intensity differences

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of sound stimuli received at the two cochlea. To accomplish this with high fidelity, the neurons in various nuclei, such as the neurons of the anterior ventral cochlear nucleus and their targets among the principal neurons of the MNTB, are capable of maintaining action potential discharge rates faithfully locked to the phase of incoming synaptic stimuli at action potential frequencies up to 600 Hz (Wu and Kelly, 1993; Taschenberger and von Gersdorff, 2000). A number of Kþ channels have been shown to have specific functions in the auditory system that allow this degree of fidelity, particularly in the MNTB. These include KV1 channels, which contribute to a rapidly inactivating low-threshold Kþ current that suppresses the generation of multiple action potentials in response to synaptic currents (Brew and Forsythe, 1995; Brew et al., 2003), and KV3 channels, which contribute to a rapidly activating higher threshold Kþ current that decreases the duration of action potentials and allows high-frequency neuronal discharge locked to synaptic inputs (Wang et al., 1998; Macica et al., 2003; Song et al., 2005). Although the greatest evidence supports roles for KV1.1 and KV3.1 as mediators of these currents, there are likely contributions from other members of these voltage-gated Kþ channel subfamilies. A tonotopic gradient in Kþ currents has also been demonstrated for neurons of the MNTB, with medial (high-frequency responding) neurons demonstrating greater total KV1- and KV3-mediated Kþ currents than neurons in the lateral part of the nucleus (lower-frequency responding cells) (Li et al., 2001; von Hehn et al., 2004; Brew and Forsythe, 2005). There is evidence for additional Kþ conductance with different tonotopic variation as well, and recent evidence suggests that KNa channels play an important role in the fidelity of neuronal action potential timing at high frequencies. In fact, a major component of Kþ current in these cells is sensitive to cytoplasmic levels of Naþ (and Cl, see below), and increasing intracellular Naþ, as well as pharmacological manipulation directed to enhancement of KNa (see below), results in an increase in the accuracy of MNTB neuronal action potential timing (Yang et al., 2007).

7.3 CLONING AND IDENTIFICATION OF GENES ENCODING KNa CHANNELS: SLACK AND SLICK 7.3.1

The Slack Channel

Although recording from native KNa channels provided evidence of the importance of these channels in neuronal regulation, identification of the molecular substrate(s) of KNa was both elusive and important, since heterologous expression would allow for the more rapid and accurate discovery of pharmacological tools. In fact, KNa was one of the last remaining physiologically identified but molecularly uncharacterized macroscopic Kþ currents. In the late 1990s, near the end of the era of rapid cloning of the Kþ channel genome by “blast” homology searching of public DNA databases, a sequence designated as Slack (Slack is an acronym for sequence like a calcium-activated Kþ channel) was cloned from a rat cDNA library (Joiner et al., 1998). A related squence (f08b12.3) is present in the genome of Caenorhabditis elegans and this was later designated as C. elegans gene Slo2 (Yuan et al., 2000). The Slack sequence has regions

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FIGURE 7.1 The proposed structures of single Slack (a) and Slick (b) a subunits; explanations for each of the structural components are found in text. KNa channels are formed by four a subunits; currently it is unknown if a KNa channel tetramer can be formed by both Slack and Slick subunits, forming KNa channels with mixed properties.

of homology with Slo1, the gene encoding the BKCa channel protein (Butler et al., 1993; Dworetzky et al., 1994; McCobb et al., 1995; Gribkoff et al., 2001a). The overall Slack sequence corresponds to a Kþ channel a subunit, with a predicted full-length protein sequence of 1237 amino acids. Like most voltage-gated Kþ channels, it had a predicted structure consisting of six transmembrane domains and a putative signature GYG pore sequence (Fig. 7.1a). Unlike the Slo1/BKCa a subunit, it does not have the S0 transmembrane domain, which characterizes the unique structure of the largeconductance Ca2þ-activated Kþ channel and is the site for interaction with a number of BKCa b subunits (see Chapter 12 in this volume). Like the BKCa channel, it has a long C-terminal region. It also has a consensus PDZ-domain sequence in this long tail, suggesting it is capable of undergoing clustering. The protein has many sites for phosphorylation and, as will be described later, its activity is modulated by protein kinases (Santi et al., 2006). Interestingly, although in other respects it resembles a voltage-gated Kþ channel, it does not have the consensus voltage sensor sequence in S4 consisting of alternating positively charged residues. While Slack has little homology with Slo1 from the N-terminus to the pore, there are discrete regions of homology in the pore region and in parts of the long C-terminal tail. Overall, the homology of the Slack channel to Slo2/f08b12.3, Slo1 or the Slo3 channel of the testis is, however, only 7%. Despite this very low degree of homology, the discrete regions of homology are in critical regions, and the channel has been considered a member of the Slo gene family. Western blot analysis indicated that Slack is found in brain as 4.5 and 7.5 Kb transcripts and is widely distributed in brain. Specifically, in situ hybridization revealed very significant signal in the cortex, hippocampus, deep cerebellar nucleus, cerebellar Purkinje neurons, pons, preoptic nucleus, substantia nigra, and in a number of brainstem nuclei, including auditory nuclei such as the ventral cochlear nuclei and the MNTB. In a subsequent study, Bhattacharjee et al. (2005) used an affinity-purified antibody against the N-terminal region of rat Slack to evaluate the distribution of Slack in more detail. It is now known that this N-terminus is specific to one splice isoform of Slack, now termed Slack-B (Brown et al., 2006). They found that the

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FIGURE 7.2 Slack-B immunoreactivity in central neurons. (a). Prominent labeling is the substantia nigra (SNR) found in the subthalamic nuclei (STh). In the substantia nigra, staining is localized to neuronal cell bodies and axonal projections (b). Scale bars, 200 mm in (a), 100 mm in (b). (c) Slack-B immunolocalization in neurons of the frontal cerebral cortex (Bhattacharjee et al., 2002).

greatest expression, as reflected in the intensity of Slack-B immunoreactivity, was in the somata and axonal processes of neurons in the component nuclei of the brainstem trigeminal system and reticular formation. Intense staining was also observed in the vestibular nuclei, oculomotor nuclei, giant presynaptic terminals of the medial nucleus of the trapezoid body, deep cerebellar nuclei, substantia nigra, amygdala, and thalamus (Fig. 7.2a and b). In the cortex, significant staining was observed only in the frontal cortex (Fig. 7.2c). Slack-B protein is therefore widespread, but not ubiquitous. Expression of Slack channels in Xenopus oocytes and transiently transfected Chinese hamster ovary (CHO) cells produced an outwardly rectifying current under voltage–clamp that activated in both expression systems at potentials positive to 60 mV and had both instantaneous and slower activating components (Fig. 7.3) (Joiner et al., 1998). The single-channel conductance, recorded from inside-out

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FIGURE 7.3 Characteristics of Slack-B and Slick channels at the macroscopic and singlechannel levels. The Whole-cell currents in Slack-B or Slick-transfected CHO cells were recorded in response to 200 ms pulses to potentials between 120 and þ120 mV. Excised inside-out single-channel records are shown at a membrane potential of þ80 mV.

membrane patches bathed in symmetrical Kþ solution, was 40–65 pS with smaller subconductance states. Increased intracellular Ca2þ produced a significant inhibition of Slack channel activity recorded in both expression systems. In this initial study, the regions of sequence similarity with the Slo1 channel suggested that the Slack a subunit might form heteromultimers with Slo1 a subunits. To test this hypothesis, mRNA for both Slack and Slo1 was coinjected into Xenopus oocytes and the whole-cell currents and single-channel currents from inside-out membrane patches were examined. Following the coinjection, the pharmacology, kinetics, and single-channel conductance of Slack homomeric channels, Slo1 homomeric channels, and putative Slack/Slo1 heteromultimeric channels were analyzed. Currents from coinjected oocytes had kinetics and activation voltage ranges that were intermediate between Slack and Slo1. In addition, Slack channels were insensitive to the BKCa channel opener NS1619 and the BKCa channel blocker iberiotoxin, whereas the Slo1 channels were sensitive to both pharmacological agents. The currents resulting from coinjection of Slack/Slo1 were equally sensitive to NS1619, but at steady state were largely insensitive to iberiotoxin. These results were consistent with the formation of Slack/Slo1 heteromultimeric channels and inconsistent with the two independent populations of Slack and Slo1 in the coinjected oocytes. In the inside-out membrane patches, intermediate-conductance channels, intermediate between the single-channel conductance of either Slack (under these conditions in oocytes 25–65 pS) or Slo1 (280 pS), were recorded with conductances between 80 and 190 pS. The channels in patches from coinjected oocytes were also activated by 1 mM Ca2þ applied to the intracellular face, similar to Slo1 channels but unlike Slack channels (which are inhibited by Ca2þ). This heteromultimerization phenomenon has also been observed with Slo2 channels from C. elegans, which are similar to Slack but like Slo1 channels are activated by Ca2þ and may form hybrid channels with Slo1 (Yuan et al., 2000, 2003). It therefore appears that the a subunits of these two channels may form channels with interesting mixed properties, both in their biophysical characteristics and pharmacology. Initially, it was thought that these results suggested that Slack may provide a substrate for the known diversity in native BKCa channels, but it is unknown whether this ever actually occurs in nature. Subsequent studies have provided

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evidence for another family member (Slick), a new alternative Slack sequence and an entirely different function for these channels as the substrate for KNa. 7.3.2

Identification of Slack as a Naþ-Activated Kþ Channel

In the initial studies following the cloning and expression of Slack Kþ channels, it was discovered that they were inhibited by [Ca2þ]in, but their sensitivity to [Naþ]in was not tested. The C. elegans ortholog, however, was found to be activated by both [Ca2þ]in and [Cl]in (Yuan et al., 2000). In 2003, a comprehensive study of both Slack and its C. elegans ortholog, Slo2, reported that Slack was activated by [Naþ]in and in the presence of elevated cytosolic Naþ, the currents closely resembled those of KNa (Yuan et al., 2003). In particular, the single-channel conductances were very similar to native KNa channels, varying significantly with different [Kþ]out and [Kþ]in. Although under the original recording conditions of Joiner et al. 1998, the rat Slack channels were reported to have single-channel conductance less that 100 pS, in this study the single-channel conductance in symmetrical 160 mM [Kþ]out/[Kþ]in was found to be 165 pS, similar to the conductance levels reported for native KNa channels (Yuan et al., 2003). The single-channel current amplitude also underwent considerable outward rectification at positive voltages. The Slack channels also exhibited multiple and prominent subconductance states, similar to native KNa channels. The whole-cell currents from Xenopus oocytes and the multichannel currents recorded from macropatches under physiological conditions were large outward currents (voltage steps between 80 and þ40 mV) with reversal potential near EK (78 mV in macropatches). The whole-cell currents did not demonstrate block at very positive voltages and had a slower activating component than was observed in macropatches. The C. elegans ortholog Slo2 was not activated by Naþ but, as reported earlier, was activated by Ca2þ. Both of these channels were also activated by [Cl]in. Although this had not been previously described for KNa channels, subsequent analyses have found Cl sensitivity of KNa channels in kidney and in MNTB neurons (Paulais et al., 2006; Yang et al., 2007). In the case of the C. elegans channel, the dependence on both [Ca2þ]in and [Cl]in is absolute, with no significant currents observed in the absence of either ion. In mammalian Slack, the dependence on [Cl]in is not absolute, and while it enhances channel activation, some degree of channel activation can be observed in Naþ alone. The effect of the two ions in Slack is cooperative or synergistic, in that the activation in the presence of both ions is greater than the sum of the activation by either ion alone. Channels in the Slo family, exemplified by the Slo1 BKCa channel, are known to possess regions in the long carboxy-terminal region that may underlie their sensitivity to ions. In the case of Slo1 BKCa channels, these consist of two RCK (regulator of Kþ conductance) domains, regions that have been previously shown in bacterial Ca2þ-activated Kþ channels to form a ring structure that responds to Ca2þ by regulating the opening and closing of the channel (Jiang et al., 2002; Parfenova et al., 2006), and a “Ca2þ bowl” believed to be the major site for the binding of Ca2þ (Schreiber and Salkoff, 1997). Slack channels also have two RCK domains (Fig. 7.1a and b) and a region homologous to the Ca2þ bowl; this latter region is highly

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homologous between Slo2 orthologs (Yuan et al., 2000, 2003). Ion sensing in this “bowl” region in Slo1 BKCa channels is accomplished by a series of negatively charged residues thought to coordinate Ca2þ, whereas in Slo2 channels, these residues are replaced by positively charged amino acids in comparable positions (Yuan et al., 2003). While these positive charges may help coordinate Cl, however, mutation of every charged residue of the bowl region of Slack to the neutral amino acid glutamine produces no change in the affinity of the channels to Cl or Naþ, indicating that this region is unlikely to control channel gating by these ions (Bhattacharjee et al., 2003). Slack has been shown to exist in at least two splice isoforms, which differ in their N-terminal domains (Brown et al., 2006). These two alternative splice variants are now known as Slack-B (the original cloned isoform) and Slack-A. As illustrated in Fig. 7.1a, the Slack-B has a longer N-terminus and results in slowly activating currents as described above, whereas the Slack-A has a shorter N-terminus and activates more rapidly, similar to Slick (see below). 7.3.3

The Slick Channel

In 2003, a second gene encoding a KNa channel was identified (Bhattacharjee et al., 2003). This channel now termed Slick (sequence like an intermediate-conductance Kþ channel) was found to be a rapidly gating Naþ- and Cl-sensitive Kþ channel with significant structural homology with Slack. Like Slack, Slick (Slo2.1) has six transmembrane domains and no apparent voltage-sensor region in S4, although, like Slack, the currents are voltage dependent. It also has a large C-terminal region with a highly conserved PDZ domain. Like Slack, Slick also has two RCK domains, and the overall homology of the channels is approximately 70% (Fig. 7.1b). The two channels (Slack-B versus Slick), however, differ significantly in several aspects, including faster activation kinetics for Slick channels than Slack-B (Fig. 7.3). Slack-B has an instantaneous component, but the majority of current is slower activating. However, as mentioned above, an alternative Slack transcript called Slack-A has now been described. Like Slick, Slack-A is more rapidly activating, and has a high degree of structural homology in its alternative N-terminal sequence with Slick’s N-terminal sequence. Unlike both Slack transcripts, Slick also has a higher sensitivity to [Cl]in, has some ability to conduct in the absence of high [Naþ]in, and, most important, has a consensus ATP binding motif in its C-terminal region. Slick is in fact an ATP-regulated channel. In the presence of 5 mM of MgATP in the recording pipettes, Slick current densities were reduced by as much as 80% at all tested voltages (Bhattacharjee et al., 2003). The effect of ATP was reproduced by slow or nonhydrolyzable analogues of ATP, and the effect was markedly reduced in the presence of ADP and abolished by mutation of the ATP binding motif. The inhibition of Slick currents by ATP was not significantly affected by the KATP channel inhibitor glybenclamide or the KATP channel opener diazoxide, suggesting that Slick represents a new type of ATP-mediated Kþ channel regulation. The distribution of Slick has been examined in the rat CNS using in situ and immunocytochemical techniques (Bhattacharjee et al., 2005). Very strong

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hybridization signals and immunoreactivity were observed in brainstem nuclei, including the MNTB, and the signal was seen in most of the areas where Slack is seen. These include the olfactory bulb, red nucleus, thalamus, facial nucleus, vestibular nucleus, pontine nucleus, oculomotor nucleus, deep cerebellar nuclei, and the substantia nigra. Most interesting, however, was the observation that Slick mRNA and immunoreactivity were found in some areas that do not express significant levels of Slack-B. These include hippocampal neurons in areas CA1, CA2, and CA3, the dentate gyrus, the supraoptic nucleus of the hypothalamus as well as other hypothalamic loci, and cortical neurons of layers II, III, and IV (Bhattacharjee et al., 2005). It is now known that Slack-B and Slick channels can form heteromultimers both in expression systems and in native neurons (Chen et al., 2006, 2007). It also appears, however, that Slick can operate independently as a KNa channel, given its solo expression in some structures. This pattern of overlapping and nonoverlapping distributions of expression of Slack and Slick can explain some or all of the observed heterogeneity in the KNa channels and currents in the literature, particularly when the alternative Slack-A transcript and possible Slack/Slick, heteromultimeric channels are factored in.

7.4 MODULATION OF KNa CHANNELS BY SECOND MESSENGERS Slack and Slick are differentially regulated by the activation of protein kinase C (Santi et al., 2006). The whole-cell Slack currents are increased by exposure of cells to the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl-2acetyl-sn-glycerol (OAG). In addition, the activation of protein kianse C slows the rate of activation of Slack currents. In contrast, the Slick currents are inhibited by the activation of protein kinase C. Each of these effects is prevented by inhibitors of protein kinase C. Similar modulation of Slack and Slick currents is observed in response to activation of Gaq-protein-coupled receptors (GqPCRs) that are coupled to activation of protein kinase C (Santi et al., 2006). When GqPCRs such as the M1 muscarinic cholinergic receptor or the mGluR1 metabotropic glutamate receptor are coexpressed with the KNa channel subunits in Xenopus oocytes, the application of receptor agonist strongly enhances Slack currents and inhibits Slick currents.

7.5 PHARMACOLOGY OF KNa CHANNELS The KNa channels are important regulators of neuronal excitability, and pharmacological agents that affect their activation can have great value as research tools and as potential therapeutic agents. As research tools, inhibitors can be used to pharmacologically dissect KNa currents from specific neurons, neuronal populations, or the whole animal, helping to determine the function of these channels in neuronal and behavioral regulation. Openers or activators of these channels can likewise be used to better understand the range and limits of KNa contributions to neuronal behavior. In the

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search for therapeutic agents that can modulate neuronal excitability (or cardiac excitability), including the response to insults such as ischemia, a well-developed pharmacology is a critical first step in the development of effective and specific drugs directed to this target. Inhibitors of specific Kþ channels have been discovered from a variety of sources over the last several decades, with specific peptide toxins providing the greatest potency and specificity. In the case of activators, which often have the most obvious therapeutic potential (by enhancing Kþ channel-mediated damping of cellular excitability), there was, however, only a single class of Kþ channel (KATP channels) for which activators were known prior to approximately 1994. We now know that many other Kþ channels can be activated (their open probability increased) by small molecules. These include BKCa/Slo1 channels and neuronal KV7 (KCNQ) channels (McKay et al., 1994; Gribkoff et al., 1996, 2001b; Cooper and Jan, 2003; Gribkoff, 2008), and new reviews of the pharmacology of both of these Kþ channel subfamilies are found in Chapters 15 and 16 of this volume. Importantly, it has been observed that openers of these different channels often (but not always) have effects on other Kþ channels. Whether this represents some common mechanism(s) of modulation, for which differently configured “receptors” exist on different channel types, is currently unknown, but this observation provides an avenue for the initial discovery of compounds affecting additional Kþ channels without the need for large-scale screening campaigns. Using small libraries of compounds enriched for Kþ channel modulators, it can prove relatively simple to discover effective initial lead compounds, when coupled with cloned channels in an appropriate expression system. Prior to the cloning of Slack and Slick channels, it had proven very difficult to discover and characterize pharmacological agents that modulate KNa channels. Using native KNa channels and currents, only two agents had previously been described to affect KNa, both producing KNa inhibition. In ventricular myocytes, the antarrhythmic drugs bepripil and quinidine (Fig. 7.4b and c), previously known to block or inhibit other ion channels, were shown to also inhibit KNa channels (Mori et al., 1998; Li et al., 1999). Following the cloning of the first KNa channels, Slack-B channels and currents were employed to determine if quinidine and bepridil also inhibited cloned KNa channels, and also to determine if the logic discussed above could be used to discover KNa activators. Using two electrode voltage clamp recording from Slack mRNAinjected Xenopus oocytes and whole-cell and excised patch-clamp recordings from a stable Slack-expressing human embryonic kidney (HEK) cell line, the effects of the two putative KNa inhibitors were directly determined (Yang et al., 2006). In both expression systems, bepridil produced significant levels of inhibition. In Xenopus oocytes, the EC50 for inhibition by bepridil was between 5 and 10 mM. The sensitivity to bepridil was apparently higher when applied to HEK cells; the estimated IC50 for inhibition of whole-cell currents was approximately 1 mM. The inhibition was readily apparent at the single-channel level as well, with marked inhibition when applied to outside-out or inside-out membrane patches, an effect that was independent of [Naþ]in, because Naþ levels were directly controlled in the excised patch experiments. The inhibition of Slack-B currents by quinidine was also studied in these expression systems. Using Slack currents expressed transiently in the CHO cells, the inhibition of Slack currents by quinidine had previously been shown using a high (1 mM)

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(a) OH

OH

Cl

S

Cl

Cl

Cl

(b)

N

CH3 O CH3

N

(c) H2C

HC

H

N H

HO H

OCH3

N

FIGURE 7.4 Structures of the KNa opener bithionol (a) and the KNa inhibitors, bepridil (b) and quinidine (c).

concentration. Using oocytes and a stable Slack-expressing cell line, it was shown that quinidine inhibited KNa currents in both expression systems, with EC50 values 100 mM in oocytes and 90 mM in Slack-expressing HEK cells (Yang et al., 2006). Similar to bepridil, quinidine produced inhibition of single-channel open probability when applied to either the intracellular or extracellular face of excised membrane patches. This study also, and for the first time, demonstrated that Slack-B channels and macroscopic currents could be enhanced by a small molecule. Bithionol, a bis-phenol antiparasitic compound (Knodell et al., 1972; Powell and Lampert, 1973) (Fig. 7.4a), was chosen for these experiments for two reasons: its previously demonstrated ability to activate another member of the Slo family, the BKCa channel

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(Boissard and Gribkoff, unpublished results), and its commercial availability. While essentially playing a hunch that the compound would also activate Slack channels, bithionol proved a good one, effectively enhancing currents in both expression systems. In Xenopus oocytes, the EC50 for activation by bithionol was 10 mM, while in Slack-expressing HEK cells, the enhancement of whole-cell currents had an EC50 of 0.7 mM. As with bepridil (but not quinidine), the sensitivity of whole-cell currents in Slack-expressing HEK cells was about 10X higher than the sensitivity observed in Slack-expressing oocytes for reasons that are not yet understood. The potency of bithionol in HEK cells (or for that matter in oocytes) expressing Slack is quite reasonable for a Kþ channel opener, suggesting that the structural motifs associated with the binding of this compound are worth pursuing further. These compounds certainly are not ideal as specific pharmacological agents and all are capable of influencing the activity of other ion channels over comparable ranges. Nevertheless, particularly with bepridil and bithionol, the inhibition and activation of Slack currents, respectively, in both systems has been sufficiently potent and certainly effective enough to use these compounds as tools in select experiments. Great care has to be taken in such situations, however, so that effects on other targets are not interpreted as an effect on KNa channels. The search for more specific and potent KNa modulators will continue, but these initial successes demonstrate that smallmolecule modulators exist and that proper screening efforts should be fruitful. It should also be noted that although these molecules, particularly the opener, have thus far been studied systematically only in using Slack-B expressing systems, they are also effective on native neuronal KNa channels (Yang et al., 2006). Nevertheless, it is not yet known if different KNa channels have identical, similar, or very different pharmacological profiles. It should prove interesting and more than of academic interest to determine the degree to which the pharmacology of the two known subtypes of KNa channels are different.

7.6 CONCLUSIONS KNa channels have been known since the 1980s to be ligand-gated neuronal Kþ channels capable of regulating the responsiveness of excitable cells by virtue of their activation following accumulation of [Naþ]in. Over the last several years, two mammalian Kþ channels, Slack and Slick, have been shown to be KNa channels, responsive to both [Naþ]in and [Cl]in, with the characteristics of slowly and more rapidly activating KNa channels, respectively. The discovery of Cl sensitivity in cloned KNa channels was surprising, but following the identification of Slack and Slick as KNa channels, examples of native Cl-sensitive native KNa channels have been found (Paulais et al., 2006; Yang et al., 2007). Currently, it cannot be said with absolute certainty that all KNa channels are encoded by Slo2 channels, but the distributions of Slack and Slick, the common effects of pharmacological agents such as bepridil and bithionol on native and cloned KNa channels, as well as the effects of other modulators such as ATP, strongly suggest that Slack and Slick are subunits comprising native KNa channels. The question of the precise makeup of KNa channels, in terms of whether

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heteromultimeric combinations of Slack and Slick form native channels, is as yet unresolved and will likely require more sophisticated pharmacological and molecular manipulations to make a final determination. The functions of KNa channels in neurons are probably numerous and are adapted specifically for each cell type in which they are expressed, but certain generalities can certainly be made. Neuronal KNa channels are involved in the control of action potential burst length by virtue of their production of summating, long-lasting AHPs following action potential trains. Since [Naþ]in will be the highest after highfrequency, prolonged trains of action potentials and high [Naþ]in is generally required for their activation, KNa channels play a prominent role in neurons wherein a combination of Kþ conductances allow periods of high-frequency discharge without significant Naþ channel inactivation and/or persistent Naþ current is induced during bursts of action potentials. KNa channels can also be activated by Naþ entry through Naþ-permeable neurotransmitter receptors such as AMPA receptors (Nanou and El, 2007). KNa is not only suited as a mechanism of terminating bursts accompanied by Naþ-dependent DAPs, but also plays a role in maintaining fidelity of high-frequency action potentials trains in auditory nuclei such as the MNTB. In this latter nucleus, bithionol, shown to be an activator of Slack-B, enhanced the native KNa current and produced an increase in the accuracy of action potential timing at high stimulation frequencies (Yang et al., 2007), providing additional evidence for native KNa channels being some combination of Slack and Slick subunits. Initial pharmacological discovery had focused on using the newly cloned Slack-B channels to investigate previous observations that some antiarrythmic compounds blocked native KNa channels in cardiac myocytes and to discover initial lead KNa openers. The results have been promising, with bepridil (and to a lesser extent quinidine) found to block Slack-B channels at reasonable potency, and the bis-phenol compound bithionol shown to open both Slack-B channels and to enhance native KNa currents in MNTB neurons. All of these agents are leads and while reasonably potent, they lack a high degree of specificity, and there is as yet little published data on their effects on Slack-A or Slick. In select experiments, however, where the contributions of other conductances can be accounted for or negated using other tools, they can be used to examine the contributions of native channels to neuronal function. The focus of the search for new compounds will be on potency and selectivity. Of course it will also be of interest to determine if there is significant therapeutic utility of openers or inhibitors of KNa channels. Openers in particular would seem to have potential utility as antiepileptic compounds as well as utility in acute neurodegenerative disorders, including stroke and other forms of neuronal ischemia and traumatic brain injury. There is significant evidence that KNa channels function in cardiac myocytes as part of the cellular response to ischemia, because the levels of [Naþ]in required for activation of the channels is seldom reached except during pathological conditions. However, even this notion may be somewhat speculative. Increasing knowledge of the structural basis for the modulation of KNa channels by phosphorylation or other influences, many of which could conceivably alter the sensitivity of KNa channels to [Naþ]in or the combination of [Naþ]in and [Cl]in, may suggest a more dynamic role for KNa channels in cardiac cells. Nevertheless, KNa channels are particularly suited to

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act as a buffer for ischemic depolarization and cellular compromise in cardiac myocytes and many neurons. The Slo2 channel of C. elegans has been directly shown to be important in providing resistance to ischemia (Yuan et al., 2003). In KNa channels where the Slick subunit is part of the molecular substrate of native KNa channels, its sensitivity to ATP (where a decrease in ATP should release the channel from inhibition) seems tailor-made for responding to ischemic conditions, where [Naþ]in levels rise accompanied by a decrease in ATP. It will be interesting to determine the degree to which openers of these channels can delay phenomena associated with neuronal ischemia, such as hypoxic/anoxic depolarization (Aitken et al., 1998; Muller, 2000). These channels are important components of the regulatory “governor” on neuronal excitability, and future experiments using newly discovered modulators would ultimately provide very useful additional information on the roles of KNa channels, their molecular makeup, and possibly important therapies for a variety of neuronal disorders.

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PART II MODULATORY MECHANISMS AND INFLUENCES ON NEURONAL VOLTAGE-GATED ION CHANNEL FUNCTION

8 ALTERNATIVE SPLICING OF NEURONAL Cav2 CALCIUM CHANNELS DIANE LIPSCOMBE1, SUMMER E. ALLEN1, ANNETTE C. GRAY1,2, SPIRO MARANGOUDAKIS1, AND JESICA RAINGO1 1 2

Department of Neuroscience, Brown University, Providence, RI 02912, USA Department of Biology, Brandeis University, Waltham, MA 02454, USA

8.1 ALTERNATIVE PRE-mRNA SPLICING 8.1.1

Why Use Alternative Pre-mRNA Splicing?

The nervous system relies on access to finely tuned receptors and ion channels to maintain normal signaling and to adjust output in response to changing external cues. Alternative pre-mRNA splicing, a major step in RNA processing, allows cells to generate multiple proteins with subtly distinct activities from a single gene. This high-resolution control of cellular function would be much harder to achieve if a single gene generated only a single protein (Schwarz et al., 1988; Lipscombe, 2005). Neurons can optimize ion channel activity by mixing and matching protein modules, building on a backbone structure comprised of essential (constitutive) exons (Chemin et al., 2001; Maniatis and Tasic, 2002). For example, cell-specific alternative pre-mRNA splicing near ion channel domains that regulate channel gating kinetics can tailor excitability and firing rates in individual neurons (Rosenblatt et al., 1997; Oberholtzer, 1999; Ramanathan et al., 1999; Baranauskas et al., 2003). By adding or subtracting a number of discrete protein modules, alternative pre-mRNA splicing could achieve incremental control over channel function and support smooth transitions between a Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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continuum of channel activities (Schwarz et al., 1988). On the other hand, the inclusion of an exon might turn a signaling pathway on or off by creating or disrupting interactions with signaling proteins and molecules. In such cases, alternative splicing has been compared to a molecular on/off switch (Mu et al., 2003; Chaudhuri et al., 2004; Raingo et al., 2007). Alternative pre-mRNA splicing is well documented: cell-specific splicing of down syndrome cell adhesion molecule (DSCAM) controls axonal targeting and cell–cell recognition during development (Schmucker et al., 2000); activity-dependent splicing of NMDA receptor RNA promotes export of NMDA receptors from the endoplasmic reticulum and controls surface expression at the synapse (Mu et al., 2003); cell-specific splicing of slo in hair cells of the inner ear underlies changes in calciumactivated potassium channels that set the resonant frequency of individual electrically tuned hair cells (Black, 1998; Shipston, 2001); the shift in alternative pre-mRNA splicing of SNAP-25 during development drives the transition from immature to mature synapses in the nervous system (Bark et al., 2004); and cell-specific pre-mRNA splicing of CaV1.3 L-type calcium channels in medium spiny neurons allows interaction with Shank and promotes channel localization at glutamatergic cortico–striatal synapses (Olson et al., 2005). Evidence shows that alternative pre-mRNA splicing is used extensively in the mammalian brain (Dredge et al., 2001; Lipscombe, 2005; Licatalosi and Darnell, 2006; Sharma and Black, 2006; Ule and Darnell, 2006; Zipursky et al., 2006). Furthermore, studies that link a specific splice isoform to an identified population of neurons and to a specific cell function support the biological importance of this process (Kollmar et al., 1997; Grabowski and Black, 2001; Baranauskas, 2004; Bell et al., 2004; Vendel et al., 2006; Raingo et al., 2007). 8.1.2

Basic Steps

Basic steps and different forms of alternative pre-mRNA splicing have been well described elsewhere (Smith and Valcarcel, 2000; Maniatis and Tasic, 2002; Black, 2003). This review will focus primarily on two common types of alternatively spliced exons: cassette exons that are included or excluded from the fully processed mRNA and mutually exclusive exons that are inserted individually but not together in the same mRNA (Fig. 8.1). The spliceosome – a complex molecular machine that controls the splicing of both constitutive and alternative exons – produces the catalytic reactions that remove introns and join exons during splicing. Whether the spliceosome causes a particular alternative exon to be included or excluded depends on the presence, absence, and/or ratios of specific splicing factors that associate with the pre-mRNA to promote specific splicing patterns (Smith and Valcarcel, 2000; Maniatis and Tasic, 2002). In turn, the cellular levels of these splicing factors depend on features such as cell type, developmental stage, and neuronal activity (Black, 2003). Splicing factors include both enhancer and repressor proteins. Splicing enhancers can recruit the spliceosome to the site of a particular alternative exon, ensuring that this exon is included. In contrast, splicing repressors can occlude a particular alternative exon from the spliceosome, causing this exon to be skipped. These enhancer and repressor factors work in concert with other RNA binding proteins to set the levels of specific mRNA splice isoforms required to support cellular function (Smith and Valcarcel,

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221

FIGURE 8.1 Two patterns of alternative pre-mRNA splicing used to generate different isoforms of CaV2.2. (a)The pattern involves the alternate expression of a cassette exon that is either included or skipped by the spliceosome. (b)The pattern involves inclusion of only one of the pair of exons (mutually exclusive). Introns are depicted with straight solid lines and exons with boxes. Constitutive exons are colored white and alternative exons are gray. When alternatively spliced exons are included and translated, they can confer unique functional properties to the protein. In (a), the inclusion of a cassette exon adds a domain that creates a new protein binding site. In (b), inclusion of either exon produces a protein binding domain, but only the second exon favors interaction with the specific target protein shown.

2000; Maniatis and Tasic, 2002; Black, 2003). Some splicing factors are cell and/or tissue specific (Fig. 8.2). Expression of these splicing factors can result in a change in the ratio of included or skipped alternative exons and can sometimes result in the expression of protein isoforms unique to specific cells or tissues (Fig. 8.2). Several RNA binding proteins involved in alternative splicing of neuronal pre-mRNAs are known, including Nova-1/2, Fox-1/2, Hu/elav family of proteins, CELF, neural PTB, KSRP (KH-type splicing regulatory protein), and NAPOR (neuroblastoma apoptosisrelated RNA-binding protein) (Min et al., 1997; Jensen et al., 2000; Polydorides et al., 2000; Ladd et al., 2001; Lisbin et al., 2001; Zhang et al., 2002; Jin et al., 2003). 8.1.3

Cell-Specific Alternative Splicing of Ion Channel Pre-mRNAs

Like all proteins, ion channels are highly sensitive to even small perturbations in their structure or chemical environment. However, for highly sensitive electrophysiological recording methods can monitor and quantify the impact of these changes on ion channel activity. Thus, those of us interested in ion channels can more readily assess the functional impact of alternative splicing on protein function and, when coupled to

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FIGURE 8.2 A large number of proteins and molecules act in concert to regulate alternative pre-mRNA splicing. In this hypothetical model of mutually exclusive splicing, all neurons express a number of regulatory proteins that either repress or enhance inclusion of one of the exon pair. In (a), repressor activity slightly exceeds enhancer activity on exon 1. Consequently, the spliceosome shows slight preference for exon 2, and the fraction of mRNAs containing exon 2 therefore slightly exceeds mRNAs containing exon 1. In (b), a repressor protein that binds to the intron upstream of exon 1 is expressed at low levels in neurons of dorsal root ganglia. This results in the spliceosome favoring exon 1 inclusion, and therefore mRNAs containing exon 1 are increased relative to total. Here, circles denote splicing enhancers, triangles denote splicing repressors, and a star denotes a tissue-specific repressor that is downregulated in dorsal root ganglia. (See the color version of this figure in Color Plate section.)

single-cell analysis, start to understand the impact of these activities within the context of specific neuronal populations (Lipscombe, 2005). Studies of alternative splicing of Slo transcripts that encode calcium-activated potassium channels nicely illustrate this point. In the chick cochlea, the pattern of alternative splicing of Slo pre-mRNAs varies with the tuning of hair cells along the basilar membrane to create a tonotopic map (Rosenblatt et al., 1997; Black, 1998; Fettiplace and Fuchs, 1999; Oberholtzer, 1999; Ramanathan et al., 1999). The correlation between the expression of Slo splice isoforms and tuning frequency in chick hair cells suggests that alternative splicing optimizes neuronal excitability over a wide range of tonal frequencies (Black, 1998; Fettiplace and Fuchs, 1999). Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) and electrophysiology have also been used to link the potassium channel splice isoform KV3.4a to the fast spiking phenotype of four different populations of neurons in the rat central nervous system (Baranauskas et al., 2003). In our studies of CaV2.2, we showed that alternative splicing in individual rat nociceptors underlies the unusually high sensitivity of calcium channels in these cells to inhibition by GABA and opioids (Raingo et al., 2007).

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8.1.4 How Many Voltage-Gated Calcium Channels are There? How Many Does the Brain Need? Currently, we can’t give precise answers to either of these questions, but we do know the numbers are high based on several lines of reasoning. First, we have genomic evidence from CaVa1 genes that encode the functional and structural core of all voltage-gated calcium channels; they span 100–800 kb of human genome sequence and each contains at least 50 exons (Soldatov, 1994; Lipscombe et al., 2002; Lipscombe and Castiglioni, 2004). Therefore, CaVa1 genes have the capacity to generate thousands of unique splice isoforms, as is the case for other genes far smaller than CaVa1 (Missler and Sudhof, 1998; Schmucker and Flanagan, 2004). To visualize the extent of alternative splicing for a given CaVa1 gene, we mapped reported sites of alternative splicing onto 2 of 10 human CaVa1 genes, CaV2.1 and CaV2.2, which encode the cores of P-type and N-type calcium channels, respectively (Fig. 8.3). From our own studies of CaV2.2 mRNAs, we know that exons e18a, e24a, e31a, and e37a/ e37b are expressed in specific regions of the nervous system or in specific types of neurons. We also know that these alternatively spliced exons uniquely influence Ntype calcium channel function (Lin et al., 1997, 1999, 2004; Pan and Lipscombe, 2000; Bell et al., 2004; Thaler et al., 2004; Gray et al., 2007; Raingo et al., 2007). Second, we have mRNA evidence, as a large number of CaVa1 subunit mRNA variants have been and continue to be isolated from a variety of tissues and cell lines (Koch et al., 1990; Perez-Reyes et al., 1990; Hui et al., 1991; Snutch et al., 1991; Diebold et al., 1992; Schneider et al., 1994; Williams et al., 1994; Marubio et al., 1996; Lin et al., 1997; Takimoto et al., 1997; Ligon et al., 1998; Mittman et al., 1999a; Cribbs et al., 2000; Chemin et al., 2001; Soong et al., 2002; Emerick et al., 2006). Third, we have functional evidence for a great diversity of voltage-gated calcium channels in the nervous system. Only 10 genes encode CaVa1 subunits, the functional core of all voltage-gated calcium channels, in mammalian genomes. Greater structural diversity than this is needed to explain the wide spectrum of cellular processes controlled by voltage-gated calcium channels (Delmas et al., 2000; Lipscombe et al., 2002; Lipscombe and Castiglioni, 2004). Fourth, genes that encode auxiliary calcium channel subunits, including all four CaVb and all three CaVa2d genes, also undergo alternative pre-mRNA splicing (Castellano and Perez-Reyes, 1994; Yamada et al., 2001; Helton and Horne, 2002; Helton et al., 2002; Lipscombe et al., 2002; Lipscombe and Castiglioni, 2004; Vendel et al., 2006). For example, alternative pre-mRNA splicing in the N-terminal domain of CaVb subunits regulates binding of synaptotagmin I and LC2 domain of microtubule-associated protein 1A (Vendel et al., 2006). The structural changes generated by alternative splicing of CaVa1 pre-mRNAs can regulate a wide range of channel properties, including biophysics, surface density, targeting, posttranslational modification, and coupling to downstream signaling pathways including G-protein-coupled receptors. Cell-specific factors coordinate alternative splicing of pre-mRNAs, and activities of these factors are influenced by tissue type, cell type, stage of development, neuronal activity, and disease (Diebold et al., 1992; Gidh-Jain et al., 1995; Angelotti and Hofmann, 1996; Lin et al., 1997, 1999; Takimoto et al., 1997; Welling et al., 1997; Vigues et al., 1998, 1999, 2002;

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FIGURE 8.3 Sites of alternatively spliced exons mapped on CaV2.1 and CaV2.2 proteins and the CaV2.2 gene. (a) and (b) illustrate the tetrameric structure and the two-dimensional transmembrane topology of CaV2.1 and CaV2.2 proteins, respectively. Approximate locations of alternatively spliced exons are highlighted with triangles and are numbered. The four main structural domains (I, II, III, and IV) are labeled and the constitutively expressed exons encoding each domain shown with black lines. (c) illustrates the human CaV2.2 gene. Exons are denoted as solid boxes and introns as lines. Constitutive exons are colored black and alternatively spliced exons colored gray. The gene structure was constructed from analysis of human genome sequence contig NT_023929.14. Small arrows indicate the location of alternative polyadenylation sites. Exons are numbered according to the previously published gene structure (Lipscombe et al., 2002). Figure adapted from Lipscombe and Castiglioni (2004). With kind permission of Springer Science and Business Media (See the color version of this figure in Color Plate section.)

Zuhlke et al., 1998; Bourinet et al., 1999; Pan and Lipscombe, 2000; Yang et al., 2000; Kaneko et al., 2002; Soong et al., 2002; Bell et al., 2004; Chaudhuri et al., 2004; Liao et al., 2004, 2005; Graf et al., 2005; Shen et al., 2006; Tiwari et al., 2006; Zhong et al., 2006; Altier et al., 2007; Gray et al., 2007; Raingo et al., 2007; Tang et al., 2007).

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Below, we briefly review alternative splicing of CaVa1 pre-mRNAs and then focus on processing of CaV2 pre-mRNAs that encode presynaptic calcium channels. 8.1.5 Which CaVa1 Subunit Domains are Modified by Alternative Pre-mRNA Splicing? Alternative splice sites exist throughout the coding regions of CaVa1 genes. At first glance, alternative splice sites appear more prevalent in the 30 halves of CaVa1 genes, but some of this bias arises from the 30 bias of sequences derived from mRNAs isolated from tissues as well as sequences derived in silico from the EST database. This is especially true for large genes such as Cava1. Certain general features of CaVa1 alternative splicing are worth pointing out. (i) Regions most divergent among different CaVa1 subunits contain the largest number of alternatively spliced exons, most notably the intracellular loops connecting domains II and III and the C-termini (Fig. 8.3). These regions play a critical role in linking the channel to target proteins and signaling molecules (Fig. 8.4). With additional information, we think alternatively spliced exons will have a similar representation in the N-terminus. (ii) Sequence differences among CaVa1 mRNA isoforms arising from alternative splicing may involve only a few nucleotides that insert a short peptide sequence (Lin et al., 1997; Bourinet et al., 1999) or hundreds of nucleotides that, if deleted, result in a frameshift and premature termination of translation (Soong et al., 2002). (iii) Similarly, the functional consequences of alternative splicing of CaVa1 pre-mRNAs may be quite subtle, such as changing channel activation kinetics (Lin et al., 2004; Murbartian et al., 2004), or profound, resulting in truncated, nonfunctional proteins (Soong et al., 2002). There are also interesting examples of alternative splicing of CaVa1 pre-mRNAs influencing channel pharmacology (Welling et al., 1997; Zuhlke et al., 1998; Beam, 1999; Bourinet et al., 1999; Fearon et al., 2000; Jimenez et al., 2000; Krovetz et al., 2000; Lacinova et al., 2000; Yatani and Kamp, 2000; Helton et al., 2002; Kaneko et al., 2002; Raingo et al., 2007). (iv) Certain sites of alternative splicing are conserved among paralogs and orthologs and are a general feature of all CaVa1 genes (e.g., cassette exons encoding part of the IVS3–IVS4 linker of CaVa1 subunits) (Lin et al., 1999; Lipscombe and Castiglioni, 2004), while other sites of alternative pre-mRNA splicing only exist in closely related CaVa1 genes (e.g., e37a and e37b in CaV2 genes (Gray et al., 2007)).

8.2 ALTERNATIVE SPLICING IN THE CaV2 GENES CaV2.1, CaV2.2, and CaV2.3 genes encode the core subunit of high-voltage-activated P-type, N-type, and R-type calcium channels respectively. CaV2 channels work together to control transmitter release from neurons of the mammalian nervous system. Their sequences andgenestructures are highly homologous.ThehumanCaV2.1gene,referred to as CACNA1A, is located on chromosome 19 at 19p13.2-p13.1; CaV2.2, CACNA1B, is located on Chromosome 9 at 9q34; and CaV2.3, CACNA1E, is on Chromosome 1 at

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FIGURE 8.4 Proteins that interact with the Cav2.2 subunit mapped onto a two-dimensional model of the channel. Several proteins interact with Cav2.2 channels, most notably in the II–III intracellular linker and C-terminus. A number of proteins associated with the synaptic machinery, and with synaptic transmission in general, interact with the II–III linker of Cav2.2, including CSP (cysteine string protein) (Miller et al., 2003), SNAP-25 (Catterall, 1999), synaptotagmin 1 (Sheng et al., 1997), syntaxin 1A (Sheng et al., 1994), huntingtin (Swayne et al., 2005), RGS12 (Richman and Diverse-Pierluissi, 2004), and b-arrestin (Puckerin et al., 2006). The asterisk (*) denotes proteins whose binding is either known or expected to be modified by alternative splicing. Binding of SNAP-25, synaptotagmin, and syntaxin is isoform dependent (Catterall, 1999). A CaV2.2 splice isoform lacking most of the II–III linker (e19 through e21) (Kaneko et al., 2002) theoretically should not interact with proteins that bind to this region. A second group of proteins interacts with the C-terminus, including 14-3-3 (Li et al., 2006), CASK (Maximov and Bezprozvanny, 2002), ENH (enigma homologue protein) (Maeno-Hikichi et al., 2003), Gaq (Simen et al., 2001), Gbg (De Waard et al., 2005), Mint1 (Maximov et al., 1999), PP2Ca (Li et al., 2005), RIM binding protein (RBP) (Hibino et al., 2002), and Tctex1 (Lai et al., 2005). CASK and Mint1 do not bind to a truncated splice isoform of CaV2.2 (Maximov et al., 1999; Maximov and Bezprozvanny, 2002). Theoretically, RIM binding protein should not bind to the truncated splice isoform of CaV2.2 described by Bezprozvanny and colleagues (Maximov et al., 1999). Gbg and CaVb subunits interact with the I–II linker (De Waard et al., 2005). Based on their sites of interaction in the I–II loop, alternative splicing at e10 (described in Section 8.2.3) should not disrupt Gbg and Cavb binding directly. The N-terminus is not known to undergo alternative splicing, thus the Gbg interacting site (Canti et al., 1999) should also not be affected by splicing. (See the color version of this figure in Color Plate section.)

1q25-q31. Access to genome sequences from a number of species, including human, has greatly facilitated our ability to confirm sites of alternative splicing in these genes and to identify new sites. Splice isoforms of all three CaV2 genes have been identified and characterized to various extents (Hui et al., 1991; Schneider et al., 1994; Williams et al., 1994; Sakurai et al., 1995; Marubio et al., 1996; Klockner et al., 1997; Lin et al., 1997,

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1999, 2004; Takimoto et al., 1997; Welling et al., 1997; Zhuchenko et al., 1997; Kamphuis and Hendriksen, 1998; Ligon et al., 1998; Pereverzev et al., 1998, 2002; Perez-Reyes et al., 1998; Soldatov et al., 1998; Vigues et al., 1998, 1999, 2002; Zuhlke et al., 1998; Beam, 1999; Bourinetet al., 1999; Ghasemzadeh et al., 1999; Hanset al., 1999; Lu and Dunlap, 1999; Mittman et al., 1999a, 1999b; Cahill et al., 2000; Cribbs et al., 2000; Krovetz et al., 2000; Lacinova et al., 2000; Monteil et al., 2000; Pan and Lipscombe, 2000; Chemin et al., 2001; Lu et al., 2001; McRory et al., 2001; Raghib et al., 2001; Staes et al., 2001; Jagannathan et al., 2002; Kaneko et al., 2002; Maximov andBezprozvanny,2002;Soongetal.,2002;Tsunemi etal.,2002;Belletal.,2004;Harry et al., 2004; Liao et al., 2004, 2005; Tang et al., 2004, 2007; Thaler et al., 2004; Graf et al., 2005; Castiglioni et al., 2006; Emerick et al., 2006; Kanumilli et al., 2006; Khanna et al., 2006; Shen et al., 2006; Vendel et al., 2006; Altier et al., 2007; Chang et al., 2007; Ikeda and Dunlap, 2007; Raingo et al., 2007). Several splice isoforms of CaV2 genes give rise to functionally and, in some cases, pharmacologically distinct channels (Bourinet et al., 1999; Hans et al., 1999; Jimenez et al., 2000; Kaneko et al., 2002; Raingo et al., 2007). We review studies of CaV2 splice isoforms according to specific structural domains of CaVa1 subunits that contain sequence encoded by alternatively spliced exons. We start at the 30 end of the channel at the C-terminus, a region that we know most about, and work forward toward the 50 N-terminus (Figs. 8.3a and b). 8.2.1

The C-Terminus

The C-terminus of CaVa1 constitutes about one fourth of the channel protein, and likely regulates various aspects of calcium channel function (Fig. 8.4) (Wei et al., 1994; Maximov et al., 1999; Zuhlke et al., 1999; Gao et al., 2000; Hering et al., 2000; Ivanina et al., 2000; Kepplinger et al., 2000; Peterson et al., 2000; Simen et al., 2001; Staes et al., 2001; Maeno-Hikichi et al., 2003; Li et al., 2004; Chen et al., 2005, 2006; Lautermilch et al., 2005; Castiglioni et al., 2006; Raingo et al., 2007; Wykes et al., 2007). CaVa1 splice isoforms differing in their C-termini have distinct kinetic properties including inactivation, activation, and deactivation (CaV1.2, CaV2.1, CaV2.2, CaV3.3) (Soldatov et al., 1997; Krovetz et al., 2000; Restituito et al., 2000; Murbartian et al., 2004; Castiglioni et al., 2006), G-protein sensitivity (CaV2.2) (Simen et al., 2001; Raingo et al., 2007), subcellular targeting (CaV1.3, CaV2.1, CaV2.2) (Maximov et al., 1999; Maximov and Bezprozvanny, 2002; Olson et al., 2005; Khanna et al., 2006), current densities (CaV2.1, CaV2.2) (Soong et al., 2002; Castiglioni et al., 2006), and sensitivity to calcium calmodulin-dependent inactivation and facilitation (CaV1.3 and CaV2.1) (Chaudhuri et al., 2004; Shen et al., 2006; Calin-Jageman et al., 2007; Chang et al., 2007; Wykes et al., 2007). The C-terminus is the most divergent domain among CaVa1 genes and alternative splicing in this region/generates differently sized forms of CaVa1 subunits (Fig. 8.3) (Hui et al., 1991; Schneider et al., 1994; Williams et al., 1994; Klockner et al., 1997; Soldatov et al., 1997; Zhuchenko et al., 1997; Ligon et al., 1998; Bourinet et al., 1999; Lu and Dunlap, 1999; Maximov et al., 1999; Hering et al., 2000; Krovetz et al., 2000; McRory et al., 2001; Xu and Lipscombe, 2001; Pereverzev et al., 2002; Calin-Jageman et al., 2007). In CaV2.1, the

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C-terminus is encoded by exons 36–47 (e36–e37) (Ophoff et al., 1996), and e37a/ e37b, e43, e44, and e47 are subject to alternative splicing (Fig. 8.3) (Zhuchenko et al., 1997; Bourinet et al., 1999; Krovetz et al., 2000; Soong et al., 2002; Chaudhuri et al., 2004; Kanumilli et al., 2006; Chang et al., 2007; Chaudhuri et al., 2007). In CaV2.2, the C-terminus is encoded by e36–e46 (Lipscombe and Castiglioni, 2004), and two putative sites of alternative splicing involving e37a/e37b and e46 have been described (Fig. 8.3) (Kaneko et al., 2002; Bell et al., 2004; Castiglioni et al., 2006; Raingo et al., 2007). 8.2.1.1 Mutually Exclusive e37a and e37b Mutually exclusive e37a and e37b are found in all three CaV2 genes (Gray et al., 2007). The lengths of e37a and e37b are identical, each containing 97 nucleotides, and they differ by 9–14 amino acids with some variation in amino acid composition of both exons among the three CaV2 genes. The e37a/e37b splice sites of CaV2.1 and CaV2.2 have been studied in most detail. Expression patterns of CaV2.1e[37a] and CaV2.1e[37b] mRNAs (Bourinet et al., 1999; Chang et al., 2007) are distinct from the expression pattern of the equivalent splice isoforms of CaV2.2 (Bell et al., 2004), suggesting that this splice site might have evolved to serve different functions in N-type and P-type channels. In CaV2.1, nine amino acids differ between e37a and e37b and they encode different forms of a helix–loop–helix structural motif that may bind calcium (EF-hand) (Bourinet et al., 1999; Krovetz et al., 2000; Soong et al., 2002; Chaudhuri et al., 2004). Both isoforms are expressed in human and rat brains and in human spinal cord (Gray et al., 2007), and selection of e37a or e37b may be under developmental control (Chang et al., 2007). The activity of CaV2.1e[37a] isoforms can be augmented via a calcium-dependent mechanism called calcium-dependent facilitation, a phenomenon that is absent in CaV2.1e[37b] isoforms. A second site of alternative splicing in the C-terminus encoding region of CaV2.1 pre-mRNA that involves e46 also modifies the calcium-dependent facilitation of CaV2.1 channels. David Yue and colleagues have shown that combinatorial alternative splicing at these two sites in CaV2.1 pre-mRNAs can generate three different forms of the Ptype channel in which calcium-dependent facilitation can be completely on, completely off, or off but ready for activation if global intracellular calcium rises (Chaudhuri et al., 2004, 2007), thereby acting as a molecular switch with three settings. Cav2.2e[37a] is enriched in dorsal root ganglia. Exons equivalent to CaV2.1e [37a] and CaV2.1e[37b] are found in CaV2.2. Alternative splicing at these sites regulates current density and G-protein signaling of the N-type channel (Fig. 8.5). CaV2.2 mRNAs expressed in most neurons contain e37b, except in dorsal root ganglia nociceptors, which express both CaV2.2e[37a] and CaV2.2e[37b] mRNAs (Bell et al., 2004). Enrichment of e37a-containing CaV2.2 mRNAs in nociceptors points to a functional role for alternative splicing of e37a pre-mRNAs in nociception. This was recently confirmed in a collaborative study led by Gerald Zamponi (Altier et al., 2007). Using splice isoform-specific small interfering RNA, Zamponi and colleagues showed that e37a-containing N-type channels contribute to basal

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CaV2.2e[37b]

GPCR

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GPCR

AP

ICa

FIGURE 8.5 Hypothesized physiological impact of alternative pre-mRNA splicing at the e37a/e37b site in CaV2.2. Hypothetical calcium currents (ICa) induced by a train of action potentials (AP) in cells expressing CaV2.2e[37b] or CaV2.2 e[37a] channels. CaV2.2e[37a] channels express at higher density compared to CaV2.2 e[37b] (Bell et al., 2004; Castiglioni et al., 2006), but they are also more sensitive to inhibition following G-protein receptor activation (Raingo et al., 2007). G-protein receptor activation is indicated by shaded regions. CaV2.2e [37b] currents are inhibited following G-protein-coupled receptor (GPCR) activation but because inhibition is purely voltage dependent, it is relieved during repetitive stimulation. CaV2.2e[37a] currents are also inhibited by GPCR activation but because inhibition of this isoform involves both voltage-dependent and voltage-independent pathways, inhibition is greater when compared to CaV2.2e[37b] currents, and it is only partially relieved during repetitive stimulation (Raingo et al., 2007). This model proposes that neurons could augment N-type current density and their sensitivity to G-protein-coupled receptors by increasing the ratio of CaV2.2e[37a]/CaV2.2e[37b] mRNAs. (See the color version of this figure in Color Plate section.)

thermal nociception and to thermal and mechanical hyperalgesia associated with inflammatory and neuropathic pain (Altier et al., 2007). More generally, this study provides evidence that CaV2.2e[37a] channels are present at presynaptic terminals of primary afferents and that they regulate synaptic transmission. N-type calcium channel blockers administered intrathecally are powerful analgesics in a variety of animal pain models (Miljanich and Ramachandran, 1995; Bowersox et al., 1996; Brose et al., 1997; Altier and Zamponi, 2004; Miljanich, 2004; Bourinet and Zamponi, 2005), validating N-type channels as targets for treating neuropathic pain. Collectively, pharmacological and siRNA studies raise the possibility that an N-type channel blocker with higher potency on CaV2.2e[37a] than CaV2.2e[37b] could have analgesic properties and fewer side effects than current therapeutics. 8.2.1.2 Alternative Splicing Controls G-Protein Signaling and Sets the Gain of the N-Type Channel What is the biological significance of e37a inclusion in CaV2.2 channels in a subset of nociceptors? We think alternative splicing of e37a and e37b in CaV2.2 pre-mRNAs is used to regulate the sensitivity of N-type channels to G protein-mediated inhibition, which in turn sets the sensitivity of nociception to neurotransmitters and drugs that act through G-protein-coupled receptors (Hille et al., 1995; Ikeda and Dunlap, 1999) (Fig. 8.5). We found that voltage-independent

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inhibition mediated by Gi/o can only gain access to the N-type channel if the CaV2.2 subunit contains e37a (Raingo et al., 2007). Combined with our previous studies that show enrichment of e37a in nociceptors (Bell et al., 2004), our data point to a molecular basis for the high susceptibility of N-type currents in sensory neurons to voltageindependent inhibition following G-protein activation (Diverse-Pierluissi et al., 1997; Ikeda and Dunlap, 2007). In addition to a higher sensitivity to G-protein-coupled receptors, CaV2.2e[37a] splice isoforms support larger N-type currents in nociceptive neurons, as compared to the ubiquitous CaV2.2e[37b] isoforms (Bell et al., 2004; Castiglioni et al., 2006). Larger currents associated with e37a-containing channels impacts the amount of calcium entering cells in response to a variety of action potential waveforms (Castiglioni et al., 2006). Overall, our studies imply that e37a effectively increases the gain of N-type channels; it supports larger N-type currents but at the same time renders them more sensitive to inhibition by G-protein-coupled receptor activation (Fig. 8.5). 8.2.1.3 Voltage-Independent Inhibition As discussed above, e37a controls Gi/o access to the N-type channel and consequently controls the level of voltageindependent inhibition. A Gi/o-dependent form of voltage-independent inhibition of N-type currents is well described in chick sensory neurons (Diverse-Pierluissi et al., 1997), consistent with enrichment of e37a in sensory neurons. Functionally, inhibition of calcium channels via this Gi/o-dependent, voltage-independent pathway is expected to be insensitive to changes in the membrane potential in contrast to the most common form of G-protein-mediated inhibition of N-type currents that is voltage dependent and mediated by G-protein Gbg dimers. Consequently, N-type currents activated by brief stimuli are well inhibited via this voltage-dependent pathway. However, with intense, high-frequency stimuli, this inhibition of N-type currents is relieved. Thus, voltage-dependent inhibition acts like a high pass filter that attenuates low-frequency signals, but has less of an effect on high-frequency signals (Ikeda and Dunlap, 1999, 2007; Elmslie, 2003). In addition to Gi/o, Gq can also inhibit N-type currents via a voltage-independent mechanism. Gq-dependent inhibition of N-type currents is well described in sympathetic neurons (Hille et al., 1995; Ikeda and Dunlap, 1999; Kammermeier et al., 2000; Elmslie, 2003) and may depend on the expression of alternatively spliced exons in CaV2.2 other than e37a. 8.2.1.4 Other Sites of Alternative Pre-mRNA Splicing in the C-Terminus In CaV2.1, alternative splicing of exon 44 regulates channel inactivation kinetics of the human spinal cord P-type channel (Krovetz et al., 2000). In this same region of the CaV2.1 gene, others have found evidence from analyses of mRNA in human brain that e43 is also subject to alternative splicing, and that together with e44, this site modifies the efficiency of P-type channel expression (Soong et al., 2002). An alternate 30 acceptor site in the intron upstream of e47 of CaV2.1 introduces a frameshift and early truncation, which deletes e47 from the C-terminus, producing a short CaV2.1 isoform. E47 is large, encoding >230 amino acids in the longer splice isoform of CaV2.1. The two C-terminus isoforms of CaV2.1 are differentially expressed in human brain (Soong et al., 2002), spinal cord (Krovetz et al., 2000), and neuroblastoma

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cells (Hans et al., 1999). An interesting consequence of e47 truncation is that the shorter form of CaV2.1 cannot contribute to spinocerebellar ataxia type-6 because the SCA6 polyglutamine expansion that underlies this disorder is located in e47 (Zhuchenko et al., 1997). Some investigators have speculated that the observed upregulation of e47 inclusion during late development might help explain why spinocerebellar ataxia phenotype has a delayed onset (Soong et al., 2002). The other well-documented functional role of the C-termini of voltage-gated calcium channel CaVa1 subunits is subcellular targeting. Splice isoform-specific interactions between two scaffold proteins, Mint1 and CASK, and the C-termini of both CaV2.1 and CaV2.2 subunits have been reported (Maximov et al., 1999) (Fig. 8.4). Mint1 and CASK only interact with the long C-terminal splice isoforms that contain e47, suggesting that alternative splicing at this site might play a role in regulating the density of calcium channels at presynaptic nerve terminals (Maximov et al., 1999). E46 in the C-terminus of CaV2.2 is equivalent to e47 of CaV2.1; it is large (>3000 nucleotides in length), it contains a putative alternative 30 acceptor site that is hypothesized to generate two differently sized forms of CaV2.2 (Williams et al., 1992; Lu and Dunlap, 1999; Maximov and Bezprozvanny, 2002), and it has three sites of polyadenylation, of which two are used to generate differently sized CaV2.2 mRNAs in the nervous system (Schorge et al., 1999). The relative abundance of the short and long forms of CaV2.2 is not known, but functional studies suggest that they localize to different subcellular compartments in neurons. The long form of CaV2.2 targets to synapses via its interaction with Mint1 and CASK, two proteins involved in subcellular targeting, and the shorter isoform of CaV2.2 predominantly localizes to soma and dendrites (Maximov and Bezprozvanny, 2002). The function of e46 beyond regulating CaV2.2 targeting needs to be investigated. 8.2.2

A Short Linker on the Outside of the Channel

The extracellular linker in the forth domain (IV) that connects the third and fourth transmembrane spanning segments (IVS3–IVS4) of most voltage-gated calcium channels contains an exon that is alternatively expressed. Alternative splicing within the IVS3–IVS4 linker is a feature of CaVa1 pre-mRNAs of all mammals studied to date, including human as well as Drosophila (Lipscombe et al., 2002). The IVS3– IVS4 region of CaV channels can therefore accommodate sequence modification (Lipscombe et al., 2002). In CaV2 genes, an alternatively spliced cassette exon (e31a) encodes part of the IVS–IVS4 linker. E31a of the CaV2.1 and CaV2.2 genes only encode two amino acids, but the length and amino acid composition of e31a of the different CaVa1 genes vary. Where function has been analyzed, e31a impacts the time course and voltage dependence of calcium channel activation (Perez-Reyes et al., 1990; Snutch et al., 1991; Starr et al., 1991; Diebold et al., 1992; Barry et al., 1995; Ihara et al., 1995; Lin et al., 1997, 1999, 2004; Ligon et al., 1998; Bourinet et al., 1999; Hans et al., 1999; Krovetz et al., 2000). E31a of CaV2.1 encodes the dipeptide sequence asparagine–proline (NP). The NP encoding exon of the human CaV2.1 gene is located within approximately 9 kb of intron sequence and its inclusion in CaV2.1 mRNAs in the mammalian nervous system

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is under regional control (Lin et al., 1997; Bourinet et al., 1999; Krovetz et al., 2000; Soong et al., 2002). The amino acids encoded by e31a in CaV2.1 are reported in some studies to slow both activation and inactivation kinetics of P-type currents (Hans et al., 1999; Krovetz et al., 2000) while others report no effect of E31a on P-type channel inactivation (Bourinet et al., 1999). However, researchers agree that the dipeptide NP encoded by E31a in CaV2.1 modulates w-agatoxin IVA sensitivity (Bourinet et al., 1999; Hans et al., 1999). E31a inclusion decreases toxin affinity 11-fold compared to CaV2.1 channels that lack the NP dipeptide. Alternative splicing of e31a in CaV2.1 premRNA may underlie some of the pharmacological differences among P-type currents in different neurons that are often referred to as P-type and Q-type currents (Sather et al., 1993; Stea et al., 1994; Randall and Tsien, 1995; Bourinet et al., 1999). The absence of e31a-containing CaV2.1 mRNAs in single Purkinje cells that express classic P-type currents with high sensitivity to w-agatoxin IVA is consistent with this hypothesis. A homologous 6 nucleotide e31a in CaV2.2 encodes glutamate–threonine (ET). E31a of CaV2.2 has been located in human, mouse, and rat genes. This exon is expressed in a tissue-specific pattern, and its presence alters N-type channel function. In rat and human, e31a-containing CaV2.2 mRNAs are preferentially expressed in neurons of the peripheral nervous system and are virtually absent from the central nervous system. Analysis of the human CaV2.2 gene reveals that e31a is contained within 14 kb of intron sequence and flanked by consensus ag–gt splice sites (Lin et al., 1997, 1999). E31a slows N-type channel activation and deactivation kinetics and shifts the voltage dependence of channel activation to slightly more depolarized voltages. Although e31a of CaV2.1 and CaV2.2 encode different dipeptides (NP and ET, respectively), they slow channel activation kinetics to a similar degree (Lin et al., 1999), consistent with the proximity of IVS3–IVS4 to the putative voltage sensor (IVS4). E31a appears to directly affect the putative voltage sensor of CaV2.2, based on our own analyses of gating currents (Lin et al., 2004). The steady-state voltage dependence of charge movement associated with N-type channel gating currents was not consistently different when e31a was present. However, “on” gating currents that precede channel opening decayed more slowly in CaV2.2 channels containing e31a when compared with gating currents measured from channels lacking e31a. A slower gating current is thought to correspond to slower movement of the charge sensor in the channel. Thus, alternative splicing in the extracellular IVS3–IVS4 linker of CaV2.2 affects the kinetics, but not the voltage dependence of N-type channel gating (Lin et al., 2004). Extracellular domains of presynaptic calcium channels have also been implicated in synapse stabilization (Nishimune et al., 2004). Postsynaptic b2 laminins bind directly to presynaptic CaV2.1 and CaV2.2 channels, thereby physically bridging the two membranes and drawing calcium channels into the synaptic cleft. This interaction between presynaptic calcium channels and postsynaptic laminins may be important for organizing neurotransmitter release sites in the active zone. It would be interesting to know if alternative splicing in extracellular S3–S4 linkers of domains III and IV influence b2 laminin binding to CaV2 channels. A positive

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finding could point to a role of e31a in synapse stabilization and might hint at why this exon is only found in N-type channels of peripheral and not central neurons. 8.2.3

The Intracellular Linker LII–III Connects Domains II and III

The intracellular linker LII–III in CaV2 channels binds presynaptic proteins and is essential for excitation–secretion coupling (Fig. 8.4). This region is encoded by e18 through e21 (Fig. 8.3) and in CaV2, LII–III connects the channel to sites of synaptic vesicle fusion by binding SNARE (soluble N-ethyl-maleimide-sensitive fusion attachment protein receptor) proteins (Catterall, 1999). LII–III also plays a role in targeting CaV2 channels to presynaptic nerve terminals (Leveque et al., 1994; Sheng et al., 1994; Mochida et al., 1996, 2003; Rettig et al., 1996, 1997; Tobi et al., 1998; Catterall, 1999; Harkins et al., 2004) and it influences the biophysical properties of CaV2 channels, particularly cumulative inactivation (Catterall, 1999; Degtiar et al., 2000; Thaler et al., 2004). CaV2 isoforms distinguished by amino acid sequence differences in their LII–III linkers are thus likely to have distinct functions. 8.2.3.1 Alternative Splicing of e18a in CaV2.2 and CaV2.3 The best studied site of alternative pre-mRNA splicing in the LII–III regions of CaV2.2 and CaV2.3 genes involves an alternatively spliced cassette e18a that lies between constitutively expressed e18 and e19. The CaV2.1 gene lacks an equivalent alternatively spliced exon in this region. Cassette e18a is found in all mammalian and chick CaV2.2 and CaV2.3 genes, implying that the sequences it encodes regulate important cellular processes (Gray et al., 2007). Indeed, the fugu CaV2.2 gene also contains a cassette e18a. In rat and human CaV2.2 genes, e18a is located within a 9 kb long genomic sequence flanked by consensus ag–gt dinucleotide splicing recognition sequences (Gray et al., 2007). The 30 intron also contains alternative dinucleotide splice acceptors, ag, separated by three nucleotides that encode the amino acid arginine (R758). We know that both splice junctions are utilized because CaV2.2 mRNAs that lack and contain this amino acid have been isolated (Coppola et al., 1994; Cahill et al., 2000; Pan and Lipscombe, 2000; Kaneko et al., 2002). However, we do not know the function of R758 in CaV2.2. E18a in CaV2.2 is 63 nucleotides long and encodes 21 amino acids. Inclusion of e18a in CaV2.2 mRNAs varies by region in the human nervous system; e18a is expressed at higher levels in spinal cord compared to whole brain (Gray et al., 2007). Similarly, in rat, we have shown that CaV2.2e[18a] mRNAs are especially prevalent in adult sympathetic ganglia (>80%), but are expressed at lower levels in rostral brain structures such as neocortex (<20%) (Pan and Lipscombe, 2000; Gray et al., 2007). E18a splicing also varies with development; CaV2.2e[18a] mRNAs are not detectable in human fetal whole brain but are upregulated late in development. A similar pattern of regulation appears in rat neuronal tissues. In sympathetic ganglia of one-day old rats, CaV2.2e[18a] constitutes only 15% of total CaV2.2 mRNAs, whereas in the same tissue in adults, 70% of all CaV2.2 mRNAs contain e18a. This striking

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developmental switch in e18a inclusion in CaV2.2 suggests that alternative splicing of e18a might play a role in neuronal differentiation and synapse maturation (Gray et al., 2007). In addition to these regional and developmental influences, e18a inclusion might depend on cell type because CaV2.2e[18a] mRNAs appear to localize with monoaminergic neurons of adult rat brains (Ghasemzadeh et al., 1999). E18a in CaV2.3 is only 25% homologous to e18a in CaV2.2; perhaps e18a in these two genes evolved to serve different functions. Alternatively, the precise amino acid sequence of e18a could be inconsequential for its function if, for example, its purpose is disruption of a ligand or protein binding domain. E18a of CaV2.3 is slightly shorter: it is 57 nucleotides long and encodes 19 amino acids (Williams et al., 1994; Pereverzev et al., 2002; Gray et al., 2007). E18a mammalian and avian orthologs are homologous, suggesting that they retain a similar function in CaV2.2 and CaV2.3 genes of different species (Gray et al., 2007). E18a inclusion in CaV2.3 mRNA is under cellular control in human tissues (Williams et al., 1994). Interestingly, the distribution of CaV2.3e[18a] mRNAs is approximately reciprocal to CaV2.2e[18a] mRNAs. As maturation proceeds, CaV2.2e [18a] mRNA levels increase while CaV2.3e[18a] mRNA levels decrease (Gray et al., 2007). There is limited information about the regional distribution of CaV2.3 mRNA isoforms, but RT-PCR analyses point to preferential expression of e18a in neocortex and cerebellum (Schramm et al., 1999; Pereverzev et al., 2002), where CaV2.2e[18a] mRNA levels are relatively low. Additional data are needed to establish if cells coordinate alternative splicing of e18a in CaV2.2 and CaV2.3 pre-mRNAs, but such experiments could point to common cellular splicing factors. 8.2.3.2 CaV2.2 Exon 18a Regulates Cumulative Inactivation E18a influences the biophysical properties of CaV2.2 and CaV2.3 channels, although the details of e18a-dependent modulation differ between channel subtypes. N-type calcium channels that contain the CaV2.2e[18a] splice isoform are less sensitive to closedstate inactivation (Pan and Lipscombe, 2000; Thaler et al., 2004) compared to channels that contain CaV2.2 isoforms lacking e18a (CaV2.2e[D18a]). In contrast, R-type channels that contain the CaV2.3e[18a] isoform exhibit increased sensitivity to calcium-dependent current enhancement (Pereverzev et al., 2002; Leroy et al., 2003) and stimulation by phorbol ester as compared to channels that contain the CaV2.3e [D18a] isoform (Klockner et al., 2004). The influence of e18a on steady-state inactivation of CaV2.2 N-type channels varies depending on the auxiliary CaVb subunit present (Pan and Lipscombe, 2000), but inclusion of e18a consistently reduces the likelihood that the N-type channel will enter closed-state inactivation (Thaler et al., 2004). Physiologically, this means that N-type channels are able to maintain calcium influx during trains of action potential-like stimuli when they contain the CaV2.2e[18a] isoform. We believe that during maturation of the nervous system, especially in peripheral ganglia, e18a is more commonly inserted into CaV2.2 mRNAs, generating N-type channels with increased fidelity of excitation–secretion coupling at the synapse. The e18a sequence is also close to the site of SNARE binding in CaV2.2 (Fig. 8.4), raising the possibility of involvement in excitation–secretion coupling. Indeed, Catterall

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and colleagues showed that LII–III isoforms of CaV2.1 (distinguished by sequence variation that cannot arise from splicing of e18a) differ in their ability to bind certain SNARE proteins (Catterall, 1999). The influence of e18a on protein binding to LII–III of CaV2.2 needs to be tested. 8.2.3.3 Large Deletions in LII–III of CaV2.2 Several LII–III mRNA isoforms of CaV2.2 lack one to three exons, resulting in large deletions from LII–III (Kaneko et al., 2002). Indeed, deletion isoforms of CaV2.2 mRNAs have been amplified from human brain that lack critical elements required for binding of synaptic proteins to LII–III. One CaV2.2 isoform lacks the amino acid sequence encoded by e19 through e21 (Kaneko et al., 2002). Analysis of mammalian CaV2.1 genes confirms that premRNA skipping e19, e20 and e21 could occur via the use of consensus gt–ag dinucleotide splice motifs. E19 is unusually large, encoding 266 amino acids. Not surprisingly, CaV2.2 isoforms that lack large segments of LII–III also have altered channel properties (Kaneko et al., 2002). The voltage dependence of inactivation for two deletion isoforms of CaV2.2 is shifted toward more depolarized potentials, and these channels recover more rapidly from inactivation, as compared to CaV2.2 channels that contain a complete LII–III (Kaneko et al., 2002). These data, along with our data showing that inclusion of e18a protects channels from closed-state inactivation, indicate that structural components of LII–III are integral to inactivation of N-type channels. The CaV2.2 isoform lacking e19 though e21 appears to be less sensitive to Conus toxins, w-conotoxin MVIIA and GVIA, than isoforms that contain these exons. However, w-conotoxin MVIIA and GVIA preferentially inhibit the inactive state of the N-type channel (Stocker et al., 1997), and therefore a reduction in toxin sensitivity could arise as a secondary consequence of the decrease in channel inactivation. 8.2.3.4 LII–III Isoforms of CaV2.1 Two LII–III variants of CaV2.1 with different expression patterns in presynaptic nerve terminals were identified using isoformspecific antibodies (Westenbroek et al., 1992; Sakurai et al., 1996; Wu et al., 1999); however, their sequence identity and molecular origins are not known. Interestingly, these LII–III isoforms interact differentially with syntaxin, SNAP-25, and synaptotagmin (Rettig et al., 1996; Kim and Catterall, 1997; Zhong et al., 1999), suggesting that they differentially influence excitation–secretion coupling. We do not know if these two CaV2.1 isoforms, called rbA and BI, originate from alternative pre-mRNA splicing (Rettig et al., 1996). Two potential sites of alternative splicing exist in the LII–III region of the human CaV2.1 gene and involve e16 and e17. However, splicing at these loci is infrequent, based on analyses of mRNAs, leaving open the question of functional relevance (Soong et al., 2002). 8.2.4

The I–II Intracellular Loop

Splicing of e10 in CaV2.1 and CaV2.2 pre-mRNAs involves the use of different 30 acceptor sites and not the insertion or exclusion of a cassette exon as discussed for e18a

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and e31a. In CaV2.1, three alternate acceptor sites are used, giving rise to three fully processed CaV2.1 mRNA variants that contain either a (i) valine-glycine (VG) insert, (ii) single glycine insert (G), or (iii) no insert (Bourinet et al., 1999; Krovetz et al., 2000; Soong et al., 2002) at the e9/e10 junction. Valine (V421) in CaV2.1 is implicated in slowing down the time course of P-type channel inactivation (Bourinet et al., 1999) and G-protein-mediated inhibition of the P-type current (Bourinet et al., 1999) as well as influencing the pharmacological sensitivity of the P-type channel to mibefradil (Jimenez et al., 2000). CaV2.1 channels that lack V421 inactivate more rapidly. The effect on inactivation also indirectly renders the P-type channel more sensitive to inhibition by mibefradil. Mibefradil, a nonselective inhibitor of voltage-gated calcium channels, preferentially binds to the inactive state of the P-type channel (Bezprozvanny et al., 1995; Jimenez et al., 2000). The different pharmacological sensitivity of these CaV2.1 splice isoforms, therefore, stems from their distinct inactivation properties and not from a direct effect of splicing on the drug binding site. Nothing is known about the cellular regulation of alternative pre-mRNA splicing in the e10 acceptor site. It does not contribute to the characteristic slow inactivation kinetics of Purkinje cell P-type currents because CaV2.1 clones isolated from Purkinje cells by single-cell RT-PCR lack V421 (Tsunemi et al., 2002). We documented a similar pattern of alternative splicing at e10 of CaV2.2 pre-mRNAs. In CaV2.2, the alternate use of a 30 acceptor site at e10 controls the inclusion of an alanine (A415) (Lin et al., 1997). We found no evidence that inclusion or exclusion of A415 in CaV2.2 modifies channel function, and also no evidence has been found that suggests splice acceptor usage is cell specific (Lin et al., 1997).

8.3 FUTURE DIRECTIONS 8.3.1

Splicing Factors

There are still several aspects of alternative pre-mRNA splicing that we know little about, but perhaps the greatest gap of knowledge relates to the splicing factors that direct this process. There have been no systematic studies of known neuronal splicing factors to determine if any splicing factor can regulate exon selection in CaVa1 pre-mRNAs. Two splicing factors, Fox-1/2 and Nova-2, have been linked to alternative pre-mRNA splicing in CaV2.2, but to date, no studies addressing function have been reported. Fox-1/2 is a known splicing regulator of neuronal exons. The Fox binding sequence in pre-mRNAs is found at high frequency <100 nt downstream of brainspecific alternative exons in several species (Minovitsky et al., 2005). There are suggestions that the Fox binding motif UGCAUG is an important switch that drives developmental and differentiation-specific changes in pre-mRNA splicing (Minovitsky et al., 2005). Fox proteins act as repressors when they bind upstream of the exon and enhancers when they bind downstream (Underwood et al., 2005). Interestingly, a Fox binding motif UGCAUG is present upstream of e18a in CaV2.2. Similarly, Nova-1 and Nova-2 (neuron-specific KH-type RNA binding proteins) can repress or enhance exon inclusion depending where they bind on the pre-mRNA relative to the

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exon (Jensen et al., 2000; Ule et al., 2006). If Nova-1/2 binds within the exon, it represses exon inclusion and if Nova-1/2 binds to an intronic motif, it enhances exon inclusion. E24 of CaV2.2 is present in a hit list of exons predicted to be regulated by Nova-2 based on a differential screen in wild-type and Nova-2 knockout mice. Nova-2 is predicted to enhance e24a inclusion in CaV2.2 (Ule et al., 2005). Consistent with this, a cluster of Nova-1/2 binding elements are located 20–105 nt downstream of the 50 splice site of e24a. These and other potential splicing factors need to be tested for their involvement in cellular control of alternative splicing of CaV2.2 and other CaVa1 premRNAs. We expect this novel line of investigation will lead to new insights into the cellular mechanisms that control calcium channel activity and channel targeting. 8.3.2

Therapeutics

Voltage-gated calcium channels in smooth muscle are the targets of classic calcium channel blockers, the most important of which are the dihydropyridines, used for over 30 years to lower blood pressure. More recently, N-type calcium channel blockers have reached the clinic to treat individuals with persistent or neuropathic pain that is otherwise unresponsive to treatment. Ziconotide, which is currently in use, is a selective inhibitor of the N-type channel and is thought to mediate analgesia by inhibiting transmitter release from primary afferents of nociceptors (Chaplan et al., 1994; Bowersox et al., 1996; Brose et al., 1997; Cox, 2000). However, this drug has actions on N-type channels in regions outside the dorsal horn, including at sympathetic synapses resulting in loss of sympathetic tone and hypotension (Miljanich and Ramachandran, 1995; Vanegas and Schaible, 2000; Ino et al., 2001). Consequently, there is interest in developing drugs that preferentially target specific splice isoforms of the N-type calcium channel, including those in nociceptors that contribute to the symptoms of disease. Obviously, the CaV2.2e[37a] isoform, which is enriched in nociceptors, is a candidate target for treating certain forms of neuropathic pain (Bell et al., 2004; Altier et al., 2007; Raingo et al., 2007). The effectiveness of e37a isoformspecific siRNA to inhibit the symptoms of neuropathic pain in rats in vivo lends credence to a drug discovery plan that incorporates isoform-selective drug screening (Altier et al., 2007). The identification of additional splice isoforms of CaV2.2, as well as other calcium channels having unique tissue distribution, could suggest novel strategies for improving the therapeutic index of existing drugs and for the development of novel agents.

8.4 CONCLUSIONS Studies of alternative pre-mRNA splicing in CaV2 channels, and voltage-gated calcium channels in general, demonstrate that CaVa1 genes have an outstanding capacity to generate thousands of distinct splice isoforms. Neurons, in turn, express the necessary splicing factors to utilize this feature, coordinating exon inclusion and suppression as needed to fine-tune calcium channel activity. Comparative analyses of splice isoforms that are cell specific and/or conserved across different species reveal regions critical for

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physiologically relevant control of channel function. Cell-specific alternative premRNA splicing has directed us to those domains of the calcium channel that regulate its function. Valuable insights into calcium channel structure, function, and pharmacology will continue as more functionally relevant splice isoforms are identified.

ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grants NS29967 and NS055251 (D.L.).

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Vigues S, Gastaldi M, Massacrier A, Cau P, Valmier J, 2002. The alpha(1A) subunits of rat brain calcium channels are developmentally regulated by alternative RNA splicing. Neuroscience 113: 509–517. Wei X, Neely A, Lacerda AE, Olcese R, Stefani E, Perez-Reyes E, Birnbaumer L, 1994. Modification of Ca2þ channel activity by deletions at the carboxyl terminus of the cardiac alpha 1 subunit. J Biol Chem 269: 1635–1640. Welling A, Ludwig A, Zimmer S, Klugbauer N, Flockerzi V, Hofmann F, 1997. Alternatively spliced IS6 segments of the alpha 1C gene determine the tissue-specific dihydropyridine sensitivity of cardiac and vascular smooth muscle L-type Ca2þ channels. Circ Res 81: 526– 532. Westenbroek RE, Hell JW, Warner C, Dubel SJ, Snutch TP, Catterall WA, 1992. Biochemical properties and subcellular distribution of an N-type calcium channel alpha 1 subunit. Neuron 9: 1099–1115. Williams ME, Brust PF, Feldman DH, Patthi S, Simerson S, Maroufi A, McCue AF, Velicelebi G, Ellis SB, Harpold MM, 1992. Structure and functional expression of an omegaconotoxin-sensitive human N-type calcium channel. Science 257: 389–395. Williams ME, Marubio LM, Deal CR, Hans M, Brust PF, Philipson LH, Miller RJ, Johnson EC, Harpold MM, Ellis SB, 1994. Structure and functional characterization of neuronal alpha 1E calcium channel subtypes. J Biol Chem 269: 22347–22357. Wu LG, Westenbroek RE, Borst JG, Catterall WA, Sakmann B, 1999. Calcium channel types with distinct presynaptic localization couple differentially to transmitter release in single calyx-type synapses. J Neurosci 19: 726–736. Wykes RC, Bauer CS, Khan SU, Weiss JL, Seward EP, 2007. Differential regulation of endogenous N- and P/Q-type Ca2þ channel inactivation by Ca2þ/calmodulin impacts on their ability to support exocytosis in chromaffin cells. J Neurosci 27: 5236–5248. Xu W, Lipscombe D, 2001. Neuronal Cav1.3alpha(1) L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines. J Neurosci 21: 5944–5951. Yamada Y, Nagashima M, Tsutsuura M, Kobayashi T, Seki S, Makita N, Horio Y, Tohse N, 2001. Cloning of a functional splice variant of L-type calcium channel beta 2 subunit from rat heart. J Biol Chem 276: 47163–47170. Yang Y, Chen X, Margulies K, Jeevanandam V, Pollack P, Bailey BA, Houser SR, 2000. L-type Ca2þ channel alpha 1c subunit isoform switching in failing human ventricular myocardium. J Mol Cell Cardiol 32: 973–984. Yatani A, Kamp TJ, 2000. Tails of the L-type Ca2þ channel: to sense oxygen or not. Circ Res 87: 535–536. Zhang W, Liu H, Han K, Grabowski PJ, 2002. Region-specific alternative splicing in the nervous system: implications for regulation by the RNA-binding protein NAPOR. RNA 8: 671–685. Zhong H, Yokoyama CT, Scheuer T, Catterall WA, 1999. Reciprocal regulation of P/Q-type Ca2þ channels by SNAP-25, syntaxin and synaptotagmin. Nat Neurosci 2: 939–941. Zhong X, Liu JR, Kyle JW, Hanck DA, Agnew WS, 2006. A profile of alternative RNA splicing and transcript variation of CACNA1H, a human T-channel gene candidate for idiopathic generalized epilepsies. Hum Mol Genet 15: 1497–1512. Zhuchenko O, Bailey J, Bonnen P, Ashizawa T, Stockton DW, Amos C, Dobyns WB, Subramony SH, Zoghbi HY, Lee CC, 1997. Autosomal dominant cerebellar ataxia (SCA6)

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associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel. Nat Genet 15: 62–69. Zipursky SL, Wojtowicz WM, Hattori D, 2006. Got diversity? Wiring the fly brain with Dscam. Trends Biochem Sci 31: 581–588. Zuhlke RD, Bouron A, Soldatov NM, Reuter H, 1998. Ca2þ channel sensitivity towards the blocker isradipine is affected by alternative splicing of the human alpha1C subunit gene. FEBS Lett 427: 220–224. Zuhlke RD, Pitt GS, Deisseroth K, Tsien RW, Reuter H, 1999. Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 399: 159–162.

9 EFFECT OF HYPOXIA/ISCHEMIA ON VOLTAGE-DEPENDENT CHANNELS XIANG Q. GU1, HANG YAO1,

AND

GABRIEL G. HADDAD1,2,3

1 Department of Pediatrics, 9500 Gilman Drive, University of California at San Diego, San Diego, CA 92093-0735, USA 2 Department of Neuroscience, 9500 Gilman Drive, University of California at San Diego, San Diego, CA 92093-0735, USA 3 The Rady Children’s Hospital-San Diego, 3020 Children’s Way, San Diego, CA 92123, USA

9.1 INTRODUCTION In mammals, oxygen is a key nutrient for life and survival. The central nervous system (CNS) is one of the most sensitive and vulnerable tissues to a hypoxic insult. Oxygen deprivation for 5–10 min leads to irreversible damage. To cope with hypoxia or anoxia, mammals have evolved and developed strategies. Some of the immediate effectors are ion channels or transporters (or ionic exchangers), and a number of membrane proteins play an important role in hypoxia and ischemia in various organs, including brain, heart, and kidneys. Hypoxia and O2 deprivation are sensed by cells in the body by various mechanisms. In spite of the fact that respiratory physiologists have advanced and supported the idea that the major sensor for low O2 is the carotid body, it is clear now that almost every cell in the organism senses a lack of O2, albeit at different levels of O2. The carotid glomus cells may sense decreasing O2 at a higher level, that is, at a level of about 55–60 torr, and may therefore be one of the earliest sensors of impending hypoxia. However,

Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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adrenal, liver, renal, nerve, lung, and muscle cells, to name a few, also sense and respond to low O2 independently (Haddad and Jiang, 1993). Ion channels play a major role in the cellular response to hypoxia. The importance of these responses lies in their timescale. Typically, ion channels allow cells to respond on the scale of seconds and minutes, and this provides some of these cells with the ability to respond rapidly for homeostatic purposes. Consider, for example, the smooth muscle cells in the pulmonary arteries: during hypoxia, certain Kþ channels are inactivated/blocked and such muscle cells consequently depolarize, activating voltage-sensitive Ca2þ channels, which, in turn, increase intracellular Ca2þ. This initiates a cascade that leads to constriction of these arteries (McMurtry et al., 1976; Harder et al., 1985). This helps in shunting blood to more oxygenated lung areas, preserving homeostasis and ventilation/perfusion ratios as close to normal as possible. The rapidity of these types of responses is clearly not limited to the lungs. Similarly, rapid responses are important in cerebral arteries as well as in nerve and glial cells. In this chapter, we describe how ion channels respond to low O2 in many tissues, including nerve and glia. In addition to ion channels, plasma membrane exchangers and transporters also participate in responses to hypoxia. For example, the Naþ/Kþ pump in nerve cells, the Naþ/HCO3 cotransporters in nerve and glia, and the Naþ/Hþ exchanger in renal tubular cells have all been shown to play significant roles in cellular responses to hypoxia or ischemia. There are both short-term and long-term effects of hypoxia on ion channels and transporters. Although there is now little doubt that membrane proteins are major contributors to the acute response of a cell to O2 deprivation, there is now also substantial evidence that changes occur in the expression or activities of such proteins over more prolonged time periods (Lipton, 1999). The nature of these changes differs from cell to cell, from one O2 level to another, and from age to age. One important unresolved question is why different cells have very different levels of susceptibility to low O2, and what are the roles of ion channels in the phenotype of tolerant versus susceptible cells. For example, within hours of the onset of hypoxic conditions, the expression of voltage-dependent Naþ channels is decreased in the plasmalemma of turtle neurons. In contrast, no such downregulation occurs in mammalian nerve cells. While these Naþ channels behave differently in the respective organisms, they are part and parcel of a whole system of response in the turtle versus the mammal. We believe that in each organism, there is coordination between various cellular compartments in such a way that ion channels, for example, behave in a way that is consistent with an overall strategy in the cell, whether in the mouse or the turtle. The cellular strategy, in turn, reflects the ecological niche occupied by the organism. In this study, we highlight the effect of hypoxia on membrane proteins and how ion channels and exchangers/transporters contribute to cellular responses. While there are still controversies in some areas, there is now extensive evidence for a role for membrane proteins in the response to conditions of O2 deprivation.

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9.2 EXPERIMENTAL MODELS FOR THE STUDY OF HYPOXIA/ISCHEMIA Oxygen deprivation resulting from cardiac arrest, cerebral stroke, or carbon monoxide poisoning can lead to profound tissue damage in the CNS. Many experimental models that simulate the pathological changes in the CNS have been developed to study the molecular and cellular pathobiological events resulting from hypoxia/ischemia. In vivo models include the constriction of blood supply to the whole brain to replicate global ischemia, such as the four-vessel occlusion model (4-VO) (Pulsinelli et al., 1982); the blockage of one major cerebral blood vessel to simulate focal cerebral ischemia, such as middle cerebral artery occlusion (MCAO) (Feustel et al., 1981); and the exposure of an animal to an artificial atmosphere in which oxygen (and CO2) can be manipulated to simulate certain stress situations, such as carbon monoxide poisoning and sleep apnea (Gu and Haddad, 2001). To facilitate more detailed studies, a number of in vitro models have also been developed to serve different purposes. Generally, brain tissues or isolated cells are used to study mechanisms of survival or cell death and these preparations are subjected to an artificial environment in which the level of oxygen can be manipulated depending on the experimental design. In addition, other components such as CO2, glucose, and ion alterations can also be set as needed (Yao et al., 2007). To study the cellular response as a function of graded oxygen level, one usually employs an experimental condition such as anoxia or hypoxia. To achieve anoxia, a “no oxygen’’ condition, an oxygen scavenger (e.g., sodium dithionate or glucose oxidase) is usually added to the argon- or nitrogen-bubbled solution. However, caution must be exercised when using oxygen scavengers since nonspecific changes could make interpretation rather difficult. The amount of oxygen under hypoxia can vary, depending on the question of interest. In any event, both anoxia and hypoxia can be easily employed in freshly prepared or cultured brain tissues (Croning and Haddad, 1998) or isolated cells for online functional studies. To study the chronic effect of constant hypoxia, intermittent hypoxia, or the combination of constant hypoxia and hypercapnia at the cellular level, one can subject cultured brain tissues or isolated cells to these environments. Another method used in this field is to freshly prepare brain slices or isolate cells from different brain regions of animals that had been exposed to different environments for different periods of time and conduct functional experiments. Together with gene profile analysis and RNA and protein quantification approaches, investigators can explore the functional change in a candidate protein, such as an ion channel, and interpret it using not only functional but also molecular tools (Ma et al., 2001). The combination of low oxygen with other pathological conditions, such as low glucose, low pH, high CO2, or ion shifts possibly simulate different pathological conditions seen in clinical settings. Oxygen glucose deprivation (OGD) is widely used to mimic the microenvironment in the ischemic infarct core during focal cerebral ischemia or in brain tissue during global ischemia. Recently we have developed an

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in vitro ischemic model to simulate cell death in the ischemic infarct penumbra during focal cerebral ischemia and investigate potential mechanisms of cell death underlying the progressive expansion of the ischemic infarct core following focal ischemia (Yao et al., 2007).

9.3 ION CHANNELS AND THEIR ROLE IN HYPOXIA 9.3.1

Overall Excitability of Neurons in the CNS

Neurons in the CNS respond to hypoxia and anoxia in a stereotypical way. In general, mature and differentiated neurons start to depolarize. If the low O2 persists for several minutes, neurons undergo a more profound depolarization (hypoxic depolarization) and eventually the membrane potential (Vm) reaches close to zero. If this condition persists, input resistance is greatly reduced, permeability of the cell membrane increases, and there is major leakage of cellular constituents out of the cells. In general, such changes become irreversible after 5–20 min of O2 deprivation in the CNS. There are a number of factors that modify such depolarization with hypoxia. We have, for example, shown that this depolarization depends on the cell type. Vagal motoneurons, hypoglossal motoneurons, hippocampal neurons, and cortical neurons behave differently during hypoxia (O’Reilly et al., 1995), although all depolarize after several minutes. Typically, hippocampal neurons hyperpolarize for the first few to several minutes (Krnjevic and Leblond, 1989), while hypoglossal neurons do not hyperpolarize before their depolarization (Haddad and Donnelly, 1990). Another important variable is the age of the animal and the neuron. Immature neurons (age below 10 days) are much more resilient to depolarization (Haddad and Donnelly, 1990). In general, it takes three–four times longer for these neurons to depolarize than mature ones (Haddad and Donnelly, 1990). Temperature, level of O2, distance from capillaries, level of glucose, ion channel expression in the plasmalemma, surface to volume ratio, and metabolic rate of specific nerve cells are all factors (Jiang et al., 1991, 1992; Xia and Haddad, 1991; Donnelly et al., 1992) that become very critical in the development of the hypoxia-induced depolarization (Jiang et al., 1991). Of major interest to a number of laboratories is the difference between anoxia-tolerant and anoxia-sensitive neurons. In the tolerant turtle, the depolarization occurs over a much slower time course, many minutes and hours (Haddad and Donnelly, 1989), rather than several minutes. This much slower depolarization in the turtle neuron occurs even at temperatures that are similar to temperatures of mammalian neurons, indicating that temperature cannot explain the slow rate of depolarization in turtle neurons. 9.3.2

Voltage-Sensitive Calcium Channels (VSCCs)

Hypoxia/ischemia is characterized by a disruption of major ionic species, especially the Ca2þ ion that accumulates intracellularly (increasing its intracellular concentration

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up to four orders of magnitude). A sustained and large increase in intracellular calcium [Ca2þ]i is harmful to cells since it can activate calcium-dependent enzymes such as lipases, proteases, endonucleases, and phosphatases. These enzymes have the potential to trigger a cell death program or otherwise promote demise of the cell. Cellular Ca2þ overload can result from multiple pathways, such as a change in the activity of membrane proteins that modulate Ca2þ influx and/or efflux, the release of Ca2þ from intracellular Ca2þ stores, or a change in Ca2þ buffering capacity. Among these, Ca2þ influx has been considered to be an important mechanism that contributes to hypoxia/ischemia-induced cell damage. Voltage-sensitive calcium channels (VSCCs) are readily activated by hypoxic depolarization and play an important role in intracellular Ca2þ increase and neurodegeneration. VSCCs are classified as T, L, N, P, Q, and R subtypes. Among them, the L-type Ca2þ channel (L-VSCC), located primarily on the neuronal soma, has been studied intensively and implicated to be part of a major pathway for Ca2þ influx and neuronal death following hypoxia/ischemia (see Chapter 1). In cortical slice preparations, for example, the dihydropyridine L-VSCC blocker nifedipine significantly attenuates OGD-induced Ca2þ influx in pyramidal neurons, while the L-VSCC agonist BAY K8644 enhances this Ca2þ entry (Pisani et al., 1998). Electrophysiological experiments demonstrated that low O2 can indeed enhance the activity of L-VSCCs. Wholecell or single Ca2þ channel recordings from inspiratory neurons show that hypoxia potentiates an L-type Ca2þ current and this effect can be blocked by both nifedipine and nitrendipine (Mironov and Richter, 1998). The involvement of L-VSCCs in anoxia-induced neuronal death was also observed in a model of chemical anoxia. In cultured cerebellar granule cells, sodium azide induces a significant amount of cell death assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Mosmann, 1983) and nifedipine alleviates this cell damage. This evidence supports the notion that low O2 enhances the channel activity of L-VSCC, which results in the influx of Ca2þ, the accumulation of [Ca2þ]i, and cell death. In addition to L-VSCCs, other subtypes such as N- and T-type Ca2þ channels have also been reported to be involved in hypoxia/ischemia-induced neuronal injury. N-type Ca2þ channels (N-VSCC) were found to be responsible for OGD-induced neuronal damage (Pringle et al., 1996). A selective blocker for N-VSCC, omega conotoxin MVIIA was found to attenuate the damage caused by OGD in this study. Another subclass of VSCC is the T-type Ca2þ channel (T-VSCC), which is activated by membrane depolarizations smaller than those required to open other types of VSCCs (Chapter 2). Although Ca2þ entry through T-VSCC does not seem to be a major contributor to the [Ca2þ]i increase following acute hypoxia, these channels may play an important role in Ca2þ influx during chronic hypoxia. In PC12 cells, for example, mRNA for the channel subunit a1H (the most abundant subunit of T-VSCC) increases significantly in response to chronic hypoxia and this change in a1H mRNA is time and dose dependent (Del Toro et al., 2003). Moreover, the density of the T-type calcium channel current also increases in parallel to the accumulation of a1H mRNA in these cells. Perhaps most importantly, this change in a1H expression is mediated by the HIF (hypoxia-inducible factor) transcription factor, which “senses’’ oxygen concentrations through the hydroxylation of two proline residues, and appears

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to be specific for T-VSCC, because it is not observed with the L-, N-, and P/Q-channel types. 9.3.3

Naþ Channels

9.3.3.1 Transient Naþ Currents Acute hypoxia depresses the transient voltagedependent Naþ current (NaT) in the CNS (Cummins et al., 1993; O’Reilly et al., 1997; Fearon and Brown, 2004). This transient Naþ current, which was first defined by Hodgkin and Huxley (1952), activates and inactivates rapidly in response to depolarization and shapes the upstroke of the action potential. Hypoxia modifies the gating properties of the transient Naþ channel by increasing the time to peak current and the time constant of inactivation and by causing a negative shift in the steady-state inactivation curve without affecting other properties such as voltage– conductance relations (O’Reilly et al., 1997; Fearon and Brown, 2004). The inhibition of the Naþ current by hypoxia is believed to be mediated by protein kinase C (PKC) because (i) a phosphorylation site in the Naþ channel was found for modulation by PKC (Numann et al., 1991; West et al., 1991), (ii) hypoxia upregulates a number of kinases, and (iii) the effects of hypoxia on channel properties were found to be attenuated by PKC blockers (O’Reilly et al., 1997). The effects of prolonged or chronic exposure to hypoxia have received experimental attention, mostly because of their implications for mountain sickness in highaltitude sojourners and patients with chronic cardiopulmonary diseases. Chronic hypoxia has been reported to augment NaT current density by as much as 50%, but without any observed effects on the time to the peak, the time constant of inactivation, or on the gating properties of the Naþ current, such as its voltage–conductance relationship or its steady-state inactivation (Fearon and Brown, 2004). In cultured chemosensitive glomus cells, the Naþ current density was reported to be increased by sixfold in 13 days following exposure to hypoxia (Stea et al., 1992). Furthermore, after prolonged exposure to hypoxia, 80% of neocortical neurons were reported to undergo a more than 20 mV depolarization in response to brief acute hypoxia, as compared with almost no effect in naive cortical neurons (Xia et al., 2000), and this effect has been attributed to the increase in Naþ channels under hypoxia. Not all brain regions respond to hypoxia in the same manner. For example, mRNA for Naþ channels has been reported to increase by 80% in cortex and hippocampus, but to decrease in the callosum with no change in the thalamus after 30 days in a hypoxic environment (Xia et al., 2000). Chronic cyclic hypoxia, which occurs in patients with sleep apnea and in other respiratory diseases, has been reported to have effects similar to those of chronic hypoxia. In an animal model of sleep apnea, alternating the level of O2 in 2-min periods in 7.5% oxygen with 3-min periods at 21% oxygen for 8 h a day for 2 weeks resulted in increased excitability in hippocampal neurons. This increased excitability was mainly due to a large increase in Naþ channel current density and a shift of the steady-state inactivation toward positive potentials, which results in an increase in available Naþ channels at the resting potential (Gu and Haddad, 2003). In contrast, with longer exposures to cyclic hypoxia, the same neurons became less

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excitable, with a lower Naþ current density (Gu and Haddad, 2001). Apparently, several factors are important in modulating the effect of hypoxia: duration of the stress, region of the brain, and age of the animal. 9.3.3.2 Persistent Naþ Currents An increase in [Naþ]i is believed to be one of the first responses to acute hypoxia, which precedes even the rise in [Ca2þ]i (Friedman and Haddad, 1994; Haigney et al., 1994). It turns out that although transient Naþ current is decreased in some neurons, an increase in overall Naþ current occurs because of an increase in a persistent Naþ current. This persistent Naþ current was found in neurons (Stafstrom et al., 1982; Gilly and Armstrong, 1984), skeletal muscles (Gage et al., 1989), and cardiac myocytes (Saint et al., 1992). The whole-cell persistent Naþ current has only 0.5–5% of the amplitude of the transient Naþ current, activating at a voltage range near the resting potential and not inactivating for up to hundreds of milliseconds. This persistent Naþ current (NaP), in contrast to NaT (transient, or classical Naþ current), is more sensitive to Naþ channel blockers TTX, lidocaine (local anesthetics) (Hammarstrom and Gage, 1998), low dosages of riluzole (an inhibitor of both voltagegated ion channels and of glutamate neurotransmission) (Faustino and Donnelly, 2006), ranolazine (an antianginal and anti-ischemic drug) (Antzelevitch et al., 2004; Belardinelli et al., 2004), and QX-314 (relatively selective blocker of NaP) (Stafstrom et al., 1982). Though NaP current is generally small, its long-lasting open state makes it an important player in the excitability of neurons, muscles, and cardiac myocytes. Since NaP differs from NaT in many of its properties, it may be expected to have a different response to hypoxia. Indeed, this appears to be the case. The amplitude, open probability of single NaP channels, and the amplitude of whole-cell NaP currents were all increased by hypoxia in ventricular myoctyes (Ju et al., 1996; Zhou et al., 2006), in hippocampal neurons (Hammarstrom and Gage, 1998), in hypothalamic neurons (Horn and Waldrop, 2000), and in neurons of the brain stem (Kawai et al., 1999). Whole-cell currents were also increased in human embryonic kidney (HEK) cells stably expressing hNav1.5 a subunits (Fearon and Brown, 2004). Hypoxia-induced increases in firing frequency have also been reported to be linked to NaP in carotid body glomus cells (Faustino and Donnelly, 2006). Interestingly, the effect of hypoxia on NaP persists even in excised inside-out patches (Hammarstrom and Gage, 2000), implying that the cytosolic components are not important for hypoxic augmentation of the NaP current. This increase in amplitude of NaP currents produced by acute hypoxia and cyanide is opposite to the effect of acute hypoxia and cyanide on NaT currents, which were both decreased. Naþ concentrations are an important component of ionic homeostasis in many cells, including neurons, and the Naþ channel is an important target for the treatment of hypoxic stress. Investigators have tried to use a variety of means to reduce injury caused by hypoxia and one of these is the use of channel blockers. Blocking Naþ channels has a protective role in hypoxia-induced injury and cell death (Prenen et al., 1988; Boening et al., 1989; Urenjak and Obrenovitch, 1996; Xia et al., 2000). For example, the Naþ channel blocker TTX was reported to attenuate depolarization induced by hypoxia (Xia et al., 2000) and reduce the decrease in

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adenosine triphosphate (ATP) during hypoxia (Kass et al., 1992). How TTX protects cells against hypoxic injury is not completely understood. One potential mechanism is through effects on apoptotic pathways as was recently demonstrated by Banasiak et al. (2004). NaP channels are the most likely targets of blockers that result in protection, because blocking NaP has been reported to be protective in reducing injury from hypoxic insults (Stys et al., 1992; Lynch et al., 1995; Taylor and Meldrum, 1995; Fung, 2000; Belardinelli et al., 2006). 9.3.3.3 Naþ Channels of Invertebrates, Reptiles, and Newborns Animals in different species and phyla, living in environments with different concentrations of O2, have evolved different responses to hypoxia. Some invertebrates and reptiles have developed specific strategies to cope with a lack of oxygen. In turtles, for example, neurons continue to fire action potentials even after 2 h in an environment totally devoid of O2 (Haddad, 1996). Turtle neurons can also depress, but not totally suppress, their electrical activity during anoxia (Feng et al., 1988). One factor implicated in this tolerance is the reduced density of voltage-gated Naþ channels in the turtle brain (Perez-Pinzon et al., 1992) and the lack of significant hypoxia-induced Naþ influx into neurons. We also found that compared to mammalian neurons, Drosophila embryonic neurons express fewer Naþ channels per unit area of membrane than neurons of mouse brain (Gu and Haddad, unpublished data). Such reduced Naþ channel density may result in lowered Naþ permeability during hypoxia, as is the case for turtle brain (Fernandes et al. 1997), and in Drosophila embryonic neurons (Gu and Haddad, 1999). Reduced entry of Naþ during hypoxia or anoxia alleviates the burden on Naþ/Kþ ATP pumps and lowers consumption of ATP. This leads to a channel and metabolic arrest state, which is articulated in the channel arrest hypothesis that provides a potential explanation for how hypoxia-tolerant animals cope with decreases in O2 (Lutz et al., 1985; Hochachka, 1986). Neonatal neurons are known to be much more tolerant to hypoxia or anoxia than their adult counterparts. Newborn rats can survive for 50 min in 100% N2 as compared to only 5 min in 15-day-old rats (mean arterial PO2 ¼ 0.2 vol%) (Fazekas et al., 1941). One of the reasons for this tolerance may be the lower Naþ channel density in the newborns. It has been reported that rat neonatal brain possesses 10-fold fewer Naþ channels than adults (Xia and Haddad, 1994). In cultured spinal cord neurons, it has been estimated that the number of Naþ channels is less than 2/mm2 in 1–2-day-old rats (MacDermott and Westbrook, 1986), which is about 30–40-fold lower than that in 14–21-day-old rats (Catterall, 1984). Whether in invertebrates, turtles, or newborn mammals, it is possible that reduced Naþ channel density confers hypoxic resistance. Although it is clear that resistance or susceptibility to low O2 in cells and tissues is much more complicated than just one factor, that is, Naþ channel availability or expression, there is little doubt that Naþ entry plays an important role in determining cell fate when tissues are deprived of O2. 9.3.3.4 Epithelial Naþ Channels Another type of Naþ channel that responds to hypoxia is the epithelial sodium channel (ENaC, also termed the sodium channel

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non-neuronal 1, SCNN1, or amiloride-sensitive sodium channel, ASSC). ENaC is located in the apical membrane of epithelial cells in the kidney, lung, and colon. It is involved in transepithelial Naþ ion transport, which it accomplishes together with the Naþ/Kþ-ATPase. ENaC plays a major role in the Naþ and Kþ ion homeostasis of blood, epithelia, and extraepithelial fluids by resorption of Naþ ions. Exposure to severe hypoxia for 3–12 h inhibits ENaC activity as measured by amiloride-sensitive 22 Naþ influx in rat ATII cells (Planes et al., 1997). Reduction of O2 to 3% for 24 h has been reported to reduce the transepithelial Naþ current and ENaC activity of alveolar epithelial cells without decreasing ENaC mRNA or protein levels (Planes et al., 2002). Exposure to normobaric hypoxia for 24 h had no effect on ENaC expression in lung (Carpenter et al., 2003). Hypoxia (up to 24 h) seems therefore to affect only the activity of ENaC without affecting the expression of the channel.

9.3.4

Kþ Channels

9.3.4.1 Delayed Rectifier Kþ Channel Delayed rectifier channels were first described by Hodgkin et al. (1949). Delayed rectifier channels are found in unmyelinated axons, motoneurons, vertebrate fast skeletal muscles, smooth muscles, and cardiac muscles. They are required for rapid recovery to the resting state after the peak of the action potential and for the maintenance of resting membrane potentials. In general, under pathophysiological conditions such as hypoxia and ischemia, delayed rectifier channels are either shut down or inhibited. For example, when studied in heterologous expression systems, currents produced by homomeric a subunits of the voltage-dependent Kþ channels, KV1.2, KV1.5, KV2.1, and KV3.1, and heteromeric a subunits of KV1.2/KV1.5 and KV2.1/KV9.3 are all decreased by hypoxia (Moudgil et al., 2006). Studies of hypoxia on channel expression yield some heterogeneous results. Prolonged exposure to hypoxia (18 h) has been reported to increase the expression of KV1.2 but not KV1.3, KV2.1, and KV3.1 in PC12 cells (Conforti and Millhorn, 1997). However, longer exposure to hypoxia (24–60 h) has been reported to decrease significantly the mRNA levels of the pore-forming KV channel a subunits, KV1.2 and KV1.5, as well as protein levels in pulmonary arterial smooth muscle cells (Wang et al., 1997). Although the pore-forming KV channel a subunits are targets of prolonged hypoxia, the mRNA levels for the modulatory/ regulatory KV channel b subunits are negligibly affected in pulmonary arterial smooth muscle cells (KVb1, KVb2, and KVb3) (Wang et al., 1997). Thus, the diminished transcription and expression of KV a subunits may reduce the number of KV channels and decrease KV currents. The nature of the exposure to hypoxia may also influence the effect on KV channels. For example, acute hypoxia after chronic hypoxia is reported to cause more inhibition of KV1.2 than acute hypoxia alone in PC12 cells (Conforti and Millhorn, 1997). Another example is that the endogenous-delayed rectifier Kþ channel in the human lung adenocarcinoma cell line A549 has been reported to be activated following reoxygenation but not during the hypoxic phase (Koong et al., 1993).

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9.3.4.2 BK Channel The BK (“big K’’ or Maxi K) channel represents one of the three classes of Ca2þ-sensitive Kþ channels: BK (large-conductance K), SK (small-conductance K), and IK (intermediate-conductance K). The name BK is derived from its large single-channel slope conductance, usually around 100– 300 pS measured in symmetrical intracellular and extracellular solutions. BK channels play important roles in cells such as in muscles, neurons, and glia in both mammals and invertebrates (see Chapter 12). Under abnormal conditions, such as hypoxia and ischemia, this channel is usually inhibited or its activity is decreased. Hypoxia inhibits BK channel activity in the plasma membranes of neurons (Liu et al., 1999a; McCartney et al., 2005), chemoreceptor cells (Peers, 1990; Riesco-Fagundo et al., 2001), recombinant HEK (Williams et al., 2004), alveolar epithelial cells (Jovanovic et al., 2003), and fetal pulmonary artery smooth muscle cells (Porter et al., 2001). Hypoxia also decreases the open probability of BK channels in the singlecell-attached mode (Liu et al., 1999a) (Figs 9.1 and 9.2) but does not affect its singlechannel slope conductance (Porter et al., 2001; Riesco-Fagundo et al., 2001; Gao and Fung, 2002; Jovanovic et al., 2003; Williams et al., 2004; McCartney et al., 2005). In inside-out patches, hypoxia does not have any effect on BK channels from cortical neurons of mouse (Liu et al., 1999a) or cells in the rabbit carotid body (Ganfornina and Lopez-Barneo, 1991) or on the open probability of BK channels from canine pulmonary arteries (Post et al., 1995). Since the effect of hypoxia is present in cell-attached patches and absent in the inside-out patches, cytosolic components appear to be necessary for the effect of hypoxia on BK (Liu et al., 1999a) (Fig. 9.2). This does not seem to be universal, because BK channel activity from inside-out patches in membrane-delimited preparations (Peng et al., 1999; Lewis et al., 2002; Hartness et al., 2003) can still be decreased by hypoxia. In addition, it has been postulated that although the a subunit of BK alone is not sensitive to hypoxia, BK channels become sensitive to hypoxia on association with an unidentified oxygen sensor and/or b subunits (Coppock et al., 2001). 9.3.4.3 SK Channel The SK channel, which has a single-channel slope conductance between 4–20 pS, is a voltage-insensitive, Ca2þ- and apamin- (a bee venom) sensitive channel. SK is sensitive to hypoxia, and, again, hypoxia generally inhibits the SK channel. For example, the SK current of chromaffin cells in sheep adrenal medulla decreases when these cells are exposed to hypoxia (Keating et al., 2001). In contrast, however, SK channels in rabbit carotid body measured in inside-out patches are not sensitive to hypoxia (Ganfornina and Lopez-Barneo, 1992), perhaps reflecting a requirement for cytosolic components or other subunits in the response to hypoxia. A recent study found that overexpression of SK3 induced abnormal respiratory responses to hypoxia and compromised parturition, suggesting that SK3 channels could be potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor (Bond et al., 2000). 9.3.4.4 IK Channel An intermediate-conductance (IK) calcium-activated channel subunit termed SK4 (Ishii et al., 1997; Joiner et al., 1997) has been found

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FIGURE 9.1 Relationship between oxygen concentrations and single-channel open probabilities of a large-conductance Kþ channel (200 pS in symmetrical Kþ solutions). The recording is from an inside-out patch of an acutely dissociated neuron of rat neocortex. This channel is calcium and ATP sensitive. Its open probability reversibly decreased with a decreased concentration of oxygen in the perfusion solution, resulting from a cytosol-independent and membrane-delimited mechanism (From Figure 2, Jiang and Haddad, 1994, with permission). The solid line represents the closed state of the channel (as is done by one means or another in Figs. 9.2 and 9.3).

to underlie the intermediate-conductance channels found in a variety of tissues. IK channels have a single-channel slope conductance of 12–80 pS, depending on the tissue and recording conditions. The SK4 channel appears to be unique in its very high affinity for Ca2þ (EC50 of 95 nM) (Joiner et al., 1997). It is minimally sensitive to apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 mM) (Ishii et al., 1997), but is inhibited by clotrimazole in the nM range. IK channels are expressed primarily in peripheral tissues, with the highest expression in smooth muscle cells (Ishii et al., 1997; Joiner et al., 1997) and in melanoma cells (Meyer et al., 1999). There are very limited reports on the effect of hypoxia on IK.

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FIGURE 9.2 Effects of hypoxia on single BK channels (>200 pS in symmetrical Kþ solutions) in a cell-attached patch (a) and an inside-out patch (b) from neocortical neurons. Note that hypoxia had an effect on the BK channel in the cell-attached patch, but and had no effect on the BK channels in the inside-out patch, indicating a cytosolic component is involved in the effect of hypoxia in these examples. (From Figure 5, Liu et al., 1999, with permission.)

9.3.4.5 Kir Channel Inwardly rectifying Kir channels, also known as “anomalous rectifier’’ channels, are expressed in skeletal and heart muscles, neurons, and glia. Current through these channels increases upon hyperpolarization. Because they open at negative voltages, Kir channels regulate the membrane potential in neurons and play a role in [Kþ] fluxes across glial cell membranes. Kir channels also control cell differentiation, modify CNS hormone secretion, modulate neurotransmitter release, CNS cell differentiation and neurogenesis, and may also regulate cerebral artery dilation (Neusch et al., 2003). Unlike other Kþ channels, Kir currents typically increase in response to hypoxia. In small-diameter (<100 mm) coronary arterial smooth muscle cells (SCASMC), hypoxia activates and increases Kir current

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density by as much as 40% under conditions of symmetrical 140 mM Kþ solutions. This increase occurs via cAMP- and PKA-dependent signaling cascades, and may at least partly explain hypoxia-induced coronary vasodilation (Park et al., 2005). Interestingly, the density and activity of Kir channels in ventricular myocytes of crucian carp (one of the most anoxia-tolerant vertebrates) are not modified by chronic hypoxia (4 weeks O2 < 0.4 mg/L, 2.68 mmHg) (Paajanen and Vornanen, 2003), indicating again that ion channel responses to hypoxia are very different in susceptible versus tolerant animals. 9.3.4.6 Background K Channel The concept of a voltage-independent “background’’ Kþ conductance in neurons, distinct from the voltage-sensitive Naþ and Kþ channels, was originally postulated by Hodgkin and Huxley (1952). In many cases such background or “leakage’’ Kþ currents appear to be carried by the two-pore domain Kþ channels (K2P), first described in yeast (Ketchum et al., 1995). Mammalian members of the K2P family include the TWIK, TREK, TASK, and TRAAK channels. The two-pore domain K channels TREK1 and TASK1 are expressed throughout the brain, but the expression patterns do not overlap significantly. They are important contributors to neuronal resting membrane potentials and excitability. They are relatively insensitive to classical Kþ channel blockers (Baker et al., 1987) and are largely voltage independent (Lesage, 2003). There are several reports that hypoxia inhibits these background Kþ channels. For example, hypoxia inhibits TREK1 and TASK1 channels (Kemp et al., 2004) and causes membrane depolarization (Plant et al., 2002). Hypoxia (PO2 5–80 torr) has also been reported to cause a partial pressure-dependent decrease in Kþ current in the presence of TEA and 4-AP in glossopharyngeal neurons, a finding that is consistent with the existence of TEA- and 4-AP-insensitive background Kþ currents (Campanucci and Nurse, 2005). TREK1, TREK2, and TRAAK can be activated by riluzole (Duprat et al., 2000), which has anti-ischemic, anticonvulsant, and sedative properties. This suggests that activation of TREK currents could contribute to the neuroprotective action of this drug (Lesage, 2003), although riluzole also inhibits Naþ currents and reduces glutamate release, actions that would also be expected to ameliorate hypoxia insults. 9.3.4.7 KATP Channel The ATP-sensitive Kþ (KATP) channel is a member of the inward rectifier Kir family of channels. It derives its name from its sensitivity to cytoplasmic ATP. These channels are open in the absence of ATP and close in the presence of ATP, the degree of closing depending on the ATP concentration. KATP channels are comprised of pore-forming subunits (Kir6.1 or Kir6.2) and a regulatory sulfonylurea receptor (SUR1 or SUR2) (Nakaya et al., 2003). KATP channels with different combinations of these subunits are present in heart, skeletal muscles, and neurons. They have been shown to have neuronal protective roles in ischemic conditions. Resistance to hypoxia–reoxygenation injury has been found to be lacking in some cells lacking KATP channels. For example, native COS-7 monkey kidney cells, which lack KATP channels, are vulnerable to hypoxia–reoxygenation injury due to cytosolic Ca2þ loading (Jovanovic et al., 1998). Conversely, cells

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normally lacking KATP channels can be rescued from hypoxia–reoxygenation injury when transfected with KATP channel genes. COS-7 cells cotransfected with the KATP channel genes, Kir6.2 and SUR1, gained resistance to hypoxia reoxygenation (Jovanovic et al., 1998). Studies performed with gene knockout techniques have yielded similar results: neurons from KATP knockout mice suffered greater damage after ischemia or anoxia (and are more susceptible to generalized seizure during hypoxia, Nakaya et al., 2003), while na€ıve neurons suffered little injury (Sun et al., 2006). Surprisingly, it appears that KATP channels do not play a major role in protecting neurons from injury in hypoxia- and anoxia-tolerant species (e.g., turtles) (Pek-Scott and Lutz, 1998). 9.3.4.8 KA Channel The transient A-current (IKA) was discovered by Nakajima (1966), Connor and Stevens (1971), and Thompson (1977), and it contributes to membrane potential repolarization in neurons following action potentials, timing of action potentials, and participating in signal integration in dendrites. These channels are found in neurons across a wide phylogenetic spectrum, from mammals to insects (Tempel et al., 1987), and were initially cloned following mutational analysis of Drosophila mutants (Schwartz et al., 1988). In mammalian neurons, these rapidly activating and inactivating currents are produced by members of three families of voltage-dependent Kþ channels: KV1.4 (Shaker family), KV3.4 (Shaw family; this channel is most densely found not only in skeletal muscles but also in neurons), and the KV4 channels (Shal family members KV4.1, KV4.2, and KV4.3). In mammalian hippocampal neurons, the A-type channels are differentially distributed regionally, but are generally divided into presynaptic KV1 and postsynaptic KV4 channels (Magee and Carruth, 1999). Again, like most Kþ channels, this current has been reported to be inhibited or decreased by hypoxia in rabbit glomus cells (Lopez-Lopez et al., 1989), in smooth muscles from canine pulmonary arteries (Post et al., 1995), and in arterial chemoreceptor cells (totally inhibited by PO2 at 90 torr) (Ganfornina and Lopez-Barneo, 1991). 9.3.5

Mitochondrial Voltage-Dependent Ion Channels

Chapter 6 of this book deals with the regulation of ion channels in mitochondria. However, because of the importance of mitochondria as the prime consumer of O2 within the cell, we shall give here a brief account of the some of the known effects of hypoxia on two different types of ion channels in the inner and outer mitochondrial membranes. 9.3.5.1 Voltage-Dependent Anion Channel (VDAC) The voltage-dependent anion channel (VDAC) is a major protein in the outer mitochondrial membrane (Shoshan-Barmatz and Gincel, 2003; Lemasters and Holmuhamedov, 2006). Despite its name, this 30 kDa protein is very different in its properties from any of the other voltage-dependent channels that we have considered. Moreover, it is a truly nonselective channel, being only marginally selective for anions over cations in its

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open state. VDAC conducts ion in its “closed’’ and “open’’ states, but when VDAC is open, it allows large molecules (e.g., 5 kDa) to pass through a pore about 3 nm in diameter (Lemasters and Holmuhamedov, 2006). In general, VDAC is a nonselective pathway for transporting anions, cations, ATP, and other metabolites into and out of the mitochondrion and plays an important role in mitochondrial physiology and pathophysiology (Shoshan-Barmatz and Gincel, 2003). Some studies have indicated that cytopathic hypoxia leads to VDAC closure (Lemasters and Holmuhamedov, 2006). In contrast, the overall channel activity of the outer mitochondrial membrane appears to be increased by hypoxia or ischemia both in rat brain (Bonanni et al., 2006) and in the mitochondria within presynaptic terminals of the squid giant synapse (Jonas et al., 2005). 9.3.5.2 MitoKATP Channel In an earlier section, we discussed the effects of hypoxia on ATP-regulated Kþ channels in the plasma membrane. A similar channel in mitochondrial membranes, the MitoKATP channel, was first identified by Inoue et al. (1991). It has a single-channel slope conductance of 9–100 pS (Ardehali, 2005) and is inhibited by a selective mitoKATP channel blocker 5-hydroxydecanoate (5-HD) (Liu et al., 1998). The cell membrane KATP channel blocker, glibenclamide, was found to nonspecifically affect mitochondrial function (Garlid et al., 1997b). Treatment of cells with diazoxide, an opener of the mitoKATP channel, has been found to provide some resistance to ischemic and hypoxic damage (Garlid et al., 1997a; Liu et al., 1999b; Deja et al., 2006). It has been suggested that transient ischemia produces H2O2, which, in turn, activates the mitoKATP channels via a PKCe-mediated pathway (Jaburek et al., 2006). Moreover, it has been proposed that mitoKATP channels, rather than plasmalemmal KATP channels, provide a major line of defense for the heart against ischemia, (Garlid et al., 1997a; Sato et al., 2000). 9.3.5.3 MitoBK Channel Channel activity similar to that of plasma membrane BK calcium-activated Kþ channels has been reported in the inner membrane of mitochondria. This mitoBK channel was initially found using electrophysiological tools in the inner mitochondrial membrane of LN229 glioma cells (Siemen et al., 1999). Subsequently, mitoBK was found in cardiac myocyte mitochondria (Xu et al., 2002) and in inner mitochondrial membranes of rat brain neurons (Douglas et al., 2006). The molecular identity of the mitoBK channel is, however, not yet clear (Ardehali, 2005). The properties of MitoBK channels (such as single-channel slope conductance, voltage dependency, Ca2þ dependency, inhibition by charybdotoxin) appear similar to those of BK channels in the plasma membrane (Siemen et al., 1999). The BK channel opener NS1619 (Olesen et al., 1994; Gribkoff et al., 1996) has been found to increase activity of MitoBK (Xu et al., 2002), as that of mitochondrial depolarization (DYm) (Debska et al., 2003; Korper et al., 2003; Sato et al., 2005). These channels are blocked by paxilline (Sanchez and McManus, 1996). Opening of this channel by NS1619 has been shown to protect cardiac myocytes from ischemic damage (Xu et al., 2002). The protection is probably through the attenuation of mitochondrial Ca2þ overload

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FIGURE 9.3 Effects of hypoxia on BK channels (>250 pS in symmetrical Kþ solutions) in mitoplast-attached patches from LN229 glioma cells under normoxic (a) and hypoxic (b) conditions. Notice that hypoxia modestly increased BK channel open probability (d), but did not have any effect on single-channel slope conductance (c). (From Figure 4, Gu et al., 2007 with permission.)

(Sato et al., 2005). The pretreatment with NS1619 reduced the O2 generation, decreased mitochondrial [Ca2þ] and normalized NADH during early ischemia and throughout reperfusion (Stowe et al., 2006). Recently, it has been shown that acute hypoxia increased the open probability of single mitochondrial BK channels in LN229 cells (Gu et al., 2007) (Fig. 9.3). 9.3.5.4 Mitochondrial Ca2þ Uniporter The mitochondrial Ca2þ uniporter, located in the inner mitochondrial membrane, has recently been recognized as a Ca2þ channel (Kirichok et al., 2004), although its properties are quite distinct from those of plasma membrane Ca2þ channels. This mitochondrial uniporter is a gateway for Ca2þ accumulation from the cytosol during intracellular Ca2þ signaling (Montero et al., 2000; Rizzuto et al., 2000; Kirichok et al., 2004). This mitochondrial Ca2þ channel has an extremely high affinity for Ca2þ, with a dissociation constant 2 nM in a low cytoplasmic Ca2þ environment (Kirichok et al., 2004). By using Ru360, a specific mitochondrial Ca2þ uptake inhibitor, Garcia-Rivas et al. 2006 were able to show a slowed uptake rate 45 Ca2þ in ischemia–reperfusion experiments of rat heart, suggesting that the uniporter is implicated in the response to ischemia reperfusion.

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Interestingly, Ca2þ uptake in rat mitochondria through the Ca2þ uniporter has been reported to be gender dependent (Arieli et al., 2004).

9.4 CONCLUSIONS Although there are many cellular and subcellular events that take place during hypoxia, we believe that hypoxia induces a temporal cascade of changes in the behavior of cellular components, such as voltage-gated ion channels, at the cell membrane. There is continuing debate in the literature concerning the sensing of a reduction in O2 and whether this is a result of events inside the cell, such as ATP depletion, or whether this takes place at the membrane level, before many of these intracellular processes have even started. For example, can ion channels directly sense and respond to a drop in partial pressure of O2? Also, is this sensing mechanism, which we have shown to exist in neurons, present and functional in other cell types? Furthermore, are there other sensing mechanisms that also start cascades that are separate from this mechanism? These questions are still not resolved. One of the first events detected at the cell membrane during hypoxia is an inward flux of Naþ resulting in an increase in intracellular Naþ; this seems to precede the rise in Ca2þ, which has been very much investigated over the past two decades (Figure 3A, Friedman and Haddad, 1993, and Figure 3, Friedman and Haddad, 1994; Haigney et al., 1994). The sources of this increase in [Naþ]i could be any of the voltagedependent channels, such as NaP, Naþ/Hþ exchangers, Naþ/Ca2þ exchangers, and/or failure of the Naþ/Kþ ATPase due to depletion of ATP (although depletion of ATP inside cells resulting from hypoxia generally occurs at a slower rate). Increases in [pH]i due to anaerobic metabolism under hypoxia, inhibition of electron transport, as well as depolarization are concomitant with the initial increase in [Naþ]i, probably via Bcl-2 family proteins (Lipton, 1999) (Fig. 9.4, see also Chapter 6). One of the next events detected during hypoxia is Ca2þ influx into cells and an increase in intracellular Ca2þ. This increase in [Ca2þ]i can result from voltagesensitive Ca2þ channel activity, NMDA receptors, or the Naþ/Ca2þ exchangers, as well as through the release of intracellularly sequestered Ca2þ from the ryanodine receptor in the ER in most cells and SR in muscle cells. Activation of kinases then follows this sustained increase in [Ca2þ]i; PKC and calcium/calmodulin-dependent kinase (CaMK) are just two kinases that may be activated and which may ultimately participate in numerous deleterious cellular processes leading to cellular compromise and death. Ultimately, persistent hypoxia leads to compromise of the bioenergetics of the cell, a relatively rapid process in neurons, leading to mitochondrial permeability changes and mitochondrial dysfunction: opening of VDAC in the outer mitochondrial membrane leads to the release of cytochrome c from the intermitochondrial space into the cytosol. Release of cytochrome c and mitochondrial dysfunction is among the first steps of the cascade leading to apoptosis. Not surprisingly, some of the membrane events have been linked to and associated with events that lead to apoptosis. Activation of certain voltage-dependent channels in

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FIGURE 9.4 Effects of hypoxia on some membrane proteins and downstream events. BAK, Bcl-2 homologous antagonist/killer; Bcl-2, B-cell lymphoma 2; CaM, calmodulin; CaMK, calcium/calmodulin-dependent kinase; Cyt C, cytochrome C; ETC, electron transport chain; PKC, protein kinase C; PTP, permeability transition pore; VDAC, voltage-dependent anion channel.

hypoxia or ischemia, including Naþ channels (Banasiak et al., 2000) and Kþ channels (Yu et al., 1997) (clearly Ca2þ overload in hypoxia is also linked to proapoptotic mechanisms), have been shown to indirectly lead to programmed cell death. Activation of some other channels in hypoxia/ischemia, such as VDAC, have also been linked to apoptosis, as has been demonstrated experimentally (Bonanni et al., 2006), probably via Bcl-2 family proteins (see Chapter 6). Neurons are highly sensitive to the effects of hypoxia/ischemia. They are highly metabolic cells, and endogenous neuroprotective mechanisms that can ameliorate the effects of hypoxia are relatively short lived. Despite over two decades of intense interest in the subject of the neuronal mechanisms of hypoxia-induced neuronal death, because of the obvious relevance to acute neurodegenerative disorders such as stroke and trauma, many questions remain unresolved. Much of the remaining mystery involves the coordination of events at the subcellular level when cells are depleted of O2 and the identification of points in hypoxia-induced cellular pathways that are amenable to therapeutic intervention. Current approaches, including proteomic analysis and the study of endogenous hypoxia-resistance mechanisms, might be helpful in elucidating more of the biochemical sequelae of neuronal (and other cellular) hypoxia and lead more rapidly to effective treatment.

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10 IN VIVO ROLES OF ION CHANNEL REGULATORY PROTEIN COMPLEXES IN NEURONAL PHYSIOLOGY AND BEHAVIOR SMITHA REDDY, MOHAMMAD SHAHIDULLAH,

AND IRWIN

B. LEVITAN

Department of Neuroscience, University of Pennsylvania School of Medicine, 215 Stemmler Hall, 3450 Hamilton Walk, Philadelphia, PA 19104-6074, USA

10.1 INTRODUCTION About five decades ago, ion channels were little more than a hypothesis. Today, ion channel proteins are well established as the fundamental elements of membrane excitability. The transition from a plausible description of entities that control passive ion transport across the membrane to fundamental fact stands upon several kinds of pioneering research. After the historical identification and characterization of the voltage-dependent sodium and potassium conductances by Hodgkin and Huxley, molecular biological, pharmacological, and electrophysiological characterization led to the identification of specialized molecules called ion channels. Ion channels are a ubiquitous class of specialized membrane proteins that, by forming hydrophilic pores in the plasma membrane, allow the movement of ions across the membrane, and thereby contribute to the electrical activity of individual neurons, and the communication between them. Such information transfer within and between neurons, mediated by ion channels, is essential for brain function and its control of behavior. It is now widely accepted that ion channels are subject to tight regulation. The opening and closing of ion channels is influenced by several factors such as Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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the membrane voltage, extracellular ligands (e.g., neurotransmitters), and intracellular messengers (e.g., calcium and cyclic nucleotides) and posttranslational modifications of the channel protein itself. Voltage-gated ion channels are a subclass of ion channels whose opening and closing are regulated by voltage across the membrane. These channels play a fundamental role in the generation and propagation of action potentials. Voltage-gated channels have an a subunit that forms the pore and also contains the voltage sensor, with which the channel can sense changes in transmembrane voltage, and thereby gate between an “open” (or conducting) state and a “closed” (or non conducting) state. When expressed alone in heterologous cells, the a subunits are generally capable of forming functional channels. Accumulating evidence shows that channel gating by these pore-forming a subunits is influenced by auxiliary (non pore-forming) subunits, variously named as b, g, and so on subunits. Modulation by these auxiliary subunits is beyond the scope of this chapter, and will not be discussed in detail here. Ion channels may also be regulated by one or more signaling proteins, which may modulate the properties of the channel by direct association with the channel itself, or via scaffolding protein(s). Proteins that associate with channels may coexist with the channel constitutively or may bind to the channel in a dynamically regulated fashion. Such association may lead to changes in channel gating, channel assembly or trafficking, or response of the channel to changes in cellular signaling events. These modulatory influences may, in effect, fine-tune channel activities to cater to the needs of the cell. In this chapter, we discuss some known examples of ion channels that are regulated by associated proteins. Although we may occasionally allude to ligandgated ion channels and voltage-gated sodium (Naþ) and calcium (Ca2þ) channels, we focus primarily on ion channel complexes associated with voltage- and/or Ca2þ-gated potassium (Kþ) channels.

10.2 MODULATION BY PROTEIN KINASES AND PHOSPHATASES Protein kinases constitute a family of proteins commonly encountered in ion channel complexes (Levitan, 2006). Phosphorylation by a kinase may increase or decrease channel activity depending on the particular kinase that is involved and the specific sites(s) that are phosphorylated. In addition, dephosphorylation of a channel by phosphoprotein phosphatases may also modulate channel properties. When kinases and phosphatases are directly associated with the channel, channel modulation by the corresponding kinase or phosphatase is sometimes preserved even in excised patches or in reconstituted lipid bilayers (Chung et al., 1991; Reinhart and Levitan, 1995). It seems likely that all ion channels are regulated by phosphorylation/dephosphorylation, but the precise mechanism of the modulatory action and the physiological outcome of such modulation may vary from channel to channel. Large conductance calcium- and voltage-gated Kþ (BK, for big potassium; also called Slowpoke) channels are no exception to such modulation. Earlier functional studies demonstrated that both phosphorylation and dephosphorylation of the a subunit can modulate the activity of Slowpoke (Slo) channels (Chung et al., 1991; White et al., 1993; Bielefeldt

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and Jackson, 1994; Reinhart and Levitan, 1995; Tian et al., 2001). Coimmunoprecipitation experiments using antibodies against protein kinases revealed that both the Src tyrosine kinase, and the active catalytic subunit (PKAc) of the cyclic AMPdependent protein kinase (PKA), bind to and phosphorylate the Drosophila Slo channel (Wang et al., 1999). Coexpression of PKAc with the Slo channel in heterologous cells results in a decrease in channel activity. Such modulation requires both the catalytic activity of PKAc and also its binding to the channel (Zhou et al., 2002). In addition to protein kinases and phosphatases, Slo channels bind to a number of other protein partners and participate with them in a dynamic regulatory complex.

10.3 IDENTIFICATION OF NEW ION CHANNEL BINDING PARTNERS BY YEAST TWO-HYBRID SCREENS The quest for new binding partners of ion channels brought to light several novel proteins that modulate ion channels. Yeast two-hybrid screens proved instrumental in the identification of new modulatory proteins of ion channels. One such screen, using the cytoplasmic amino-terminal domain of the Kv4 subfamily of Kv channels as bait, identified the Kþ channel interacting proteins (KChIPs) that specifically interact with Kv4 channels and modulate their membrane density and gating kinetics (An et al., 2000). Kþ channel-associated protein (KChAP), also identified in a two-hybrid screen, shares homology with transcription factor interacting proteins. Although first identified as a Kvb1.2 binding partner, KChAP interacts with selected a subunits, and increases their current density (Wible et al., 1998; Kuryshev et al., 2000; Kuryshev et al., 2001). Yeast two-hybrid screens also identified the ubiquitous Ca2þ sensor protein, calmodulin (CAM), as a KCNQ (KV7) Kþ channel binding protein (Wen and Levitan, 2002; Yus-Najera et al., 2002). CAM binds constitutively to specific sequence motifs in the C-terminal domain of KCNQ2 and KCNQ3 channels and is required for the Ca2þ-independent generation of KCNQ2/KCNQ3 current in heterologous cells (Wen and Levitan, 2002) and rat hippocampal neurons (Shahidullah et al., 2005). Intriguingly, CAM also mediates the Ca2þ-induced inhibition of KCNQ2/KCNQ3 current (Gamper and Shapiro, 2003). Tethered CAM also modulates several other ion channels such as the small (SK) and intermediate (IK) conductance Ca2þ-activated Kþ channels, voltage-gated Naþ and Ca2þ channels, NMDA receptors, and cyclic nucleotide-gated channels in a variety of different ways (Levitan, 1999; Levitan, 2006). Two other yeast two-hybrid screens using either a piece (Xia et al., 1998) or all (Schopperle et al., 1998) of the carboxy terminal tail of the Drosophila Slowpoke channel (dSlo) as the bait revealed two binding partners of dSlo, SLIP1 and Slob. SLIP1 (for Slo Interacting Protein) specifically interacts with dSlo and human Slo and reduces the insertion of dSlo into the cell membrane (Xia et al., 1998). The other protein, named Slob (for Slo binding), has no homology to any previously described protein, except for a kinase-like domain and a leucine zipper domain. Slob and dSlo coimmunoprecipitate from adult fly heads and heterologous cells, suggesting that the dSlo/Slob proteins interact in vivo. In addition, Slob also coimmunoprecipitates with

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the Drosophila ether-a0 -go-go (Eag) channel, but not with mouse Slo or Shaker or a rat Kv1 channel (Schopperle et al., 1998). Electrophysiological recordings using a heterologous expression system showed that Slob modulates the activity of the dSlo channel (Schopperle et al., 1998; Zeng et al., 2005, 2006).

10.4 MODULATION BY 14-3-3 PROTEINS 14-3-3 proteins constitute a ubiquitous family of highly conserved cytosolic proteins that bind specifically to a wide variety of target proteins, usually at phosphoserine sites, and thereby regulate their function. Members of the 14-3-3 family exist as dimers, and each monomeric subunit is capable of binding to specific recognition sequences in target proteins. The binding of 14-3-3 results in such functional consequences as changes in structural conformation, subcellular targeting, or cross-bridging of two proteins. Thus, 14-3-3 proteins function as intracellular regulatory or adaptor proteins in diverse cellular functions. There are seven mammalian genes that encode different isoforms of the 14-3-3 family with varying tissue distribution patterns (Fu et al., 2000). Two of these isoforms, 14-3-3z and e, are also present in Drosophila. A number of studies in the past decade or so demonstrated association of 14-3-3 proteins to several ion channels including Ca2þ-activated Kþ channels, Ca2þ-activated chloride (Cl) channels, GABA-B receptors, a2-adrenergic receptors (Craparo et al., 1997; Prezeau et al., 1999; Chan et al., 2000; Couve et al., 2001). For instance, 14-3-3z binds to and modulates dSlo. This interaction is mediated by the dSlo-binding protein Slob (Zhou et al., 1999, 2003). The interaction of 14-3-3 with Slob is regulated by the type II Ca2þ/ CaM-dependent protein kinase (CaMKII)- mediated phosphorylation of two specific serine residues in Slob. Flies engineered to have either elevated or suppressed CaMKII activity in their neurons exhibit a marked change in the extent of 14-3-3 binding to Slob (Zhou et al., 1999). These results imply that the modulation of dSlo channel activity by these binding partners may itself be under dynamic control. The dSlo/Slob/14-3-3z complex has been shown to exist in adult heads and in the presynaptic terminals at the larval neuromuscular junction (NMJ) in Drosophila (Zhou et al., 1999). Thus, Slob and 14-3-3z, via dynamic regulation of dSlo, can potentially modulate neuronal excitability and thereby tune synaptic transmission in the CNS and at the NMJ. A recent study showed that 14-3-3 binds to and modulates the inactivation properties of Cav2.2 channels. Furthermore, interference with 14-3-3 binding in hippocampal neurons speeds up inactivation of Cav2.2 channels and enhances short-term synaptic depression (Li et al., 2006b). It is worthy of note in this context that mutant alleles that reduce the 14-3-3z isoform in flies lead to defects in olfactory learning and memory and synaptic plasticity at the neuromuscular junction (Skoulakis and Davis, 1996; Philip et al., 2001). These reports raise the interesting possibility that the effects of 14-3-3 on synaptic and behavioral plasticity may be mediated by modulation of ion channels. On another note, 14-3-3 binding facilitates surface expression of some channels as in the case of the KATP channel a subunit Kir6.2 (Yuan et al., 2003) and two-pore domain acid-sensitive potassium channels (TASK) (Rajan et al., 2002). Modulation of ion channels by 14-3-3 is not restricted to neurons. For instance, binding of 14-3-3e accelerates and enhances human ether-a-go-go-related (HERG)

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channel activation, an effect that is dependent on phosphorylation of HERG by PKA (Kagan et al., 2002). HERG encodes the pore-forming subunit of the delayed rectifier Kþ current (IKr) in the heart and is responsible for repolarizing the cardiac action potential, thereby playing a critical role in the regulation of heart rhythm. Mutations in the HERG gene are associated with congenital long-QT syndrome (LQTS). Stressinduced stimulation of b-adrenergic receptors leads to elevated cAMP levels, which in turn regulate HERG channel activity, either directly or via PKA-dependent phosphorylation of the channel. The binding of 14-3-3 to the HERG channel amplifies and prolongs this effect of adrenergic stimulation on HERG channel activity, thus providing a unique mechanism for plasticity in the regulation of cardiac rhythm (Kagan et al., 2002).

10.5 MODULATION BY A-KINASE ANCHORING PROTEINS (AKAPs) AKAPs are scaffolding proteins that bind to the regulatory subunit of PKA and target it to a specific substrate or cellular compartment. In addition to PKA, AKAPs bind to other kinases and phosphatases and target them to specific residues in a multiprotein signaling complex. There are over 20 AKAPs, at least some of which interact with ion channels (Li et al., 2006a). For example, a particular AKAP protein interacts with the inwardly rectifying Kþ channel Kir2.1 and increases its sensitivity to cAMP regulation (Dart and Leyland, 2001). There are now several reports of AKAPs that associate with members of the KCNQ family of Kþ channels (Marx et al., 2002; Hoshi et al., 2003; Kurokawa et al., 2004).

10.6 PHYSIOLOGICAL SIGNIFICANCE OF ION CHANNEL COMPLEXES Clearly, ion channels play a critical role in a multitude of behaviors. For example, in Drosophila, flies carrying mutations in the Kþ channel genes shaker, slo, and eag rapidly shake their legs in response to ether-induced anesthesia (Kaplan and Trout, 1969; Atkinson et al., 1991; Warmke et al., 1991). A similar neurological phenotype was noted for a mutation in hyperkinetic (Kaplan and Trout, 1969), which encodes the auxiliary b subunit for Shaker. Shaker mutants have also been demonstrated to sleep less and have a shorter life span (Cirelli et al., 2005). A mutation in another Kþ channel gene, seizure, causes convulsive seizures at high temperature (Jackson et al., 1984). Studies carried out with BK channel null mutants demonstrated an important role for these channels in the circadian regulation of behavioral rhythms in both flies and mammals (Ceriani et al., 2002; Meredith et al., 2006). Emerging evidence in the past decade or so indicates a role for these and other ion channels in circadian rhythms. Finally, in their survey of mutations that affect Kþ and Naþ channel genes, Fergestad et al. found that both mutations that increase membrane excitability and those that decrease membrane excitability can cause neurodegeneration. Many, but not all of these mutations, are also associated with a decrease in life span (Fergestad et al., 2006). An increasing number of human neurodegenerative diseases associated with malfunctioning ion channels are coming to light (Graves and Hanna, 2005).

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As is evident from the studies described above, genetic approaches are very useful in addressing questions related to the in vivo physiological roles of ion channels, because they allow the study of channels in an intact native environment. Given that membrane excitability is a key mechanism for transmission of neuronal information, it comes as no surprise that ion channels, the key molecules for membrane excitability, play a critical role in the regulation of diverse physiological processes and/or behaviors. While these attributes of ion channels make them interesting to study, it makes their specific contribution to particular behaviors difficult to assess. There are numerous examples of a mutation in a single ion channel gene causing defects in diverse physiological and/or behavioral outputs. On the contrary, recruitment of compensatory mechanisms, thereby resulting in no obvious phenotype, is a confounding problem in gene deletion studies of ion channels. To get around these shortcomings in the study of in vivo functional roles of ion channels, one strategy may be to turn to channel modulatory proteins, especially those that bind specifically to only one channel but not others. In an effort to explore the physiological significance of dSlo channel modulation in flies, we investigated how the ion channel modulator, Slob, can influence physiology and behavior in an intact organism. Recent studies demonstrated that there are multiple Slob variants, all of which bind to dSlo in heterologous cells (Jaramillo et al., 2006). All Slobs also modulate dSlo, but in distinct ways (Zeng et al., 2005; Zeng et al., 2006). Immunohistochemistry on fly heads showed that the predominant Slob, Slob57, is expressed prominently in (but not restricted to) the neurosecretory pars intercerebralis (PI) neurons in the dorsal brain (Jaramillo et al., 2004). The PI region of the brains of many insects, including crickets, locusts, cockroaches, and fruit flies contains neurosecretory neurons that participate in the control of a variety of physiological functions that include feeding and metabolism (Zaretsky and Loher, 1983; Krauthamer, 1985). In Drosophila, a subset of the PI neurons express the Drosophila insulin-like peptides (dilps) (Ikeya et al., 2002; Rulifson et al., 2002). Ablation of these dilp-secreting PI neurons in adult flies leads to increased resistance to oxidative and starvation stress and a longer life span (Ikeya et al., 2002; Rulifson et al., 2002; Broughton et al., 2005). In view of the fact that Slob expression is prominent in the metabolically relevant PI neurons, we hypothesized that slob may participate in the regulation of feeding behavior. slob mutants with dramatically decreased Slob expression show prolonged survival under complete food-deprivation conditions (Fig. 10.1). Furthermore, knockdown of Slob expression by RNA interference in the whole fly, or in the PI neurons, increases resistance to starvation-induced death, thus indicating that the feeding phenotype in slob mutants maps to the PI neurons. Consistent with our findings in heterologous cells, whole-cell patch-clamp recordings from intact PI neurons demonstrate a decrease in whole-cell outward currents in Slob57 overexpressors and an increase in current in slob mutants. Pharmacological blockade experiments and single-channel recordings indicate that these effects on whole-cell outward currents ensued by genetic manipulation of Slob expression levels are mediated by the modulation (or lack thereof) of dSlo (Fig. 10.2; Shahidullah et al., manuscript in preparation). On the basis of these findings, we propose that Slob, via modulation of dSlo in the PI neurons, regulates neurosecretion of dilps from the PI neurons and

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FIGURE 10.1 Enhanced starvation stress resistance in slob mutant flies. Survival curves showing the percentage of flies that are alive over a period of 3–4 days of starvation. As compared to wild-type control (^) flies, several slob mutant (slob mutant 1-& slob mutant 2-~) flies live significantly longer under starvation conditions.

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FIGURE 10.2 Slob modulates dSlo channels in vivo. Representative single-channel current traces at þ60 mV from Drosophila PI neurons, in the brains of living flies, demonstrate modulation of the dSlo channel by Slob. As compared to wild-type control, dSlo channel open probability is dramatically decreased in Slob57 overexpressor flies and increased in slob mutant flies.

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thereby affects feeding behavior. Previous studies that demonstrated enhanced starvation resistance in flies with decreased insulin signaling are consistent with this hypothesis (Clancy et al., 2001; Kramer et al., 2003). It is important to mention in this context that Ca2þ-activated Kþ channels have been reported to be important for the regulated release of insulin from pancreatic b cells (Gopel et al., 1999; Goforth et al., 2001; Goforth et al., 2002), as well as for the release of neurotransmitters and neuropeptides in molluscs, flies, and rodents (Gorman et al., 1981; Gho and Ganetzky, 1992; Bielefeldt and Jackson, 1994).

10.7 CONCLUSIONS Neuromodulation is of critical importance in controlling complex behaviors in diverse organisms. Since ion channels are key molecules that participate in neuromodulation, understanding the molecular mechanisms underlying the modulation of neuronal ion channels and how such modulation leads to changes in neuronal physiology and behavior is of fundamental importance. Although there are many examples of ion channel signaling complexes as described above, the physiological significance of ion channel modulation has been reported in only a few cases. Changes in membrane excitability mediated by ion channels have been shown to underlie many physiological and pathophysiological conditions, but how these changes are integrated at the systems level remains an important goal for future channel enthusiasts.

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11 REGULATION OF NEURONAL ION CHANNELS BY G-PROTEIN-COUPLED RECEPTORS IN SYMPATHETIC NEURONS MARK S. SHAPIRO1 AND NIKITA GAMPER2 1

Department of Physiology, University of Texas Health Science Center at San Antonio, MS7756, San Antonio, TX 78229, USA 2 Institute of Membrane and Systems Biology, Faculty of Biological Sciences, The University of Leeds, Leeds LS2 9JT, UK

11.1 INTRODUCTION For many years, cultured sympathetic neurons (isolated mainly from rat, mice, chicken, and frogs) have served as reliable native models in the studies of modulation of neuronal ion channels by G-protein-coupled receptors (GPCRs). The features of sympathetic neurons that make them such good objects for experiments includes simplicity of culturing, robust and reliable endogenous expression of numerous GPCRs and ion channels, and, perhaps most importantly, the relative phenotypic homogeneity of the noradrenergic neurons within the ganglia (compared to neurons from the central nervous system or sensory ganglia). These cells have been extensively used in the studies of GPCR modulation of neuronal voltage-gated ion channels, including high-threshold Ca2þ channels (historically classified as N-, P/Q-, and R-types (Nowycky et al., 1985), now also called CaV2.1, CaV2.2, and CaV2.3, respectively) and “M-type” Kþ channels. Both channel types are of paramount

Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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physiological importance. Thus, influx of Ca2þ through Ca2þ channels drives exocytosis and release of neurotransmitter at nerve terminals and mediates Ca2þdependent pacemaking or bursting via KCa channels at the soma (Tsien, 1983); in turn, M-type Kþ channels, due to their unique biophysical properties (see below), play a dominant role in controlling the excitability and neuronal discharge properties of the many neurons that express these channels (Jones et al., 1995; Wang et al., 1998; Peretz et al., 2005; Gamper et al., 2006; Yue and Yaari, 2006; Zaika et al., 2006). Hence, there is a large literature on the modulation of these two currents in sympathetic neurons, particularly those of the rat superior cervical ganglion (SCG). In this chapter, we focus on studies using those cells in the modulation of N-type Ca2þ and M-type Kþ channels, with particular attention to signaling pathways mediated by the Gq/11 class of Gprotein, and downstream actions of its activation, such as altered phosphatidylinositol 4,5-bisphosphate (PIP2) abundance, protein kinase C (PKC) activation, and [Ca2þ]i signals. Although other types of intriguing modulatory influences for both channel types have been well described, such as the effects of various b subunits (Cooper et al., 2000; Richards et al., 2004), regulation of trafficking to the membrane (Jarvis and Zamponi, 2007), RGS proteins (Jeong and Ikeda, 2000), and vesicle release proteins such as syntaxin and cysteine string protein (Jarvis and Zamponi, 2005), we do not focus on them here.

11.2 MODULATION OF M-TYPE Kþ CHANNELS 11.2.1

Historical Overview

The M-current was discovered by Brown and Adams (1980) as a slowly activated Kþ current in sympathetic neurons that is suppressed by the stimulation of certain GPCRs, including the namesake muscarinic receptor, and also bradykinin, P2Y, and angiotensin II receptors, and potentially by any other GPCR that is coupled to the Gq/11 subtype of G-proteins (reviewed in Delmas and Brown, 2005). M-channels are coded for by the KCNQ group of Kþ channel genes (KCNQ1–5, which are translated into Kv7.1–7.5 channel subunits), and most M-currents in neurons are made by heteromultimers of Kv7.2 and Kv7.3 (Wang et al., 1998; Shapiro et al., 2000; Selyanko et al., 2002). Summarized in Fig. 11.1a are the biophysical properties of heterologously expressed Kv7.2/7.3 channels. The channels have slow activation and deactivation kinetics (time constants for activation and deactivation at 0 mV and 60 mV, respectively, are in the range of 100–250 ms, depending on the subunit composition), a relatively negative threshold for activation (below 60 mV) and little inactivation under physiological conditions (Selyanko et al., 2000; Shapiro et al., 2000; Gamper et al., 2003). Due to these distinctive features, M-channel activity has a strong control over neuronal excitability. Thus, their negative threshold for activation allows a fraction of M-channels to be open near the resting membrane potential, conferring strong control over the threshold for action potential (AP) firing; their slow gating kinetics and the absence of inactivation, in turn, provide a mechanism for control over AP firing (e.g., accommodation of firing rate, Fig. 11.1b). As illustrated in Fig. 11.1c,

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FIGURE 11.1 M-current in sympathetic neurons. (a) Biophysical properties of heterologously expressed M-channels. Kv7.2/7.3 heteromultimers were expressed in CHO cells and recorded under whole-cell voltage-clamp. Currents were evoked by a family of 800 ms square voltage pulses from a holding potential of 70 mV to a range of potentials between 80 and þ40 mV (10 mV increments). The lower panel shows the activation curve from the current traces shown above. Plotted are the tail current amplitudes versus the test-pulse voltage. The tail-current amplitudes reflect the fractional activation of the channels at the end of the preceding test pulses. (b) Superimposed are the current-clamp record from an SCG neuron (black) and the voltage-clamp trace of Kv7.2/7.3 current activation from a CHO cell expressing the channels (red). Both traces are synchronized for timescale. The overlay shows that the accommodation of action potential firing in the SCG neuron (induced by the depolarizing current injection) temporally correlates with the degree of M-current activation. (c) Two further examples of the effect of M-current on the excitability of SCG neurons. Upper traces show the current-clamp experiment in which an SCG neuron was treated with the M-channel opener, retigabine (10 mM). The drug reversibly inhibited AP firing in response to a 100 pA depolarizing current pulse. Below is the similar experiment but the M-channel blocker, XE991 (10 mM), was applied instead. The blocker induced continuous firing, which is reversed by washing the drug off. The lower trace is reproduced from Zaika et al. (2006). (See the color version of this figure in Color Plate section.)

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manipulation of M-channel activity (either via physiological inhibition or pharmacological enhancement) has a strong effect on firing output. Accordingly, lossof-function mutations within M-channel genes often result in forms of epilepsy (for review, see Jentsch, 2000), whereas M-channel openers have antiepileptic (Rundfeldt, 1999; Rundfeldt and Netzer, 2000) and antiexcitotoxic (Gamper et al., 2006) effects. Given its strong effect on neuronal excitability, it is not surprising that GPCRcoupled modulation of M-current has been intensively studied. It was soon discovered that the modulation is mediated by Gaq or Ga11 subunits (Haley et al., 1998, 2000) and their usual downstream effector, PLCb. In an important experiment done by Alex Selyanko in David Brown’s group, it became apparent that external application of muscarinic agonist inhibits M-channels isolated in cell-attached patches from SCG neurons. Based on the distinction first made by Soejima and Noma (1984) between “membrane-delimited” and “diffusible messenger” signaling, such inhibition must require an intracellular messenger that is able to diffuse between the receptors situated outside the patch and the channels within it (Selyanko et al., 1992). Summarized in Fig. 11.2 is the canonical motif of Gq/11-mediated signaling. Triggering of

FIGURE 11.2 Gq/11-coupled signaling in mammalian cells. Depicted is the canonical pathway triggered by the activation of Gq- or G11-coupled receptors in neurons: activation of phospholipase C (PLC) results in hydrolysis of membrane phosphatidylinositol 4,5bisphosphate (PIP2, brown shapes) and release of inositol triphosphate (IP3, brown octagons) and diacylglycerol (DAG, green rectangle). The former may trigger release of Ca2þ (green spheres) from IP3-sensitive stores, whereas the latter can activate protein kinase C (PKC, ochre shape) or be further degraded to phosphatidic (PA, pink ovals) or arachidonic (AA, green ovals) acids. Each of these intermediates is capable of triggering diverse downstream signaling pathways. (See the color version of this figure in Color Plate section.)

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Gq/11-coupled GPCR activates PLCb, which, in turn, hydrolyzes PIP2 to release diacylglycerol (DAG) and IP3. The latter may (or as we shall see later, may not) induce release of Ca2þ from IP3-sensitive intracellular stores, whereas the former can activate PKC and be further metabolized to produce other second messenger molecules, such as arachidonic acid (AA) or phosphatidic acid (PA). Throughout the 1990s, many candidate products of PLC activation were exhaustively tested as being the “mystery” messenger, but that period of study did not resolve the mystery (Robbins et al., 1993; Hille, 1994; Marrion, 1997). 11.2.2

Modulation by Intracellular Ca2þ

Among the possible downstream effectors of Gq/11-coupled receptor stimulation, intracellular Ca2þ seemed to be one of the most probable candidates: it can freely diffuse through the cytosol; it regulates many membrane ion channels; and it is a ubiquitous signal downstream of PLC activation (discussed subsequently). Yet, the sensitivity of M-channels to such Ca2þ rises remained controversial for a long time. In excised patches from mammalian SCG neurons, M-channels were sharply inhibited by Ca2þ (Selyanko and Brown, 1996a, 1996b); however, in whole-cell experiments on amphibian neurons, M-channels responded to intracellular Ca2þ in a more complex way (activation by small Ca2þ rises and inhibition by strong Ca2þ elevations) (Marrion et al., 1991; Yu et al., 1994; Tokimasa, 1996; Tokimasa et al., 1996, 1997). Guided by the identification of calmodulin (CaM) as the Ca2þ sensor mediating Ca2þ-dependent inactivation of L-type Ca2þ channels and activation of SK-type Kþ channels (Xia et al., 1998; Zuhlke and Reuter, 1998; Peterson et al., 1999; Qin et al., 1999), we tested whether CaM served that role for M-channels as well. Overexpressed in Chinese hamster ovary (CHO) cells, mammalian Kv7.2/7.3 channels displayed little-to-no sensitivity to physiologically relevant [Ca2þ]i rises unless coexpressed with functional CaM (Gamper and Shapiro, 2003). The maneuver bestowed to the channels high inhibitory sensitivity to [Ca2þ]i (in the range comparable to that observed for native Mchannels in mammalian SCG neurons by the Brown group), whereas coexpression of the channels with dominant negative CaM rendered the Kv7.2/7.3 channels totally Ca2þ insensitive. Several labs have shown CaM to be a partner molecule to M-channels (Wen and Levitan, 2002; Yus-Najera et al., 2002; Gamper and Shapiro, 2003; Gamper et al., 2005). Although we interpret this partnership as a mechanism conferring Ca2þsensitivity to M-channels, an alternate, independent role of CaM in M-channel assembly has also been suggested (Wen and Levitan, 2002; Shahidullah et al., 2005). Does intracellular Ca2þ play a role in Gq/11-mediated inhibition of M-current in SCG neurons? The answer is definitely yes for some of the receptors and definitely no for the others. Thus, bradykinin-induced depression of M-current is mainly abolished by maneuvers that disrupt [Ca2þ]i rises, such as Ca2þ chelation or store depletion (Cruzblanca et al., 1998; Bofill-Cardona et al., 2000), or that render M-channels Ca2þ insensitive, such as overexpression of dominant negative CaM (Gamper and Shapiro, 2003). The mode of action seems similar for M-current inhibition by P2Y receptors in the same cells (Bofill-Cardona et al., 2000; Zaika et al., 2007). However, all those maneuvers do little to the depression of M-current by muscarinic or angiotensin II

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FIGURE 11.3 Bradykinin, but not muscarinic, receptors induce release of Ca2þ from intracellular stores in cultured SCG neurons. Cells were bath loaded with fura-2 as the AM ester for 30 min at 37 C and ratiometric Ca2þ signals were recorded using the TILL Photonics imaging system on an inverted Nikon TE300 microscope. The muscarinic agonist oxotremorine (10 mM) failed to induce Ca2þ transients, whereas application of bradykinin (250 nM) induced such a transient that was especially prominent in the neuronal processes.

receptor stimulation – correlating with the lack of [Ca2þ]i rises in SCG neurons provoked by stimulation of these receptors (Fig. 11.3) (Wanke et al., 1987; Beech et al., 1991; Shapiro et al., 1994a; Cruzblanca et al., 1998; Delmas et al., 2002). Thus, while relevant to bradykinin and P2Yreceptor-mediated M-current modulation, Ca2þ signaling cannot be considered as a ubiquitous mechanism mediating Gq/11- and PLC-coupled inhibition of M-channels. 11.2.3

Modulation by Membrane PIP2

The first hints that the signal mediating receptor-induced M-channel suppression was transduced not by a release of some channel inhibitor but by the withdrawal of

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something needed for normal M-channel activity came from the groups of Bertil Hille (Suh and Hille, 2002) and Diomedes Logothetis (Zhang et al., 2003). In the latter study, it was established that the rundown of M-current observed upon excision of giant membrane patches from Xenopus oocytes overexpressing Kv7.2/7.3 channels can be facilitated by the application of PIP2 scavengers or reversed by direct PIP2 application (Zhang et al., 2003). In another type of experiment done by both groups, it was also demonstrated that the recovery of M-current after muscarinic depression requires PIP2 resynthesis. The logic in these experiments was that if receptor-induced M-channel inhibition is mediated by PIP2 depletion, then the recovery from such inhibition should require PIP2 resynthesis. Indeed, block of PI4-kinase, a key enzyme of PIP2 synthesis, prevented current recovery after muscarinic inhibition (Suh and Hille, 2002; Zhang et al., 2003). Later, the same maneuver was shown to be effective for the case of angiotensin II AT1 receptors (Zaika et al., 2006). Many other groups have used alternative approaches to gain further support for the idea of PIP2 depletion as the signal underlying Gq/11-coupled modulation of Mchannels (Ford et al., 2003, 2004; Li et al., 2005; Winks et al., 2005; Robbins et al., 2006; Zaika et al., 2006, 2007). One such approach is to manipulate tonic PIP2 levels in the plasma membrane by overexpressing PIP2-scavenging or PIP-producing constructs and observing changes in the tonic current densities of the cloned or native M-channels. To this end, we overexpressed several constructs that sequester, cleave, or produce PIP2. One such construct consists of the pleckstrin homology domain of PLCd (PLCd-PH) fused to GFP; this construct sequesters PIP2, making it unavailable for other interactions (Stauffer et al., 1998; Raucher et al., 2000). As discussed below, PLCd-PH also binds to IP3 with an affinity more than 10-fold higher (Hirose et al., 1999). Another such construct is a PIP2-specific 50 phosphatase fused with the membrane-localization tag of Lyn kinase and GFP (Raucher et al., 2000). This construct dephosphorylates, rather than hydrolyzes, PIP2, and so does not produce any downstream second messengers. Overexpression of either of these constructs in CHO cells reduced M-current density by two–threefold (Li et al., 2005). Furthermore, overexpression of a phosphatidyl-4-phosphate 5-kinase [PI(4) 5-kinase], which elevates tonic PIP2 levels (Shyng et al., 2000; Bender et al., 2002), increased current density of Kv7.2 expressed in CHO cells (Li et al., 2005) and blunted muscarinic depression of native M-current in SCG neurons (Winks et al., 2005). Recently, an elegant system was developed in which a PIP2 phosphatase or PI (4)5-kinase can be acutely recruited to the plasma membrane and activated using the mechanism of chemically induced dimerization (CID) (Suh et al., 2006; Varnai et al., 2006). Such recruitment of the former robustly inhibited overexpressed Kv7.2/ 7.3 channels, whereas recruitment of the latter blunted muscarinic depression of the current. A similar idea of manipulation of PIP2 levels was used in experiments with palmitoylated membrane-targeted peptides (palpeptides) that can be applied from the outside of the neuron. Application of a PIP2 binding palpeptide suppressed Mcurrent in SCG neurons (Robbins et al., 2006). All of these experiments are consistent with PIP2 levels controlling M-channel activity, and with its depletion underlying its inhibition by muscarinic agonists. Another advantage of the PLCdPH construct is that since it has GFP attached, it can be used as an optical reporter of

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PIP2 hydrolysis. In unstimulated cells, the membrane has much PIP2, but cytosolic [IP3] is low, and so the probe localizes to the membrane. Upon activation of PLC and PIP2 hydrolysis, the probe translocates to the IP3 accumulating in the cytosol, a process that can easily be optically monitored. Such translocation experiments demonstrated the kinetics of M-current inhibition and PIP2 hydrolysis induced by muscarinic stimulation to be virtually identical (Horowitz et al., 2005; Winks et al., 2005). Further direct evidence of the PIP2 sensitivity of cloned M-channels has been obtained in single-channel experiments (Li et al., 2005). We directly applied a soluble PIP2-analogue to the inner side of the membrane containing a single Kv7 channel in inside-out patch experiments. The open probability (Po) of all Kv7 channels tested (Kv7.2–7.4) ran down rapidly upon excision (presumably due to the loss or degradation of PIP2), but increasing concentrations of applied PIP2 analogue progressively increased the Po of these channels, in some cases to unity (Li et al., 2005). Interestingly, the apparent affinity to PIP2 was seen to be quite divergent among the different Kv7 subunits, and as a result, the tonic Po of the Kv7 subunits in cell-attached patches varies as well. From the dose–response relations of PIP2 versus Po for the different Kv7 subunits appeared a pattern in which Kv7.2 and Kv7.4 have low apparent PIP2 affinity (IC50 200 mM); Kv7.3 has an apparent affinity some 100-fold higher (IC50 2 mM), whereas Kv7.2/7.3 heteromultimers have an intermediate PIP2 affinity (IC50 40 mM), in accordance with their being heteromeric channels containing both types of PIP2 binding subunits. Accordingly, the tonic Po (at saturating voltages) of Kv7.2 and Kv7.4 is very low (0.1), the Po of Kv7.3 is near unity and the Po of Kv7.2/7.3 heteromultimers is in the range of 0.3 (Selyanko et al., 2001; Li et al., 2004a, 2005). Thus, it seems that the tonic activity of a given Kv7 subunit is in direct correlation with its apparent PIP2 affinity and the tonic concentration of PIP2 in the plasma membrane; for that reason, the maximal Po of Kv7 channels is directly governed by membrane PIP2 abundance. In line with these ideas, overexpression, or sudden activation via CID, of PI (4)5-kinase dramatically increased the currents of Kv7.2, but not of Kv7.3 (Li et al., 2004a; Suh et al., 2006). The different intrinsic affinity for PIP2 of different Kv7 channels implies that neuronal M-channels assembled from different KCNQ gene products should respond to neurotransmitter stimulation with different sensitivities, thus providing an additional mechanism of specificity in neuronal signaling (also see below). Currently, it is believed that the apparent PIP2 affinity of M-channels is a dynamic entity that can itself be the subject of modulation. Thus, it has been suggested that binding of Ca2þ/CaM may decrease the PIP2 affinity, decreasing channel activity at tonic PIP2 levels or enhancing its sensitivity to PIP2 depletion (Delmas and Brown, 2005). All the experiments described above convincingly establish the fact that Mchannels are sensitive to PIP2 depletion and that stimulation of several Gq/11-coupled receptors induces robust PIP2 hydrolysis. But is such stimulation sufficient to significantly reduce PIP2 levels in the plasma membrane to the point where the reduction is sensed by M-channels? Indeed, translocation of the PLCd-PH construct is a good readout of PIP2 hydrolysis, but not of PIP2 depletion, since the construct may translocate following IP3 release induced by PLC activity, even if there is still much PIP2 in the membrane. In support of this concern, PLCd-PH translocation is blocked

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when accumulation of IP3 in the cytosol is prevented by overexpression of an IP3 5-phosphatase, which prevents cytosolic IP3 accumulation (Hirose et al., 1999; Horowitz et al., 2005; Suh et al., 2006). To probe the actual changes in PIP2 abundance upon muscarinic modulation, we and others used high-performance liquid chromatography (HPLC) analysis of the total pools of anionic phospholipids (Nasuhoglu et al., 2002) in membrane extracts from CHO cells overexpressing M1 receptors (Horowitz et al., 2005; Li et al., 2005). In these experiments, it was found that approximately 90% of total PIP2 is degraded within the first minute of muscarinic stimulation. Similar PIP2 depletion was observed in CHO cells overexpressing AT1 receptors (Zaika et al., 2006). In native SCG cells, Winks et al. (2005) titrated the loss of membrane localization of the PLCd-PH probe as a function of dialysis of a range of IP3 concentrations to mathematically quantify the extent of PIP2 depletion in the neurons by muscarinic stimulation, and found similarly that muscarinic agonists can reduce global membrane PIP2 by >80%. To summarize this section, the activity of M-channels in sympathetic neurons is tightly governed by plasma membrane PIP2 abundance, and PLC-induced PIP2 depletion underlies muscarinic and angiotensin II suppression of these channels. In contrast, bradykinin and P2Y receptor stimulation depress M-current via [Ca2þ]i signals. Why should bradykinin or P2Y receptors use another route to inhibit M-channels? We will try to answer this question in section 11.3.6 of this chapter. 11.2.4

Other Modes of Modulation

Other signaling pathways affect M-current activity in SCG neurons either directly or indirectly. Thus, different M-channel subunits are subject to multiple phosphorylations that either directly affect channel gating or change its sensitivity to GPCR modulation. Thus, Kv7.3, 7.4, and 7.5 are phosphorylated by Src (Gamper et al., 2003; Li et al., 2004b) or receptor tyrosine kinases (such as the EGF receptor) (Jia et al., 2007) at two tyrosines equivalent to Y67 (N-terminus) and Y349 (C-terminus) in Kv7.3 (Li et al., 2004b; Jia et al., 2007), an effect resulting in the decrease of current amplitude and slowing of kinetics. In addition, Kv7.2 can be phosphorylated at S541 and S534 in the channel C-terminus by PKC, which is recruited to the channel by the A-kinase anchoring protein AKAP79/150 (Hoshi et al., 2003). These phosphorylations result in an increase of channel sensitivity to muscarinic, but not bradykinin, inhibition (Hoshi et al., 2003, 2005), presumably by decreasing the affinity of PIP2 for the channel (similar to suggested mechanism of action of Ca2þ/CaM; Delmas and Brown, 2005). Consistent with this, muscarinic depression of M-current in AKAP150 knockout mice was substantially attenuated (Hoshi et al., 2005). Finally, M-channels can be inhibited by cyclic ADP-ribose (Bowden et al., 1999; Higashida et al., 2000) and augmented by physiological concentrations of H2O2 (Gamper et al., 2006), and M-current is somewhat pH sensitive (Prole et al., 2003), although it is not clear if any of these M-channel properties are used in intracellular signaling pathways.

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11.3 MODULATION OF VOLTAGE-GATED Ca2þ CHANNELS 11.3.1

Overview

Voltage-gated Ca2þ channels (VGCCs) form a large family of Ca2þ-selective ion channels that are expressed in all types of excitable cells and mediate release of neurotransmitters from synaptic terminals, secretion of neuromediators and hormones by neurons and neuroendocrine cells, excitation–contraction coupling, and Ca2þ-dependent gene expression (Catterall, 2000). There are five types of VGCC currents (ICa): high-voltage-activated L-, N-, P/Q-, and R-types, and low-voltage-activated T-type. The pore-forming subunit of VGCC, called a1, has 24 transmembrane domains (TMD) organized in four 6-TMD repeats. a1 Subunits are coded by three gene subfamilies: Cav1–3. Cav1.1–1.4 code for the L-type channels; Cav2.1 for P/Q-, Cav2.2 for N-, Cav2.3 for R-, and Cav3.1–3.3 for T-type channels, respectively. VGCC are usually assembled with auxiliary subunits: b, a2d, and, in some cases, g. In rat SCG cells, ICa is >80% of the w-conotoxin GVIA-sensitive N-type (Plummer et al., 1989), but in murine SCG, ICa is a mixture of about 50% N-type, 20% L-type, 25% P/Q-type, and the rest R-type (Shapiro et al., 1999). The N-type channels in particular have been shown to be expressed in a number of alternatively spliced forms (see Chapter 8; Lin et al., 1999), perhaps accounting for some of the variability in the pharmacological profile and modulatory sensitivities of Cav2.2 channels in the nervous system.

11.3.2

A Fast and Direct Modulatory Pathway

The early work of Dunlap and Fischbach 1978 showed that several neurotransmitters, including norepinephrine (NE), 5-HT, and GABA could trigger depressions of ICa in chick ganglia via G-protein actions (Holz et al., 1986). In rat SCG neurons, a similar and rapid depression of ICa by muscarinic (Wanke et al., 1987), somatostatin (Ikeda and Schofield, 1989), adrenergic (Plummer et al., 1991), and peptidergic (Foucart et al., 1993) receptor stimulation was observed. This action is mediated by pertussistoxin (PTX)-sensitive (Go/i) G-proteins. This type of signal was shown to result from a direct action of G-protein (Forscher et al., 1986; Lipscombe et al., 1989; Bernheim et al., 1991; Shapiro and Hille, 1993) with the channels, resulting in a shift in the voltage dependence of activation (Marchetti et al., 1986; Ikeda et al., 1987). The biophysics was nicely analyzed by Bruce Bean, who described the G-protein unbound and bound channels as “willing” and “reluctant,” respectively, such that “reluctant” channels only open at much more depolarized potentials than do “willing” ones (Bean, 1989). The “willing/reluctant” model correctly predicts that G-protein binding itself to the channel should be voltage dependent (favored at hyperpolarized potentials, disfavored at depolarized ones), such that upon a physiological depolarization, only the (unbound) “willing” channels can initially open, but a maintained depolarization will induce unbinding of G-protein, conversion of “reluctant” channels to “willing” ones, and a seeming biphasic or “slowed” activation time course (Boland and Bean, 1993). Indeed, as a quick and easy assay of Ca2þ channel modulation, many investigators

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simply look for this signature of slowed activation. As for Go/i activation of GIRK inward rectifier channels (Logothetis et al., 1987), it was soon realized that the bg dimer of the G-protein was responsible for this direct action on the Ca2þ channel as well (Herlitze et al., 1996; Ikeda, 1996; Delmas et al., 1998b). A number of other labs also contemporaneously explored this issue, using other neurons or receptor types (Gross et al., 1989; Kasai and Aosaki, 1989; Bley and Tsien, 1990; Elmslie et al., 1990; Schofield, 1990; Penington et al., 1991; Scholz and Miller, 1991; Ikeda, 1992; Golard and Siegelbaum, 1993). The topic has also been nicely reviewed (Hille, 1994; Ikeda and Dunlap, 1999). 11.3.3

A Slower and Indirect Pathway

The use of other G-protein mechanisms of modulation of these channels, however, soon became clear. The best example remains that of muscarinic agonists in SCG cells, for which the same agonist initiates two distinct signals acting on the same channels, but via different receptor subtypes and modes of action. The first uses the M2 or M4 subtypes of receptors (Bernheim et al., 1992; Shapiro et al., 1999), PTXsensitive Go/i G-proteins, and the voltage-dependent “willling/reluctant” mechanism discussed above (Beech et al., 1992). Due to its direct nature, its top speed is very fast; at <400 ms, it compares with rapid activation of GIRK Kþ channels (Surprenant and North, 1988). The second uses M1 receptors (Bernheim et al., 1992), coupled to PTX-insensitive Gq/11 proteins (Beech et al., 1992; Delmas et al., 1998a) and a downstream intracellular messenger (Bernheim et al., 1991), whose actions on the Ca2þ channels are wholly voltage independent (Beech et al., 1992). Since a second messenger molecule is involved, the speed of action is much slower, requiring 30 s for full effect (Zhou et al., 1997). If both M2/4 receptor-mediated “fast” and M1 receptor-mediated “slow” pathways are concurrently triggered, one observes a biphasic depression of ICa, manifested as an abrupt inhibition of 50%, followed thereafter by a more gradual developing inhibition of another 50% (Beech et al., 1992; Shapiro et al., 1994b). An early observation was that this slower pathway was blocked if intracellular Ca2þ were reduced to levels <10 nM by dialysis with Ca2þ chelators (Beech et al., 1991), an effect now understood to originate from the need of PLCb for permissive [Ca2þ]i for activation (Horowitz et al., 2005). Since the canonical Gq/11-mediated signal in a variety of cells indeed consists of PLCb activation and triggering of its downstream pathways (Fig. 11.2), much work tested the involvement of IP3, DAG, and downstream protein kinase C, but (as in the case for M-current) results were negative (Hille, 1994). Although cytosolic IP3 production widely induces release of Ca2þ from intracellular stores (Berridge, 2005), we already knew that such [Ca2þ]i signals could not be the messenger, since muscarinic stimulation in SCG neurons does not produce observable [Ca2þ]i signals (Wanke et al., 1987; Beech et al., 1991), although stimulation of other Gq/11-coupled receptors in the same cells does (see below). Stimulation of angiotensin II receptors of SCG cells, which are also coupled to Gq/11, suppresses ICa via the same mechanism (Shapiro et al., 1994a). We note here that similar dual pathways of N- and P/Q-type Ca2þ channel inhibition have been observed in other

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neurons, such as sensomotor pyramidal cells (Stewart et al., 1999) and striatum (Howe and Surmeier, 1995), and in reconstituted systems (Melliti et al., 2001). 11.3.4

Intracellular Mechanisms: PIP2

The question of mechanism for Gq/11-mediated muscarinic suppression of ICa has much in common with that acting on M-type Kþ channels (see above), including the same M1 receptors, coupling to Gq/11, need for robust PLCb activity and similar time course. Hence, the two fields soon become intertwined (reviewed in Hille, 1992, 1994). The breakthrough came with the discovery that the M-channels are inhibited via the receptor-induced PIP2 depletion (as discussed above). Thus, the M-channels joined an expanding list of other PIP2-sensitive ion channels and transporters (Suh and Hille, 2005; Gamper and Shapiro, 2007a). Our group was motivated by the report that cloned P/Q-type channels also require PIP2, and that stimulation of receptors heterologously expressed in oocytes inhibits those channels by depletion of PIP2 (Wu et al., 2002). That work also suggested a role of PIP2 in the “willing/reluctant” mechanism discussed above, but this facet is still unclear. Guided by that report and the apparent commonality of the Gq/11 signals toward M and Ca2þ channels, we asked whether N-type channels of SCG neurons were also sensitive to PIP2 abundance, and if the second messenger mechanism underlying depression of ICa used by M1 receptors was mediated by depletion of PIP2 (Gamper et al., 2004), as for M-current. Indeed, we found that cloned N-type channels expressed in oocytes are also sensitive to PIP2 abundance, with channels excised in inside-out patches rapidly running down in the absence, but not in the presence, of PIP2 in the cytoplasmically facing bath solution. We then performed a number of experiments on PTX-treated SCG neurons. Similar to the experiments with M-current, inhibition of PI4-kinase with wortmannin blocked recovery of ICa from muscarinic suppression, indicating that PIP2 resynthesis is required for the recovery and that PIP2 depletion mediates the inhibition. Another test was the slowing and attenuation of ICa suppression by dialysis of PIP2 into the cytoplasm from the whole-cell pipettes (providing an inexhaustible supply). In another set of experiments, we used the fluorescently tagged PLCd-PH construct discussed above. As a PIP2-sequestering agent, this construct lowers available plasmalemmal PIP2 and buffers changes in its abundance. Thus, PTX-treated SCG neurons transfected with PLCd-PH using the biolistic method (Gamper and Shapiro, 2006) displayed reduced ICa amplitudes and blunted muscarinic suppression. Similarly, overexpression in neurons of a PIP2 5-phosphatase sharply reduced ICa amplitudes and attenuated the modulation. Both of these experiments are consistent with the functional interaction of the Ca2þ channels with PIP2 and with PIP2 depletion as the underlying mechanism of Gq/11-mediated muscarinic depression of ICa (Gamper et al., 2004). The time course of PLCd-PH translocation in SCG neurons in response to muscarinic stimulation was again nearly identical to that of ICa depression (Zhang et al., 2003; Gamper et al., 2004; Winks et al., 2005). As a control, expression of a construct consisting of EGFP fused with the PH domain of Akt, which binds not to PIP2 but to (PI3,4P2) and PIP3 (Franke et al., 1997), had no effect on tonic ICa amplitudes, nor on muscarinic suppression.

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11.3.5

303

Other Intracellular Mechanisms

Another intracellular mechanism put forth as underlying the depression of ICa by M1 receptor stimulation of sympathetic neurons involves arachidonic acid. This is plausible considering that AA can be liberated upon receptor stimulation via G-protein activation of phospholipase A2 (PLA2), or more plausible in the case of Gq/11 signaling via conversion of DAG to AA by DAG lipase (see Irvine, 1982; Axelrod et al., 1988 for reviews). In addition, a number of reports suggest direct or indirect AA mediation in the modulation of a number of ion channels (Ordway et al, 1991; Piomelli, 1994 for reviews). Keyser and Alger 1990 showed the indirect depression of L-type ICa in hippocampal neurons by AA, acting via PKC and reactive oxygen species (Keyser and Alger, 1990), and Schmitt and Meves (1995) suggested a direct AA-induced suppression of ICa in neuroblastoma cells (Schmitt and Meves, 1995). The Rittenhouse lab has pursued a line of investigation whose core is the sensitivity of L- and N-type channels to AA, and has suggested that AA production is the primary signal mediating M1 receptor-mediated inhibition of those channels. The effects of bath-applied AA were seen at the singlechannel and whole-cell levels, and were prevented by application of bovine serum albumin (BSA, thought to be able to scavenge free AA) (Liu and Rittenhouse, 2000; Liu et al., 2001). Blockade of PTX-insensitive depression of N-type ICa was seen by inhibitors of PLA2 or by BSA (Liu and Rittenhouse, 2003), although ourgroup found no effect of the PLA2 inhibitor (Gamper et al., 2004). A recent study documents involvement of AA of PTX-insensitive depression of the smaller L-type ICa in these neurons. The evidence included similar pharmacological tests as above, but also included PLA2 phosphorylation assays, PLA2 antibodies, and PLA2 knockout mice (Liu et al., 2006). For both N-type and L-type inhibitions, these authors find the modulation to require Gq/11a and PLC activity as well. They suggest that AA may compete for PIP2 binding to the channels, or that downstream activation of PKC by DAG production phosphorylates and turns on PLA2 (Liu et al., 2004, 2006). Our group believes the major signal for Gq/11-mediated muscarinic inhibition of N- and P/Q-type channels to be depletion of PIP2, but we have no data regarding actions on L-type channels. Given the clear dynamic nature of PIP2 affinities for many channels, including Kir3 channels (Gbg increases, Naþ decreases) and M-channels (PKC, Ca2þ/calmodulin decrease), we hypothesize that AA may similarly play a role in tuning the sensitivity of Ca2þ channels to PIP2 abundance (alters affinity), so that smaller, or spatiotemporally restricted, depletions of PIP2 have the intended modulatory effect on the channels. This hypothesis could be rigorously tested with biochemical measurements of channel affinity for PIP2, with values compared in the presence or absence of AA. A similar hypothesis of AA antagonism of PIP2 binding has been suggested for the regulation of inactivation of Kv channels (Oliver et al., 2004) and Gq/11-mediated inhibition of GIRK Kþ channels in hippocampal neurons (Sohn et al., 2007). We have put forth a “coincidence detector” hypothesis for channel modulation as a mechanism for ensuring fidelity in receptor signaling toward the plethora of channels and transporters that are being shown to be sensitive to PIP2 abundance (Delmas et al., 2005; Gamper and Shapiro, 2007b). Several actions of protein kinases on N-type channels have been documented. The adrenergic depression of N-type ICa in avian dorsal root ganglion (DRG) neurons

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requires PKC activity (Rane and Dunlap, 1986; Rane et al., 1989), a result also seen for the Gq/11-mediated muscarinic inhibition of cloned R-type channels (Bannister et al., 2004), but not for N-type channels in rat SCG cells (Bernheim et al., 1991; Barrett and Rittenhouse, 2000). The PKC-mediated action in avian DRG was suggested to be mediated by bg dimers liberated from Gi, but not Go, activation of PLC (by Gbg) and generation of DAG, but was not manifested by “kinetic slowing” (Diverse-Pierluissi et al., 1995). As widely seen for A-kinase anchoring proteins (Beene and Scott, 2007), the enigma homologue (ENH) scaffolding protein was shown to recruit the atypical PKCe isoform to N-type, but not P/Q-type, channels and to potentiate their upregulation by phorbol esters (Maeno-Hikichi et al., 2003), perhaps underlying the reports of PKC action on N-type channels. Work in hippocampal neurons suggests a glucocorticoid-induced suppression of N-type and L-type ICa via Go/i and PKC (ffrenchMullen, 1995). Dopamine receptor-induced inhibition of N-type and P/Q-type channels via protein kinase Awas reported in striatum (Surmeier et al., 1995). Another line of investigation indicates modulation of avian DRG Ca2þ channels by tyrosine phosphorylation mediated by Go (Diverse-Pierluissi et al., 1997) via direct tyrosine kinase interaction with the channels (Schiff et al., 2000; Richman et al., 2004). Analyses of alternatively spliced forms of N-type channels indicate a tyrosine present in one of them being the site of the Src-mediated modulation (Raingo et al., 2007). Such differences in signaling across neuronal and species types may indicate evolutionary divergence between avian and mammal, or cell-specific divergence in signaling mechanisms between autonomic and sensory ganglia. 11.3.6 Receptor Specificity in Modulation of IM and ICa in Sympathetic Neurons As alluded to above, the M1 type is not the only Gq/11-coupled receptor in SCG neurons; the cells also express the bradykinin B2, angiotensin II AT1, and purinergic P2Y6 types. As already mentioned, the AT1-receptor stimulation inhibits N-type Ca2þ and M-type Kþ channels via the intracellular mechanism as do M1 receptors (Shapiro et al., 1994; Zaika et al., 2006). Although the stimulation of B2 and P2Y receptors also provokes strong PIP2 hydrolysis, as measured by translocation of the PLCd probe (Gamper et al., 2004; Winks et al., 2005) (Fig. 11.4), these receptors do not induce appreciable suppression of ICa (Gamper et al., 2004; Zaika et al., 2007). The reason seems to be due to their ability to release intracellular Ca2þ from stores and to generate subsequent [Ca2þ]i signals (Cruzblanca et al., 1998; Delmas et al., 2002; Zaika et al., 2007), unlike M1 or AT1 receptor stimulation (Wanke et al., 1987; Beech et al., 1991; Shapiro et al., 1994a). It is very important to note that this receptor specificity in Ca2þ signaling occurs in neurons, but not in heterologous systems in which stimulation of any overexpressed Gq/11-coupled receptor induces large [Ca2þ]i transients, and all receptors seem to have the same effects. It has been proposed that in sympathetic neurons, the specificity is due to specific clustering of certain receptors, but not others, with IP3 receptors, such that the IP3 produced by PLC activity is in the right microdomain to cause Ca2þ release (Delmas et al., 2002). This microdomain theory is akin to the proposed mechanism of many other neuronal processes and functions,

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FIGURE 11.4 Purinergic stimulation provokes PIP2 hydrolysis, but does not depress ICa. (a) Plotted are normalized Ca2þ current amplitudes recorded from a superior cervical ganglion (SCG) neuron under perforated patch voltage-clamp. UTP (10 mM), BK (250 nM), oxo-M (10 mM), or CdCl2 (100 mM) were bath applied during the periods shown by the bars. Representative current traces are shown in the inset. (b) Using the “gene gun,” SCG neurons were transfected with the PLCd-PH-EGFP construct that is an optical reporter of PIP2 hydrolysis. Before purinergic stimulation (control), the probe is localized to the membrane as expected, since tonic [IP3] is low and membrane [PIP2] is substantial. However, upon stimulation of the P2Y receptors by UTP (10 mM) and activation of PLCb, the probe translocates to the cytosol. Images were acquired using the Nikon swept field confocal (SFC) system in the Department of Physiology core facility at UTHSCSA. (See the color version of this figure in Color Plate section.)

most notably the subcellular organization of the synaptic zone (Delmas et al., 2004). Via binding to frequenin in Drosophila, or to its mammalian homologue, neuronal calcium sensor-1, Ca2þ signals powerfully stimulate PI4-kinase activity (Hendricks et al., 1999; Koizumi et al., 2002; Zheng et al., 2005), resulting in increased PIP2 production that we hypothesize compensates for PIP2 depletion by PLC activity that otherwise occurs (Delmas et al., 2005). Indeed, in neuroblastoma cells, careful measurements show PIP2 levels to initially increase following bradykinin receptor stimulation, followed by a decline, the former attributed to receptor-mediated stimulation of both PI4- and PI(4)5-kinases (Xu et al., 2003). Thus, our model supposes that muscarinic or angiotensin stimulation, neither of which provoke [Ca2þ]i signals and subsequent stimulation of PI4-kinase, induce PIP2 depletion inhibiting Ca2þ, M-type (see above), and any other PIP2-sensitive channels. Whether such depletions are necessarily global (Suh et al., 2004) or locally restricted to microdomains (Cho et al., 2005) is an open question recently discussed

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(Gamper and Shapiro, 2007b). On the other hand, ICa is usually not depressed by bradykinin or P2Y-receptor stimulation, due to concurrent stimulation of PIP2 synthesis, which does not permit PIP2 levels to fall appreciably. Interestingly, another lab does find bradykinin stimulation to depress ICa in the same SCG cells via lowered PIP2 (Lechner et al., 2005), possibly due to variations in tonic rates of PI-kinases and phosphatases,variable tonic [PIP2] or to variable store Ca2þ load manifested by cell preparations between labs. Also of note is the lack of depression by bradykinin stimulation, of the highly PIP2-sensitive Kir3 (GIRK) channels heterologously expressed in SCG cells, consistent with bradykinin not causing PIP2 depletion in these neurons (Winks et al., 2005) (again, local versus global issues may apply). The idea of concurrent activation of PIP2 synthesis by those Gq/11-coupled agonists that raise [Ca2þ]i is confounded, however, with the evidence discussed above that prevention of IP3-mediated Ca2þ rises blocks suppression of M-channels by bradykinin or purinergic agonists (Cruzblanca et al., 1998; Bofill-Cardona et al., 2000; Zaika

FIGURE 11.5 Schematic representation of bipartite mode of action of Gq/11-coupled receptors in sympathetic neurons. Both “Mode 1” and “Mode 2” receptors activate Gq/11-type G-proteins, phospholipase C (PLC), and PIP2 hydrolysis. “Mode 1” receptors do not stimulate PIP2 synthesis and suppress M-current (IM) and Ca2þ current (ICa) by depletion of PIP2. Mode 1 does not elicit intracellular Ca2þ [Ca2þ]i signals. “Mode 2” receptors do induce [Ca2þ]i signals and concurrently stimulate PIP2 synthesis via neuronal Ca2þ sensor-1 (NCS-1), preventing PIP2 depletion. Thus, “mode 2” receptors depress neither the ICa nor the PIP2-sensitive Kir3 (GIRK) channels (Winks et al., 2005). The released Ca2þ provoked by “mode 2” receptors binds to NCS-1 and to calmodulin (CaM). Ca2þ/CaM binds to M-channels, resulting in their inhibition (see text). (Adapted from Zaika et al., 2007.)

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et al., 2007). It is indeed not clear why suppression of such Ca2þ rises would not disrupt stimulation of PIP2 synthesis, which, in turn, might allow depletion of PIP2 and Mcurrent suppression. Perhaps the PIP2 synthesis machinery is better coupled to IP3sensitive stores either via affinity mechanisms or by further spatial coupling. These issues are currently under investigation in our laboratories (Fig. 11.5).

11.4 CONCLUSIONS Modulation of voltage-gated Kþ and Ca2þ channels in neurons provides a powerful mechanism for the regulation of neuronal output. Compared to the sub-millisecond timescale of the fast ionotropic action of neurotransmitters, the modulatory actions, on which our focus is, are much slower. However, these pathways direct the nervous response on the timescale of a thought, a movement, or a heartbeat, making them a prime mechanism for the response of the individual to a constantly changing environment and for the control of internal homeostasis. The G-protein pathways can be rapid and localized, as is the case for the direct action on Ca2þ channels that is complete under a second, or can be more slowly developing and more broadly acting, as is the case of Gq/11-mediated regulation of Ca2þ channels and M-channels. The latter is particularly well suited to the regulation of neuronal excitability and discharge patterns, whereas the former probably regulates the release of neurotransmitter at specific varicosities. Over and above these signaling pathways are even longer lasting modulatory signals, such as the modulation of Kv7 channels by tyrosine kinases that likely act over hours or days, and even longer term control over neuronal function at the transcriptional level, in which individual neurons, for example, decide which M-type subunits to express, so as to achieve the appropriate sensitivities to the various neurotransmitter inputs, in accordance with the agenda of the neuronal type or brain region.

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Selyanko AA, Hadley JK, Wood IC, Abogadie FC, Jentsch TJ, Brown DA, 2000. Inhibition of KCNQ1-4 potassium channels expressed in mammalian cells via M1 muscarinic acetylcholine receptors. J Physiol (Lond) 522 (Part 3): 349–355. Selyanko AA, Hadley JK, Brown DA, 2001. Properties of single M-type KCNQ2/KCNQ3 potassium channels expressed in mammalian cells. J Physiol 534: 15–24. Selyanko AA, Delmas P, Hadley JK, Tatulian L, Wood IC, Mistry M, London B, Brown DA, 2002. Dominant-negative subunits reveal potassium channel families that contribute to M-like potassium currents. J Neurosci 22: RC212. Shahidullah M, Santarelli LC, Wen H, Levitan IB, 2005. Expression of a calmodulin-binding KCNQ2 potassium channel fragment modulates neuronal M-current and membrane excitability. Proc Natl Acad Sci USA 102: 16454–16459. Shapiro MS, Hille B, 1993. Substance P and somatostatin inhibit calcium channels in rat sympathetic neurons via different G protein pathways. Neuron 10: 11–20. Shapiro MS, Wollmuth LP, Hille B, 1994a. Angiotensin II inhibits calcium and M current channels in rat sympathetic neurons via G proteins. Neuron 12: 1319–1329. Shapiro MS, Wollmuth LP, Hille B, 1994b. Modulation of Ca2þ channels by PTX-sensitive Gproteins is blocked by N- ethylmaleimide in rat sympathetic neurons. J Neurosci 14: 7109– 7116. Shapiro MS, Loose MD, Hamilton SE, Nathanson NM, Gomeza J, Wess J, Hille B, 1999. Assignment of muscarinic receptor subtypes mediating G-protein modulation of Ca2þ channels by using knockout mice. Proc Natl Acad Sci USA 96: 10899–10904. Shapiro MS, Roche JP, Kaftan EJ, Cruzblanca H, Mackie K, Hille B, 2000. Reconstitution of muscarinic modulation of the KCNQ2/KCNQ3 Kþ channels that underlie the neuronal M current. J Neurosci 20: 1710–1721. Shyng SL, Barbieri A, Gumusboga A, Cukras C, Pike L, Davis JN, Stahl PD, Nichols CG, 2000. Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase. Proc Natl Acad Sci USA 97: 937–941. Soejima M, Noma A, 1984. Mode of regulation of the ACh-sensitive K-channel by the muscarinic receptor in rabbit atrial cells. Pflugers Arch 400: 424–431. Sohn JW, Lim A, Lee SH, Ho WK, 2007. Decrease in PIP2-channel interactions is the final common mechanism involved in PKC- and arachidonic acid-mediated inhibitions of GABAB-activated Kþ current. J Physiol 582: 1037–1046. Stauffer TP, Ahn S, Meyer T, 1998. Receptor-induced transient reduction in plasma membrane PtdIns(4,5) P2 concentration monitored in living cells. Curr Biol 8: 343–346. Stewart AE, Yan Z, Surmeier DJ, Foehring RC, 1999. Muscarine modulates Ca2þ channel currents in rat sensorimotor pyramidal cells via two distinct pathways. J Neurophysiol 81: 72–84. Suh B-C, Hille B, 2002. Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron 35: 507–520. Suh B-C, Hille B, 2005. Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr Opin Neurobiol 15: 370–378. Suh B-C, Horowitz LF, Hirdes W, Mackie K, Hille B, 2004. Regulation of KCNQ2/KCNQ3 current by G-protein cycling: the kinetics of receptor-mediated signaling by Gq. J Gen Physiol 123: 663–683. Suh B-C, Inoue T, Meyer T, Hille B, 2006. Rapid chemically induced changes of PtdIns(4,5) P2 gate KCNQ ion channels. Science 314: 1454–1457.

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12 BK CHANNELS: REGULATION OF EXPRESSION AND PHYSIOLOGICAL IMPACT PALLOB KUNDU1, ABDERRAHMANE ALIOUA1, YOGESH KUMAR1, RONG LU1, JIMMY W. OU1, ENRIQUE SANCHEZ-PASTOR1, MIN LI1, ENRICO STEFANI1,2,4,5, AND LIGIA TORO1,3,4,5 1

Department of Anesthesiology, Division of Molecular Medicine, University of California, Los Angeles, USA 2 Department of Physiology, University of California, Los Angeles, USA 3 Department of Molecular and Medical Pharmacology, University of California, Los Angeles, USA 4 Cardiovascular Research Laboratory, University of California, Los Angeles, USA 5 Brain Research Institute, University of California, Los Angeles, USA

12.1 BK CHANNELS: MEMBERS OF THE SLO FAMILY The BK channel a subunit was the first identified member of the Slo gene family (Table 12.1) and is now referred to as Slo1 or KCNMA1 (Fig. 12.1). Four Slo1 subunits are needed to make a functional channel. The first steps toward Slo1 cloning were the pioneering patch-clamp studies with muscle cells from the lethargic Drosophila mutant (slo) lacking calcium-dependent outward potassium currents that pinpointed this mutant as key to identify the genetic loci responsible for BK currents (Elkins et al., 1986). Successful cloning and functional expression identified the Drosophila Slo gene as the essential Kþ conducting component of BK channels (Atkinson et al., 1991; Adelman et al., 1992), followed by cloning of mouse (Elkins et al., 1986; Butler et al.,

Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

317

318

KCa4.2, Slick, SLO2.1

Slack, KCa4.1

KCNMC1, KCa5.1, Kcnma3

Slo2.1

Slo2.2

Slo3

?, unknown.

BK channels, KCa1.1, MaxiK

Alternative names

Channel

Slo1

Slo Family

TABLE 12.1

KCNU1

KCNT1

KCNT2

KCNMA1

Gene 2þ

Spermatocytes and mature spermatozoa

Brain, Kidney

Voltage, Naþ, Cl Internal pH

Brain

Ubiquitous

Tissue Distribution

Voltage, Naþ, Cl, ATP

Voltage and Ca

Activation

Blood flow, airway tone, neurotransmission, neural firing, motor coordination, uresis, hearing, male potency, circadian rhythms Neuroregulation, firing properties of auditory neurons Neuroregulation, firing properties of auditory neurons ?

Functional Implications

BK CHANNELS: MEMBERS OF THE SLO FAMILY

319

Slo1 (KCNMA1) gene (a)

* **

5'-UTR

1

2

*

3 4 5

6 7

8

* *

*

9 10 11 12 13 14 15 16 17 18 19 20 21

22 23 24 25

* 26

27 3'-UTR TGA

ATG

400 bp

M1 M2 M3 M4 mSlo1 MANGGGGGGGSSGGGGGGGGGSGLRMSSNIHANHLSLDASSSSSSSSSSSSSSSSSSSSSVHEPKMDALIIPVTMEVP hSlo1 MANGGGGGGGSSGGGGGGGG-SSLRMSSNIHANHLSLDASSSSSSSSSSSSSSSSSSSSSSVHEPKMDALIIPVTMEVP

(b)

*

N

3,4

+

+ S5 S1 S2 S3 S4

S0

+

2,3

* * 1,2

SSQA(V)DG RCK1

S6 DRD(G)DV

9,10

*

18,19

*

*

16,17

S7

S8

S10

S9

C RCK2 23,24

*

* “Ca2+ bowl”

FIGURE 12.1 Gene map and protein topology of Slo1 (KCNMA1) gene. (a) Gene map showing constitutive exons (gray boxes, at scale) and introns (broken lines, not to scale) of human Slo1. Known splice sites in various species are marked with an arrow and a star. Enlarged region shows probable translation start sites (methionine, M1–M4) in human and mouse and high degree of homology between species, which applies for the rest of the protein (not shown). Most of studies have used clones beginning at M3 (red). (b) Slo1 protein topology. Seven transmembrane domains are marked as S0–S6. Cytoplasmic hydrophobic domains are marked as S7–S10. Regulator of conductance of Kþ domains, RCK1 (blue) and RCK2 (pink) positions (Yusifov et al., 2008). Ca2þ bowl, calcium sensing domain (green). Arrowheads mark junctions of translated constitutive exons; numbers of corresponding exons are only given for known alternative splice sites (*) (e.g., 1,2 marks where the translated protein of exon 1 joins the translated protein of exon 2). Red boxes and sequences mark human mutations linked to generalized epilepsy and paroxysmal movement disorder (D/G), and autism (A/V). (See the color version of this figure in Color Plate section.)

1993), human (Pallanck and Ganetzky, 1994; Wallner et al., 1995), and other mammalian orthologues. Later, analysis of Caenorhabditis elegans genome sequences led to the discovery of Slo2 (Wei et al., 1996), which is not only activated by Ca2þ but also shows dependence on Cl, while EST and human genomic sequences helped in the identification of Slo2.1 (Slick) (Bhattacharjee et al., 2003), Slo2.2 (Slack) (Joiner et al., 1998), and Slo3 (Schreiber et al., 1998). Mammalian Slo2 channels are sensitive to Naþ and Cl (see Chapter 7), while Slo3 responds to changes in pH. Both

320

BK CHANNELS: REGULATION OF EXPRESSION AND PHYSIOLOGICAL IMPACT

Slo2 channels are expressed in brain and controlled by neuromodulators through Gaq-protein-coupled receptors in opposite ways: Slo2.1 is strongly inhibited, whereas Slo2.2 currents are strongly activated (Santi et al., 2006). Interestingly, Slo2 channels regulate the accuracy of timing of neurons from the medial nucleus of the trapezoid body, suggesting their role in the auditory system (Yang et al., 2007). Among the Slo family members, Slo3 is the least characterized, and unlike Slo1, which is ubiquitous, Slo3 expression is highly tissue specific and found only in testis. Although from the same family, Slo1, Slo2, and Slo3 have extensive sequence dissimilarities (global alignment gives 20% identity between Slo1 and Slo2 and 40% identity between Slo1 and Slo3) that are reflected, as mentioned above, in distinct sensitivity to intracellular ions, channel kinetics, and functions. In this review, we will focus on the Slo1 channel because of its ubiquitous expression, diverse physiological function, and extensive characterization.

12.2 MOLECULAR DIVERSITY OF SLO1 CHANNELS: FUNCTIONAL CONSEQUENCES In humans, the Slo1 gene is mapped to chromosome 10 (10q22.3 locus) and is at least 768 kb long. In mouse, it is found in chromosomal location 14A3 and is at least 705 kb long. The mRNA is assembled from 27 constitutive exons in both organisms. Exon structure and sequences are fairly conserved among species. From this single gene, complex diversity and functions of the channel can be generated due to (a) mRNA processing, (b) posttranslational modifications, and (c) association with partners.

12.2.1

mRNA Processing

In general, transcript diversity can arise from expression of different: (i) 50 -untranslated region (UTR), (ii) alternative usage of pre-mRNA polyadenylation signals that generate 30 -UTR of variable lengths, and/or (iii) pre-mRNA alternative splicing. Slo1 appears to use all three of these mechanisms. The first insight into the diversity of Slo mRNA expression was obtained from the extensive analysis of Drosophila Slo (dSlo) gene. dSlo is expressed from multiple transcription start sites and multiple tissue-specific promoters (Becker et al., 1995; Brenner and Atkinson, 1996; Brenner et al., 1996; Bohm et al., 2000) and can also be subject of substantial alternative splicing events (Adelman et al., 1992). Likewise, we recently demonstrated that mouse Slo1 (mSlo1) is a multitranscription start site type of gene with at least two promoters (Kundu et al., 2007). Consistent with our results, UniGene database shows a variety of Slo1 mRNAs with different 50 -UTR (and 30 -UTR). 50 -UTR The mSlo1 gene has at least two promoters that can initiate transcription from multiple sites. Transcripts with 184, 186, 804, 826, and 1187 nt long 50 -UTR were easily detectable along with several others of similar lengths. Primer extension data from different tissue samples indicated that there is a tendency of preferential

MOLECULAR DIVERSITY OF SLO1 CHANNELS: FUNCTIONAL CONSEQUENCES

321

expression of a particular transcript in a specific tissue. For example, a transcript with 826 nt long 50 -UTR is more abundant in bladder compared to uterus or intestine (Kundu et al., 2007). These observations suggest that tissue-specific transcription factor–promoter interactions must occur. 30 -UTR Mammalian Slo1 genes also contain variable 30 -UTR. In mouse aorta, we have detected 351 nt and 626 nt long 30 -UTRs, while in uterus, only 351 nt 30 -UTR appeared to be present (unpublished data). Human Slo1 mRNAs also have 30 -UTRs with variable lengths as found in several EST clones in GenBank sequences. Thus, one can easily imagine that a large number of different Slo1 transcripts can be generated from the combinatorial assembly of UTRs of variable lengths. At present, the role of distinct 50 and 30 -UTRs in mammalian Slo1 gene is unknown, but one can speculate that different 30 -UTRs could differentially affect Slo1 transcript stability, while different 50 -UTRs could alter its translational efficiency, ultimately affecting Slo1 expression and function. 12.2.1.1 Alternative Splicing Splicing of the Slo1 gene can alter the biophysical, regulatory responses to phosphorylation and hypoxia, as well as cellular traffic of Slo1 channels (Table 12.2). Slo1 gene has very long introns where many potential exonic sequences can be embedded. In fact, since the initial cloning process, it was made evident that numerous isoforms of Slo1 might be generated as a result of splice variation (Butler et al., 1993; Pallanck and Ganetzky, 1994; Tseng-Crank et al., 1994; Wallner et al., 1995; McCobb et al., 1995). Subsequent publications described several alternative splicing events at multiple sites in Slo1, and analysis of their functional significance indicated that fine tuning of channel properties could be obtained from these events. A well-characterized example is the STREX splice exon first identified in PC12 pheochromocytoma cells, which adds 58 amino acids to the channel. Inclusion of STREX results in a more voltage-sensitive channel and currents with slower deactivation kinetics (Saito et al., 1997; Chen et al., 2005). Most importantly, this exon switches the channel from a state of protein kinase A (PKA)-mediated activation to one of PKA-mediated inhibition (Tian et al., 2001), and its inclusion can be modulated by hormones such as estrogen, testosterone, and corticosterone (Mahmoud and McCobb, 2004; Zhu et al., 2005; Lai and McCobb, 2006). Furthermore, STREX-exon containing channels can be reversibly inhibited by hypoxic conditions in a Ca2þ-dependent manner (McCartney et al., 2005). Two more splice inserts mk44, a 44-amino acid insert (Korovkina et al., 2001), and e22 (Chen et al., 2005), a 29-amino acid insert, first described in human brain (hBR5 variant) (Tseng-Crank et al., 1994) affect the voltage sensitivity of the channel in opposite ways; mk44 decreases while e22 increases it. Remarkably, splice variation can also affect the number of channels at the surface membrane by modulating channel traffic properties. In this regard, a 33-amino acid insert SV1 inhibits channel migration to the plasma membrane by retention in the endoplasmic reticulum via its CVLF motif (Zarei et al., 2001, 2004). Interestingly, SV1 expression increases with aging in rat corpora, correlating with a decrease of BK protein at the cell membrane and increased retention in the cytoplasm (Davies et al., 2007).

322

mk44

SV1

No name No name

hbr2

hbr4 –

1

2

3 4

5

6 7

16 and 17 16 and 17

16 and 17

3 and 4 9 and 10

2 and 3

1 and 2

Flanking Exons

Chicken, human, mouse, quail, turtle

Human Turtle

36 þ 4 27 þ 4

Chicken Chicken, rat

Rat

Human

Species

4

8 31

33

44

Amino Acid Length

Slo1 Channel Splice Variants

Splice Insert/ Variant

TABLE 12.2

2þ

– –

Lower apparent affinity for Ca2þ

Decrease in voltage and Ca sensitivities in heterologous systems. In uterine smooth muscle, it is cleaved leading to intracellular retention of the pore-forming C-termini and membrane localization of N-termini ER retention, dominant negative via CVLF signal – –

Channel Property

(Rosenblatt et al., 1997) (Navaratnam et al., 1997; Langer et al., 2003) (Tseng-Crank et al., 1994; Rosenblatt et al., 1997; Jones et al., 1998; Ramanathan et al., 2000; Chen et al., 2005) (Tseng-Crank et al., 1994) (Jones et al., 1998)

(Zarei et al., 2001, 2004)

(Korovkina et al., 2001, 2006)

References

323

STREX

No name

gBK (gliomaBK) De23

No name

No name

Combination of #5 þ #9 þ #13

9

10

11 12

13

14

15

Within 27

18 and 19 Skipping exon 19 23 & 24

18 and 19

18 and 19

18 and 19

Human, mouse rat

Quail

4 þ 58 þ 27

Chicken, quail, rat

Human Mouse

Chicken, human, mouse, turtle

Chicken, human, mouse, quail, rabbit, rat, turtle

Human, mouse

8, 18, 7/8, 33, 37/38, 60/61

27

62 4, stop

3

58–61

29

Deactivated 20-fold more slowly when combined with b subunit

Reduced surface expression of 60/61 aa insert

Increased sensitivity to Ca2þ Dominant negative for surface expression –

–

Greater apparent Ca2þ affinity, switches PKA-dependent phosphorylation output

Increased sensitivity to voltage and intracellular Ca2þ

Exon numbering is based on the human reference sequence (NM_002247). ER, endoplasmic reticulum; (–), unknown.

e22/hbr5

8

(Rosenblatt et al., 1997; Ramanathan et al., 2000; Langer et al., 2003) (Ferrer et al., 1996; Langer et al., 2003; Ma et al., 2007) (Ramanathan et al., 2000)

(Tseng-Crank et al., 1994; Liu et al., 2002; Chen et al., 2005) (Ferrer et al., 1996; Xie and McCobb., 1998; Jones et al., 1998; Ramanathan et al., 2000; Tian et al., 2001; Chen et al., 2005) (Tseng-Crank et al., 1994; Rosenblatt et al., 1997; Jones et al., 1998; Chen et al., 2005) (Liu et al., 2002) (Chen et al., 2005)

324

BK CHANNELS: REGULATION OF EXPRESSION AND PHYSIOLOGICAL IMPACT

Besides exon alternative splicing, Slo1 splice variation also includes exon duplication (our unpublished results) and exon skipping (Beisel et al., 2007). Slo1 splice variants can differ significantly in their tissue distribution and expression pattern in developmental stages (Chen et al., 2005). Certain physiological conditions like aging (Davies et al., 2007) or disease conditions like diabetes (Davies et al., 2006) can also change splicing patterns. Thus, pre-mRNA processing events of Slo1 provide a mechanism for generation of physiologically and pathophysiologically diverse isoforms of BK channels. 12.2.2

Posttranslational Modifications: Phosphorylation

Phosphorylation by several cellular kinases can produce Slo1 channels with different types of activities (Schubert and Nelson, 2001). In general, cGMP-dependent protein kinase G (PKG) phosphorylation produces Slo1 channels with increased apparent sensitivity to Ca2þ, whereas protein kinase C phosphorylation inhibits the channel. Regulation by PKA phosphorylation can be defined by the Slo1 isoform being expressed. For example, the majority of Slo1 channels expressed in pregnant uterus are activated by PKA, whereas most of the channels are inhibited in the nonpregnant uterus (Perez and Toro, 1994). This tuning can be explained by STREX decrease in pregnancy (Zhu et al., 2005), as this exon favors inhibition of the channel by PKA (Tian et al., 2001). At the molecular level, inhibition of Slo1 activity requires phosphorylation of only a single STREX insert containing subunit (at a STREX PKA consensus site) provided a serine in the backbone of the protein, S869, is dephosphorylated, whereas activation of Slo1 by PKA requires phosphorylation of the same constitutive S869 site in all four subunits (numbers are as in GenBank U11058) (Tian et al., 2004). Other protein kinases also play significant roles in channel functional diversity and physiological outcomes. Ca2þ/calmodulin-dependent protein kinase II (CaMKII)mediated phosphorylation of Slo Thr107 can mediate tolerance to alcohol (Liu et al., 2006); tyrosine kinase c-Src can directly phosphorylate the channel, inhibiting its activity, which leads to vasoconstriction (Alioua et al., 2002). Proline-rich tyrosine kinase 2 (Pyk2), a calcium-sensitive tyrosine kinase, associates and enhances Slo1 channel activity (Ling et al., 2004). Moreover, Slo1 phosphorylation events can be developmentally regulated (Lin et al., 2005) as well as related to pathophysiological functions like vascular hyporesponsiveness following hemorrhagic shock in rat (Zhou et al., 2005). 12.2.3

Protein Partners

Slo1 channels can associate with a number of proteins resulting in phenotypic alterations that increase their diversity, influencing from cell-signaling pathways to cell excitability. 12.2.3.1 b Subunits Slo1 associates with regulatory b subunits (b1–b4, also named KCNMB1–B4) in a relatively tissue-specific manner: b1 is abundant in smooth muscles (Jiang et al., 1999; Brenner et al., 2000b), while b4 is abundant in brain (Meera

MOLECULAR DIVERSITY OF SLO1 CHANNELS: FUNCTIONAL CONSEQUENCES

325

et al., 2000; Brenner et al., 2005). b2 and b3 are more ubiquitously distributed. b2 mRNAs have been detected in smooth muscle containing tissues, brain, adrenal gland, ovary, pancreas, and kidney where it is particularly abundant in fetal stages (Wallner et al., 1999; Uebele et al., 2000), and b3 (a–d splice variants) mRNAs are detected in a variety of tissues with higher expression in testis, pancreas, spleen, placenta, heart, adrenal medulla and cortex, stomach, and liver (Brenner et al., 2000a; Uebele et al., 2000; Xia et al., 2000). All four subunits affect biophysical and pharmacological properties of Slo1 channels, with the exception of b3d isoform, which has no apparent effects. In particular, b2 and b3a–c cause different degrees of inactivation of Slo1 currents: b3b producing inactivation that is so rapid that currents appear to activate fast and rectify (Wallner et al., 1999; Uebele et al., 2000; Xia et al., 2000) and b3a displaying time-dependent inactivation (Zeng et al., 2007) that might play a role in the regulation of cell firing. The most prominent effect on Slo1 pharmacology is imprinted by b4 subunit, which makes the channel resistant to iberiotoxin, though other b subunits also increase toxin resistance but their effectiveness is less pronounced (Meera et al., 2000). b1 causes a large increase in the apparent Ca2þ affinity of Slo1. This favors Ca2þ negative feedback regulation of smooth muscle contractility by allowing a significant increase in channel open probability by local Ca2þ increases, leading to hyperpolarization and thus playing a key role in the control of vascular tone (Meera et al., 1996; Brenner et al., 2000b). Recent studies have highlighted new roles of b1 and b2 subunits in the regulation of Slo1 trafficking. Both subunits accelerate endocytosis of Slo1 via leucine-based signals in their carboxyl termini, providing new mechanisms for regulation of channel surface expression (Toro et al., 2006; Zarei et al., 2007). 12.2.3.2 Slo Members Tissue expression pattern of Slo1 overlaps with Slo2.2 in brain and kidney. In heterologous systems, when Slo1 and Slo2.2 subunits were coexpressed, a channel of intermediate conductance and activation by Ca2þ was recorded (Joiner et al., 1998). Thus, it has been suggested that, in vivo, these two channels can form a heteromultimer with an amalgamate property. It would be interesting to determine whether Slo1 could form heteromultimers with Slo3 that could perhaps result in a pH and Ca2þ-sensitive channel complex. One can speculate that Slo1 could favor association with Slo3 over Slo2.2, as Slo3 has similar topology to Slo1 with seven transmembrane domains. 12.2.3.3 Other Transmembrane Partners Slo1 channels interact with other transmembrane proteins besides b subunits or Slo family members. Initial studies in lipid bilayers of reconstituted smooth muscle plasma membranes hinted at the concept that Slo1 channels were in complex with b-adrenergic receptors, thromboxane A2 receptors, and G-proteins (Toro et al., 1990; Scornik and Toro, 1992; Scornik et al., 1993). Along this line of thinking, Slo1 channels have been found to form a large signaling complex in rat brain that includes the b2 adrenergic receptor, the cytosolic A-kinase-anchoring protein (AKAP79/150), PKA, and the L-type Ca2þ channel (Liu et al., 2004). In addition to L-type Ca2þ channels, electrophysiological and immunoprecipitation studies have demonstrated that N-type Ca2þ channels can

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also be located in close proximity to Slo1 (Grunnet and Kaufmann, 2004; Loane et al., 2007). The geographical proximity of Slo1 channels with distinct G-protein-coupled receptors and Ca2þ channels would define the functional role of Slo1 channel in a given cell. 12.2.3.4 Cytoplasmic Partner Proteins The long cytoplasmic carboxyl terminal domain seems to serve as a hub where a number of proteins can land to exert distinct functions. Besides PKA, which associates indirectly with Slo1 via a leucine zipper motif at the C-terminus of Slo1 (Tian et al., 2003), Slo1 channels can also interact with a number of cytoplasmic or internally membrane-bound proteins, such as caveolin-1 and actin in the vasculature and uterus (Brainard et al., 2005; Wang et al., 2005). In the brain, the phosphatase calcineurin (Loane et al., 2006), ankyrin-repeat family protein (ANKRA) (Lim and Park, 2005), cereblon (Jo et al., 2005), and syntaxin1A (Ling et al., 2003; Cibulsky et al., 2005) can associate with Slo1 and regulate neuronal excitability through modulation of channel activity. Associations with proteins, such as cPLA2 (c-phospholipase A2) in GH3 cells and RACK1 (receptor for activated C kinase 1) in vitro, have been described, although the exact nature and functional correlates of these associations are not clear yet. Of special interest is the interaction of Slo1 with hemeoxygenase-2 (HO-2) as it defines Slo1 channels as oxygen sensors. In fact, inhibition of Slo1 channels by hypoxia is dependent on HO-2 expression and is increased by HO-2 stimulation (Williams et al., 2004). For a comprehensive list of proteins that can interact with BK channels, refer to Lu et al. (2006a).

12.3 REGULATION OF SLO1 EXPRESSION Regulation of Slo1 channel expression can occur at different points in time during the life of Slo1 channels from their transcription, intracellular trafficking, and membrane localization until their degradation (Figs. 12.2 and 12.3). Studies about the promoter and transcriptional module of the mammalian Slo1 genes are limited to the human and mouse (Dhulipala and Kotlikoff, 1999; Kundu et al., 2007), while mechanisms that regulate their transcription, trafficking, and degradation are beginning to emerge. 12.3.1

Transcriptional Regulation

12.3.1.1 Slo1 Promoter-Regulatory Motifs mSlo1 has at least two promoters, a TATA-less GC-rich proximal promoter and another distal promoter. Because mSlo1 50 -UTR shares 90% homology with hSlo in the first 600 nt (Kundu et al., 2007), it seems safe to predict that the proximal promoter region is conserved among species. However, the distal promoter region of Slo1 may have evolved differently based on the needs of particular organisms. It is worth mentioning that most of the ubiquitous mammalian promoters are GC rich, which is also a characteristic of Slo1. Similar to dSlo (Brenner et al., 1996; Chang et al., 2000), different promoters of mSlo1 may have tissue-specific expression patterns. Analysis of human and mouse Slo1 promoter

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ATG (M3)

100%

mSlo1 versus hSlo1

50% -5

-4

-3

Kb

-2

-1 -826 -1187 -804

(b)

Homology

(a)

0 -186 -184ATG(M3) mSlo1 CDS

-344

(c)

ATG(M3) hSlo1 CDS

ERE

1/2 ERE

15

Luc/Rluc

(d)

Sp1

AP-1

Myo D

MEF2

SRF

mSlo1 promoter stimulation

10

5

0 0.1 Estrogen (nM)

10

FIGURE 12.2 Human (h) and mouse (m) Slo1 promoter-regulatory regions: induction by estrogen. (a) Homology plot of mouse and human sequences 50 of initiation codon encoding M3 shows high degree of homology (areas in pink) in the first Kb. VISTA browser and LAGAN algorithm were used. (b and c) Schemes of mSlo1 (b) and human (c) sequences 50 to ATG (A is þ1) of M3 marking positions of transcription start sites (arrows) and of transcription factor binding sites. (d) Estrogen-mediated upregulation of mSlo1 gene promoter is dose dependent. Luc/RLuc, promoter activity of a 2924 nt segment reported by firefly luciferase (Luc) normalized to the activity of a transfection control vector reported by Renilla luciferase (RLuc). 17 b-estradiol, estrogen.

regions depicted the presence of multiple smooth muscle-specific transcription factor binding sites (e.g., serum response factor, myocyte enhancer factor 2, and MyoD elements) that could explain high smooth muscle expression (Fig. 12.2) (Dhulipala and Kotlikoff, 1999; Kundu et al., 2007). 12.3.1.2 Estrogen Regulation The regulation of Slo1 expression by hormones was first approached by early studies examining the changes in uterine mRNA (and protein) levels during pregnancy. In the rat, Slo1 mRNA levels are downregulated in late pregnancy (Song et al., 1999), while in mouse they are upregulated (Benkusky

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FIGURE 12.3 A simplified scheme of Slo1 channel transcriptional regulation by estrogen, channel maturation, and degradation. Estrogen binds to the estrogen receptor a after crossing the plasma membrane, causing dimerization of the receptor. Dimerized receptor enters the nucleus and binds to estrogen response elements (ERE) present in Slo1 gene enhancing transcription. mRNA migrates to the cytoplasm and interacts with the translational machinery to form polyribosomes. Slo1 protein is inserted to endoplasmic reticulum (ER) membrane where it assembles into tetramers and associates or not associates with b subunits (depending on the cell type). Tetrameric channels are transferred to the cell membrane. Splice variant 1 insert (SV1) retains insertless Slo1 and b1 subunit in the ER. Surface expression of the channel can be regulated by b subunits via endocytosis. Direct binding of estrogen to Slo1 induces channel degradation by the proteasome. Dotted arrows indicate probable events: phosphorylation favoring forward traffic and association with partners in the cytosol during traffic to the membrane. (See the color version of this figure in Color Plate section.)

et al., 2000; Eghbali et al., 2003). Although opposite transcription mechanisms seem to be in place in these orthologues, the end result was similar, a decreased expression of protein at the surface of both rat and mice myocytes. In mouse, the excess production of protein by increased mRNA levels was retained in intracellular compartments (Eghbali et al., 2003). The role of estrogen was uncovered by treatment of normal mouse with 17b-estradiol, as it mimicked the increase in Slo1 mRNA and protein levels observed in late pregnancy (Holdiman et al., 2002).

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Likewise, estrogen treatment mimicked the decrease in Slo1 expression in rat myometrium, which can be blocked by the selective estrogen receptor antagonist ICI 182780. Interestingly, estrogen can also modulate alternative splicing of Slo1. In particular, estrogen decreases STREX-variant mRNA levels in the rat uterus (Zhu et al., 2005). Different transcriptional mechanisms seem to govern Slo1 expression not only among species but also in different tissues within species. For example, in rat, estrogen decreases Slo1 mRNAs in uterus but does not significantly affect Slo1 expression in aorta (Tsang et al., 2004; Zhu et al., 2005). Another example is the guinea pig, where estrogen upregulates Slo1 mRNA (and protein) in aorta but has no apparent effect on the pituitary, preoptic area, ventral hypothalamus, hippocampus, and amygdala (Jamali et al., 2003). These studies underscore the need to understand the transcriptional and tissue-specific mechanisms governing the regulation of the human Slo1 gene by estrogen. As a starting point, we have compared human, mouse, and rat Slo1 promoter regions and found that they have in common the housing of multiple elements that can mediate estrogen responsiveness. The three promoters are GC rich, a characteristic needed to attract Sp-factors for binding and facilitate estrogen receptor alpha (ERa)stimulated transcription (Bjornstrom and Sjoberg, 2005). Also, they contain several half estrogen response element (ERE) sites that could mediate estrogen response. However, only mSlo1 contains two semiperfect EREs palindromic sequences. We recently demonstrated that these two EREs are important for direct binding of ERa to the mSlo1 promoter and upregulation of transcription in an estrogen-dependent manner (Fig. 12.2) (Kundu et al., 2007). Interestingly, the rat sequence has a similar ERE in an equivalent position, but not the human gene. It therefore seems that different species have evolved different mechanisms for regulation by estrogen. 12.3.1.3 Other Steroid Hormones In addition to estrogen, other steroid hormones affect Slo1 mRNA expression and splicing. Early work using hypophysectomized rats demonstrated a decrease in the inclusion of STREX into Slo transcripts of adrenal chromaffin cells, which could be prevented by subcutaneous injection of adrenocorticotropin (ACTH) (Xie and McCobb, 1998). This result suggested a role for ACTH-induced hormones like glucocorticoids or adrenal androgen dehydroepiandrosterone in the upregulation of STREX expression. In fact, dehydroepiandrosterone, as well as testosterone, increased the amount of STREX containing variants. However, opposite to the prediction, glucocorticoids negatively affected STREX inclusion (Lai and McCobb, 2002). The opposing actions of glucocorticoids and androgens on STREX inclusion in chromaffin cells in vitro resemble those reported for estrogen and progesterone in the uterus in vivo where progesterone increases Slo1 mRNA and STREX inclusion while estrogen decreases their expression (Zhu et al., 2005). Interestingly, progesterone concentration has a peak in midpregnancy that coincides with increased expression of Slo1 channels at this stage, favoring uterine quiescence, while maximum estrogen levels at the end of pregnancy coincide with the lowest expression of Slo1 channels, a condition that should favor parturition. Thus,

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Slo1 expression and splicing are differentially regulated by the concentration- and time-dependent actions of steroid hormones with important implications in organ physiology. 12.3.1.4 Hypoxia Hypoxia or low oxygen tension can upregulate the expression of Slo1 and b1 mRNAs both in vitro in fetal pulmonary artery myocytes and in vivo in adult lungs (Resnik et al., 2006). Moreover, usage of the hypoxia-mimetic deferoxamine that blocks HIF-1a (hypoxia inducible factor-1a) degradation resulted in a 63% increase in Slo1 expression. Thus, hypoxia-induced Slo1 transcriptional up-regulation is likely mediated by HIF-1a. Because perinatal pulmonary vasodilation is mediated via Slo1 channels (Cornfield et al., 1996) it is possible that low O2 tension in the fetus prepares the pulmonary circulation for an increased expression of Slo1 channels needed at birth. Interestingly, b1-subunit promoter activity was found to be stimulated by hypoxia (Resnik et al., 2006). It would be interesting to determine whether the Slo1 promoter is induced by hypoxia or whether increased mRNA levels are also due to changes in mRNA stability by O2 levels. 12.3.2

Traffic Regulation and Degradation

Little is known about Slo1 channel trafficking mechanisms. However, accumulating evidence shows the importance of the C-terminal domain of the channel for this purpose. Initial studies in epithelial Madin-Darby canine kidney (MDCK) cells demonstrated that Slo1 channel traffic to the plasma membrane was dependent on the presence of the intracellular C-terminus, as a construct lacking this domain and containing up to transmembrane domain S6 (hSlo1–323, numbering in all sequences of Slo1 as in GenBank accession no. U11058) failed to reach the membrane (BravoZehnder et al., 2000). Elegant studies using iberiotoxin binding and fluorescently labeled iberiotoxin to detect properly folded channels and surface expression have circumscribed hSlo1–343 (containing NH2-terminus-S0-S6 plus additional 20 amino acids) as the domain containing sufficient information for cell surface expression, tetramerization, and coassembly with the b1 subunit (Schmalhofer et al., 2005), whereas the exoplasmic amino terminus and S0 are necessary for b1 functional effects (Wallner et al., 1996). Even though hSlo1–343 sequences are sufficient for cell surface expression, other regions in the carboxyl terminus seem to play roles in surface targeting (e.g., 1047DLIFCL1052) (Kwon and Guggino, 2004), indicating that specific conformations or mechanisms may be required for proper and specialized traffic of the channel. In this regard, we know little about polarized traffic mechanisms or degradation pathways utilized by Slo1 in different types of cells. In MDCK cells, apical expression of Slo1 channels uses a lipid raft mechanism (Bravo-Zehnder et al., 2000), and in cortical collecting duct epithelial cells, the C-terminus sequence 1058 NAGQSRA1064 seems to play a role in apical sorting (Kwon et al., 2004). With respect to degradation mechanisms, Slo1 channels can be subject to estrogen-induced proteasomal degradationvia the PEST (rich in Pro, Glu, Asp, Ser, and Thr) degradation motif—RLEDEQPSTLSPKK—in its carboxyl terminus (Korovkina et al., 2004). In

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this case, a direct binding of estrogen to Slo1 (facilitated by the b1 subunit) has been proposed to trigger the degradation cascade in HEK 293 cells. It is noteworthy to mention here that besides inducing proteasomal degradation, micromolar estrogen is also a direct regulator of Slo1 channel electrical activity via binding to b1 subunit (Valverde et al., 1999; Dick and Sanders, 2001). However, in this case, instead of causing decreased activity (due to degradation), estrogen increases channel activity at low intracellular Ca2þ. Other channel trafficking mechanisms include selective expression of splice variants, association with b subunits, and regulation by protein kinases. Studies with ciliary ganglion neurons showed that the p38 mitogen-activated protein kinase pathway may negatively affect channel trafficking beyond the Golgi apparatus preventing channels from reaching the surface (Chae and Dryer, 2005). An actinmediated mechanism has been proposed, but it would be interesting to determine whether the Slo1 channel itself is phosphorylated by p38 as part of the kinase downsizing effect. Several splice variants modulate Slo1 channel surface expression (Table 12.2). As stated earlier, SV1, a 33-amino acid insert discovered in rat myometrium, makes the first intracellular loop longer and acts as a dominant negative regulator of insertless Slo1 channel surface expression (Zarei et al., 2001). This downregulation of surface expression does not involve misfolding (and quality control) of the channel but occurs via a novel endoplasmic retention signal (CVLF) present in the spliced exon that retains channels, though correctly folded, in the endoplasmic reticulum. Because SV1-containing channels also retain Slo1 regulatory b1 subunit in the endoplasmic reticulum, it is likely that Slo1 and b1 subunits coassemble in this organelle (Zarei et al., 2004). In line with this view, the artificially made hSlo1–651 construct that does not reach the membrane but stays in the endoplasmic reticulum properly folded is also able to associate with b1 subunit (Schmalhofer et al., 2005). Dominant negative Slo1 isoforms are also generated from several splicing events at the extreme end of the channel (Ma et al., 2007; Kim et al., 2007a) and by skipping exon 19 (which generates a stop codon in the intracellular domain) (Chen et al., 2005). Recently, both human b1 and b2 subunits of the channel were shown to downregulate Slo1 surface expression via endocytotic mechanisms regulated by leucine-based motifs in the b subunits (Zarei et al., 2004; Toro et al., 2006). However, the avian b1 subunit can also increase surface expression of a C-termini splice variant (VEDEC) (Kim et al., 2007b). Therefore, multiple mechanisms regulate Slo1 channel surface expression possibly in a tissue-specific manner.

12.4 SLO1 CHANNELOPATHIES AND KNOCKOUT MODELS 12.4.1

Channelopathies

In humans, a form of epilepsy and paroxysmal movement disorder have been related to a mutation in the regulator of conductance for Kþ (RCK1) domain of Slo1 (Fig. 12.2). Out of 13 individuals affected with generalized epilepsy and paroxysmal movement

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BK CHANNELS: REGULATION OF EXPRESSION AND PHYSIOLOGICAL IMPACT

disorder, all carried the mutation that resulted in the substitution of a negatively charged aspartic acid to a neutral glycine residue within the RCK1 domain (D369–G; numbering as in GenBank U11058). After heterologous expression, this mutant channel showed a greater macroscopic current and higher sensitivity to Ca2þ. Thus, more channels would open and increase the firing rate of the neurons by accelerating the repolarization of action potentials (Du et al., 2005). In another study with patients, the Slo1 channel has been associated with autism and mental retardation. In one patient, a chromosomal translocation event separated the Slo1 coding sequence from its promoter region resulting in silencing of one copy of the gene and consequently reducing expression of Slo1 protein. In another patient, a substitution of alanine with valine (A73–V; numbering as in GenBank U11058) in the first intracellular loop was found. However, this mutant was not characterized (Laumonnier et al., 2006).

12.4.2

Slo1 Channel Knockouts

Two different groups have developed Slo1 channel knockout mice by targeting different regions of the channel. Slo/ generated by deleting the pore-forming exon resulted in mice with deficiencies in motor function coordination, abnormal locomotion, and loss of eye-blink conditioning regardless of the growth stage and gender of the animal. These conditions indicated cerebellar dysfunction, and in line with this, a dramatic reduction of spontaneous activity of cerebellar Purkinje neurons was observed. Subsequent studies with these mice linked Slo1 channel function with blood pressure regulation in small arteries (Sausbier et al., 2005), hearing loss with progression of age due to the dysfunction of outer hair cells (Ruttiger et al., 2004), and reduced sensitivity toward cholinergic stimulation in airway smooth muscle (Sausbier et al., 2007). Slo/ mice generated by targeting exon 1 (encoding part of 50 untranslated region, the NH2-terminus, S0, and part of first intracellular loop, Fig. 12.1) display modest ataxia. In addition, these mice showed an enhanced spontaneous contraction of urinary bladder and thus increased urination frequency (Meredith et al., 2004) and increased resistance to noise-induced hearing loss (Pyott et al., 2007). This null mouse model also linked Slo1 channels to erectile function and circadian rhythm. Erectile dysfunction in Slo1/ was related to reduction of nerveevoked relaxations and increased basal contractility of the corpus cavernosum smooth muscle, which under normal conditions should relax to allow increased blood flow into the penile corpora and erection (Werner et al., 2005). On the contrary, deletion of the circadian-regulated Slo1 gene (Panda et al., 2002; Pitts et al., 2006) correlates with an increased high-frequency firing at night of neurons in the suprachiasmatic nucleus (the master pacemaker where circadian timing of behavior is regulated), indicating that Slo1 channels are normally involved in diminishing high-frequency activity at night (Meredith et al., 2006). It is noteworthy to mention that similar results are produced by inhibiting Slo1 channels with iberiotoxin in nighttime neurons (Pitts et al., 2006). Thus, Slo1 channels, besides being functionally rhythmic, may participate in the generation of spike frequency rhythms in the suprachiasmatic nucleus, and thus might play a role in circadian behavior.

PERSPECTIVES

12.4.3

333

b Subunit Knockouts

b1 and b4 subunit knockouts have been engineered, giving valuable information on the role of these subunits. b1 deletion produces hypertension and demonstrates the role of b1 in the efficient coupling of Ca2þ sparks to Slo1 channel activity in smooth muscle (Brenner et al., 2000b). With respect to the b4 subunit knockout, despite b4 high transcript expression in the brain, deletion of this subunit produced mice with temporal lobe seizures but without overall/severe damage to brain function. Dentate gyrus granule cells of b4/ mice display a gain-of-function phenotype as Slo1 acquires fast gating activity and the density of functional channels per membrane patch increases. These characteristics coincide with increased firing frequencies and seizures in this animal model, suggesting a protective role of b4 from temporal lobe seizures (Brenner et al., 2005).

12.5 PERSPECTIVES To date a number of human diseases such as epilepsy, paroxysmal movement disorder, autism, mental retardation, hypertension, and cancer cell (breast and prostate) proliferation are linked to Slo1 channel function (Du et al., 2005; Sausbier et al., 2005; Laumonnier et al., 2006; Bloch et al., 2007; Coiret et al., 2007). In addition, when human coronary smooth muscle cells age, Slo1 channel numbers decrease (Marijic et al., 2001) and when cultured in the presence of high glucose, mimicking diabetic conditions, Slo1 current density decreases and activation kinetics slows down (Lu et al., 2006b). Studies with animal models also showed that aging affects Slo1 channel function in rat corporal smooth muscle cells (Davies et al., 2006, 2007). Despite these correlative data, a number of questions remain: First, do observed Slo1 modifications lead to or counteract the disease or condition? Second, are there ways of controlling Slo1 channel-related disease states? Promising approaches like injecting Slo1 cDNA into corporal tissue or bladder of animal models (Christ et al., 2001; Melman et al., 2003; Christ et al., 2004) are now beginning to be scrutinized in clinical trials (Melman et al., 2006). Another clinically relevant condition where Slo1 channel-directed therapies may be useful is in the regulation of vascular tone, especially under conditions of artificially supplied steroid hormones. Of particular interest is estrogen, which is well known for its cardiovascular protective effects (Zhu et al., 2004; Pepine et al., 2006). However, the Heart and Estrogen/Progestin Replacement Study (HERS) and the Women’s Angiographic Vitamin and Estrogen (WAVE) Trial challenged this concept and reported an increased risk of cardiovascular events (e.g., coronary heart disease, stroke, and peripheral arterial disease) by hormone replacement therapy in women with previous heart disease (Grady et al., 2002; Waters et al., 2002). Recently, however, the WHI-Coronary Artery Calcium Study reported that estrogen treatment reduced coronary artery calcium plaques in younger postmenopausal women (50–59 years old) (Manson et al., 2007). Eminently, there is a need to understand basic mechanisms of estrogen long-range actions in the

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vasculature (and other organs). Given the fact that Slo1 has multiple functional roles including the regulation of vascular tone, investigating basic mechanisms of estrogen-induced Slo1 gene expression and splicing is essential and of clinical relevance. Although some progress has recently been achieved (Kundu et al., 2007), a comprehensive knowledge of the regulation of the Slo1 gene would greatly hasten the discovery of Slo1 gene regulatory mechanisms and provide new opportunities for future therapies. Small-molecule modulators of Slo1 channels, the subject of the contribution by Starrett (Chapter 16), may also provide an avenue for disease treatment.

ACKNOWLEDGMENTS This work was supported by NIH grants HL54970 (LT), HD046510 (ES), and AHA National Center grants 0435084N (AA) and 0830145N (PK).

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13 STRUCTURAL BASIS FOR AUXILIARY KChIP MODULATION OF KV4 CHANNELS KEWEI WANG1 AND JIJIE CHAI2 1

Neuroscience Research Institute and Department of Neurobiology, Key Laboratory for Neuroscience of the Ministry of Education, Center for Protein Sciences, Peking University Health Science Center, 38 Xueyuan Road, Beijing, 100083, China 2 National Institute of Biological Sciences No. 7 Science Park Road, Beijing 102206, China

13.1 INTRODUCTION The action potential constitutes the fundamental unit of information encoded in neurons. The shape of the action potential is of critical importance in the regulation of neuronal excitability and signaling. Rapidly inactivating A-type Kþ currents shape the action potential by regulating its waveforms and duration. The A-type inactivation leads to reduction or elimination of potassium currents that can be regulated through a dynamic process by a variety of intracellular factors such as neurotransmitters, kinases, second messengers, and b subunits, resulting in a diverse mechanism for control of membrane excitability. Native voltage-gated potassium channels (Kv) are in a form of complex composed of a tetrameric core of pore-forming a subunits and additional auxiliary subunits. Binding of auxiliary subunits can change the functional properties of Kv channels at the membrane surface as well as their intracellular trafficking and gating kinetics (Abbott and Goldstein, 1998; Trimmer, 1998; Shibata

Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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et al., 2003; Birnbaum et al., 2004; Rhodes et al., 2004). Cytosolic Kv channelinteracting proteins KChIPs (216–256 amino acids) that belong to neuronal calcium sensor (NCS) family of calcium binding EF-hand proteins coassemble with Kv4 (Shal) a subunits to form a native complex that encodes somatodendritic A-type Kþ current ISA in neurons and transient outward current ITO in cardiac myocytes (An et al., 2000; Kuo et al., 2001; Holmqvist et al., 2002; Nadal et al., 2003). The specific binding of KChIPs to the Kv4 N-terminus enhances surface expression, facilitates subunit assembly, and regulates the functional gating properties of Kv4 channels (An et al., 2000; Holmqvist et al., 2002; Shibata et al., 2003; Kunjilwar et al., 2004). The KChIP enhancement of channel expression has been attributed to the trafficking effects of KChIPs as a result of protein redistribution to the cell surface. Interesting questions arise concerning how the local interactions between the N-terminal of Kv4 and KChIPs are translated into a variety of functional changes, such as enhanced current density and altered gating properties of Kv4 channels. Biophysical studies combined with mutagenesis have identified two N-terminal regions of Kv4.2 necessary for KChIP1 modulation and interaction with Kv4.2 channels (Scannevin et al., 2004). Structural efforts from several studies have recently solved individual crystal structures of KChIP1 and the Kv4.2 N-terminus, as well as the 3D structure of Kv4.2/KChIP2 complex by electron microscopy (EM) at 21 A resolution, in an attempt to understand the underlying mechanism of Kv4 and KChIPs interaction (Kim et al., 2004; Scannevin et al., 2004; Zhou et al., 2004). More recently, the cocrystal structure of Kv4.3 N-terminus and KChIP1 complex, solved independently, revealed a unique clamping action of the complex, in which a single KChIP1 molecule laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner, forming an octamer. This chapter focuses on the significance of the structural findings that translate into functional modulation of Kv4 channels by KChIPs. Understanding molecular mechanisms of Kv4 and KChIP protein interaction may have therapeutic potential for the treatment of membrane excitability-related disorders. 13.2 KV4 a SUBUNITS In 1987, the Shaker channel was the first voltage-dependent Kþ channel cloned, and in this case from a hyperexcitable mutant gene of the fruitfly Drosophila melanogaster that causes flies to shake when exposed to the anaesthetic either (Kamb et al., 1987; Papazian et al., 1987). Following the identification of the Shaker channel, subfamilies of Shaker-related genes from Drosophila that bear 40% sequence homology with Shaker were subsequently cloned in 1990, known as Shab, Shaw, and Shal (Wei et al., 1990). For Shaker-related mammalian counterparts, they are defined as Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw), and Kv4 (Shal) (Dolly and Parcej, 1996; Tempel et al., 1988). The Kv4 (Shal) subfamily is comprised of three distinct genes: Kv4.1, Kv4.2, and Kv4.3. These channel protein a-subunits encoded by the three genes are highly homologous within the six transmembrane regions, with divergent amino- and carboxy termini. The voltage-gated Kv4 channel sequences display several structural elements that are present in most Kþ channels with hallmarks including the

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intracellular amino-terminal cytoplasmic domain, the T1 tetramerization domain, the six transmembrane a-helical domains (S1–S6), voltage-sensing domains (S1–S4) and seven positive charges in the S4 voltage sensor region, the pore domain (P-loop) between S5 and S6, and an intracellular carboxyterminal cytoplasmic domain. Kv4 a subunits form a tetramer with members of their own subfamily through the cytoplasmic amino-terminal portion of the channel known as the tetramerization domain, or T1 domain. Heterologous expression of Kv4 demonstrates that Kv4 channels activate at subthreshold membrane potentials, inactivate rapidly, and recover quickly from inactivation. Such a typical profile of fast inactivating currents is known as A-type (Connor and Stevens, 1971). The rapidly inactivating A-type Kv4 currents are concentrated in somatodendritic compartments of neurons as ISA (transient subthreshold A-type Kþ current) and in cardiac myocytes as ITO (transient outward current).

13.3 STRUCTURAL FEATURES OF THE T1 DOMAIN The Kv4 N-terminus features intersubunit Zn2þ binding sites in the cytoplasmic amino-terminal portion known as the tetramerization domain or T1 domain, a region that is highly conserved among Kv channels (Li et al., 1992; Jahng et al., 2002; Scannevin et al., 2004). The T1 domain consists of 130 amino acids directly preceding the first transmembrane domain. The structure of the T1 domain reveals four zinc ions bound to the T1 tetramer, and each Zn2þ located at the interface between two adjacent monomers is involved in intersubunit contacts. The Kv4 intersubunit zinc binding site, critical for T1 domain interaction in early channel formation, is composed of three cysteines and one histidine in the characteristic sequence where Cys131, Cys132, His104 come from one monomer and Cys110 comes from another monomer. The Zn2þ is only required for Shab-, Shal-, or Shaw-like channels, but not Shaker channels, suggesting that T1 assembly of non-Shaker channels differs from Shaker channels (Bixby et al., 1999; Jahng et al., 2002). In addition, the T1 domain shows a loop (amino acids 70–90) that extends from the surface of the molecule (Li et al., 1992; Jahng et al., 2002; Scannevin et al., 2004). Mutations in Kv4.2 Zn2þ binding sites that disrupt T1 domain interaction and tetrameric assembly fail to form functional channels, resulting in entrapment of the protein within the endoplasmic reticulum (Kunjilwar et al., 2004). However, at least two members of KChIPs, KChIP1 (Wang et al., 2007) and KChIP3 (Kunjilwar et al., 2004), are shown to be capable of rescuing the function of Zn2þ mutations by driving the mutant subunits to form tetramers and stabilizing the tetrameric assembly (Kunjilwar et al., 2004; Wang et al., 2007).

13.4 GATING PROPERTIES OF KV4 CHANNELS Kv channels autoregulate their closing by a process known as inactivation, which is a key component of the channel’s function. The gating mechanism of Kv channels has

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been a subject for intensive investigations. In voltage-gated Shaker family channels, two inactivation mechanisms are well characterized and described. One mechanism of inactivation is termed N-type or “ball-and-chain”-type inactivation, which involves a positively charged inactivation particle (the a-ball) in the aminoterminus on a tether (chain) binding to the intracellular entrance of the pore (Hoshi et al., 1990; Zagotta et al., 1990). The other slower type of inactivation is known as C-type inactivation and involves the constriction of the outer mouth of the channel pore (Hoshi et al., 1991). Biophysical studies show that Kv4 channel gating features N-type inactivation. Results supporting the concept that Kv4 channels undergo N-type inactivation show that soluble Kv4.2 N-terminal peptide causes channel block (Gebauer et al., 2004). The effect of KChIP1 binding of the proximal N-terminus of Kv4 on inactivation indicates that the Kv4 N-terminus behaves like an inactivating ball peptide (Wang et al., 2007). In a background of Kv2.1 channels that inactivate over a much slower timescale, chimeric Kv2.1/Kv4.2NT, where the N-terminal Kv2.1 is replaced by the corresponding Kv4.2 N-terminus, causes a faster inactivation. All these findings suggest that the Kv4 N-terminus is involved in fast inactivation, manifesting the N-type inactivation. On the contrary, N-terminal deletions of Kv4 channels only partially eliminate or slow down the fast phase of inactivation, rather than a complete loss of inactivation as observed in Shaker channel, suggesting that Kv4 inactivation may differ from the classical Shaker N-type inactivation. In support of this, double-mutant cycle analysis and pharmacology experiments with tertaethylammonium (TEA) indicate that there are differences between Kv4 and Shaker N-type inactivation (Gebauer et al., 2004).

13.5 FUNCTION OF KChIPs The gating kinetics of Kv4 pore-forming a subunits significantly differs from native A-type currents, suggesting unknown intracellular modulators or factors that might regulate the gating properties of Kv4 channels. Using the cytoplasmic N-terminal domain (amino acids 1–180) of rat Kv4.3 as a bait, three cytosolic Kv channelinteracting proteins (named KChIPs) were identified from a rat brain library in yeast two-hybrid (YTH) screens (An et al., 2000). Similarly, KChIP4 of the mouse and human was accidentally cloned by using C-terminal 43 amino acid residues of presenilin 2 (PS2, amino acids 406–448) as a bait (Morohashi et al., 2002) in the YTH system. KChIPs1-4 (216–256 amino acids) can coimmunoprecipitate and colocalize with either Kv4 cotransfected cells or Kv4a subunits from tissues, thus constituting the integral components of the native Kv4 channel complex. KChIPs1-4 all have a conserved carboxy-terminal core region that contains four EF-hand-like calcium binding motifs, but a variable amino-terminal region that has been proposed to cause diverse modulation on Kv4 function (Fig. 13.1). KChIP3 is encoded by the same gene locus as two previously characterized proteins, DREAM (downstream regulatory element antagonist modulator) and calsenilin (Spreafico et al., 2001). DREAM has been described as a Ca2þ-regulated DNA binding protein that represses transcription of prodynorphine genes (Carrion et al., 1999). KChIP4, also known as a calsenilin-like

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GVVNEETFKQIYAQFFPHGDASTYAHYLFNAFDTTQTGSVKFEDFVTALSILLRGTVHEK GIVNEENFKQIYSQFFPQGDSSNYATFLFNAFDTNHDGSVSFEDFVAGLSVILRGTIDDR GLVDEDTFKLIYSQFFPQGDATTYAHFLFNAFDADGNGAIHFEDFVVGLSILLRGTVHEK GVVNEETFKEIYSQFFPQGDSTTYAHFLFNAFDTDHNGAVSFEDFIKGLSILLRGTVQEK EF hand 1 EF-hand EF hand 1 EF-hand

LRWTFNLYDINKDGYINKEEMMDIVKAIYDMMGKYTYPVLKEDTPRQHVDVFFQKMDKNK 186 LSWAFNLYDLNKDGCITKEEMLDIMKSIYDMMGKYTYPALREEAPREHVESFFQKMDRNK 222 LKWAFNLYDINKDGYITKEEMLAIMKSIYDMMGRHTYPILRKDAPLEHVERFFQKMDRNQ 226 LNWAFNLYDINKDGYITKEEMLDIMKAIYDMMGKCTYPVLKEDAPRQHVETFFQKMDKNK 199 EF-hand 3

DGIVTLDEFLESCQEDDNIMRSLQLFQNVM DGVVTIEEFIESCQQDENIMRSMQLFDNVI DGVVTIDEFLETCQKDENIMSSMQLFENVI DGVVTIDEFIESCQKDENIMRSMQLFENVI EF-hand 4

rKChIP1 rKChIP2 rKChIP3 mKChIP4

rKChIP1 rKChIP2 rKChIP3 mKChIP4

rKChIP1 rKChIP2 rKChIP3 mKChIP4

FIGURE 13.1 Amino acid alignment of KChIPs1-4 (216 to 259aa). Core regions of KChIPs are conserved with variable N-termini and four EF-hands (shaded). Accession numbers of KChIPs1-4 genes listed here are EDM04076, NP_064480, NP_064480 and NP_671712, respectively.

216 252 256 229

-------------KDKIEDDLEMTMVCHRPEGLEQLEAQTNFTKRELQVLYRGFKNECPS 66 --------------NSVEDEFELSTVCHRPEGLEQLQEQTKFTRRELQVLYRGFKNECPS 102 ------------GSDSSDSELELSTVRHQPEGLDQLQAQTKFTKKELQSLYRGFKNECPT 106 ------------LEDSVEDELEMATVRHRPEALELLEAQSKFTKKELQILYRGFKNECPS 79

rKChIP1 rKChIP2 rKChIP3 mKChIP4 126 162 166 139

-------------------------------------MGAVMGTFSSLQTKQRRPS---- 19 MRGQGRKESLSESRDLDGSYDQLTGHPPGPSKKALKQRFLKLLPCCGPQALPSVSE---- 56 MQRTKEAMKASDGSLLGDPGRIPLSKREGIKWQRPRFTRQALMRCCLIKWILSSAAPQ-- 58 MNLEGLEMIA-------------------------VLIVIVLFVKLLEQFGLIEAG---- 31

rKChIP1 rKChIP2 rKChIP3 mKChIP4a

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STRUCTURAL BASIS FOR AUXILIARY KChIP MODULATION OF KV4 CHANNELS

protein (CALP), binds to presenilin-2, which is known to facilitate intramembranous g-cleavage of b-amyloid protein precursor (bAPP) (Morohashi et al., 2002). KChIPs1-3 increase Kv4 current amplitude, moderately slow down channel inactivation, and accelerate recovery from inactivation. By contrast, KChIP4 does not promote Kv4 surface expression and functions as a suppressor for inactivation without effect on recovery from inactivation (An et al., 2000). Figure 13.1 shows the amino acid alignment of KChIPs1-4 (216–259aa) Biophysical studies have shown that KChIPs affect the inactivation kinetics of Kv4 channels differently. KChIP1 speeds up inactivation of Kv4.1 (Beck et al., 2002). Coexpression of KChIP1-3 with Kv4.2 or Kv4.3 slows down fast inactivation and causes a complete steady-state inactivation, resulting in a crossover of inactivation kinetics (An et al., 2000). KChIPs1-3 consistently increases the recovery rate from inactivation of all Kv4 channels. By binding and covering ER retention signals localized in the proximal N-terminal domain of Kv4, KChIP1 also increases macroscopic current by promoting surface expression, without altering singlechannel current (Beck et al., 2002). This is consistent with the observation that N-terminal deletion of the first 20 or 40 residues results in an increase in current amplitude (Bahring et al., 2001), although the exact retention signal of the Kv4 N-terminus remains elusive. Coexpression of KChIP1-3 results in higher expression of Kv4 channel proteins with a level typically 5–10 times that of Kv4 alone, as well as redistribution of channel proteins to the cell surface. In contrast, KChIP4, which differs only in its N-terminus from the other KChIPs, eliminates the fast inactivation without affecting surface expression and recovery from inactivation (Holmqvist et al., 2002).

13.6 STRUCTURAL BASIS FOR KChIP MODULATION OF KV4 FUNCTION Kv4 modulation by multifunctional KChIP proteins raises an important question concerning how local interactions between the Kv4 N-terminus and KChIPs can be translated into altered gating properties and surface expression of Kv4 channel proteins. Using site-directed mutagenesis and deletion approaches combined with electrophysiology, functional mapping of Kv4 N-terminus and KChIP interaction has identified two N-terminal domains, the proximal N-terminal residues 7–11 and an internal region of 71–90 amino acids that are critical for the KChIP modulation of Kv4 (Scannevin et al., 2004). Structural efforts from several groups have recently attempted to address the underlying mechanism of Kv4 and KChIPs interaction (Kim et al., 2004; Scannevin et al., 2004; Zhou et al., 2004). The crystal structure of KChIP1 shows that KChIP1 can be divided into N- and C-terminal lobes with two Ca2þ ions mapped to the last two of four EF-hands (EF3- and EF-4) (Scannevin et al., 2004; Zhou et al., 2004). Five a helices (H1–H5) are located in the hydrophilic N-terminal lobe, and helices H6–H10 form the hydrophobic C-terminal lobe. By creating a tandem complex of KChIP1 with the first N-terminal 30 amino acids of Kv4.2, Zhou et al. (2004) reported the crystal

STRUCTURAL BASIS FOR KChIP MODULATION OF KV4 FUNCTION

349

structure of a clamp-shaped dimeric assembly, with the first 20 residues of Kv4.2 colinear with KChIP1 in a head-to-tail fashion. Kim et al. (2004) purified the intact functional Kv4.2–KChIP2 complex from mammalian cells and determined the 3D structure of the channel complex by electron microscopy (EM) at 21 A resolution. The EM structure reveals a 4:4 complex, where KChIP2 forms four peripheral columns (offset by 45 ) that appear to attach laterally to the four inner windows of the T1 gondola, suggesting a unique structural arrangement of Kv4.2–KChIP2, compared with the previously characterized T1–Kvb2 complex in which Kvb2 is located right below the gondola (Gulbis et al., 2000; Orlova et al., 2003). More recently, two labs independently solved the cocrystal structure of the KChIP1/ Kv4.3 N-terminus complex, a result that has shed light on the mechanism of the protein–protein interaction of the complex (Pioletti et al., 2006; Wang et al., 2007). This structure reveals that there exists a clamping mode of the complex in which a single KChIP1 molecule as a monomer laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner, with two contact interfaces being involved in the interaction (Fig. 13.2). In the first interface, the proximal N-terminal hydrophobic peptide (residues 6–21) of Kv4.3 reaches out into a deep elongated groove and is thereby sequestered by this

FIGURE 13.2 The overall architecture of the KChIP1–Kv4.3N complex. All three panels have the same color codes with some secondary structural elements labeled specifically. (a) One KChIP1 molecule in gold interacts simultaneously with two Kv4.3Ns in blue. In the complex, each KChIP1 molecule not only binds to the N-terminal peptide of one Kv4.3 but also interacts with an adjacent Kv4.3 T1 domain forming two contact interfaces, the first interface shown in red frame and the second interface shown in blue frame. (b) The 4:4 complex of KChIP1– Kv4.3N shown on this panel is generated from the complex on panel A through symmetric operations. (c) KChIP1–Kv4.3N complex in 4:4 showing the clamping effect of KChIP1 molecule on the tetramer of Kv4.3. (See the color version of this figure in Color Plate section.)

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STRUCTURAL BASIS FOR AUXILIARY KChIP MODULATION OF KV4 CHANNELS

FIGURE 13.3 Significant structural rearrangements occur to KChIP1 upon Kv4.3 N-terminal binding. Surface representations are shown to an isolated KChIP1 (a) and Kv4.3-bound KChIP1 (b). The two KChIP1 molecules are in the same orientation and can be aligned with each other with root mean square deviation of 1.86 A . Note the striking hydrophobic groove on the Kv4.3-bound KChIP1 with a size of about 30 16 12 A , depicted in (b). (c) Structural alignment between the isolated KChIP1 (cyan) and the Kv4.3-bound KChIP1 (gold). For clarity, the N-terminal peptide of Kv4.3 in the Kv4.3-bound KChIP1 is not shown. The a helix 10 is labeled in red rotating about 40 outward in the Kv4.3-bound KChIP1, whereas the helices a2 and a8 have translated 2.5 and 1.5 A , respectively. (See the color version of this figure in Color Plate section.)

hydrophobic pocket on the surface of KChIP1, which is important for the physical suppression of Kv4 inactivation by KChIPs, as evidenced by electrophysiological studies. Within the first interface, the most striking conformational change in the Kv4.3bound KChIP1 occurs in H10, which is completely replaced by the N-terminal peptide of Kv4.3 and swings outward about 40 from the hydrophobic groove (Fig. 13.3), creating a clear-cut hydrophobic groove on the surface of KChIP1 that is hidden in the free KChIP1 structure (Pioletti et al., 2006; Wang et al., 2007). Interestingly, KChIP1 was unexpectedly shown to interact with two neighboring Kv4.3 T1 domains in the 4 : 4 complex, suggesting that KChIP1 may stabilize the tetramer of Kv4.3 channels. Indeed, biochemical studies show that KChIP1 can rescue the tetramer of a tetramerization-disrupting Kv4.3 mutant, whereas the mutants that disrupt the interaction at either of the interfaces fail to do so, indicating that both interfaces in the complex play an important role in stabilizing the tetramer of Kv4.3 (Wang et al., 2007). The significance of the tetramer-stabilizing effect of KChIP1 on Kv4.3 is demonstrated by electrophysiology, which showed that coexpression of wild-type KChIP1 rescued the function of the mutant Kv4.3 C110A. Strikingly, such a

THE FIRST INTERFACE OF KV4.3N–KChIP1 COMPLEX

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rescuing capacity of KChIP1 is significantly compromised by the mutants either in KChIP1 or in Kv4.3 that disrupt the second interface. Collectively, these results demonstrate that KChIP1 likely functions as a molecular clamp to modulate the Kv4.3 channel properties.

13.7 THE FIRST INTERFACE OF KV4.3N–KChIP1 COMPLEX IS RESPONSIBLE FOR INACTIVATION The first contact interface features the Kv4.3 N-terminal peptide binding to a deep hydrophobic pocket on the surface of KChIP1, revealing the physical suppression of Kv4 inactivation by KChIPs (Figs. 13.2 and 13.3). Although the overall positioning of the T1 domain of Kv4.3 remains essentially unchanged upon binding to KChIP1 (Fig. 13.3c), some regions of KChIP1 have undergone significant structural rearrangements compared to the isolated KChIP1, as indicated by an RMSD 1.86 A over all the aligned Ca atoms (Fig. 13.3c). The most striking conformational change in the Kv4.3-bound KChIP1 occurs in H10, which is completely replaced by the N-terminal peptide of Kv4.3 and swings outward about 40 from the hydrophobic groove (Fig. 13.3c). In addition, H2 and H8 have translated outwardly about 2.5 and 1.5 A, respectively, thus further contributing to the configuration of the peptide binding pocket on the surface of KChIP1 (Fig. 13.3c). As a result, all these structural changes create a clear-cut hydrophobic groove on the surface of KChIP1 (Fig. 13.3b) that is completely hidden in the free KChIP1 structure (Fig. 13.3a). It is noteworthy that binding of Kv4.3N does not cause the relative rotations between the N- and C-terminal lobes of KChIP1, as observed in complexes composed of other EF-hand proteins and their target proteins (Lewit-Bentley and Rety, 2000). The N-terminal peptide of Kv4.3 forms an a-helix followed by an extended segment and binds to the deep pocket of KChIP1. The interaction around this region is predominantly mediated by hydrophobic contacts. Almost all of the hydrophobic residues from the N-terminal peptide of Kv4.3 are involved in the extensive interaction with their neighboring residues from KChIP1, resulting in a 2352 A2 burial of exposed surface. W8 and W19, two highly conserved bulky residues among the Kv4 members (Fig. 13.4c), are positioned at both ends of the deep groove and can be best characterized as two pillars to support the whole peptide on the surface of KChIP1 (Fig. 13.4a). W19 contributes to the interaction between Kv4.3N and KChIP1 by docking into the deep hydrophobic groove, making van der Waals contacts with seven residues Y134, I150, Val151, Y155, I154, F178, and the Ca atom of H174 in KChIP1. On the N-terminus of the peptide, the two conserved bulky hydrophobic residues W8 and F11 are tightly fitted into another hydrophobic cavity built by nine residues I77, Y78, F74, L94, F111, L115, L118, F60, and the Ca atom of G59 in KChIP1. Interestingly, although the orientation of the N-terminal peptide of Kv4.2 fused to the C-terminus of KChIP1 is the reverse of that of Kv4.3 observed in the current structure (Zhou et al., 2004), the position of W8 is almost identical in both structures (not shown). To further strengthen the hydrophobic interaction, residues L9, P10, A12, A15, I17, and P21 in the inactivation peptide of Kv4.3 make hydrophobic contacts with

352

STRUCTURAL BASIS FOR AUXILIARY KChIP MODULATION OF KV4 CHANNELS

FIGURE 13.4 The N-terminal inactivation peptide of Kv4.3 is completely sequestered in a hydrophobic groove on the surface of KChIP1. (a) The N-terminal hydrophobic peptide of Kv4.3 binds to an elongated hydrophobic groove on the surface of KChIP1. KChIP1 is shown in the surface representation, and the N-terminal inactivation peptide of Kv4.3 is shown in ribbon. The Kv4.3 N-terminal residues involved in hydrophobic contacts with KChIP1 are colored in magenta. (b) An SDS-PAGE gel shows the pull-down results for the point mutants of both KChIP1 and the N-terminal peptide of Kv4.3. The single mutation of KChIP1 Y134E and the triple mutation W8E, P10E, and A15E in Kv4.3 completely abolish their interactions with their respective wild-type partners. GST-pull-down assay is used to detect the interaction between WT Kv4.3N and KChIP1 mutants (left panel), whereas various His-tagged Kv.3 mutants are immobilized on Ni-resin and WT KChIP1 is allowed to flow through Ni-resin (right panel). In both cases, the resin is visualized on SDS-PAGE followed by coomassie staining. (c) Sequence alignments of the N-terminal peptide of Kv4-family proteins among different species. (d) Effects of KChIP mutation within the first interface on Kv4.3. The representative traces recorded from oocytes injected with cRNAs of WT Kv4.3 alone, WT Kv4.3 þ WT KChIP1, and WT Kv4.3 þ KChIP Y134E. The left side panel depicts currents recorded from oocytes held at 80 mV by a family of pulses from 60 to 40 mV in 10 mV increments for 1 s, and the right panel shows recovery from inactivation for varying lengths of time at step from 80 to þ40 mV. (e) The representative traces of Kv4.3 triple mutant (W8E-P10E-A15E) alone and the triple mutant þ WT KChIP1 recorded from oocytes under the same protocol as in (d). (See the color version of this figure in Color Plate section.)

THE SECOND INTERFACE MEDIATES INTERSUBUNIT INTERACTION

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residues mainly from the two sidewalls of the pocket, whereas A14 directly contacts the bottom of the pocket (Fig. 13.4a). The two conserved residues G18 and P21 serve as hinge points that allow the Nterminus of Kv4.3 to make two sharp turns, thus resulting in the segment of residues 22–40 antiparallel to its preceding residues (6–17) bound to the pocket (Fig. 13.2). This structural feature of the “U”-shaped peptide suggests that the length of the residues 23–40 may be important to maintain the Kv4 inactivation (Baldwin et al., 1991), because a shortened peptide would lead to a steric clash of Kv4.3 and KChIP1 due to the spatial restrictions around this region. In addition to completely covering the proximal N-terminal residues (6–17), residues 23–40 in Kv4.3N also interact with H10 of KChIP1 through hydrophobic contacts and hydrogen bonds, as deletion of H10 in KChIP1 led to loss of its binding to the T1 domain of Kv4.3. This is in agreement with the previous studies, which showed that truncation of H10 in KChIP1 abolished its interaction with Kv4.2 (Zhou et al., 2004). A single mutation of Y134E in KChIP1 and a triple mutation (W8E-P10E-A15E) in Kv4.3N disrupt their interaction with Kv4.3N or KChIP1, respectively (Fig. 13.4b, left and right panels), indicating that these residues within the first interface are critical in mediating the specific interaction. By sequence comparison, the N-terminal hydrophobic residues of the Kv4-family are highly conserved across species, suggesting that these residues mediate hydrophobic–hydrophobic interactions (Fig. 13.4c). To further confirm the functional significance of the KChIP1 Y134E and the triple mutant of Kv4.3, the effect of these mutants on the modulation of Kv4.3 was tested. As expected, coexpression of WT Kv4.3 and WT KChIP1 resulted in a significant increase in current amplitude, slowed inactivation, and accelerated recovery from inactivation compared to Kv4.3 alone (Fig. 13.4d). In contrast, the KChIP1 Y134E mutation that abolishes its interaction with Kv4.3N showed no effects on modulation of Kv4.3 channel function (Fig. 13.4d). As a control, the KChIP1 mutation L115E that interacts with Kv4.3N (Fig. 13.4b) still retained its ability to modulate the Kv4.3 channels in a manner similar to the WT KChIP1. All these results show that the first interface mediates the functional interaction between Kv4.3 and KChIP1, as disruption of this interface by mutations in either Kv4.3 or KChIP1 resulted in the loss of Kv4.3 modulation by KChIP1. The physical immobilization of the Kv4.3 N-terminal peptide by KChIP1 suggests that the peptide is involved in inactivation gating, likely providing a structural basis for the elimination of the open-state inactivation of Kv4.3 by KChIP1 (Beck et al., 2002; Gebauer et al., 2004).

13.8 THE SECOND INTERFACE MEDIATES INTERSUBUNIT INTERACTION Besides sequestering the N-terminal peptide of Kv4.3, KChIP1 was unexpectedly shown to interact with a neighboring Kv4.3N, constituting a second interface between them. The interaction around this interface is mainly mediated by H2 of KChIP1 and Kv4.3 residues 70–78 (Fig. 13.5a and b). Within the second interface, the F73 that is conserved among Kv4 members but not in other Kv channels (Fig. 13.5c) fits tightly

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STRUCTURAL BASIS FOR AUXILIARY KChIP MODULATION OF KV4 CHANNELS

FIGURE 13.5 The second contact interface between KChIP1 and Kv4.3 T1 domain. (a) The interaction within the second interface is mediated by both hydrophobic contacts and salt bridges. The KChIP1 molecule and the Kv4.3 peptide (residues 70–78) are shown in surface representation and dark green ribbon, respectively. The residues of Kv4.3 colored in magenta are involved in hydrophobic interaction with KChIP1. The blue, red, and white represent the positive, negative, and hydrophobic surface of Kv4.3, respectively. (b) A close-up view of the interaction within the second interface between KChIP1 and Kv4.3N. The KChIP1 molecule is shown in light green ribbon and colored in green, and the residues involved in the interaction with Kv4.3N are highlighted in yellow. The O and N atoms are shown as red and blue, respectively. The salt bridges are indicated by black dashed dots. (c) Sequence alignment of Kv4.3 peptide (70–78) with corresponding sequences of other Kv proteins. The residues F73 and F74 of Kv4-family labeled in magenta square on the top form hydrophobic contacts with KChIP1 molecule and those with blue squares make salt bridges with KChIP1. (d) The representative traces are recorded from oocytes injected with cRNAs of Kv4.3 double mutant (E70A, F73E) that disrupts the second interface, in the presence or absence of WT KChIP1 or the KChIP1 the triple mutant (L39E, Y57A, K61A). The left panel depicts currents from oocytes held at 80 mV by a family of pulses from 60 to 40 mV in 10 mV increments for 1 s, and the right panel shows recovery from inactivation for varying lengths of time at step from 80 to þ40 mV every 8 s. (See the color version of this figure in Color Plate section.)

into a hydrophobic cavity from residues L39, L42, L53, Y57, and F108 in KChIP1 (Fig. 13.5a and b). Apart from the hydrophobic interactions, salt bridges also seem to play important roles in the interaction within the second interface. Two consecutive acidic residues, E77 and D78 from Kv4.3N, form salt bridges with residues K50 and R51 of KChIP1, respectively (Fig. 13.5b). E70, another conserved residue among the Kv4 members (Fig. 13.5c), also makes a salt bridge with K61 in KChIP1 (Fig. 13.5b). This basic residue is conserved among the KChIPs, but not in other EF-hand proteins like frequenin and recoverin, suggesting a role of salt bridges in the specific recognition of Kv4 by KChIPs.

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To assess the functional significance of the residues crucial for the interaction within the second interface, a double Kv4.3 mutant E70A-F73E was examined for its modulation by KChIP1. This double mutation alone yielded functional channels, although it had faster inactivation kinetics when compared with WT Kv4.3 (Fig. 13.5d). When coexpressed with the Kv4.3 double mutant, WT KChIP1 increased the current amplitude about threefold and slowed down the fast time constant of inactivation with slight effects on the slow time constant of inactivation and little effect on recovery from inactivation, compared with the double mutant alone (Fig. 13.5d). In contrast, when coexpressed the KChIP1 triple mutant (L39E-Y57A-K61A) completely abolished the increase of the Kv4.3 double mutant, compared to the effect of WT KChIP1 on the Kv4.3 double mutant (Fig. 13.5d). Both the double mutation of Kv4.3 and the triple mutation of KChIP1 within the second interface still retained the KChIP1 modulation of inactivation but not current amplitude, suggesting a role of the second interface in channel surface expression. These results also indicate that disruption of either of the interfaces can result in loss of Kv4.3 modulation by KChIP1 (Figs. 13.4 and 13.5), suggesting that the two interfaces act in concert to facilitate the proper assembly of the channel complexes.

13.9 STABILIZATION OF KV4.3 TETRAMER BY KChIP1 In the clamping mode of the complex, KChIP1 can be depicted as a clamp that holds two Kv4.3 molecules firmly together (Fig. 13.2b and c). Thus, a more stable tetrameric Kv4.3 can be expected upon KChIP1 binding. Indeed, it has been shown that KChIP3 can rescue the Zn2þ mutant that disrupts the tetramerization of Kv4.2, which strongly suggests that KChIP3 plays a role in stabilizing Kv4.2 channel assembly (Kunjilwar et al., 2004). If the tetramer of Kv4.3 is indeed strengthened by the binding of KChIP1, then disruption of the interaction at either of the interfaces will eliminate this stabilizing effect exerted by KChIP1. To confirm this, the ability of the KChIP1 triple mutant L39E-57A-K61A was examined for rescuing the tetramerization of the Zn2þ mutant (C110A) of Kv4.3N using a gel-filtration assay. As expected, this Zn2þ mutant of the T1 domain is eluted as a monomer at higher fractions of low molecular weight (Fig. 13.6a, left panel) and is significantly shifted to the position of the tetramer after addition of WT KChIP1 (Fig. 13.6a, right panel). In contrast, the Zn2þ mutant C110A of Kv4.3 remains as a monomer with almost no shift to the tetrameric position after the addition of the KChIP1 triple mutant (Fig. 13.6b, right panel), although it still retains its ability to interact with the WT Kv4.3N due to the intact first interface. These results indicate that the association of KChIP1 with Kv4.3N within the second interface plays an important role in stabilizing the tetramer of Kv4.3. To further confirm the role of the second interface in stabilizing the Kv4.3 tetramer, electrophysiological analysis was used to test whether the KChIP1 triple mutant could rescue the Kv4.3 Zn2þ mutant C110A. Consistent with the previous observation (Kunjilwar et al., 2004), the Kv4.3 C110A mutant alone did not express functional A-type current (Fig. 13.6c), and coexpression of WT KChIP1 rescued the function of Kv4.3 C110A Zn2þ mutant with increased current amplitude, characteristic A-type

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STRUCTURAL BASIS FOR AUXILIARY KChIP MODULATION OF KV4 CHANNELS

FIGURE 13.6 Disruption of the second interface by KChIP1 mutation fails to rescue the tetrameric mutant of Kv4.3 Zn2þ mutant C110A that destabilizes tetrameric assembly by gelfiltration assay. (a) The gel bands show the molecular size of protein fractions collected for the tetrameric mutant of Kv4.3 (C110A), WT KChIP1, and the complex between them. Proteins from each fraction are detected by Coomassie staining following SDS-PAGE. The Kv4.3 mutant is eluted at the position of monomer (left panel) and shifts to the position of tetramer (right panel) when in complex with WT KChIP1. (b) The KChIP1 triple mutant at the second interface losses its ability to rescue the tetrameric mutant of Kv4.3 Zn2þ mutant (C110A). The triple mutant of KChIP1 (L39E, Y57A, K61A) formed a complex with the Kv4.3 mutant (C110A) due to the existence of the first interface, but this complex is much smaller than the one formed between WT KChIP1 and the same Kv4.3 mutant, as it is eluted at the fractions of 30–32. (c) The triple mutant of KChIP1 (L39E, Y57A, K61A) at the second interface could not rescue the functional expression of Kv4.3 Zn2þ mutant C110A that disrupts T1 domain interaction and tetrameric assembly. The triple mutant of KChIP1 has no effect on recovery from the inactivation of Kv4.3 Zn2þ mutant C110A.

gating kinetics, and faster recovery from inactivation (Fig. 13.6c). In contrast, coexpression of this KChIP triple mutant can only rescue a limited degree of function of Kv4.3 C110A with a small increase in current amplitude, but not recovery of inactivation, compared to the case of coexpression of WT KChIP1 with Kv4.3 C110A (Fig. 13.6c), suggesting that these residues in KChIP1 from the second interface play a critical role in promoting the tetrameric assembly of functional channels. These functional tests, coupled with the results of gel filtration, suggest that one of the fundamental functions of KChIPs binding to Kv4 proteins is to stabilize the channel complex and thus promote their tetrameric assembly or trafficking to the membrane surface (Kunjilwar et al., 2004). This notion of molecular clamping may present a unique mechanism by which KChIP proteins regulate the current density of Kv4 channels through the second interface clamping, which is in contrast to known a–b interactions such as Kv1.2/Kvb2, in which Kvb2 itself as a tetramer may function by stabilizing channel assembly and promoting surface expression of Kv1.2 (Gulbis et al., 1999; Shi et al., 1996; Long et al., 2005). Therefore, it will be interesting to determine

STRUCTURAL COMPARISON BETWEEN KV4.3N–KChIP1 AND KVa–KVb COMPLEXES

357

whether tetramerization of Kvb2 is required for its function in augmenting the surface expression of Kv1.2. 13.10 STRUCTURAL COMPARISON BETWEEN KV4.3N–KChIP1 AND KVa–KVb COMPLEXES The crystal structures of Kv4.3 and Kv1.2 T1 domains show a striking similarity with an RMSD of 1.05 A over the aligned Ca atoms, although their sequences share only 24.6% identity. The tetramers assembled from two distinct T1 domains of Kv4.3 and Kv1.2 also resemble each other. The fact that the membrane-spanning domains of the two channel proteins have much higher sequence identity (46.0%) suggests that the overall architectures of Kv4.3 and Kv1.2 are similar. We therefore modeled the Kv4.3– KChIP1 channel complex, in which transmemebrane segments of Kv1.2 are used in the modeling, and then compared it with the Kv1.2–Kvb complex (Long et al., 2005). KChIP1 laterally attaches two Kv4.3 N-termini (Fig. 13.7a and b, left panels). In contrast, with Kvb2, a subunit of Kv1.2 channels, the molecule is immediately underneath one T1 domain, aligning well with the Kv1.2 tetramer and resulting in a significant increase in the height of the Kv1.2–Kvb channel complex (Fig. 13.7a,

FIGURE 13.7 Comparison of the modeled Kv4.3–KChIP1 channel complex with Kv1.2– Kvb2. (a) Side views of Kv1.2/Kv4.3 T1-KChIP complex in which Kv4.3 T1 domain fused with transmembrane-spanning domains of Kv1.2 (left panel) and Kv1.2-Kvb2 complex (right panel). The tetrameric subunits of Kv1.2 channels are labeled with colors of cyan, yellow, pink, and green, respectively. KChIP1 and Kvb2 are labeled in blue and wheat, respectively. (b) Top views of panel A, showing KChIPs positioned between two adjacent T1 domains (left panel) and Kvb2 beneath the T1 domains (right panel). (See the color version of this figure in Color Plate section.)

358

STRUCTURAL BASIS FOR AUXILIARY KChIP MODULATION OF KV4 CHANNELS

right panel). By residing in between two adjacent T1 domains, KChIP1 firmly fortifies the Kv4.3 tetramer so that binding of KChIP1 to the T1 domain essentially does not change the height of the Kv4.3 channel (Fig. 13.7a and b), which is consistent with the EM structure of the Kv4.2–KChIP2 complex (Kim et al., 2004). Kvb2 exists as a tetramer and may have a role in stabilizing channel assembly and promoting the surface expression of Kv1.2 (Carrion et al., 1999; Bahring et al., 2001; Beck et al., 2002). Therefore, it will be interesting to determine whether tetramerization of Kvb2 is required for its function in augmenting the surface expression of Kv1.2. The clamping mode of KCHIP1 on Kv4 provides the structural basis for understanding how KChIP1 increases Kv4 surface expression, regulates gating kinetics, and possibly regulates trafficking.

13.11 SUMMARY The rapidly inactivating (A-type) potassium channels regulate membrane excitability, which defines the fundamental mechanisms of neuronal functions such as pain signaling. The cytosolic Kv channel-interacting proteins KChIPs, which belong to the neuronal calcium sensor (NCS) family of calcium binding EF-hand proteins, coassemble with Kv4 (Shal) a subunits to form a native complex that encodes major components of neuronal somatodendritic A-type Kþ current ISA in neurons and transient outward current ITO in cardiac myocytes. The specific binding of auxiliary KChIPs to the Kv4 N-terminus results in modulation of gating properties, surface expression, and subunit assembly of Kv4 channels. The recent solution of the cocrystal structure of the Kv4 N-terminus/KChIP complex revealed a unique clamping mode of Kv4 and KChIP interaction, with two-contact interfaces being involved in the complex interaction. The clamping mode has provided a structural framework for understanding the effects of KChIP1 on Kv4 channel gating and possible surface expression. However, many interesting questions still remain to be answered. For example, does the second interface affect the channel trafficking in any way besides promoting the tetrameric assembly? Since all KChIPs share the core regions but vary in their N-termini, do other KChIPs form a structure similar to KChIP1? Can the clamping structure of KChIP1 account for the functional differences among other KChIPs (KChIP4, for instance) on Kv4 function? Understanding the molecular mechanism of Kv4 and KChIP interaction may lead to a better understanding of the channel biology and function of native A-type Kv4 Kþ currents. Because of the relevance of these findings to neuronal excitability, they offer the potential for the development of treatments for human disease conditions.

ACKNOWLEDGMENTS The preparation of this manuscript is supported by research grants from the National Science Foundation of China, 30630017 and the Ministry of Science Technology of China, 2006AA02Z183 and 2007CB512100 to KWW.

REFERENCES

359

ABBREVIATIONS KChIPs Kv DREAM CALP YTH ISA

(Kv channel-interacting proteins) (voltage-gated potassium channels) (downstream regulatory element antagonist modulator) (calsenilin-like protein) (yeast two-hybrid) (transient subthreshold A-type Kþ current)

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Hoshi T, Zagotta WN, Aldrich RW, 1990. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science 250: 533–538. Hoshi T, Zagotta WN, Aldrich RW, 1991. Two types of inactivation in Shaker Kþ channels: effects of alterations in the carboxy-terminal region. Neuron 7: 547–556. Jahng AW, Strang C, Kaiser D, Pollard T, Pfaffinger P, Choe S, 2002. Zinc mediates assembly of the T1 domain of the voltage-gated K channel 4.2 J Biol Chem 277: 47885–47890. Kamb A, Iverson LE, Tanouye MA, 1987. Molecular characterization of Shaker, a Drosophila gene that encodes a potassium channel. Cell 50: 405–413. Kim LA, Furst J, Gutierrez D, Butler MH, Xu S, Goldstein SA, Grigorieff N, 2004. Threedimensional structure of Ito; Kv4.2-KChIP2 ion channels by electron microscopy at 21 Angstrom resolution. Neuron 41: 513–519. Kunjilwar K, Strang C, DeRubeis D, Pfaffinger PJ, 2004. KChIP3 rescues the functional expression of Shal channel tetramerization mutants. J Biol Chem 279: 54542–54551. Kuo HC, Cheng CF, Clark RB, Lin JJ, Lin JL, Hoshijima M, Nguyen-Tran VT, Gu Y, Ikeda Y, Chu PH et al., 2001. A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of Ito and confers susceptibility to ventricular tachycardia. Cell 107: 801– 813. Lewit-Bentley A, Rety S, 2000. EF-hand calcium-binding proteins. Curr Opin Struct Biol 10: 637–643. Li M, Jan YN, Jan LY, 1992. Specification of subunit assembly by the hydrophilic aminoterminal domain of the Shaker potassium channel. Science 257: 1225–1230. Long SB, Campbell EB, Mackinnon R, 2005. Crystal structure of a mammalian voltagedependent Shaker family Kþ channel. Science 309: 897–903. Morohashi Y, Hatano N, Ohya S, Takikawa R, Watabiki T, Takasugi N, Imaizumi Y, Tomita T, Iwatsubo T, 2002. Molecular cloning and characterization of CALP/KChIP4, a novel EFhand protein interacting with presenilin 2 and voltage-gated potassium channel subunit Kv4. J Biol Chem 277: 14965–14975. Nadal MS, Ozaita A, Amarillo Y, Vega-Saenz de Miera E, Ma Y, Mo W, Goldberg EM, Misumi Y, Ikehara Y, Neubert TA, Rudy B, 2003. The CD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type Kþ channels. Neuron 37: 449–461. Orlova EV, Papakosta M, Booy FP, van Heel M, Dolly JO, 2003. Voltage-gated Kþ channel from mammalian brain: 3D structure at 18A of the complete (alpha)4(beta)4 complex. J Mol Biol 326: 1005–1012. Papazian DM, Schwarz TL, Tempel BL, Jan YN, Jan LY, 1987. Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila. Science 237: 749–753. Pioletti M, Findeisen F, Hura GL, Minor DL Jr, 2006. Three-dimensional structure of the KChIP1-Kv4.3 T1 complex reveals a cross-shaped octamer. Nat Struct Mol Biol 13: 987– 995. Rhodes KJ, Carroll KI, Sung MA, Doliveira LC, Monaghan MM, Burke SL, Strassle BW, Buchwalder L, Menegola M, Cao Jet al., 2004. KChIPs and Kv4 alpha subunits as integral components of A-type potassium channels in mammalian brain. J Neurosci 24: 7903–7915. Scannevin RH, Wang K, Jow F, Megules J, Kopsco DC, Edris W, Carroll KC, Lu Q, Xu W, Xu Z, et al., 2004. Two N-terminal domains of Kv4 Kþ channels regulate binding to and modulation by KChIP1. Neuron 41: 587–598.

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Shi G, Nakahira K, Hammond S, Rhodes KJ, Schechter LE, Trimmer JS, 1996. Beta subunits promote Kþ channel surface expression through effects early in biosynthesis. Neuron 16: 843–852. Shibata R, Misonou H, Campomanes CR, Anderson AE, Schrader LA, Doliveira LC, Carroll KI, Sweatt JD, Rhodes KJ, Trimmer JS, 2003. A fundamental role for KChIPs in determining the molecular properties and trafficking of Kv4.2 potassium channels. J Biol Chem 278: 36445–36454. Spreafico F, Barski JJ, Farina C, Meyer M, 2001. Mouse DREAM/calsenilin/KChIP3: gene structure, coding potential, and expression. Mol Cell Neurosci 17: 1–16. Tempel BL, Jan YN, Jan LY, 1988. Cloning of a probable potassium channel gene from mouse brain. Nature 332: 837–839. Trimmer JS, 1998. Regulation of ion channel expression by cytoplasmic subunits. Curr Opin Neurobiol 8: 370–374. Wang H, Yan Y, Liu Q, Huang Y, Shen Y, Chen L, Chen Y, Yang Q, Hao Q, Wang K, Chai J, 2007. Structural basis for modulation of Kv4 Kþ channels by auxiliary KChIP subunits. Nat Neurosci 10: 32–39. Wei A, Covarrubias M, Butler A, Baker K, Pak M, Salkoff L, 1990. Kþ current diversity is produced by an extended gene family conserved in Drosophila and mouse. Science 248: 599–603. Zagotta WN, Hoshi T, Aldrich RW, 1990. Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB. Science 250: 568–571. Zhou W, Qian Y, Kunjilwar K, Pfaffinger PJ, Choe S, 2004. Structural insights into the functional interaction of KChIP1 with Shal-type Kþ channels. Neuron 41: 573–586.

PART III DRUG DISCOVERY TARGETS AND TECHNOLOGY

14 SODIUM CHANNEL BLOCKERS FOR THE TREATMENT OF CHRONIC PAIN MARK R. BOWLBY

AND

EDWARD KAFTAN

Discovery Neuroscience, Wyeth Research, Princeton, NJ 08543, USA

14.1 INTRODUCTION Voltage-dependent sodium (Naþ) channels (Navs) are integral for the initiation and propagation of action potentials in neurons and other electrically excitable cells. Activated by depolarization of the membrane, they selectively allow Naþ passage into cells, creating further depolarization that propagates along the nerve fiber. Voltagegated Naþ channels (VGSCs) are large, multimeric complexes composed of a and b subunits. The a subunit contains the ion pore and all the essential elements of sodium channel function. Coexpression of b subunits can modulate the kinetics of gating and expression levels. Naþ channels are well known for their rapid opening and subsequent inactivation, effectively cutting off ion flow. Channels return to the closed and primed state upon membrane repolarization. Spontaneous, high-frequency trains of impulses are rarely observed in normal sensory afferents, but they are prominent in many neuropathic pain conditions. The firing of bursts of ectopic action potentials has been shown to underlie the initiation, and possibly the maintenance, of tactile allodynia in some chronic pain models (Liu et al., 2000a, 2000b). The ability of known Naþ channel blockers to selectively block high-frequency activity, while leaving low-frequency impulses essentially unaffected, has been demonstrated in many preclinical and clinical studies and generally results in a reduction in pain. Such frequency-dependent or “use-dependent” block is a property

Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

365

366

SODIUM CHANNEL BLOCKERS FOR THE TREATMENT OF CHRONIC PAIN

common to many ion channel blockers and can be explained by a preferential affinity of the drug for a particular conformational state (“state-dependency”) of the channel (Hille, 2001). This is usually either the open state or the inactivated state in the case of Naþ channels (Ragsdale et al., 1996). Many of the commonly studied Naþ channelblocking drugs exhibit some degree of state dependency of their effects on channel function. This property is crucial since these drugs would be far less tolerated were it not for frequency-dependent block. A preference for inactivated channels provides greater potency and a wider therapeutic window for these drugs. By stabilizing the hyperexcitable neurons along the abnormal pathway while having minimal effects on normally polarized tissue, the block of nerve conduction in normal fibers is minimized. The clinical effectiveness of agents that act primarily through a use-dependent block of Naþ channels is well known. These agents fall into multiple therapeutic classes in the neuroscience and cardiac areas, for example, local anesthetics, type 1b antiarrhythmics, anticonvulsants (Catterall, 1987), and antihyperalgesics for the treatment of chronic, neuropathic pain (Backonja, 1994; Tanelian and Victory, 1995). The development of Naþ channel blockers for the treatment of chronic pain has generated great activity in the pharmaceutical industry as Naþ channel blockade offers a proven mechanism for a condition that has traditionally been very difficult to treat. Drugs such as lidocaine, mexiletine, and carbamazepine have long been used to treat chronic pain; however, they are nonselective Naþ channel blockers that can lead to multiple adverse events and dose-limiting side effects. The side effects generally fall into the categories of central nervous system (CNS) (nausea, dizziness, ataxia, drowsiness, blurred vision) or cardiac-related symptoms (Jarvis and Coukell, 1998; Jensen, 2002; Walia et al., 2004; Kalso, 2005; Tremont-Lukats et al., 2005). One obvious method to potentially minimize CNS and cardiac side effects, while maintaining therapeutic efficacy, is to discover and develop subtype selective Naþ channel blockers. It is thought that Naþ channels such as Nav1.3, 1.7, 1.8, 1.9 are all potential targets for pain due to their predominant expression in peripheral neurons, and that selectivity over the cardiac Nav1.5, CNS-expressed Nav1.1, 1.2, and skeletal muscle Nav1.4 channels would be desirable.

14.2 PHARMACOLOGY The primary division of the Nav channel family is based upon sensitivity to tetrodotoxin (TTX). Most Nav proteins are sensitive to TTX (TTX-S), with the exceptions being the peripherally expressed Nav1.8/1.9, which are TTX resistant (TTX-R), and the cardiac Nav1.5, which possesses moderate TTX sensitivity (IC50 ¼ 300 nM)(Lei et al., 2004). TTX blocks all TTX-S Nav channels at low nanomolar concentrations in a nonuse-dependent fashion. This division by TTX sensitivity, however, says little about the known or expected pharmacology of small-molecule blockers of these channels. This difference between the toxin-related and small molecule-based pharmacology is common in many channel families, and relates largely to the different binding sites for the two classes of blockers. The dominant site to which previously characterized (and most recently discovered) small-molecule modulators block Nav channels is via the

PHARMACOLOGY

367

“local-anesthetic” site located partway into the internal vestibule. The compounds blocking in this manner are detailed in Tables 14.1 and 14.2 and in the “local anesthetics” and “antiepileptics” sections below. In contrast, the various blocking and gating toxins bind to extracellular sites or to internal sites near the edges of the internal vestibule. For a detailed review of this literature, see (Ogata and Ohishi, 2002). 14.2.1

TTX and Pain Relief in Animal Models

Mechanistic investigations into the relationship of TTX-S versus TTX-R Nav types in pain relief have utilized TTX directly to block pain behaviors. In the spinal nerve ligation model (SNL) of neuropathic pain, direct application of TTX to the L5 dorsal root ganglia (DRG) significantly reduced pain behaviors. A significant, but milder, reduction was found with epidural application of TTX, while no effect was seen with i.p. administration (Lyu et al., 2000). Interestingly, recording of compound action potentials generated in the sciatic nerve were not blocked by therapeutically effective concentrations of TTX; thus, while the site of action may be at the DRG/spinal nerves, complete block of action potential conduction was not required for pain relief. 14.2.2

Conotoxins

Conotoxins from marine cone snails provide a rich source of peptide toxins, which often target ion channels of various classes. The m- and d-conotoxins, part of the M-family, six-cysteine type of cone snail toxin, are of special interest in the current context, as they selectively block Nav channels in patterns different from TTX (Wood et al., 2004). The m-conotoxin PIIIa is a toxin that blocks Nav1.2, but not Nav1.7, in recombinant cell lines and PC12 cells. Most interesting for the pain field, however, are the discoveries of toxins capable of selectively blocking the TTX-R Nav channels. The first of these, SmIIIA, is a m-conotoxin that has been reported to irreversibly block TTX-R Nav current in DRG neurons, presumably via blockade of Nav1.8/1.9 (West et al., 2002; Keizer et al., 2003). Most recently, the earliest described family of conotoxins (mO-conotoxins) MrVIB (McIntosh et al., 1995) has been shown to block Nav1.8 10x more potently than any of the other Navs, including Nav1.9 (Ekberg et al., 2006). The effect of intrathecal injection of this peptide relieved both neuropathic (partial ligation of the left sciatic nerve) and chronic inflammatory (24 h post-complete Freund’s adjuvant (CFA)) pain. Allodynia and hyperalgesia were both reduced by intrathecal infusion of MrVIB (0.03–3 nmol), whereas motor side effects occurred only at 30-fold higher doses. Subcutaneous administration of the synthetic MrVIB toxin has also been shown to block rat Nav1.8 sodium channels and have potent and long-lasting local anesthetic effects when tested in two pain skinflinch sensitivity assays and in postincision allodynia in rats. Furthermore, MrVIB can block propagation of action potentials in A and C fibers in sciatic nerve as well as in an isolated skeletal muscle preparation from rat (Bulaj et al., 2006). This latter feature is due to its activity on Nav1.4 channels expressed in muscle (Zorn et al., 2006), indicating that the toxin is not completely selective for Nav1.8, at least in tissue preparations.

368

Zonisamide

Lamotrigine

Carbamazepine

Mexiletine

Lidocaine

Name

Cl

H 2N

N

O

Cl

H2N

N

N

H2N

O

O

N

N H

O

N

N

O

Structure

S

O

NH2

NH2

(Guay, 2003; Sobieszek et al., 2003)

(Eisenberg et al., 2005; Wiffen and Rees, 2007)

(Ambrosio et al., 2002; Wiffen et al., 2005)

(Jarvis and Coukell, 1998)

(Kalso et al., 1998; Tremont-Lukats et al., 2005)

Review References

Epilepsy

Epilepsy

Epilepsy

Local anesthetic

Local anesthetic

Original Therapeutic

TABLE 14.1 Clinically Used Compounds that Block Naþ Channels (Nonselectively)

62 (Nav1.2)

28 (Nav1.2)

2.3–4 (Frog skeletal muscle)

5 (Nav1.5)

Use-Dependence

Nerve injury models of chronic pain

Acute thermal

Weak “neuropathic” activity in humans

Formalin (phase 2) model

Human trigeminal neuralgia, diabetic neuropathy

Formalin (phase 2) model

Formalin (phases 1 and 2), SNI Human peripheral nerve damage, diabetic neuropathy

Human amputation, SCI, postherpetic neuralgia, and others

Formalin, SNL (thermal hyper algesia)

Pain Type

369

Imipramine

Amitriptyline

Lacosamide

O

N

N

N H

O

O

H N

N

(Kvinesdal et al., 1984; Joss, 1999; Mochizucki, 2004)

(Joss, 1999; Mochizucki, 2004)

(Beyreuther et al., 2007b)

Depression

Depression

Epilepsy

32 (Nav1.7) (Dick et al., 2007)

354 (Nav1.7) (Dick et al., 2007)

Enhances Nav slow inactivation

Model: formalin (phase 2) Human: diabetic neuropathy Postherpetic neuralgia

Human: diabetic neuropathy

Model: SNL

Formalin, carrageenan, CFA, STZ, MIA, SCI, ION

370

CDA54

BPBTS

Ralfinamide (NW-1029)

Name

N

H N

F

O

O

SO2NH2

O

SO2NH2

O

O

NH

NH

Structure

S

HN

O

OCF3

S

NH2

Potency (mM)

(Shao et al., 2005; Brochu et al., 2006)

(Priest et al., 2004; Shao et al., 2005)

EP Ki ¼ 0.18 (Nav1.8)

EP Ki ¼ 0.15 (Nav1.7)

(Faravelli et al., 2000; EP IC50 ¼ 55 Veneroni et al., 2003; (TTX-R) and 22 Stummann et al., 2005) (TTX-S) at 70 mV

References

TABLE 14.2 New Small-Molecule Nav Blocking Agents Active in Pain Models In Vivo

Nav1.8–Nav1.5 > Nav 1.7 > Nav1.2

Nav1.5 > Nav1.7–Nav 1.2 > TTX-R

Nonselective

Selectivity (Most to Least Potent)

25

9

Yes

UseDependent Ratio

SNL, CCI, CFA

Formalin (phases 1 and 2)

Formalin (phases 1 and 2), CFA, CCI

Pain Models (In Vivo)

371

11

BA 42730A

Crobenetine (BIII 890 CL)

13B

13,

OH

N

OCF3

N

OH

O

O

O

N

S

OCH3

OCH3

O

NH

N

O

N

S

Cl

(Liang et al., 2005)

(McCullough et al., 1987; Liang et al., 2005)

(Carter et al., 2000; Laird et al., 2001)

(Ok et al., 2006)

VIPR ¼ 1.3, EP Kr ¼ 10, Ki ¼ 0.14 mM

VIPR IC50 ¼ 4

Binding Ki ¼ 49 nM, IC50i ¼ 77 nM, IC50r ¼ 18 mM

EP IC50 ¼ 0.64, 0.36 (Nav1.7)

Nav1.7 block, others unknown

Nav1.7 block, others unknown

Nonselective

Unknown

71

Unknown

230

Unknown

(Continued)

Formalin

Formalin (phase 2); s.c. only

CFA

CFA

372

A-803467

4030W92

M58373

12

Name

CN

Cl

TABLE 14.2

Cl

N

Cl

O

H2N

O

HCl

N

O

H

N

N

O

NH

N

OH

S

Structure

(Continued )

F

N

O

NH2

O

O

Cl

(Jarvis et al., 2007)

(Clayton et al., 1998; Collins et al., 1998; Trezise et al., 1998)

(Akada et al., 2006)

(Liang et al., 2005)

References

EP IC50 10 nM

EP IC50 ¼ 22 at 60 mV (TTX-R) and 5 at 70 mV (TTX-S)

Binding Ki ¼ 0.7

VIPR ¼ 1.0 EP Kr ¼ 6.7, Ki ¼ 0.16 mM

Potency (mM)

Nav1.8 Nav1.5, Nav1.2, Nav1.7

Nonselective

Unknown

Nav1.7 block, others unknown

Selectivity (Most to Least Potent)

Formalin

Pain Models (In Vivo)

10

Yes

CFA, CCI, capsaicin, SNL, SNI. inactive in formalin

Formalin (phase 2), carrageenan, CFA, CCI

Unknown Formalin, CCI

42

UseDependent Ratio

373

#47

PPPA

Cl

N

O

NH

O O

F

O

O

NH

F3CO

N

N

O

NH2

(Hoyt et al., 2007)

(Ilyin et al., 2006)

EP IC50 ¼ 35 nM Ki ¼ 40 nM, Kr >3 mM

EP Ki ¼ 0.041, Kr ¼ 22 mM

Nav1.7 block, others unknown

Nonselective

>75

537

SNL tactile allodynia (10 mg/kg p.o.)

Partial sciatic nerve ligation, CFA, postincisional (mechanical endpoints)

374

14.2.3

SODIUM CHANNEL BLOCKERS FOR THE TREATMENT OF CHRONIC PAIN

Local Anesthetics

Lidocaine (lignocaine, xylocaine) was first synthesized in 1943 and is still the most widely used local anesthetic. Although lidocaine is a relatively weak blocker of Naþ channels (IC50 generally 50 mM), it does block in a state-dependent manner (approximately fivefold more potent (Smith et al., 2006)), which allows neuronal stabilization without blocking conduction (see Table 14.1). Lidocaine also preferentially blocks TTX-R currents (Roy and Narahashi, 1992; Chevrier et al., 2004; Weiser, 2006), which are predominantly expressed in primary afferent neurons, a feature that may contribute to lidocaine’s discrimination between sensory and motor effects. Due to its poor oral bioavailability, lidocaine must be injected locally or infused as an intravenous or epidural solution. In rat pain models, systemic lidocaine has been shown to be effective at preventing pain behaviors following formalin injection and also in reversing thermal hyperalgesia associated with sciatic nerve ligation without affecting processing on the uninjured side (Abram and Yaksh, 1994). Lidocaine has been extensively studied in human clinical trials (for reviews see Kalso et al., 1998; Tremont-Lukats et al., 2005) and has been shown to be broadly effective against pain associated with amputation (Wu et al., 2002), spinal cord injury (Finnerup et al., 2005), and postherpetic neuralgia (intravenous) (Rowbotham et al., 1991) among others. Lidocaine (Lidoderm) administered topically as a patch is only one of the five drugs currently approved by the Food and Drug Administration (FDA) for the treatment of chronic pain. Although the lidocaine patch is approved for postherpetic neuralgia, pilot studies suggest that it may also be effective for alleviating the pain and potentially other symptoms associated with osteoarthritis, lower back pain, and diabetic neuropathy (Argoff et al., 2004; Burch et al., 2004; Gimbel et al., 2005). The nonselective nature of the Naþ channel block by lidocaine, however, can lead to adverse events and doselimiting side effects (Challapalli et al., 2005; Tremont-Lukats et al., 2005). Mexiletine was developed as an orally available analogue of lidocaine. It is also a state-dependent (three-fourfold) (De Luca et al., 2000) and relatively weak blocker of Naþ channels with in vitro potencies in the mM range, although it is slightly more potent than lidocaine. Mexiletine is currently approved by the FDA only for the treatment of life-threatening ventricular arrhythmia and not for the treatment of pain. Mexiletine is effective in reducing mechanical allodynia induced by peripheral nerve injury in animal models of neuropathic pain (Erichsen and Blackburn-Munro, 2002; Lindia et al., 2005) and in reducing flinching behavior in both phase 1 and phase 2 of the formalin test (Blackburn-Munro et al., 2002). There are human studies examining the efficacy of oral mexiletine in chronic pain compared to lidocaine (usually i.v.), and the results are mixed. Mexiletine has been shown to be effective in pain associated with peripheral nerve damage (Chabal et al., 1992) and diabetic neuropathy (Dejgard et al., 1988), but ineffective against central pain associated with spinal cord injury (ChiouTan et al., 1996), HIV-related painful peripheral neuropathy (Kemper et al., 1998), and others (Ando et al., 2000; Wallace et al., 2000). Side effects and adverse events associated with systemic mexiletine in these studies were generally mild and included nausea, gastrointestinal disturbances, dizziness, and tremor.

PHARMACOLOGY

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Antiepileptics

Many antiepileptic drugs such as carbamazepine and lamotrigine have Naþ channel blocking activity, which contributes to their antiepileptic activity (see Table 14.1). Generally these are weak but use-dependent blockers of Naþ channels, although other activities of these compounds, such as block of voltage-gated Ca2þ channels (Stefani et al., 1996), may contribute to their in vivo profile. Carbmazepine and lamotrigine block both TTX-S and TTX-R Naþ channels in DRG neurons with low affinity but show a strong preference for the inactivated state (inactivated state affinities of 28 mM and 111 mM, respectively, for carbamazepine) (Ilyin et al., 2006). In the rat formalin model, both carbamazepine and lamotrigine reduce phase 2 flinching behavior (but not phase 1) at doses that do not affect motor coordination (Blackburn-Munro et al., 2002). Results for carbamazepine and lamotrigine in animal models of neuropathic pain are mixed and appear to be model dependent and sedative, or ataxic side effects can be seen at or near the efficacious doses (Ilyin et al., 2006). Clinically, carbamazepine is primarily used as an antiepileptic, although it is also approved by the Food and Drug Administration for the treatment of pain, where it is the first line treatment for trigeminal neuralgia. Evidence from clinical trials also suggests the utility of carbamazepine in the treatment of painful diabetic neuropathy (Gomez-Perez et al., 1996; Vinik, 2005). Overall the clinical data on lamotrigine is inconclusive and lamotrigine is only considered a fourth line treatment for neuropathic pain (Moulin et al., 2007). Zonisamide is another antiepileptic drug that has proven useful in treating chronic pain. A number of activities have been attributed to zonisamide including Naþ channel blockade, Cav channel blockade, potassium channel potentiation, nitric oxide synthase inhibition, and nitric oxide free radical scavenger activity. Many of these activities may contribute to antinociceptive action, making it difficult to assign a clear mechanism of action to zonisamide in in vivo studies. Given this caveat, systemically administered zonisamide has been shown to alleviate acute thermal pain in mice (Sakaue et al., 2004) and is effective at relieving thermal hyperalgesia and tactile allodynia in nerve injury models of chronic pain (Hord et al., 2003; Tanabe et al., 2005). There are a number of clinical case studies and open-label reports suggesting that zonisamide is effective in alleviating chronic pain in humans; however, data from few randomized controlled trials have been published (Guay, 2003). Data from one randomized, placebo-controlled study examined 42 patients with painful diabetic neuropathy. Pain scores for the zonisamide-treated group decreased more than the placebo group, although these differences did not reach statistical significance (Atli and Dogra, 2005). The most unusual member of this category may well be lacosamide (harkoseride or ADD 234037), a functionalized amino acid that was specifically designed as an anticonvulsant. Unlike the other Naþ channel blocking compounds, lacosamide selectively enhances slow inactivation of Naþ channels (and binds collapsin-response mediator protein 2 or CRMP-2) without affecting fast inactivation (Errington et al., 2006; Beyreuther et al., 2007b). Some other anticonvulsant compounds such as zonisamide are reported to enhance fast and slow inactivation (Schauf, 1987). In animal models of pain, lacosamide is generally without effect in acute pain models, but

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inhibits chronic inflammatory and neuropathic pain. Activity has been reported in diverse thermal and mechanical endpoints in the inflammatory carrageenan and CFA models and in the models of streptocotozin-induced diabetic neuropathic pain, arthritic pain, and spinal cord and infraorbital injury-induced pain (Beyreuther et al., 2006, 2007a, 2007c; Hao et al., 2006; Stohr et al., 2006). Thus, lacosamide has broad antinociceptive activities in chronic pain models. Clinical experience has been encouraging as well; patients with painful diabetic neuropathy have reported strong and sustained pain reduction in several multicenter, randomized, double-blind, placebo-controlled trials (McCleane et al., 2003; Shaibani et al., 2006; Wymer et al., 2006; Rauck et al., 2007).

14.3 NEW SMALL-MOLECULE PHARMACOLOGY Many small-molecule Nav blockers have been identified as binding to the “local anesthetic” binding site of Naþ channels. It is apparent that this site is relatively promiscuous with respect to the binding of small drug-like molecules and is partially conserved across all the Nav subtypes. Many investigators have used the available pharmacological agents such as batracotoxin (BTX), veratridine, and other tools which open or prevent inactivation of Nav channels, enabling assays for Nav channel inhibitors on multiwell higher throughput platforms where membrane potential cannot be tightly controlled. Compounds that bind to the local anesthetic site, as it turns out, also allosterically inhibit BTX binding to its site, thus providing the basis for a binding assay for the discovery of blocking compounds (Postma and Catterall, 1984; Sheldon et al., 1994). This fact has likely contributed to the large variety of modulators discovered in past years, as classical ligand binding assays based on the displacement of radiolabeled BTX have proven useful for identifying “hits.” One limitation of this method, however, is that most studies have used brain or heart tissue/channels; thus, the compounds identified are not selective for channels specifically involved in pain states. Another limitation is that such a method discovers modulators that act in a manner similar to previous compounds, in this case those that inhibit BTX binding and are therefore likely binding to the local anesthetic site. Nevertheless, the newer pharmacological agents have generally evolved from these nonselective starting points, and other plate-based screens have been performed utilizing the pain-related Naþ channels Nav1.7 and Nav1.8, minimizing the first of these limitations. The activity of these new agents on Nav channels and in subsequent pain models has built a compelling picture for the effectiveness of Naþ channel blockers in treating pain in recent years and is detailed in the following sections and in Table 14.2. 14.3.1

Ralfinamide (NW-1029)

A series of largely nonselective Nav blockers that inhibit binding to the batrachotoxinin site (neurotoxin 2) in native DRG cells is exemplified by NW-1029 (ralfinamide). NW-1029 blocks TTX-S and TTX-R channels in DRG neurons in a use-dependent

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fashion, although its potency is unimpressive. At physiological membrane potentials of 70 mV, the IC50 ¼ 55 mM for TTX-R and 22 mM for TTX-S channels, but 50 mM is able to shift the V1/2 for steady-state inactivation by 11 and 15 mV for TTX-R and TTX-S channels, respectively (Stummann et al., 2005). Ralfinamide strongly blocked the phase 2 (neuropathic-like) pain in the formalin model (10–40 mg/kg p.o.) and produced some decrease in phase 1 (acute inflammatory) pain at 40 mg/kg p.o. In the inflammatory CFA-induced mechanical allodynia model, activity was observed with an ED50 of 0.5 mg/kg i.p. or p.o. In the chronic constriction injury (CCI) rat model of chronic inflammatory pain, mechanical allodynia was blocked with ED50s of 0.9 and 0.7 mg/kg i.p. and p.o., respectively (Veneroni et al., 2003). No effect was observed in acute pain models of hot-plate and tail-flick tests in the rat, whereas in the rotarod test of motor coordination, TD50s of 245–470 mg/kg p.o. predict a wide therapeutic index for this molecule. Suppression of seizures was also demonstrated for NW-1029 and its related molecules in a variety of anticonvulsant models (Pevarello et al., 1998), thus supporting the link between hyperexcitability in chronic pain and epilepsy models. In rats, ralfinamide was effective in reducing spontaneous neuropathic pain behavior, via its suppression of self-amputation (autotomy). Compared with vehicle-treated rats, ralfinamide significantly delayed the autotomy onset day in a dose-dependent manner over a 42-day study. Autotomy levels in rats treated with a 120 mg/kg/day dose remained significantly suppressed until the end of the experiment (postoperative day 63). In a parallel electrophysiological study, it was shown that ralfinamide blocked ectopic neuroma discharges generated in sensory fibers without affecting normal nerve conduction (Seltzer et al., 2006). Clinical experience with this compound has progressed as far as phase 2, where ralfinamide produced a significant improvement in pain scores as measured by the visual analogue scale of pain intensity (VASPI; 26% improvement versus baseline). The trial enrolled 18 patients who were administered ascending oral doses of ralfinamide (80, 160, 320 mg/day) for 4 weeks. Pain relief was experienced in >70% of patients in at least one category. Pin-prick allodynia improved in 67% of patients (12/18) and in seven patients (39%) evoked pain was reduced by 50%. Improvements were also observed in several other global pain scales and measures (Gustorff and Palmas, 2005). Avery structurally similar compound safinamide is also a Naþ channel blocker, but it also possesses monoamine oxidase-B and Cav inhibitory activity. This compound has been investigated as an antiepileptic and Parkinson’s disease treatment, but may be effective for pain as well (Fariello et al., 1998; Pevarello et al., 1998; Salvati et al., 1999). 14.3.2

BPBTS, CDA54

High-throughput screening for blockers of the peripherally expressed and pain implicated Nav1.7 have been performed using a fluorescent, functional assay designed to detect the voltage change in a Nav1.7-expressing cell line induced upon addition of Naþ channel openers. Using FRET and VIPR technologies yielded a compound termed “BPBTS” (Felix et al., 2004; Ok et al., 2006). BPBTS is

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significantly more potent than other Naþ channel blockers currently used to treat neuropathic pain, although like these other agents, its block is largely nonselective across the different Nav subtypes (Priest et al., 2004). Block by BPBTS was found to be voltage and use dependent, with binding preferentially to open and inactivated states of the channel, which causes a dose-dependent hyperpolarizing shift in the steady-state availability curves for all the sodium channel subtypes tested. Although BPBTS has poor pharmacokinetic characteristics, it was still able to reduce the early and late phase pain behavior in the formalin model (Priest et al., 2004; Shao et al., 2005). Medicinal chemistry efforts to optimize BPBTS have led to CDA54, a more metabolically stable analogue that also blocks Nav1.8/1.7, but in an even greater usedependent manner (Shao et al., 2005). The potency is similar to BPBTS, but block of the pain-related Nav1.8 is enhanced (although block of cardiac-expressed Nav1.5 is still prominent) (Brochu et al., 2006). To characterize its activity in a chronic nerve injury model, the CCI model was examined. In this model, the induced injury leads to spontaneous action potential firing in the sciatic nerve, and block of these ectopic discharges is thought to at least partially underlie relief from neuropathic pain. CDA54 was able to block these spontaneous action potentials at 10-fold lower concentrations than those associated with conduction block in A or C fibers, thus dissociating potential analgesic and antihyperalgesic effects. Pain relief is also produced in CCI as well as in the SNL neuropathic model, at 10 mg/kg p.o. in both models. Unlike most other compounds of this type, when dosed in vivo, the distribution of CDA54 is largely restricted to peripheral tissues, yet it is still active in the formalin model, reducing pain behaviors to a similar level as that observed with mexiletine. Consistent with the spinal nerve data, no effect was found on acute nociception, motor coordination, or cardiac electrophysiology, except at higher concentrations (Shao et al., 2005; Brochu et al., 2006). Recently, further improvements in BPBTS and CDA54 have been made, especially targeting the metabolic instability of the two former compounds upon incubation with human liver microsomes. The more metabolically stable compounds 13 (racemate) and 13B (utamer, i.e., most active) were described to have similar potency as CDA54, better selectivity versus the hERG cardiac potassium channel, and thus were tested in vivo. The acute inflammatory CFA pain model was examined, where CDA54, 13, and 13B were all shown to have similar antiallodynic activity at 3 mg/kg p.o., thus indicating the therapeutic potential for these blockers (Ok et al., 2006). 14.3.3

Crobenetine (BIII 890 CL)

Discovered by investigators at Boehringer Ingelheim and the University of Washington, the compound crobenetine (BIII 890 CL) reduced binding of [3H]BTX A-20abenzoate at the neurotoxin receptor site 2 of the mixed Naþ channels expressed in rat brain synaptosomes (IC50 ¼ 49 nM) and TTX-R Nav channels in DRG (Carter et al., 2000; Dekker et al., 2005). A strong preference for the inactivated state (ratio of 230 in the IC50s for the resting and inactivated states) and use dependence has been described

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for its action. A further exploration of the binding site has been performed using sitedirected mutagenesis of cloned channels, where an alteration in compound binding occurred when channels were mutated in the local anesthetic site, indicating that binding occurs to this site (Carter et al., 2000). In rat cortical and striatal brain slices, 0.3 mM crobenetine inhibited veratridine-induced glutamate release, whereas in serum-free cultures of cortical neurons, it inhibited veratridine-induced glutamate release and neurotoxicity/cytotoxicity (Hamasaki et al., 1996; Carter et al., 2000). Crobenetine is active at blocking inflammatory pain in the CFA model in rats, with mechanical hyperalgesia and joint stiffness being significantly reduced after 5 days of treatment, with no effect on edema (Laird et al., 2001). These results were similar to those observed with mexiletine. Neuroprotective effects have also been reported at doses as low as 3 mg/kg s.c. in transient and permanent models of ischemic stroke in rats, whereas no effects were observed with 30 mg/kg s.c. in the rotarod test of motor coordination (Carter et al., 2000). 14.3.4

BA 42730A

A blocker of cardiac Naþ current described in an abstract (McCullough et al., 1987) as BA 42730A was shown recently to also block Nav1.7 and have activity in phase 2 of formalin-induced pain (Liang et al., 2005). This compound, however, was not selective for Nav channels, as it blocked other channels including hERG. Improved analogues (compounds 11 and 12) (Liang et al., 2005) were thus produced, which contained little hERG activity while retaining Nav1.7 block. These analogues produced significant activity in the formalin model of pain. Their potency on other Nav channels is unknown, but it is unlikely that these compounds are selective for Nav1.7. 14.3.5

M58373

In the veratridine-induced cytotoxicity assay (Hamasaki et al., 1996), M58373 was identified as a potent inhibitor of cell death (Akada et al., 2006). Determined to have an affinity (Ki) of 0.7 mM in the [3H]BTX binding assay subsequent experiments determined that M58373 blocked veratridine-induced release of substance-P from DRG neurons. In the formalin test, oral M58373 (0.3–10 mg/kg) reduced nociceptive behaviors in phase 2, while in the CCI chronic pain model, oral M58373 (1–10 mg/kg) attenuated mechanical allodynia and thermal hyperalgesia without affecting normal responses in the uninjured paw (Akada et al., 2006). 14.3.6

4030W92

Starting with the antiepileptic drug lamotrigine, the novel compound 4030W92 has been described as a voltage- and use-dependent Naþ channel inhibitor (Trezise et al., 1998). Both TTX-R and TTX-S channels are blocked in rat DRG small-diameter neurons in vitro, showing a preferential affinity (five–seven fold) for the slow inactivated state of the channel. Although a weak blocker with only micromolar activity, potential apparently exists for the treatment of chronic inflammatory (Clayton et al., 1998) and neuropathic

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(Collins et al., 1998) pain, although much of the data has been published only in abstract form. This compound was examined in a clinical trial of 41 subjects with chronic neuropathic pain with prominent allodynia (Wallace et al., 2002). 4030W92 (25 mg/day) reduced allodynia on day 1 but not on days 7 or 14 of a 2 week trial. 14.3.7

A-803467

A new compound A-803467, discovered and characterized collaboratively by Abbott and ICAgen, has been described recently (Jarvis et al., 2007). It is the most selective compound described for DRG-associated Nav1.8 channels, use-dependently blocking with an IC50 of 10 nM while only blocking other Nav channels (Nav1.2, 1.5, 1.7) with IC50s of 3–10 mM. This activity profile translates into a dose-dependent reduction of mechanical allodynia in both neuropathic and inflammatory models (SNL, SNI, capsaicin-induced secondary mechanical allodynia and thermal hyperalgesia following CFA). A-803467 was inactive in the formalin, acute and postoperative pain models, and also showed no deficits in spontaneous exploratory behaviors or motor coordination. Thus, specific Nav1.8 blockade appears to result in broad activity across neuropathic and inflammatory models, while showing little or no effect on motor coordination or exploratory activity and no effects on other behavioral measures of peripheral nerve activity, including acute pain. Thus, pharmacological blockade of Nav1.8 appears to be an efficacious and tolerated method for the relief of chronic pain, at least in these animal models. 14.3.8

PPPA

V102862 was originally described as an anticonvulsant (Dimmock et al., 1993, 1996) and subsequently discovered to be a broad-spectrum, state-dependent blocker of voltage-gated Naþ channels (Ilyin et al., 2005). In an effort to obtain control over pain states and a better therapeutic index, broad-spectrum Naþ channel blockers with higher potency, faster onset kinetics, and greater state dependence than existing drugs were the goal of an optimization effort around V102862 (Shao et al., 2004; Yang et al., 2004). The resultant compound, PPPA, met these criteria, displaying greater than 1000-fold better potency and binding kinetics and 10-fold more state dependence than the comparators carbamazepine and lamotrigine (Ilyin et al., 2006). This activity translated into activity in the partial sciatic nerve ligation, CFA, and postincisional pain models using mechanical endpoints, although efficacy was similar to carbamazepine and lamotrigine. With a therapeutic index >10 when measured by ataxia in the rotarod model, PPPA demonstrated better tolerability than the comparator compounds, thus indicating that this approach may prove effective for identifying new Nav channel blockers for pain. 14.3.9

Merck Compound 47

Last, a potent, state-dependent Nav1.7 blocker termed #47 (Hoyt et al., 2007) has been described in a medicinal chemistry-focused paper. Neuronal firing in vivo

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was inhibited in the rat peripheral axotomy model with 2 mg/kg i.v., while oral activity (10 mg/kg) was observed in the SNL tactile allodynia model. Further optimization of pharmacokinetic and pharmacodynamic parameters reportedly continues.

14.4 ANTIDEPRESSANTS Tricyclic antidepressants (TCAs) such as amitriptyline have long been used to treat chronic pain conditions (see Table 14.1), either alone or as adjuvant treatment for opioid therapy. Administration of antidepressants has been shown to reduce pain in patients with diabetic neuropathy (Max et al., 1987, 1992a), post herpetic neuralgia (Watson et al., 1982), and other chronic pain conditions (Magni, 1991; Onghena and Van Houdenhove, 1992; Finnerup et al., 2005). Drugs in this class are usually thought to alleviate pain by increasing levels of norepinephrine and serotonin, thereby stimulating descending inhibitory pain pathways. However, many of the compounds in this class also exhibit Nav, Cav, and ionotropic glutamate receptor-blocking activities, which may contribute to their antinociceptive profile. Amitriptyline is the most widely prescribed TCA for the treatment of chronic pain. Many reports describe the inhibitory action of amitriptyline on Naþ channels in vitro and it is generally described as a nonselective, use-dependent blocker with moderate potency (IC50 from 5–80 mM). In isolated rat DRG neurons, amitriptyline inhibited both the fast (TTX-S) and slow (TTX-R) sodium currents with similar potency (IC50 20 mM) (Pancrazio et al., 1998). A recent paper (Dick et al., 2007) examined the blockade of the peripheral sodium channel Nav1.7 by amitriptyline in detail, and reports that the affinity for the inactivated state of the channel is 0.24 mM, a value that is less than the clinical plasma concentration for amitriptyline. Efficacy in the in vivo neuropathic SNL model has been demonstrated (Mochizucki, 2004), which has translated into clinical efficacy in diabetic neuropathy (Joss, 1999) and postherpetic neuralgia (Watson et al., 1982; Watson and Evans, 1985). Imipramine, another widely prescribed TCA, has long been known to have Naþ channel blocking properties (Schauf et al., 1975). Imipramine is a use-dependent blocker that preferentially binds to and stabilizes the inactivated state of Naþ channels (Dick et al., 2007), although an additional open-channel blocking mechanism has been described (Yang and Kuo, 2002). Imipramine has been demonstrated to be effective in certain human experimental pain models (Bromm et al., 1986; Poulsen et al., 1995; Enggaard et al., 2001) and in patients with painful peripheral polyneuropathy, including diabetic neuropathy (Kvinesdal et al., 1984; Sindrup et al., 2003). Although numerous reports demonstrate the clinical efficacy of TCAs against chronic pain, the preclinical efficacy data for both amitriptyline and imipramine are mixed. Imipramine was shown to be ineffective at reducing tactile and cold allodynia in a number of models of neuropathic pain (Hama and Borsook, 2005) and the

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hot-plate model of acute thermal pain (Otsuka et al., 2001), although it was shown to be analgesic in an electrical shock model of acute pain (Abdel-Salam et al., 2003). There are numerous reports showing efficacy of imipramine and other TCAs in the rat formalin model. The effects of TCAs in the formalin model are largely reversed by noradrenergic and serotonergic antagonists, suggesting that Naþ channel blockade, however, is not the dominant mechanism of action for these compounds in this model (Otsuka et al., 2001; Yokogawa et al., 2002). The effectiveness of TCAs in pain conditions prompted the examination of other antidepressants in pain. Serotonin-norepinephrine reuptake inhibitors (SNRIs) are generally effective in chronic pain models (Iyengar et al., 2004; Mochizucki, 2004) and clinically in diabetic neuropathy (Max et al., 1992). In contrast, selective serotonin reuptake inhibitors (SSRIs) have been shown to be ineffective clinically in chronic pain conditions (Watson and Evans, 1985; Max et al., 1992b). Recently, however, all three types of antidepressant have been shown to block Naþ channels in a use-dependent manner (Huang et al., 2006). The therapeutic concentrations (for their effective use in depression) of the SSRIs, however, are lower than their affinity for the inactivated state of Nav1.7, whereas that of the TCAs and NRIs are greater than their affinity for Nav1.7 (Dick et al., 2007). Thus, the activity of antidepressants in pain conditions may be more related to their Naþ channel blocking activity than to their activity at inhibiting monoamine reuptake. This supports the role of Naþ channel blocking activity in the pain efficacy displayed by other antidepressants.

14.5 CONCLUSIONS Experience over the past half-century has demonstrated repeatedly the therapeutic value of small-molecule blockers of Naþ channels for many disorders of neuronal hyperexcitability. Recent years have seen a resurgence of interest in Nav channel blockers for chronic pain conditions, especially focused around Nav1.3 and the peripherally expressed Nav1.7–1.9 varieties. While many programs have focused on obtaining strongly use-dependent blockers as a means of targeting hyperexcitable cells selectively, recent successes have been notable for their ability to discover Nav subtype selective blockers, a feat previously thought unattainable. The recent recognition that commonly used antidepressants also contain Naþ channel blocking activity may point to another potential use for this class of molecules in future therapeutic settings. The “best” Nav subtype to target for pain conditions has implicated Nav1.3, Nav1.8/1.9, and Nav1.7, but it is likely that broader testing in animal models and in human pain conditions will be the ultimate factor in deciding which subtype(s) are best to target for a particular type or category of pain condition. Indeed, if successful, multiple types of Nav blockers with different combinations of selectivity and use dependence will be needed clinically to allow for choice by individual patients and their medical providers, similar to how epilepsy and depression are treated currently. If this goal is realized, the efforts of many scientists will be a success and bring immeasurable benefits to many patients in need.

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ABBREVIATIONS TTX-S TTX-R DRG Kr Ki VIPR EP IC50i IC50r MIA STZ ION CFA CCI SNI SNL TD50 Cav CRMP-2 FRET

(tetrodotoxin-sensitive) (tetrodotoxin-resistant) (dorsal root ganglia) (association rate constant, resting state) (association rate constant, inactivated state) (voltage ion probe reader) (electrophysiology) (IC50 for inactivated state) (IC50 for resting state) (monosodium iodoacetate) (streptocotozin) (infraorbital nerve) (complete Freunds adjuvant) (chronic constriction injury) (spinal (spared) nerve injury) (spinal nerve ligation) (toxic dose for 50% of subjects) (voltage-dependent Ca2þ channel) (collapsin-response mediator protein 2) (fluorescence resonance energy transfer)

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15 NEURONAL KV7 POTASSIUM CHANNELS AS EMERGING TARGETS FOR THE TREATMENT OF PAIN Steven I. Dworetzky and Valentin K. Gribkoff Knopp Neurosciences Inc., 2100 Wharton Street, Suite 615, Pittsburgh, PA 15203, USA

15.1 INTRODUCTION Potassium (Kþ) channels are the most diverse class of ion channels. Kþ currents are found in most cells and are associated with a wide range of functions, including the regulation of the electrical properties of excitable cells. The primary, pore-forming (a) subunits of these highly selective cation channels are divided into three primary structural classes based on the number of transmembrane (TM)-spanning regions and pore (P) regions: currently there are known to be 6TM/1P, 2TM/1P, and 4TM/2P Kþ channels. An additional 7TM representative that is a variant of the 6TM motif class has also been characterized (the BK channel, Slo1 or KCa1.1). Depending on the type of Kþ channel, the open probability (Popen) or activity of the particular voltagedependent Kþ channel can be regulated by transmembrane voltage (VM), protein phosphorylation and interaction with companion proteins as well as by a host of second messengers and endogenous ligands including ionic species such as calcium (Ca2þ), sodium (Naþ), and hydrogen (Hþ). The KV7 subfamily of voltage-gated Kþ channels consists of five homologous pore-forming a subunits, KV7.1–7.5, which have a structure typical of voltage-gated Kþ channels, with 6TM-spanning regions (S1–S6) flanked by intracellular N-terminal and C-terminal domains, a typical voltage-sensor domain located in S4 comprised of alternating positively charged

Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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residues, and a single P region between S5 and S6 of each subunit (Fig. 15.1a). A subunit interaction and assembly complex is located in the cytoplasmic C-terminal domain (Howard et al., 2007; Wehling et al., 2007). The channels are formed as tetramers of the primary subunits, either as homotetramers or heterotetramers. The degree of homology between the members of the subfamily is shown in Fig. 15.1b.

FIGURE 15.1 (a) Illustration of the structure of a neuronal KV7 channel subunit. The KV7 protein has six transmembrane domains (S1–S6), a pore-forming P-loop, and a voltage sensor in the S4 domain indicated by the positively charged residues (þþþ). The channel is a tetramer of either homomultimers of the same subfamily member, such as KV7.2 channels comprising the substrate of neuronal IKS, or heteromultimeric mixtures of subfamily members, as is the case with M-channels that are heteromultimers of KV7.2/7.3 or KV7.5/7.3. The four alpha subunits (a1–a4) are indicated to illustrate the tetramerization of the channel complex, and the subunit interaction domains are also shown. Also illustrated is the relationship between neuronal KV7 channels and some receptor-mediated mechanisms of channel regulation. The (.) symbol in S5 marks the conserved amino acid between KV7.2–7.5 that is one known site of small-molecule interaction. (b) The table presents pair wise distances, shown as percent identity for purposes of illustration only, determined from the alignment of the human KV7.1–KV7.5 protein sequences. Factors that might affect these values include the inclusion of splice variants and the type of programs used to generate the alignment and the calculation of the sequence similarities.

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At least one member of the subfamily of KV7 channels is known to be associated with modulatory b subunits, an association that significantly affects the biophysical characteristics of the resulting currents and defines their physiological functions. KV7.1 associates with the b subunit KCNE1 to form the slow, depolarization-activated cardiac IKs current (Barhanin et al., 1996; Sanguinetti et al., 1996). The association of KV7.1 mutations with cardiac long-QT syndrome in humans resulted in its original designation as the KVLQT1 channel. In contrast, when the KV7.1 a subunit associates with the b subunit KCNE3 in intestinal crypt cells, the biophysical characteristics change to a constitutively open channel (Schroeder et al., 2000). The combination of KV7.1 with the b subunit KCNE2 in the gastric epithelium results in the channel responsible for coupling of Kþ to gastric acid secretion via colocalization of the channel with the gastric H-K-ATPase (Lambrecht et al., 2005). While several KCNE b subunits are found in the central nervous system (CNS), their association with neuronal KV7 channels has not been fully explored and will not be discussed in this review. Neurons are known to express KV7 channels comprised of KV7.2–7.5 a subunits. Some of these gene products may be exclusively neuronal, while others such as KV7.5 can be found in other tissues, such as the skeletal muscle. The first of the central nervous system KV7 genes, KV7.2 and KV7.3 (KCNQ2 and KCNQ3), were discovered using two different approaches. The first approach identified an expressed sequence tag (EST) from public access gene databases that was homologous to the cardiac KVLQT1 (KV7.1; KCNQ1) channel. The EST contained the hallmark pore signature, a GYG amino acid sequence, of Kþ channels and was found only in brain cDNA libraries. Screening of a brain cDNA library with a probe derived from the EST sequence led to the identification of a full-length gene sequence that demonstrated noninactivating Kþ currents upon expression in Xenopus laevis oocytes (Yang et al., 1998). The second approach utilized linkage analysis to identify a chromosomal region in families with inherited epilepsy of newborns and identified the candidate genes by positional cloning. These cloning experiments led to the discovery of two genes; mutations in these genes were responsible for benign familial neonatal convulsions (BFNC). These genes were found to encode KV7 channels and were localized to chromosome 20 for KV7.2 and chromosome 8 for KV7.3 (Biervert et al., 1998; Charlier et al., 1998; Singh et al., 1998). Subsequently, two additional neuronal KV7 family members, KV7.4 (KCNQ4) (Coucke et al., 1999; Kubisch et al., 1999) and KV7.5 (KCNQ5) (Kananura et al., 2000), were identified and cloned. Collectively, these genes were assigned to a subfamily of voltage-gated Kþ channels, the KV7 channel subfamily, by the International Union of Pharmacology (IUPHAR). The original name for these channels was KCNQ, a name assigned by the HUGO Gene Nomenclature Committee (HGNC) (Gutman et al., 2005). In this chapter, as has already been mentioned, we will use the KV7 nomenclature. The chapter will focus only on KV7.2, 7.3, and 7.5 channels. Although KV7.4 is also a neuronal channel, the function and localization of these channels are more restricted. Their most notable known function is their involvement in cochlear hair cell regulation in the inner ear (Coucke et al., 1999; Kubisch et al., 1999; Van et al., 2000; Beisel et al., 2005; Kharkovets et al., 2006). Therefore, this review will be restricted to KV7.2, KV7.3, and,

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to a lesser extent, KV7.5, and they will be referred to collectively as neuronal KV7 channels.

15.2 NEURONAL KV7 CHANNELS: IDENTIFICATION AS M-CHANNELS AND DISTRIBUTION IN THE CENTRAL NERVOUS SYSTEM After the initial cloning, and as studies delineating the localization and expression of the neuronal KV7 channels were published, the potential importance of KV7 channels as regulators of neuronal excitability was immediately apparent, particularly when mutated neuronal KV7 channels, as mentioned above, were shown to cause BFNC (Biervert et al., 1998; Charlier et al., 1998; Singh et al., 1998; Yang et al., 1998; Biervert and Steinlein, 1999). Shortly thereafter, pharmacological and biophysical identity was established between KV7.2/7.3 (and likely KV7.5/7.3) heteromultimers and the elusive “M”-channel (Wang et al., 1998; Brown and Yu, 2000), providing significant new evidence for their importance in neuronal regulation. Native M-channels and the corresponding macroscopic M-current were first characterized in amphibian sympathetic neurons (Brown and Adams, 1980; Adams et al., 1982a). M-channels were notable as they were slowly activating and noninactivating, active at membrane potentials at or near the resting membrane potential of neurons, and muscarinic cholinergic agonists produced a reduction in the Mcurrent, demonstrating a direct and inhibitory link between G-protein-coupled receptors (GPCRs) and a physiological Kþ current. Inhibitory linkages to other receptors were also found, indicating a propensity for channel inhibition by one or more second messenger systems initiated by this class of ligand–receptor interactions (Adams et al., 1982b, 1983 ; Shapiro et al., 1994; Schweitzer, 2000; Wallace et al., 2002; Filippov et al., 2006; Jia et al., 2007). The GPCR-mediated inhibition of neuronal KV7 channels was later found to result from the hydrolysis of membraneassociated phosphatidylinositol 4,5-bisphosphate (PIP2), secondary to the activation of phospholipase C (PLC) (Suh and Hille, 2002; Zhang et al., 2003). This finding has been much extended recently. These data will be discussed briefly here; however, these have been extensively dealt with in Chapter 11 of this book. In the evaluation of emerging drug targets, the putative target’s known contribution to cellular physiology, the distribution of the gene products, and their possible direct linkage with disease via mutation analysis are criteria that add significant weight to the rationale for pursuing a particular small-molecule modulator discovery program. The primary pore-forming components of M-channels, KV7.2, KV7.3, and (probably) KV7.5, are widely distributed in brain and spinal cord. Prior to the discovery of their molecular correlates, M-channel-mediated Kþ currents were found in many neuronal populations by virtue of their characteristic inhibition by activation of certain G-protein-coupled receptors and the relaxation of their current following hyperpolarization. After the cloning of neuronal KV7 channels and their identification as the molecular substrates of M-channels, studies focused on regional gene expression and immunocytochemical analyses of CNS KV7 channel distributions. These studies confirmed the widespread distribution of M-channels, with cortical, thalamic,

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cerebellar, and hippocampal neurons expressing among the highest levels of various combinations of KV7.2, KV7.3, and KV7.5 mRNAs (Lerche et al., 2000; Geiger et al., 2006; Liang et al., 2006; Kanaumi et al., 2007). KV7 mRNA and channel density generally increase in late embryonic and early postnatal development in the rodent and human central nervous system (Smith et al., 2001; Geiger et al., 2006; Kanaumi et al., 2007), after which there may be a regional reduction in channel density. The presence and importance of neuronal KV7/M-channels in regulating neuronal excitability has been well documented in sensory ganglia (Passmore et al., 2003; Rivera-Arconada et al., 2004; Rivera-Arconada and Lopez-Garcia, 2005), an important contributor to their potential utility in pain therapy (see discussion below). In general, the distribution of these channels, both regionally and developmentally, as well as their biophysical characteristics support their role in providing enduring resistance to depolarizing excitatory influences. Mutation analyses demonstrated their involvement in BFNC and suggested their utility as targets for antiepileptic drugs (AEDs) (Rogawski, 2006). Recognition of some forms of chronic pain as a form of neuronal hyperexcitability disorder involving neurons in the sensory/pain pathways (Waxman, 1999), neurons that were known to express KV7 channels as well as other voltage-gated ion channels, suggested that these channels may have value as targets for the discovery of pain therapeutics. Initial studies suggested that the subcellular distribution of KV7 channels was primarily somatodendritic. Recent studies have shown that their subcellular distributions, and therefore the role of the channels, are likely to be more complex. KV7.2/ KV7.3 heteromultimeric M-channels are preferentially localized on the surface of axons and enriched in axon initial segments (Chung et al., 2006; Vervaeke et al., 2006; Yue and Yaari, 2006). Distribution of the channel complex to these loci is differentially regulated. Axon initial segments, an ideal locale for control of spike after hyperpolarization, are mediated by ankyrin-G binding motifs of both KV7.2 and KV7.3 channel subunits, whereas more distal axonal surface expression is regulated by domains within the KV7.2 channel C-terminal region (Chung et al., 2006). Devaux et al. (2004) examined a KV7.2 mutation that produces myokymia and the distribution of KV7.2 and KV7.3 subunits in axons by confocal microscopy. The R207W KV7.2 channel has reduced charge in the S4 voltage sensor (Fig. 15.1a) and shifts the activation voltage curve in the positive (depolarizing) direction (Dedek et al., 2001). Its association with myokymia suggested some form of functional control of motor axon excitability by these channels. Immunocytochemical analyses using polyclonal antibodies to both KV7.2 and KV7.3 channel proteins demonstrated that KV7.2 was found at nodes of Ranvier in these peripheral nerves as well as in the spinal cord and in axon initial segments in much of the CNS (Devaux et al., 2004). KV7.2 and KV7.3, however, were not found to colocalize at these nodes (KV7.3 was primarily found in nodes of smaller diameter fibers), directly demonstrating that while these two channels often form heteromultimeric M-channels, they do not always have overlapping distributions and can have important independent functions. A subsequent study showed that the slow nodal Kþ current IKS, which participates in the pronounced spike-frequency adaptation observed in motor nerve fibers, is mediated exclusively by KV7.2 in large diameter fibers of sensory nerves (Schwarz et al., 2006). Clearly, unlike early simplistic views of

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FIGURE 15.2 (a) Immunocytochemical analysis of KV7.2 protein expression in a dorsal root ganglion. Staining, using an affinity-purified primary antiserum raised against a KV7.2 peptide, reflects the degree of KV7.2 channel expression in neurons of all sizes within the DRG. (b) Staining with a preimmune serum is negative, as is staining in the presence of excess peptide (not shown), demonstrating specificity of the staining shown in panel A.

the function of KV7 channels in neuronal network dynamics, their physiologic roles are complex and only partially understood. For example, it is not known if they have a significant direct role in the regulation of neurotransmitter release at the level of the synaptic bouton, and their role in the regulation of dendritic excitability is also less clear. Our group used in situ hybridization and immunocytochemistry to examine the distribution of KV7.2 channel protein throughout the brain with particular focus on pain pathways. As shown in Fig. 15.2 (Goldstein and Dworetzky, unpublished observations), KV7.2 protein is expressed at highest levels in the large cell and medium cell populations of sensory neurons in the dorsal root ganglion (DRG), with moderate to low levels of expression in the small cell population. Double-label immunofluorescense studies (not shown) demonstrated that some of the large to medium diameter neurons that express the highest levels of KV7.2 protein also express the neuroactive peptide calcitonin gene-related peptide (CGRP). The smaller diameter neurons, which expressed lower levels of KV7.2 protein, all coexpressed substance P. In a study of trigeminal circuitry, RT-PCR techniques applied to dissociated neurons of the trigeminal ganglion gave detectable mRNA signals for KV7.2, KV7.3, and KV7.5 subunits, with the most abundant signal for KV7.2. Further localization studies by in situ hybridization demonstrated the presence of neuronal KV7 channels in the trigeminal nucleus caudalis. Given that the release of CGRP has been closely linked to the onset of migraine pain and both CGRP and substance P have been associated with inflammatory pain (Henry, 1993; Sluka, 1996; Durham, 2006; Doods et al., 2007), modulating KV7 channels in these key regions could significantly impact these pain modalities. These studies are supported by another report examining KV7 members in the nociceptive sensory system (Passmore et al., 2003). Using RT-PCR, they demonstrated the presence of KV7.2, 7.3, and 7.5 mRNA in both small and large DRG neurons. Moreover, confocal microscopy revealed signal in the somata and neuronal

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processes of these neurons (Passmore et al., 2003). An additional localization study has reported the presence of KV7.2, 7.3, and 7.5 in rat visceral sensory neurons isolated from the nodose ganglia (Wladyka and Kunze, 2006). Collectively, these results indicate that neuronal KV7 channels are localized and play an important role in key pain circuitry. It follows from this that openers of these channels could possibly reduce the release of neuroactive peptides involved in pain and/or hyperpolarize these neurons, alleviating either pain initiation and/or transmission of painful stimuli.

15.3 INFLUENCE OF KV7 CHANNELS ON NEURONAL BEHAVIOR Native M-channels and cloned KV7 channels activate at membrane potentials that are close to the resting potential of neurons, unlike most voltage-gated potassium channels. They are relatively slow to activate upon depolarization and slow to deactivate, and, perhaps most important in terms of their functional significance, they are noninactivating. Therefore, in the absence of external modulatory influences, these channels can have enduring effects on membrane excitability within their activation–voltage ranges. Under physiological conditions, as was demonstrated with native M-channels (Brown and Adams, 1980; Constanti and Brown, 1981; Adams et al., 1982a; Selyanko et al., 1992; Stansfeld et al., 1993; Costa and Brown, 1997; Zhu et al., 2000), they can be very effective at regulating the subthreshold excitability of certain neuronal populations, with significant roles in regulating the frequency and ultimately the pattern of action potential discharge in many types of neurons. Their importance in neuronal regulation was punctuated by the discovery that neuronal KV7 mutations lead to BFNC (Biervert et al., 1998; Charlier et al., 1998; Biervert and Steinlein, 1999; Lerche et al., 1999; Hirose et al., 2000; Singh et al., 2003), indicating that reduction or removal of the influence of KV7.2 and KV7.3 channels can dramatically alter neuronal excitability. Most mutations leading to BFNC are autosomal dominant negative mutations, with little or no effect on remaining wild-type KV7 channels. This indicates that the phenotype of BFNC in most cases was caused by a reduction in current, a haploinsufficiency, rather than an alteration in the biophysical characteristics of mutant channels. This was further evidence that neuronal KV7/Mchannels contribute significantly to the maintenance of electrical homeostasis in many neurons, and that modest alteration of the level of expressed current can have profound effects in some systems depending on the stage of development of the organism. As the name implies, BFNC is largely benign and resolves quickly during development. While there remains some increased risk of seizure disorder and even more serious complications in later life, it is clear that the role of KV7 channels is complex and that it changes with development. Members of at least one family with KV7 mutation-related BFNC also have a concomitant form of myokymia (Dedek et al., 2001), an involuntary repetitive series of muscle contractions, indicating additional functions of at least one member of this ion channel subfamily. Knockout animals have not significantly contributed to our understanding of the involvement of these channels in disease states or their potential as targets for the development of treatments due to homozygous lethality or other complicating issues.

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One study disrupted the mouse Kv7.2 gene and heterozygous mutant mice had decreased Kv7.2 expression and demonstrated hypersensitivity to the proconvulsant pentylenetetrazole (Watanabe et al., 2000). Incidentally, there was no mention of a change in nociceptive behavior. Conclusions concerning the physiological roles of neuronal KV7 channels and the importance of development in defining these functions have been greatly strengthened recently by transgenic studies. Peters et al., (2005) used transgenic M-channel-deficient mice conditionally expressing a dominant negative pore mutant construct (hQ2-G279S) in a comprehensive study of the electrophysiological, morphological, developmental, and behavioral consequences of M-channel deficiency. The results were complex; pyramidal cells were lost in mutantmale mice, but therewas muchless cell loss in female mice, demonstrating X-chromosomalinheritance and a different pattern of expression of the mutant channel. These morphological changes in male mice were observed only when the channels were suppressed during early development (approximately from birth to 3 weeks postnatal). While conditional suppression of M-channels following this critical period resulted in altered hippocampal physiology, the morphology was largely unchanged. Under whole-cell voltageclamp, hippocampal pyramidal neurons from male mice expressing the mutant channel (either throughout life or conditionally) had a significant reduction in M-channel current and increased resting input resistance, and responded to depolarizing current pulses with significantly greater numbersofactionpotentials (reduced spike-frequency adaptation), all in keeping with the known functions of these channels. They also had undetectable levels of medium-duration after-hyperpolarizations following action potentials and a reduction in subthreshold electrical resonance behavior in the “theta” frequency range (3–5 Hz). Theta frequency resonance in hippocampal neurons has long been associated with neuronal correlates of learning (Gray and Ball, 1970; Staubli and Xu, 1995; Kinney et al., 1999; McNaughton et al., 2006); and Hu et al. (2002) had recently demonstrated an M-channel contribution to hippocampal theta resonance. This discussion of KV7 channel function is intended to convey some of what may be contributing to the difficulty in readily translating our knowledge of both the physiology and pharmacology of these channels into useful therapeutics. In thinking about the discovery and development of drugs targeting KV7 channels, modulator compounds have usually been thought of in almost a unitary sense, when clearly the channels themselves have multiple and complex roles in many different neurons. In at least two dimensions, they are a multimodal target; they are a subfamily of KV channels, and the same members are likely to have different functions in different neurons and possibly at different loci in the same neurons, and their distributions and functions may change with development. A further layer of complexity possibly affecting the utility and side effect profile of neuronal KV7 modulators is provided by the following discussion of biochemical substrates of KV7 channel regulation.

15.4 REGULATION OF KV7 CHANNELS Transmembrane voltage (Vm) is the primary controller or regulator of neuronal KV7 current. However, well before the molecular entities comprising M-channels had been

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cloned and characterized, this class of Kþ current was known for, and in fact defined by, its modulation by endogenous ligand–receptor interactions (Brown and Adams, 1980; Shapiro et al., 1994; Marrion, 1997). The relatively hyperpolarized voltage– activation ranges of KV7/M-channels means that these channels can contribute significantly to resting levels of neuronal excitability as well as countering some forms of physiologically relevant depolarization. GPCR-mediated or other physiologically relevant inhibition of M-current in neurons produces slow depolarizations, resulting in long-term increases in neuronal excitability, a major contributor to the integration of synaptic influences in many cells. The discovery and the subsequent characterization of the underlying biochemical pathways that connect receptor activation and M-channel inhibition are among the most important and interesting findings associated with these channels. In rat superior cervical ganglion (SCG) neurons, Marrion et al. (1989) demonstrated pharmacologically that muscarinic cholinergic suppression of M-current was probably mediated by receptors of the M1 receptor subtype. Shapiro et al., (2001) later confirmed this, when they found that M1 muscarinic receptor knockout mice lacked muscarinic modulation of M/KV7 channels. The latter group also demonstrated that they could create a functional KV7.2/KV7.3 modulatory system in vitro by coexpressing the channels in tsA-201 cells along with muscarinic M1 receptors, an effect that depended on some unknown intracellular factor but was not a result of intracellular Ca2þ transients (Shapiro et al., 2000, 2001). A diffusible messenger had been implicated in muscarinic suppression of M channels as early as 1992 (Selyanko et al., 1992), and hints of the identity of this factor came from both an increasing awareness of the role of membrane phospholipids in ion channel regulation (Hilgemann et al., 2001), and the earlier work implicating the involvement of phoshotidylinositol 4,5-bisphosphate (PIP2) hydrolysis in bradykinin receptor activation-mediated depolarization of neuroblastoma–glioma hybrid cells, an effect attributable to M-current suppression (Higashida et al., 1986). In 2002, Suh and colleagues used this heterologous coexpression system to examine the recovery of M-current following its suppression by muscarinic cholinergic receptor activation. They found that recovery was dependent on intracellular ATP and was independent of G-protein activity (Suh and Hille, 2002). The need for hydrolysable ATP indicated involvement of one or more kinases in the recovery of native M-current or recombinant KV7-mediated current, so they tested a number of kinase inhibitors in both SCG cells and in the tsA-201 heterologous system. Wortmannin, an inhibitor of Pl 4-kinase (a lipid kinase that, with ATP, replenishes PIP2), as well as other pharmacological inhibitors of PIP2 generation, greatly reduced or eliminated recovery of the current, strongly suggesting PIP2 involvement in M-current recovery (Suh and Hille, 2002). Zhang et al. (2003) directly tested this hypothesis using PIP2 and a number of analogues to determine if PIP2 could both activate M/KV7 channels inhibited by GPCR activation as well as re-activate channels that have run down following insideout patch excision. Rapid channel rundown in excised membrane patches had been a problem since the discovery of M-channels and inhibited single-channel analysis of cloned KV7 channels. Most single-channel work therefore relied on the use of cell-attached recordings, where manipulation of the intracellular and extracellular

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milieu is problematic. Zhang et al. (2003) found that PIP2 and analogues could reestablish current following rundown and the following receptor-mediated inhibition, and that all neuronal KV7 channel subtypes were similarly modulated. These results have been confirmed and extended by several other groups (Post-Munson et al., 2003; Li et al., 2005; Suh et al., 2006; Suh and Hille, 2007). Zhang et al. (2003) also mutated a positively charged amino acid residue in a proximal C-terminal region of KV7.2 that is homologous to PIP2 binding domains in other ion channels; coexpression of these mutated KV7.2 subunits (H328C-KV7.2) with wild-type KV7.3 subunits produced currents of significantly lower amplitude that were much less sensitive to PIP2 and more sensitive to receptor-mediated inhibition. Li et al. (2005) also examined the role of PIP2 in neuronal KV7 channel open probability. While their experiments confirmed the relationship of muscarinic cholinergic suppression of these channels to regulation of PIP2 and the re-activation of rundown channels in excised patches, they also discovered that different subfamily members had different open probabilities when recorded in cell-attached patch configurations (PIP2 system intact). This apparently intrinsic correlation of open probability (at a given voltage) with KV7 subtype was found to correlate with their sensitivity to PIP2, suggesting that open probability is not only a function of the PIP2 available to the channel, but also of the channel’s inherent PIP2 sensitivity. Another study has used palmitoylated peptides (palpeptides) to dynamically probe the intramembrane regulation of neuronal KV7 channels (Robbins et al., 2006). These peptides were designed to interrupt associations such as the coupling of G-proteins to a receptor and receptor activation to PIP2 hydrolysis; their PIP2 palpeptide (palmitoyl-HRQKHFEKRR) was designed based on the assumption that H328 was a C-terminal residue involved in PIP2 binding, as reported previously (Zhang et al., 2003). Robbins and colleagues found that their PIP2 palpeptide rapidly and reversibly reduced current and sensitized currents to muscarinic receptor activation, additional evidence of the importance of PIP2 to neuronal KV7 regulation. It is currently unknown whether PIP2’s regulation of these channels is involved in the activity of small-molecule modulators. We have found that retigabine does not reactivate KV7.2/7.3 heteromultimeric channels after the rundown in inside-out excised membrane patches, but an effect of the compound is usually re-established after PIP2 partially reactivates the channel (Gribkoff, Post-Munson, Boissard, and Dworetzky, in preparation). This suggests that retigabine activation of these channels requires some degree of PIP2 occupancy of its binding site. Intracellular Ca2þ was shown long ago to suppress M-channels (Tokimasa, 1985; Marrion, 1996; Selyanko and Brown, 1996a, 1996b), and Ca2þ/calmodulin (CaM) has been shown to be an important regulator of neuronal KV7 channels (Yus-Najera et al., 2002; Gamper et al., 2005; Shahidullah et al., 2005), with identified binding domains in the C-terminus (Wen and Levitan, 2002; Yus-Najera et al., 2002; Shahidullah et al., 2005). This appears not only to be the case in amphibian neurons and cloned KV7 channels, but also in mammalian central neurons, where it was recently shown that hippocampal neurons expressing a CaM binding motif fusion protein had lower density of M-current; the lower M-current density was associated with a change in neuronal discharge characteristics, including increased action potentials evoked by the depolarizing current and an increase in action potential after depolarizations

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(Shahidullah et al., 2005). These findings are also relevant to receptor-mediated regulation of neuronal KV7 current, since increases in intracellular Ca2þ can be mediated through IP3-coupled receptors, including bradykinin and P2Y receptors, and the effects of activation of these receptors, unlike muscarinic receptors, depend on CaM (Villarroel, 1996; Cruzblanca et al., 1998; Gamper and Shapiro, 2003; Zaika et al., 2007). Gamper et al. (2005) have shown that coexpression of KV7.2, KV7.4, and KV7.5 with CaM produces current suppression, but that coexpression does not suppress KV7.3 (or KV7.1, the cardiac channel). Interestingly, they also found that the KV7 current enhancement produced by the cysteine-modifying reagent N-ethylmaleimide (NEM) was reduced by CaM overexpression, and that the C-terminal domains were involved in the binding/activity of each overlap. Phosphorylation of neuronal KV7 channels via Src tyrosine kinase also produces channel suppression, distinct from GPCR-mediated inhibition (Gamper et al., 2003). Src tyrosine kinase associates with KV7.2–7.5, but appears to actually produce phosphorylation only of KV7.3–7.5; mutation of two tyrosines (tyr-67 and tyr-349) produced additive inhibition (Li et al., 2004). Recently, a physiological role for this additional mechanism of KV7 channel inhibition was demonstrated; activation of epidermal growth factor receptors, a member of the class of receptor tyrosine kinases, was found to produce biphasic inhibition. In a recombinant expression system and in SCG neurons, the faster of the two inhibitory components resulted from PIP2 hydrolysis, while a slower form of inhibition was found to result from tyrosine phosphorylation (Jia et al., 2007). These results serve to indicate the multiple routes of receptor-mediated inhibition of KV7 channel currents, and help to suggest mechanisms of pharmacological modulation that may both explain the actions of known small-molecule modulators as well as suggest additional routes to synthesis of new molecules. Additional phosphorylation sites have recently been identified, one of which is found in the S4–S5 linker region of all neuronal KV7 channels, producing channel inhibition when mutated (Surti et al., 2005). While there are additional mechanisms of neuronal KV7 channel modulation that have recently been elucidated, these examples are sufficient to indicate the importance of understanding the role of biochemical regulation of neuronal KV7 channel function and possibly drug discovery.

15.5 NEURONAL KV7 CHANNEL PHARMACOLOGY 15.5.1

Modulators

Some reference has been made in earlier sections of this chapter to neuronal KV7 channel modulators such as retigabine. At the outset, we think it is important to operationally define some of the terminology used in our description of the pharmacology of these voltage-gated ion channels. Unlike established pharmacological terminology for GPCRs, the mode of action of Kþ channel modulators, in particular, compounds that activate the channel, is still being refined. The application of voltageclamp techniques to the study of ion channel pharmacology enabled detailed biophysical studies of either whole-cell currents or single channels, allowing some

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characterization of the nature of compound–channel interactions, but this has not prevented ongoing confusion around the terminology. The term opener or activator is commonly used throughout the literature, but does not adequately describe the mode of action of all these “positive modulator” compounds. In general, openers or activators are expected to increase the open probability of the channel or increase macroscopic current amplitude, but this nomenclature is really too simplistic. For example, retigabine, the first publicly disclosed KV7 opener, has a complex and interesting profile in that it has inhibitory activity at higher membrane potentials. In our Kþ channel opener programs, we typically found a number of different types of positive modulators in addition to those with the voltage-dependent opener-inhibitor profile of retigabine. As per our experience, neuronal KV7 channel openers may work in concert with the activity of a channel over the “normal” activation–voltage range, enhancing currents without significantly affecting the activation threshold, while others can significantly alter the activation threshold. In addition, some openers appeared to remove the voltage dependence of activation entirely. Whether these effects represent some continuum is currently unclear, since the effects are often concentration dependent. Clearly, the modes of compound interaction that can increase channel current are complex and in most cases not well understood, and the implications of these profiles on neuronal responsiveness and systems physiology remain to be elucidated. Much the same confused terminology concerning compounds that decrease channel current also exists. Pore-blocking peptides, for example, occlude the pore and directly limit ion flux, and are therefore clearly current blockers. However, small molecules that inhibit channel current may not actually block, but rather in many respects act like inverse agonists, reducing current by some means other than directly blocking ion flow through the pore. For the purpose of this study, the term opener is used to describe molecules that increase ion channel current over physiologically relevant levels of Vm, and inhibitor will be used to describe molecules doing the opposite. Almost immediately after the cloning of neuronal Kv7 channels, it became widely accepted that these channels may have significant value as drug targets. The initial publicly disclosed opener of these channels was the antiepileptic drug candidate retigabine (Fig. 15.3) (Main et al., 2000; Wickenden et al., 2000; Schroder et al., 2001; Tatulian et al., 2001), an analogue of the approved pain medication flupirtine (Katadolon) (Mastronardi et al., 1988; Ringe et al., 2003; Boscia et al., 2006). Retigabine is modestly potent and is not highly specific, but it is a very effective opener of KV7.2, KV7.5, and heteromultimeric KV7 channels (Main et al., 2000; Wickenden et al., 2000; Schroder et al., 2001; Tatulian et al., 2001; Tatulian and Brown, 2003; Schenzer et al., 2005). Its effects are characterized by a significant increase in channel current over a narrow voltage range. As mentioned above, at more positive voltages, the opener is less effective, and under some conditions, channel current significantly decreases at more positive voltages relative to control currents (this “crossover” voltage dependence of opener action is a characteristic of many neuronal KV7 channel openers). This effect is also concentration dependent and is more pronounced at higher concentrations. Recent evidence has pinpointed probable sites of interaction of this compound within KV7 channels (Schenzer et al., 2005).

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FIGURE 15.3 Structures of KV7 openers discussed in text, and associated with the experimental or clinical treatment of pain: (a) Marketed compounds, (b) retigabine is in clinical development (the structure of ICA-105665 has not been publicly disclosed), and (c) preclinical discovery compounds used for proof-of-concept studies in animal models.

In 1997, our group initially used the expression of mouse KV7.2 channels, and later human KV7.2/7.3 heteromultimers, to initiate a program targeting these channels as regulators of neuronal excitability. We found that some openers of another important Kþ channel, the large-conductance calcium-activated Kþ (maxi-K, BK, KCa1.1) channel, were also openers of neuronal KV7 channels; this is typified by the fluoroxindole compound BMS-204352, a potent maxi-K channel opener (Gribkoff et al., 2001) whose KV7 channel activity has been cited by others (Schroder et al., 2001, 2003; Dupuis et al., 2002; Jensen, 2002). Over the past 10 years, a number of synthetic programs have been initiated to design neuronal KV7 channel openers (Munro and Dalby-Brown, 2007). It has actually proven quite easy to discover openers of these channels. Openers such as zinc pirithione (Xiong et al., 2007) and the structurally related compounds such as meclofenamic acid and diclofenac have been discovered by academic laboratories (Fig. 15.3) (Peretz et al., 2005). A large number of compounds resulted from our program at Bristol-Myers Squibb (Wu et al., 2003, 2004a, 2004b; Hewawasam et al., 2004; L’Heureux et al., 2005), and additional compounds have been offered or further characterized by teams at Icagen (Wickenden et al., 2008) and NeuroSearch (Schroder et al., 2001, 2003; Dupuis et al., 2002; Jensen, 2002). While other companies have had programs in this area, there has been little public disclosure of specific compounds of interest along with data sufficient to evaluate their potency, efficacy, and specificity. A major struggle with most compounds has been their relative lack of specificity, either between Kþ channel families, as is the case with BMS-204352, or between members of the KV7 subfamily, or both.

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A compound recently reported from the group at Icagen, however, provides some degree of discrimination between neuronal KV7 subfamily members, and reflects their understanding of this issue and their commitment to discovering more subtypespecific modulators. The compound ICA-27243 (N-(6-chloro-pyridin-3-yl)3,4-difluoro-benzamide; Fig. 15.3) (Wickenden et al., 2008) produces a leftward shift in the voltage dependence of KV7.2/7.3 channel activation with an EC50 of 1–2 mM and demonstrates some degree of selectivity, with greater potency and possibly efficacy against KV7.2/7.3 heteromultimeric channels versus either KV7.4 homomultimers or KV7.3/7.5 heteromultimers. In addition, ICA-27243 did not have significant effects on several ion channels and receptors that are the target of current AEDs, such as g-amino butyric acid (GABA) receptors, native high-voltageactivated Ca2þ channels, and a Naþ channel (NaV1.2). As discussed above, in contrast to ICA-27243; retigabine, Bristol-Myers Squibb compounds BMS-204352 and the acrylamide, known publicly as [S]-1 (Fig. 15.3), do not discriminate significantly between subfamily members and appear to require a conserved tryptophan (at position 236 in KV7.2, position 265 in KV7.3, position 242 in KV7.4, and position 235 in KV7.5) in the S5 transmembrane domain to maintain efficacy (Schenzer et al., 2005; Wuttke et al., 2005; Bentzen et al., 2006), as well as possibly other residues in the S6 domain identified as critical for gating (Wuttke et al., 2005). This site of action may not allow big discrimination between neuronal KV7 subfamily members, but it does allow discrimination between neuronal KV7 channels and the cardiac KV7 channel KV7.1, since this site in S5 is not conserved in this subfamily member. Since mutations of this channel (KV7.1) are associated with long QT syndrome (Peroz et al., 2008), its exogenous modulation is to be approached with extreme caution, if at all, and, incidentally, any putative KV7 channel modulator should be assessed for activity at this channel. It is currently unknown whether ICA27243 interacts with the critical tryptophan residue in S5 in neuronal KV7 channels, or whether it has an entirely novel site of interaction that is not conserved between all of the subfamily members. Regardless of the molecular substrate, the compound represents some degree of progress toward subtype specificity and still exhibits activity in models of neuronal hyperexcitability. As mentioned previously, inhibitors of these channels have also been described, most notably linopirdine and XE-991, compounds that originated in a Dupont program for cognition enhancement (Costa and Brown, 1997; Zaczek et al., 1998; Wang et al., 2000). Linopirdine (AVIVA) was the first inhibitor to be tested in clinical trials. The mixed results from these trials and the subsequent removal from further development were attributed to the short and variable half-life in humans and poor brain penetration in animal studies. While additional inhibitors have been discovered in the course of synthetic programs targeting openers, the lack of therapeutic targets for KV7 inhibitors, other than cognition enhancement, has largely relegated these compounds to the status of research tools. This stands in contrast to the situation with openers of neuronal KV7 channels, where localization, functional data, and results of animal studies and, recently, human experimentation have continued to support the idea that the right molecule should have value in several widespread and important neurogenic conditions and diseases, including pain.

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Therapeutic Targets

The spectrum of therapeutic targets for modulators of KV7 channels has not significantly changed since initial descriptions, primarily focusing on chronic pain and epilepsy (Cooper and Jan, 2003; Gribkoff, 2003; Munro and Dalby-Brown, 2007). Some evidence now exists to support anxiety as a target for neuronal KV7 openers (Korsgaard et al., 2005; Hansen et al., 2008), and there is some suggestion that modulators may have utility in the treatment of addiction (Hansen et al., 2007) and dystonia (Richter et al., 2006). In general, these therapeutic targets were determined based on the initial channelopathy (BFNC) findings, the distributions of the channels, the known effects of reference compounds, and the theoretical linkage of disorders other than the epilepsies with selective neuronal “hyperexcitability,” such as migraine pain and other chronic and particularly neuropathic pain (Wall, 1991; Welch et al., 1993; Waxman, 1999). Neuronal KV7 channel inhibitors may have therapeutic value as candidate cognition-enhancing compounds; however, their current value remains as research tools for attenuating or blocking the effects of opener compounds and for pharmacological excision of these currents to study their physiological roles, in particular, neurons and neuronal networks. As mentioned above, the first significant modulators of KV7 channels described publicly were the AED retigabine and the candidate cognition-enhancer compounds linopirdine and XE-991 (Fig 15.3). From the perspective of providing some degree of proof-of-concept in human disease for AEDs (retigabine) and pain (flupirtine, diclofenac), these retrospectively discovered neuronal KV7 modulators have proven useful for increasing confidence in these channels as therapeutic targets.

15.5.3 Neuronal KV7 Modulation and its Effect on Pain Circuitry and Experimental Pain Our direct experience with therapeutic applications for neuronal KV7 modulators has been in the areas of chronic pain and migraine headache. Pain is a broad term used to describe the discomfort, distress, or suffering resulting from the excessive activation of specific subsets of sensory neurons. Acute pain can have significant survival value for an organism by promoting flight or other responses that will minimize further injury. In this vein, acute pain is not usually considered a disorder, although therapeutics directed to acute pain clearly have utility in medical and dental procedures, among others. On the other hand, chronic or semichronic pain syndromes (such as neuropathic pain or cancer pain), or chronic–episodic forms of pain such as migraine can be severe, life altering, and are often of no obvious value to an organism, although in and of themselves they are not really diseases either, but are usually reflections of other conditions. Inflammatory pain occurs after tissue injury and is mediated by the release of multiple proinflammatory factors that result in tissue swelling and the activation of specialized peripheral pain receptors or “nociceptors.” Neuropathic pain often (but not always) results from a lesion or injury to nerves in the peripheral or central nervous system, and is a common and often incapacitating clinical condition largely resistant to treatment with commonly prescribed analgesics. Painful peripheral neuropathies can

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have many etiologies such as trauma and metabolic disorders, but in most cases, there is no known cause. Neuropathic pain is characterized by spontaneous pain, mechanical and thermal hyperalgesia (exaggerated response to a painful stimulus) and allodynia (pain response to an innocuous stimulus). Symptoms are correlated with and may result from the development of “central sensitization” in the spinal cord, which may be initiated by unusually high-frequency and sustained (“ectopic”) spontaneous peripheral nerve activity and a decrease in inhibitory tone; as such it is an example of neuroplasticity run amok (Ossipov et al., 2000; Campbell and Meyer, 2006). In what is no doubt a serious but nevertheless useful oversimplification of pain pathophysiology, central sensitization associated with pain syndromes results from neuronal hyperexcitability and, in turn, produces hyperexcitability in more central components of pain pathways. This phenomenology thus opens the door for treatment of these forms of pain by decreasing this excessive neuronal activity (while hopefully sparing “normal” activity). This logic is identical to that used to ameliorate epileptiform neuronal activity, and suggests that chronic pain can be approached pharmacologically by many of the same drugs such as the AED and neuronal KV7 opener retigabine. There is a growing body of evidence suggesting that neuronal KV7 channels are important regulators of excitability in neurons of pain pathways. For example, the opener retigabine and inhibitors XE-991 and linopirdine were used to investigate the roles of KV7 channels in pain transmission using spinal cord preparations as well as recordings from single spinal neurons. It was shown that motor neurons and neurons in the deep dorsal horn express functional M-currents; application of retigabine produced decreases in the excitability of these neurons, resulting in depression of transmission of nociceptive information, effects that were blocked by XE-991 (Rivera-Arconada et al., 2004; Rivera-Arconada and Lopez-Garcia, 2005, 2006). In another study, both small- and large-diameter-cultured DRG neurons were demonstrated to have M-currents that were enhanced by retigabine and blocked by linopirdine (Passmore et al., 2003). Retigabine was shown to inhibit C and Ad fiber-mediated responses; this effect was blocked by the administration of linopirdine. While the specifics of the neuronal circuitry and neurochemistry underlying pain can be somewhat species specific, acute pain is so important to the survival of organisms that one has to drop to the lowest branches of the phylogenetic tree to convincingly locate animals that do not display behavior reflecting (albeit perhaps somewhat anthropomorphically) acute pain-like responses to potentially harmful stimuli. Chronic pain is somewhat more problematic, but rodents can be shown to respond to some experimental manipulations by the development of exaggerated sensory responses, indicative of the development of chronic neuropathic pain. Some human pain modalities such as migraine, however, remain challenging. Similar to the modeling of psychiatric disorders in animals, some forms of pain like migraine do not have any obvious mechanism for their independent measurement. Nevertheless, in a research setting, both neuropathic pain and inflammatory pain can be modeled in animals for the characterization of pain therapeutics, although there have been some questions concerning the predictive validity of these models (Whiteside et al., 2008). However, with proper care, particularly with respect to the determination of the contribution of side effects (such as lethargy, somnolence, or anesthesia) to apparent

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analgesic effects in particular models with specific compounds, useful and reasonably predictive information can be obtained. Neuropathic pain models range from the use of various types of nerve constrictions or ligations to the use of the antitumor drug streptozotocin to induce diabetes and its attendant neuropathic pain. This latter drug’s toxicity to pancreatic cells is the basis of both its utility in pancreatic cancer and its use in producing experimental diabetes. Changes in pain thresholds due to any of these manipulations are measured in a number of ways. For example, they can be measured mechanically by testing the threshold of tolerated pressure on a paw or the response to thermal pain can be tested by assessing the animal’s tolerance to local changes in skin temperature. Inflammatory pain is induced by injecting (usually) the animal’s paw with formalin, carrageenan, or other chemical stimuli to induce paw flinching, biting, licking, and other responses and the results quantified in terms of an acute response phase, measured within the first 10–15 min, and a persistent response phase, measured between 10–15 min and 90 min after injection. Demonstration of the archetypal broad-spectrum neuronal KV7 channel opener retigabine’s analgesic activity has been reported in several pain models. Retigabine was shown to have analgesic activity in a behavioral model of inflammatory pain produced by intrapaw injection of carrageenan, an effect that was reversed by the application of the inhibitor XE-991 (Passmore et al., 2003). In a spinal nerve ligation model of neuropathic pain in rats, retigabine significantly and dose-dependently increased the pain threshold, an effect that could be diminished with the coadministration of linopirdine (Dost et al., 2004). In a model of neuropathic pain induced by chronic constriction injury as well as in the model of persistent pain induced by formalin, retigabine demonstrated efficacy under some test conditions (BlackburnMunro and Jensen, 2003). With respect to modeling different types of pain and understanding the broad utility of neuronal KV7 modulation, visceral sensory neurons of the nodose ganglion have been shown to express KV7.2/7.3/7.5 proteins, and M-currents could be recorded from these cells. Flupirtine, an analogue of retigabine approved in Europe for the treatment of pain, increased M-current and hyperpolarized neuronal resting membrane potentials in these cells in culture (Wladyka and Kunze, 2006). Consistent with this, in a capsaicin-induced visceral pain model, retigabine dose-dependently suppressed visceral pain behavior (Hirano et al., 2007). In a study of pain of musculoskeletal origin, retigabine produced antinociceptive effects in a model of acid-induced muscle allodynia (Nielsen et al., 2004). Taken together, these data demonstrate a high degree of proof-of-concept for the role of neuronal KV7 channel modulation in the treatment of various neuropathic and inflammatory pain conditions in animals. At the Bristol-Myers Squibb Company, we had an extensive program targeting pain by modulating neuronal KV7 channels. Many of these compounds proved to be analgesic at doses that produced no significant effects on independent measures of general motor disruption and somnolence. One such compound, the cinamamide (S)-3(2-fluoro- phenyl)-N-(1-[3-(pyridine-3-yloxy)-phenyl]-ethyl)-acrylamide (Fig. 15.3), was tested in several pain models using i.v. administration. In the formalin test of inflammatory pain, the compound produced a significant reduction in both the early and late phases of the response profile using flinching as the measure of pain response

NEURONAL KV7 POTASSIUM CHANNELS AS EMERGING TARGETS

Threshold (g)

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**

**

15

** ** **

10

Gabapentin (100 mg/kg)

* BMS-Cinnamamide ex. (10 mg/kg)

5 PEG400 (0.5 mL/kg) i.v.

0 –15 0

30

60

90

Time (min)

FIGURE 15.4 Effects of a neuronal KV7 opener in a rat model of neuropathic pain. The BMScinnamamide induced a significant attenuation of neuropathic pain behavior at 10 mg/kg i.v., peaking 15 min after injection. A lesser but significant effect was also observed at 3 mg/kg i.v. (not shown). This particular cinnamamide has a modest duration of action due to either rapid metabolism or high clearance. Gabapentin (100 mg/kg i.v.) has a slower onset of action but longer duration of effect. (n ¼ 8–10 rats per group. *P < 0.05, **P < 0.01 versus PEG-400, Dunnett’s test).

(not shown). In a spinal nerve ligation model, the compound significantly reduced tactile allodynia, although its duration of action was limited (Fig. 15.4). The compound was also active in the streptozotocin model of diabetic neuropathy (not shown). Retigabine, like the Bristol-Myers Squibb compounds discussed above (and below for migraine), is a broad-spectrum opener of neuronal KV7 channels. Retigabine also interacts with other potential pharmacological targets. Collectively, these compounds leave room for improvement in understanding the potential contribution of neuronal KV7 channels in general and specific subfamily members in particular to analgesia related to their activation. The Icagen compound introduced previously (ICA-27243; Fig. 15.3) is a much more discriminating opener of KV7.2/7.3 channels relative to its effects on other subfamily members, and was reported to show efficacy in a wide range of anticonvulsant models (Wickenden et al., 2008). ICA-27243 also demonstrated analgesic efficacy in the rat carrageenan model of inflammatory pain, the rat formalin model of chronic pain, and also antihyperalgesia and antiallodynic efficacy in the rat chronic constrictive injury model of neuropathic pain. Icagen has recently advanced a compound, ICA-105665 (the structure has not been disclosed), into phase 1 clinical trials to assess its safety and pharmacokinetic profiles, with a stated goal of advancing the compound into clinical trials as an AED and as a pain therapeutic (www.Icagen.com). As already mentioned, the use of KV7 openers for the treatment of migraine pain is another interesting therapeutic indication for these compounds. In part, this was based on the localization of these channels within neuronal populations associated with migraine headache (such as the trigeminal nuclei) and on the concept that this condition falls within the broad spectrum of “hyperexcitability disorders.” Migraine is characterized by unilateral throbbing head pain that is often associated with visual disturbances and is considered to be a chronic or chronic–episodic disorder. It has been suggested that cortical spreading depression may underlie various prodromes

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that precede the onset of migraine headache, particularly visual aura (Lauritzen, 1994). Cortical spreading depression is defined as a wave of neuronal excitation followed by long-lasting inhibition that spreads from a focal point. Clinical neurological migraine prodromes proceed in a temporal fashion that is correlated with the expected rate of spreading depression. These neurological symptoms, with associated changes in blood flow, correlate well with spreading depression phenomena (Lauritzen, 1994). More recently, spreading depression was visualized during triggered migraine attacks in humans using functional magnetic resonance imaging (Cao et al., 1999). This evidence suggests that spreading depression may underlie the visual aura and possibly other prodromes that precede migraine and cause the ensuing migraine attack and accompanying pain (Hardebo, 1992). Similar to the modeling of neuropathic and inflammatory pain for the testing of preclinical drug candidates, it is possible to produce cortical spreading depression in laboratory animals. In view of these findings, attenuation of cortical spreading depression induced by placing crystals of KCl onto the cortex has become a currently accepted practice to evaluate and compare novel compounds for their potential in the prophylactic treatment of migraine headache. We standardized this model using valproic acid, a drug sometimes used for the prophylactic treatment of migraine headache. Using the BMS-acrylamide (S)-1 KV7 opener (Fig. 15.3), we found that the compound produced a reduction in the total number of cortical spreading depressions induced by the KCl. The potency compared to valproic acid was greater, while efficacy was reduced relative to this reference compound, thus providing additional support for the broad utility of this target in the treatment of pain (Wu et al., 2003).

15.6 SUMMARY AND DISCUSSION We have presented a review of previous work, including our own contributions, in which we characterize this new subfamily of important voltage-dependent Kþ channels and introduce their pharmacology. We believe that these channels represent interesting targets for the development of important new therapeutics in a number of areas, and we have most closely focused on their potential utility in the treatment of serious and chronic pain syndromes. There remains a large unmet medical need for the treatment of pain, and developing compounds against new targets is a good strategy for attempting to circumvent the shortcomings of existing medications. Many lines of evidence support the hypothesis that neuronal KV7 channel openers would be useful for the treatment of different types of pain, particularly chronic neuropathic pain and migraine. The neuronal KV7 channels are localized in key neuronal circuitry involved in pain pathways; the key role the M-current plays in controlling cell firing is well documented and preclinical proof-of-concept studies demonstrate their efficacy in a wide array of pain models with at least three different neuronal KV7 channel openers. This is an area that is currently seeing significant activity, including clinical trials of at least one opener of these channels. Valeant pharmaceuticals is currently testing retigabine in phase 3 trials for the adjunctive treatment for partial-onset seizures after it

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was demonstrated to be efficacious in reducing monthly seizure rates in a phase 2 trial (Porter et al., 2007). In addition, Valeant has filed an IND application for retigabine (as an immediate release formulation) in the treatment of pain associated with postherpetic neuralgia (www.Valeant.com). In addition, both flupirtine and diclofenac are effective and approved for use in human pain (Mastronardi et al., 1988; Ringe et al., 2003; Moore, 2007). None of these compounds are specific, however, they can have a number of side effects, depending on the dose. Most important, their other pharmacology may very well contribute as much or more to their efficacy in pain as their ability to open KV7 channels (among other things diclofenac is a COX inhibitor and flupirtine is an NMDA receptor antagonist). It is unclear whether new molecules such as ICA-27243 represent a significant advance in terms of therapeutic potential, as opposed to the demonstrated progress in molecular pharmacology. Determining this will require comprehensive analyses of new compounds’ effects in animal models of disease, compared to activity profiles in models predictive of dose-limiting side effects. Finally, we will have to compare these profiles with those obtained under similar conditions with other molecules not displaying this degree of subfamily and molecular target specificity. To approach pain as it has been approached with sodium and calcium channels, additional key data will be required. For example, we know that alternative splice variants exist for neuronal KV7 channel members (Smith et al., 2001), however, there has been little or no work to determine if there is any functional (modal) significance to their expression. Specifically, are there defined sensory neuronal populations expressing specific KV7.2–7.5 variants, which could be specifically targeted for pain, as may be the case for N-type (CaV2.2) Ca2þ channels (see Chapter 8) (Bell et al., 2004; Altier et al., 2007)? Also, are channel splice variants sufficiently distinct to provide a substrate for differential pharmacology, something that has not yet been convincingly or usefully shown for other voltage-gated ion channels? Are there functional neuronal KCNE beta subunits, and are they involved in changing the biophysical and pharmacological characteristics of neuronal KV7 channels as they are with peripheral KV7.1 channels? Most important, is there something about the voltage dependence of KV7 modulators, a feature that is readily apparent in the actions of many openers, which can be exploited to provide state dependency of drug action in a mode favoring therapeutic utility? Drug discovery efforts directed toward detection and exploitation of these more subtle aspects of drug action have greater promise to produce effective pain medications with minimal side effects.

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16 SMALL-MOLECULE MODULATORS OF LARGE-CONDUCTANCE, CALCIUM-ACTIVATED (BK) CHANNELS John E. Starrett Jr. Discovery Chemistry, Bristol-Myers Squibb Co., 5 Research Parkway, Wallingford, CT 06492, USA

16.1 INTRODUCTION Large-conductance, calcium-activated potassium (maxi-K, BKCa, big K, or BK) channels are present in a wide variety of excitable and nonexcitable cells, including neurons (Meech, 1978), pancreas (Findlay et al., 1985), skeletal muscles (Lerche et al., 1995), and mammalian smooth muscles such as trachea (McCann and Welsh, 1986), colon (Carl et al., 1992), and bladder (Suarez-Kurtz et al., 1991; Zografos et al., 1992). They are also found within the vasculature, including coronary (Sargent et al., 1993) and cerebral vessels (Holland et al., 1996). BK channels have single-channel conductance values in excess of 100 pS, yet maintain a high degree of selectivity for potassium (Latorre et al., 1989). Because of their widespread tissue distribution and dependence on voltage and calcium for activation, BK channel regulation represents an attractive approach to treat a number of diseases associated with abnormalities of smooth muscle and neurons. Due to their large conductance and gating by the critical intracellular messenger calcium, modulation of relatively few BK channels can have profound effects on cell and tissue functions. As knowledge of

Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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BK channel regulation by small molecules is growing, so is the number of reports of the utility of those small molecules. In model systems, openers have demonstrated activity for the treatment of smooth muscle disorders associated with erectile dysfunction, urinary incontinence, irritable bowel syndrome, and hypertension. BK blockers may be of utility for the treatment of glaucoma and malignant glioma. This review will focus on small-molecule regulators of BK channels that are reported to directly modulate the BK channel. There are also reports of molecules that regulate BK channels via second messenger cascades, such as those induced by stimulation of canabinoid, protinoid, and kinase receptors, which may impact diseases such as neurogenic inflammation of the airway, hypertension, stroke, and glaucoma; however, these approaches are outside the scope of the current study. There have been several reviews focusing on small-molecule regulators of BK channels (Starrett et al., 1996; Gribkoff et al., 1997; Calderone, 2002; Nardi et al., 2003); the current study will concentrate on small-molecule BK channel modulators that have been reported since 2002.

16.2 BK CHANNEL OPENERS Ohwada et al. (2003) have studied the effects of analogues of dehydroabietic acid (Fig. 16.1a) for their ability to open cloned maxi-K channels in human embryonic kidney (HEK) 293 cells. Introducing chlorines at the 12 and 14 positions provided the analogue depicted in Fig. 16.1b, which significantly increased channel-opening activity. The carboxylic acid appears to play an important role for activity in this class of molecules; reduction of the acid to afford the alcohol, depicted in Fig. 16.1c, resulted in complete loss of activity. A more detailed examination of the properties of 12,14-dichloro-dehydroabietic acid (Fig. 16.1b) revealed that it is a potent opener of BK channel a subunits expressed in HEK cells, as measured in inside-out patches via changing the voltage and calcium sensitivity of the channel (Sakamoto et al., 2006). It has an inverse voltage dependence for BK channel activation, but may not have a significant interaction with the BKb1 subunit. The potentiation of BK channels was significantly larger at negative potentials as well as at lower calcium concentrations.

FIGURE 16.1

The dehydroabietic acids and alcohol openers of BK channels.

BK CHANNEL OPENERS

FIGURE 16.2

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Benzimidazolone and diphenyl hetereocyclic BK openers.

A series of diphenyl-substituted heterocycles were synthesized and evaluated by electrophysiological techniques as openers of cloned BK channels expressed in Xenopus oocytes (Romine et al., 2002). Deannulation of the prototypical benzimidazolones NS-004 and NS-1619, shown in Fig. 16.2a and b, respectively, afforded derivatives such as the trifluoromethylphenyl oxadiazole, shown in Fig. 16.2c, which opened BK channels to 138% of control when evaluated at 20 mM. For comparison, under these conditions, NS-004 and NS-1619 opened BK channels to 132% and 116% of control, respectively. Decreasing the electron density of the phenyl ring by the addition of another trifluoromethyl group afforded the structure depicted in Fig. 16.2d, which opened BK channels to 159% of control. The structure–activity relationship that emerged from this study indicated a lipophilic pocket exists adjacent to the binding region of the channel. The presence of a hydrogen bond donor on the heterocyclic ring was found to be of paramount importance for BK channel activity in these series of compounds. For example, the triazole (Fig. 16.2e) was devoid of activity. The presence of an alcohol capable of hydrogen bonding to a carbonyl is a recurring theme in a variety of BK openers (Starrett et al., 1996). Calderone and coworkers have published a series of papers examining the vasodilatory activity of a series of bis-aryl heterocycles and bis-aryl amides. In these papers, compounds were evaluated for their ability to relax thoracic aortic rings of male normotensive rats precontracted with potassium chloride. Electrophysiologic data were not presented, but the authors speculated that the relaxant properties of these compounds are mediated through BK channels by structural homology to known BK openers such as NS-1619 and the triazolone shown in Fig. 16.2d. In a series of

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FIGURE 16.3 Bis-phenyl amide and triazole BK openers.

bis-phenyl amides, the phenol shown in Fig. 16.3a demonstrated a pIC50 ¼ 7.56 and an efficacy (Emax %) ¼ 86 (Biagi et al., 2004b). Examination of a series of 1,2,3 triazoles revealed the ester illustrated in Fig. 16.3b as the most potent analogue, with a pIC50 ¼ 6.55 and an efficacy (Emax %) ¼ 56 (Biagi et al., 2004a). By comparison, NS-1619 had a pIC50 ¼ 5.18 and an efficacy (Emax %) ¼ 100. Extension of the 1,2,3 triazoles to bis-benzylated versions (Fig. 16.3c) resulted in a phenol with a pIC50 ¼ 7.56 and an efficacy (Emax %) ¼ 95 (Calderone et al., 2005). Replacement of a phenyl group in the bis-phenyl amide series uncovered a pyridine and thiophene, as shown in Fig. 16.3d and e, respectively, as the most active analogues, with full efficacy (100%) and pIC50 ¼ 4.93 for both compounds (Calderone et al., 2006b). Similar to previous reports, replacement of the phenol with a methoxy group resulted in a marked decrease in efficacy and potency. Reversing the position of the amide functionality of bis-phenyl amides (Fig. 16.3a) provided the chlorophenol, shown in Fig. 16.3f, with pIC50 ¼ 6.16 and full efficacy (Calderone et al., 2006a). The plant-derived sapogenin, diosgenin, depicted in Fig. 16.4, increased the activity of BK channels in human cortical neurons (HCN-1A cells) when applied to the external surface of the cells, with an EC50 ¼ 25 mM (Wang et al., 2006). The increased current amplitude occurred without altering the single-channel conductance. Application of diosgenin to the intracellular surface had no effect on BK channels.

16.3 CENTRAL NERVOUS SYSTEM (CNS) THERAPEUTICS 16.3.1

Stroke/Traumatic Brain Injury

Following a stroke or traumatic brain injury, neurons may undergo membrane depolarization, leading to increased intracellular calcium levels and neurotoxicity.

CENTRAL NERVOUS SYSTEM (CNS) THERAPEUTICS

FIGURE 16.4

427

The BK opener diosgenin.

Opening BK channels will lead to membrane hyperpolarization, blocking the further opening of voltage-activated calcium channels. Employing pharmacologic activators of BK channels may activate them at lower levels of intracellular calcium and membrane depolarization, thereby providing a neuroprotective effect. A series of 4-phenyl-3-aminoquinolin-2ones derivatives were synthesized and evaluated as activators of cloned BK channels express in Xenopus oocytes (Hewawasam et al., 2002a). The most active compound to emerge from this study was the trifluoromethyl sulfonamide, shown in Fig. 16.5a, which opened channels to 343% of control at the test concentration of 20 mM. The acidity of the proton plays an important role in the activity of the molecule, as indicated by the weaker increases in current exhibited by the methylsulfonamide, shown in Fig. 16.5b, and the acetamide, shown in Fig. 16.5c (164% and 120% of control, respectively). For

FIGURE 16.5 4-Phenyl-3-amino quinolinone BK openers.

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SMALL-MOLECULE MODULATORS

the purpose of treating stroke, the sulfonamide depicted in Fig. 16.5a could not be advanced further due to undetectable brain levels of the compound following i.v. administration of 5 mg/kg to rats. The parent 3-aminoquinolinone (Fig. 16.5d), which opened BK channels to 186% of control, was determined to achieve brain levels of 1.4 mM and a brain/plasma ratio of 5.3 at 2 h following i.v. administration. To determine the ability of the parent 3-aminoquinolinone to reduce cell loss resulting from neuronal ischemia, a standard rodent model of focal ischemia was employed, which involved permanent occlusion of middle cerebral artery in spontaneously hypertensive rats (MCAO model). When administered i.v. 30 min postMCAO at a dose of 1 mg/kg, it induced a significant reduction (14%, p < 0.05) in neocortical infarct volume. A series of 1,3,4 oxadiazoles were studied in Xenopus oocytes as BK openers (Romine et al., 2007). Similar to the bis-aryl heterocycles discussed above, deannulation of NS-004 and optimization led to BMS-191011, as shown in Fig. 16.6a. When evaluated at 1 mM, BMS-191011 opened cloned BK channels to 126% of control. In rats, BMS-191011 demonstrated a brain/plasma ratio of 21 at 15 min following i.v. administration. In a permanent occlusion stroke model, BMS-191011 produced a significant reduction in infarct volume when administered 2 h following occlusion of the middle cerebral artery in rats. One shortcoming of BMS-191011 was the low aqueous solubility (<1 mg/mL), which hampered its administration in an intravenous formulation. A variety of watersoluble prodrugs of BMS-191011 were synthesized and evaluated for solution state stability and rate of conversion to BMS-191011 in rat and human plasma (Hewawasam et al., 2003a). The optimal prodrug to emerge from these studies was the deoxycarnitine ester prodrug, shown in Fig. 16.6b, with an aqueous solubility >25 mg/mL. Incubation in rat and human plasma indicated cleavage of the prodrug to afford BMS-191011 and the prodrug was also cleaved in rats following i.v. administration to provide BMS191011 in blood and brain. After stirring for 24 h in PEG400/water, >97% of the prodrug remained intact. When evaluated in a permanent occlusion stroke model, the prodrug significantly reduced cortical infarct volume following i.v. administration to rats.

FIGURE 16.6 BMS-191011.

The BK channel openers BMS-191011 and BMS-204352, and a prodrug of

CENTRAL NERVOUS SYSTEM (CNS) THERAPEUTICS

429

The fluorooxindole BMS-204352 (Maxipost, Fig 16.6c) opens cloned BK channels to 247% of control at 10 mM in HEK 293 cells (Hewawasam et al., 2002b). In a rat permanent occlusion model of stroke, BMS-204352 significantly reduced infarct volume when administered 2 h following the occlusion (Gribkoff et al., 2001). In a rat model of traumatic brain injury, administration of 0.1 mg/kg BMS204352 improved neurological motor function 1 and 2 weeks postinjury and reduced the extent of cerebral edema in the ipsilateral hippocampus, thalamus, and adjacent cortex (Cheney et al., 2001). Administration of 0.03 mg/kg of BMS-204352 reduced cerebral edema in the ipsilateral thalamus. BMS-204352 underwent a series of clinical trials (the potassium-channel opener stroke trials; POST), including two phase III efficacy trials, which were multicentered, worldwide clinical trials, enrolling 1500 patients (Starrett et al., 2004). The drug was administered at doses of 1 mg/day or 0.1 mg/day for 4 days following a stroke. The trials were methodologically sound and showed a positive effect for TPA; however, BMS-204352 was not statistically different from placebo. Due to the low aqueous solubility of BMS-204352, a Tween-80 formulation was employed to administer the drug intravenously. Dose escalation in POST was limiting because the formulation became hypotensive. It was concluded that alternative formulations or a prodrug would be needed to safely administer higher doses to adequately test the hypothesis of utilizing a BK opener as a neuroprotectant following a stroke. Cilostazol (Fig. 16.7a) has been approved in the United States as a cyclic nucleotide phosphodiesterase 3 inhibitor for reduction in intermittent claudation. By elevating cyclic AMP, it acts as a vasodilator and antiplatelet aggregator (Hong et al., 2006). In patients with recurrent cerebral infarction, cilostazol demonstrated a reduced risk of stroke. Subsequent to the clinical findings, cilostazol was found to have BK channel opening properties (Hong et al., 2003). In SK–N–SH cells, cilostazol opened BK channels in a concentration-dependent manner as determined in whole-cell patches. The opening could be blocked by the BK channel blocker IbTx, but not by the KATP blocker glibenclamide. In a rat model of ischemia, cilostazol significantly reduced infarct volume when administered 1 h after the completion of a 2 h occlusion of the middle cerebral artery. In in vitro studies, cilostazol, as well as BMS-204352,

FIGURE 16.7

The BK channel openers cilostazol, KR 31378, and zonisamide.

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prevented a TNF-a-induced decrease in viability with suppressed DNA fragmentation, along with an increase in CK2 phosphorylation and a decrease in PTEN phosphorylation. A downstream effect of this BK-mediated second messenger system was an increase in Akt/CREB phosphorylation, increase in Bcl-2 protein, and decrease in Bax protein, leading to prevention of cell death. The benzopyran KR 31378, illustrated in Fig. 16.7b, has also demonstrated BKopening and anti-apoptotic properties via a similar pathway as cilostazol (Kim et al., 2004). When administered to rats i.p. at 30 or 50 mg/kg at 5 min, 4 h, and 8 h after the completion of 2 h of ischemia of the middle cerebral artery, KR 31378 demonstrated a significant reduction in cerebral infarct as well as a reduction in TUNEL-positive cells, a decrease in DNA fragmentation, an increase in Bcl-2, and decreases in Bax and cytochrome C expression (Hong et al., 2002). 16.3.2

Epilepsy/Bipolar Disorder

The benzisoxazole sulfonamide zonisamide, illustrated in Fig. 16.7c, is approved in the United States for the treatment of epilepsy. The mechanism of action of zonisamide is not well understood, but there are reports that it blocks voltage-dependent sodium channels and T-type calcium channels. A recent report showed that clinically relevant brain concentrations (30 mM) of zonisamide can activate BK channels (Huang et al., 2007). In neuron-derived H19-7 cells, zonisamide increased the activity of BK channels in a concentration-dependent manner by significantly increasing the channel open probability. The authors speculate that it is reasonable to assume that BK channels present in neurons are relevant targets of the action of zonisamide. In addition to its action as an anticonvulsant, zonisamide has also demonstrated clinical efficacy in bipolar disorder (Ghaemi et al., 2006). In an 8-week study conducted in bipolar outpatients, a significant improvement in the depressive symptoms was observed.

16.4 SMOOTH MUSCLE THERAPEUTICS 16.4.1

Erectile Dysfunction (ED)

Among the various types of potassium channels present in corporal smooth muscle, BK channels are the most prominent subtype. Opening of BK channels in corporal smooth muscle may lead to membrane hyperpolarization, resulting in smooth muscle relaxation, an increase in corporal blood flow, and an improvement in erection function. Several different groups have studied the effects of BK channels and their role in ED. Hewawasam et al. (2003b) have described several series of 4-aryl quinolinones demonstrating in vivo activity in a rat model of erectile dysfunction. The compounds were initially characterized for their ability to increase potassium-mediated wholecell outward currents using two-electrode voltage-clamp recording from Xenopus oocytes expressing cloned hSlo BK channels. Structure–activity relationship studies were conducted in a series of 3-aryl-6-trifluoromethyl quinolinones, depicted in

SMOOTH MUSCLE THERAPEUTICS

FIGURE 16.8

431

Quinolinone BK channel openers.

Fig. 16.8a, in which the C-3 alkoxy substituent (R2) was optimized. In all examples, the phenol (R1 ¼ H) was significantly more active than the corresponding methyl ethers (R1 ¼ CH3). The most effective opener identified in this series was ethyl alcohol BMS223131, shown in Fig. 16.8b. At the test concentration of 20 mM, it opened BK channels to 378% of control. In phenylepherine-contracted rabbit cavernosum strips, BMS-223131 produced an 83% inhibition of isomeric force. The relaxation of rabbit cavernosum strips induced by BMS-223131 could be reversed by iberiotoxin (IbTX), a selective BK channel blocker, indicating that the tissue relaxation induced by BMS223131 was mediated through the opening of BK channels. In an in vivo rat model of erectile function, BMS-223131 was administered i.v. at a dose of 1 mg/kg. It had no effect on basal intracavernous pressure, but produced a significant potentiation of electrically evoked increases in intracavernous pressure 50 min postdose, indicating that BK openers such as BMS-223131 may be of value in treating humans with erectile dysfunction. Further evaluation of BMS-223131 revealed that it may exist as two stable atropisomers (Vrudhula et al., 2007). Separation of BMS-223131 by chiral highperformance liquid chromatography (HPLC) resulted in isolation of (þ)-BMS223131 and ()-BMS-223131, whose absolute stereochemistry was determined by single X-ray crystallography. Electrophysiological evaluation of the atropisomers determined the () and (þ) isomers that opened cloned BK channels to 292 and 214% of control, respectively, at the test concentration of 10 mM. At this concentration, racemic BMS-223131 opened BK channels to 259% of control. In an effort to extend the activity observed in alcohol BMS-223131, a series of 3-quinolinyl mercaptans were studied (Hewawasam et al., 2004). The amino

432

SMALL-MOLECULE MODULATORS

alcohol, shown in Fig. 16.8c, opened BK channels to 192% of control in Xenopus oocytes expressing hSlo. It also demonstrated a considerable improvement in aqueous solubility compared to related analogues, with water solubility of >1.9 mg/mL. At the test concentration of 10 mM, it inhibited 55% of isometric force in isolated rabbit corpus cavernosum precontracted with phenylepherine; at a concentration of 1 mg/kg i.v., it produced a significant increase in electrically stimulated intracavernous pressure in a rat model of erectile function. Further work in the 3-quinolinyl mercaptans by Boy et al. (2004) identified compounds extremely efficacious at opening cloned BK channels expressed in oocytes. The N-methyl piperazine, depicted in Fig. 16.8d, opened BK channels to 474% of control at the test concentration of 20 mM and the N,N-diethyl amine, shown in Fig. 16.8e, opened channels to 192% of control. Although numerous efficacious openers were identified in this study, in many cases the opening did not translate into relaxation of phenylepherine-contracted rabbit corpus cavernosum strips. The authors speculate that this disconnect in activity may be due to differences in tissue penetration issues, or may be due to differences in the channels expressed in oocytes versus those expressed in cavernosum strips. The most efficacious compound in the rabbit strips in this series was the amine, shown in Fig. 16.8e, which produced a 57% relaxation of rabbit cavernosum strips at the test concentration of 10 mM. Studies conducted by Nelson and coworkers have examined the effects of BK channels on erectile function in BK channel knockout mice (Slo/) (Werner et al., 2005). In cavernosum tissue from (Slo/) mice, precontracted with phenylepherine, a fourfold increase in phasic contractions was observed. In electrically stimulated cavernosum tissue from (Slo/) mice, relaxation was reduced to 50%, similar to the reduction observed in wild-type cavernosum tissue treated with the BK blocker iberiotoxin. Intracavernous pressure in vivo exhibited significant oscillations in (Slo/) mice, but not wild-type mice. Electrically stimulated increases in intracavernous pressure were reduced by 22% in (Slo/) mice. The authors strongly support the idea that the BK channel could be an important target in treating patients with erectile dysfunction. Melman et al. (2006) have taken the concept of treating erectile dysfunction by opening BK channels into the clinic by treating patients with injections of hMaxiK, a naked DNA plasmid carrying the human cDNA encoding hSlo. A gene transfer approach that overexpresses BK channels in the corpus cavernosum may overcome malfunctioning or underexpressed endogenous BK channels in men with ED. In a phase I study, 11 patients were given a single-dose caverosum injection of hMaxi-K. The injection was well tolerated with no adverse events or clinical laboratory abnormalities. This trial was not designed to study efficacy; however, one patient in each of the high-dose groups reported clinically significant and sustained improvement in erectile function. The authors suggest that further clinical trials should be conducted with larger patient groups to confirm the safety and efficacy of this approach. The gene transfer therapy approach may also be applicable to other smooth muscle disorders such as urinary incontinence, irritable bowel syndrome, and asthma.

SMOOTH MUSCLE THERAPEUTICS

16.4.2

433

Irritable Bowel Syndrome

BK channel openers may also be of utility for the treatment of irritable bowel syndrome. BK channels are expressed in the gastrointestinal track where they influence the action potentials of the vagal motor neurons (Pedarzani et al., 2000) as well as primary afferent neurons responsible for normalizing stretch-induced excitation (Kunze et al., 2000) and secretomotor reflexes (Clerc and Furness, 2002). Opening of BK channels may therefore result in an increase in transit time in the gut. In addition to demonstrating activity in a rat model of erectile dysfunction (discussed above), BMS-223131 (Fig. 16.8b) has also shown activity in a rat model of irritable bowel syndrome (Sivarao et al., 2005). Following i.p. administration of 3, 6, and 20 mg/kg, BMS-223131 markedly inhibited fecal output in response to stress as well as demonstrated a significant reduction in moisture content. A reduction in stressinduced colonic motility as well as in visceral nociception was also observed. 16.4.3

Urinary Incontinence

Indoles, as shown in Fig. 16.9 are claimed to open BK channels in freshly isolated bladder smooth muscle cells as well as relax isolated rabbit urinary bladder strips precontracted with potassium chloride (Lee et al., 2005). Specific data were not provided, but the inventors claim that the compounds in the patent application are useful for the treatment of urinary incontinence or overactive bladder. 16.4.4

General Smooth Muscle Relaxants

Examination of a series of derivatives of ketoconazole, as shown in Fig. 16.10a, for their ability to open BK channels expressed in bovine smooth muscle cells has identified single variations on the scaffold which changed the characteristics from a BK channel blocker to an opener (Power et al., 2006). The bromophenyl analogue, depicted in Fig. 16.10b, produced a 95% blockade of BK currents at 30 mM, whereas the aminophenyl analogue, shown in Fig. 16.10c, produced a 566% opening at the same concentration. The imidazole is not required for opening activity as demonstrated by the 235% opening of the racemic mixture of 1:1 cis:trans isomers of the methyl analogue, illustrated in Fig. 16.10d. Mallotoxin (Fig. 16.11a; rottlerin; MTX) is a natural product capable of opening BK channels, with the unusual property of opening BK channels in a voltagedependent and calcium-independent manner (Zakharov et al., 2005). Mallotoxin has been reported to inhibit protein kinase C d with an IC50 ¼ 36 mM; however,

FIGURE 16.9

An indole BK channel opener.

434

SMALL-MOLECULE MODULATORS

FIGURE 16.10 The BK channel modulators ketoconazole and derivatives of ketoconazole.

activation of BK channels by mallotoxin was observed at 100 nM. As a potential smooth muscle relaxant, this study relates the folk remedy of kamala for the treatment of tapeworm. Mallotoxin is purified from kamala, suggesting that the antihelmenthic effects may be a result of BK channel activation by mallotoxin, relaxing smooth muscle in the tapeworm, resulting in a decrease in egg-laying behavior. The authors suggest that the Mallotoxin scaffold may be utilized to enhance channel activity. In patch-clamp experiments, the benzofuran KB 130015, shown in Fig. 16.11b, activated BK channels formed by hSlo (a) subunits in HEK 393 cells (Gessner et al., 2007). The channels were reversibly opened by shifting the open-probability/voltage relationship by about 60 mV. The shift was considerably stronger (90 mV) with the addition of the Slo b1 subunit. Similar to mallotoxin, activation of BK channels was voltage dependent and calcium independent. In porcine pulmonary arteries contracted with prostaglandin F2a, KB 130015 induced endothelium-independent vasorelaxation, half-maximal at 43 mM.

FIGURE 16.11 The BK channel openers mallotoxin and KB 130013.

BK BLOCKERS/INHIBITORS

435

16.5 BK BLOCKERS/INHIBITORS 16.5.1

Anesthesia/Anesthetics

Deng and coworkers have studied the effects of the general anesthetic agent isoflurane, shown in Fig. 16.12 in oocytes expressing mslo1 (Wang et al., 2004). Using outside-out patches at different command potentials, it was determined that clinically relevant concentrations of isoflurane inhibited BK channels in a reversible, voltage-dependent manner. 16.5.2

Glaucoma

Ocular hypertension without optic nerve damage is believed to be a prelude to glaucoma, a condition in which sustained intraocular pressure results in optic nerve damage and irreversible loss of visual function. If elevated intraocular pressure is treated before nerve damage occurs, it may be possible to prevent the loss of visual function associated with glaucoma. BK channel blockers have been claimed in the patent literature to diminish fluid in the eye, thereby lowering intraocular pressure. Researchers at Merck have claimed a series of 70 paxilline (Fig. 16.13a) –like natural products as BK blockers for the treatment of glaucoma (Goetz et al., 2003). The compounds block BK channels in CHO cells constitutively expressing the a subunit, or in HEK 293 cells constitutively expressing both a and b1 subunits at compound concentrations at, or below, 100 nM. The inventors describe an in vivo model to measure intraocular pressure; however, no data on compound activity were provided. Another patent application claiming BK blockers for the treatment of glaucoma disclosed a series of indazoles, as illustrated in Fig. 16.13b (Doherty et al., 2004) The compounds blocked BK channels in transiently transfected TsA-201 cells as well as decreased BK currents in human nonpigmented ciliary epithelial cells. No biological data were presented that was specific to glaucoma. In a follow-up patent application to the indazoles, a series of tetrahydrocarbazoles, exemplified by Fig. 16.13c, were claimed as BK channel blockers, which lower intraocular pressure and therefore would be of use for the treatment of glaucoma (Gao and Shen, 2006). No other biological data were presented in this application.

FIGURE 16.12 The BK channel blocker isoflurane.

436

SMALL-MOLECULE MODULATORS

FIGURE 16.13 BK channel blockers to treat glaucoma.

16.5.3

Malignant Glioma

BK channel blockers may have utility in the treatment of glioma (primary brain tumors believed to have originated from normal glial cells) (see Chapter 4). Gliomas account for 20% of all brain malignancies (Weaver et al., 2004), and are generally very difficult to treat with poor prognosis. In at least 50% of human gliomas, the epidermal growth factor receptors (erbB) are upregulated. The erbB receptors are tyrosine kinases that activate downstream signaling, resulting in an increase in intracellular calcium concentrations and activation of BK channels. The change in potassium conductance that results from the calcium-induced opening of BK channels has been shown to accompany the proliferation of numerous cell types. Examination of malignant glioma biopsy tissue revealed a significant expression of BK channel protein, which was subsequently identified as a novel splice variant of the hSlo gene with enhanced calcium sensitivity and named gBK (Liu et al., 2002; Ransom et al., 2002). Modulation of glioma BK channels was demonstrated by disruption of constitutive erbB2 activation. Application of Tyrphostin AG825, as shown in Fig. 16.14, a specific erbB2 inhibitor, induced a 30 mV positive shift in gBK channel activation in a dose-dependent manner in cell-attached patches (Olsen et al., 2005). The modulation appeared to occur via changes in intracellular calcium levels. A similar decrease in BK channel activation produced by a specific small-molecule blocker of these channels could therefore have utility in preventing glioma proliferation.

FIGURE 16.14 The glioma BK channel modulator tyrphostin AG 825.

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16.6 CONCLUSION BK channel modulation remains an elusive target for specifically developing drugs to treat human diseases. Although many compounds have demonstrated efficacy in animal models of diseases thought to be regulated by BK channels, to date none have reachedthemarket.However,thatisnottosaythattherearenoBKchannelmodulatorson the market. Drugs such as zonisamide and cilostazol were approved based on their demonstrated clinical efficacy in the treatment epilepsy and intermittent claudation, respectively. After their approval, further evaluation revealed the BK-modulating properties of both drugs, which may play a role in their mechanism of action. With the increasing knowledge of BK channel regulation, it should only be a matter of time before a drug specifically targeting BK channels reaches the market for one of the indications discussed in this chapter, or another yet uncharacterized utility.

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17 HIGH-THROUGHPUT SCREENING TECHNOLOGIES IN ION CHANNEL DRUG DISCOVERY Edward B. Stevens, Andrew D. Whyment, and J. Mark Treherne Pfizer Global Research and Development, Sandwich Laboratories (IPC 351), Sandwich, Kent, CT13 9NJ, UK

17.1 INTRODUCTION Ion channels regulate many diverse physiological processes including the electrical impulses that underlie sensory and motor functions in the brain; the control of contractile activity of the heart, skeletal, vascular, and visceral smooth muscle; and nutrient uptake, hormone secretion, cell replication, and development. There is accumulating genetic evidence for the importance of ion channels in many disease pathologies, and so the modulation of their function may be of value in treating an increasing variety of diseases. Consequently, there is considerable further potential in future ion channel drug discovery to uncover novel therapies that address unmet medical need. Selecting the most appropriate ion channels for drug discovery research is based upon the potential therapeutic relevance and toxicological implications of modulating the function of that channel. Analysis of the molecular classification of drug targets found that all known orally bioavailable drugs only bind to approximately 500 distinct molecular targets (Drews, 2000). Only around 5% of these molecular targets, however, were classed as ion channels. This relatively low percentage (e.g., compared with enzymes) probably has resulted from a lack of suitable starting points for medicinal chemistry and difficulties

Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright 2009 John Wiley & Sons, Inc.

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in configuring relevant screens to identify novel chemical series. The uses of existing ion channel drugs span many therapeutic areas, and this observation suggests that there is considerable further potential for ion channel drug discovery with new and existing targets. As the selective permeability and gating of ion channels are responsible for maintaining cell membrane potential and controlling cellular excitability (Armstrong and Hille, 1998), which is a key role in the functioning of all cells, it is not surprising that ion channels are such important drug targets (Ashcroft and Roper, 1993). It is estimated that approximately 300 pore-forming ion channel subunits are encoded in the human genome, giving rise to many more functional channels by combination with various auxiliary subunits. Potassium channels, for example, represent the largest and most diverse subgroup of ion channels and play a central role in regulating the membrane potential of cells (Kaczorowski and Garcia, 1999; Coghlan et al., 2001). Consequently, potassium channel inhibitors and openers offer significant therapeutic opportunities in cardiac, smooth muscle, neuronal, immune, and secretory systems (Liu et al., 1998). Where there has been an existing starting point for medicinal chemistry, ion channels have proved to be a highly tractable class of drug targets for the pharmaceutical industry (Treherne, 2002). Nevertheless, ion channels remain a relatively underexploited class of protein targets, mainly because of the absence of adequate screening technologies that enable pharmaceutical and biotechnology companies to evaluate focused libraries of compounds against potential ion channels of interest and then support medicinal chemistry to optimize the active compounds. Consequently, the progress of ion channel drug discovery is currently being constrained by the ability to identify lead compounds that can provide tractable starting points for medicinal chemistry. Recent advances in laboratory automation have brought significant opportunities to increase screening throughput for ion channel assays, but careful assay configuration to model drug–target interactions in a physiologically relevant manner is an essential consideration when selecting an assay for screening. Spectrums of ion channel screening platforms are described in this chapter, and some of these new technologies are transforming ion channel screening. A particular emphasis is placed on the mechanistic basis of drug interaction with the channel. For example, screening techniques should be able to detect compounds that interact with the channel by a preferred mode of action, so that novel “state-dependent” blockers, for example, can be discovered. This requires the assay system to generate relevant data from the compounds screened at the earliest stage of the drug discovery process. Ion channels should be fully integrated throughout the drug discovery process and not just introduced at the end of discovery to work out the mechanism of action of the drug and to identify unexpected toxicological implications. Advances in laboratory automation, instrumentation, and assay miniaturization have brought significant increases in the potential for screening throughput. For ion channel assays, however, careful assay configuration is more important than is generally required for, say, enzyme assays. A number of ion channel screening platforms are described in this review to provide some insights into the variety of formats available for high-throughput screening (HTS), together with some of their inherent advantages and limitations. Particular emphasis is placed on the mechanistic

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basis of drug–target interaction in the design of HTS approaches and how this relates to ion channel function. For example, an important aspect of the development of ion channels as drug targets is their ability to exist in multiple conformational states (e.g., open, closed, and inactivated) and show “state-dependent” drug sensitivity (Liu et al., 1998). This property of “state-dependent” blockade can be exploited therapeutically to discover drugs that selectively target the forms of the channel present under “pathological” conditions, while sparing normal channel functioning. The benefits of being able to select “state-dependent” ion channel modulators from screens can be of key importance in the discovery of novel medicinal chemistry and in avoiding potential toxicity complications. In this chapter, the focus is to update the reader on specific screening technologies and also attempt to illustrate how the technologies can be used to discover ion channel drugs.

17.2 ION CHANNEL HTS TECHNOLOGIES High-throughput, low false-positive hit rate, no false negatives, reliability, reproducibility, detection of fast channel activation and inactivation, good correlation with electrophysiology, low cost, and amenability to miniaturization are all characteristics that would make the ideal ion channel assay platform. Although there have been some very exciting developments over the last decade, still no single high-throughput ion channel assay platform is available that meets all these rigorous criteria. Current ion channel assay platforms can be broadly separated into several categories: ligand binding, ionic flux, fluorescence, luminescence, single-cell electrophysiology, and intact tissue electrophysiology. These different areas will be reviewed in the next sections. 17.2.1

Ligand Binding Assays

Many ligands have been identified that bind to ion channels or channel accessory proteins to activate or modulate their function. Typically, the ligand is radiolabeled and compounds are identified that competitively displaced or allosterically effected ligand binding to the ion channel. This approach usually identifies only compounds that behave in a mechanistically similar way to the labeled ligand. However, such techniques have been successfully used to discover calcium channel blockers and NMDA receptor antagonists. Ligand binding assays have also been used for discovering and validating some of the first-generation (e.g., tolbutamide, chlorpropamide, acetohexamide, and tolazamide) and second-generation (e.g., glibenclamide, glipizide, and glibornuride) sulphonylureas. Sulphonylureas stimulate insulin secretion by binding to sulphonylurea receptors leading to the closure of the ATP-sensitive potassium channel. Consequently, many sulphonylureas that were used in the treatment of maturity-onset diabetes were discovered by using binding assays. However, the ligand binding approach has limited success in discovering novel chemical series, as compounds that modulate ion channel activity independently to sulphonylurea binding go undetected. More recently, new improved classes of compound, unrelated

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to sulphonylureas, have been discovered using functional assays rather than the traditional ligand binding assays (Emilien et al., 1999). Ligand binding assays have also been used for identifying compounds with undesirable pharmacological properties. For example, compounds displaying toxicity toward the human ether-a-go-go-related gene (hERG) potassium channel may prolong the QT phase of a typical cardiac electrocardiogram (De Ponti et al., 2000). Long QT can result in the onset of ventricular arrhythmia and occasional sudden death. Dofetilide is a drug used to treat cardiac arrhythmias that functions by blocking hERG. Due to its hERG blocking capabilities, radiolabeled dofetilide can be used to identify other compounds that modulate hERG activity by performing radioligand binding assays, which has been patented. Radiolabeled dofetilide is incubated with whole cell or membrane preparations containing hERG, and compounds are then added and assessed for their ability to displace bound dofetilide (Diaz et al., 2004). However, compounds that modulate hERG activity via an alternative mechanism to dofetilide are not detected in this type of assay. This is a serious disadvantage considering the structural diversity of known compounds that modulate hERG activity (Vandenberg et al., 2001). Accordingly, functional electrophysiological assays for hERG safety pharmacology profiling are preferred when configuring a useful screen. Throughput of binding assays has been increased by moving away from filter binding assays toward homogenous assays that are more amenable to automation, for example, scillintation proximity assays (Zhang et al., 2006). The lower sensitivity of homogenous HTS assays has resulted in the development of radioligands with higher specific activity than tritiated radioligands, for example, [35S]MK-0499 for hERG binding (Raab et al., 2006). Fluorescence polarization (FP) is a technique based on the reduced tumbling rate of a fluorescent compound upon binding to the protein target, resulting in a polarized emission signal. FP binding assays have been adapted to GPCR HTS, providing a low cost, homogenous nonradiometric assay, especially in combination with “redshifted” fluorescent probes that reduce compound interference and “false-postive” rates (Allen et al., 2000). This technology is slowly being adapted to ion channel binding assay; for example, a hERG FP binding assay has been developed using a Cy3B labeled dofetilide derivative that shows a high correlation with data from [3H]-dofetilide binding (Deacon et al., 2007). However, further implementation of ion channel FP binding assays is limited by availability and development costs of highaffinity fluorescent probes. In summary, the application of ligand binding assays to ion channel pharmacology, although useful in certain specific examples, is relatively unattractive because of the limited information content and the inability to identify compounds that modulate ion channel activity independently by the mechanism of the labeled ligand. Further disadvantages include difficulties in modifying or synthesizing the ligand of interest and the influence of numerous parameters known to affect ligand binding. These include state-dependent ligand binding, voltage-dependent ligand binding and the binding of the ligand to multiple binding sites. In addition, some ion channels are not amenable to ligand binding assays, as there may be no known selective high-affinity ligands available. Applications outside of ion channel pharmacology and traditional screening include ion channel localization studies, isolation and identification of a

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ligand binding partner, and drug discovery platforms for the identification of compounds that may up- or downmodulate ion channel expression in the cell membrane (Ford et al., 2002). Nevertheless, where applicable, ligand binding assays are compatible with very high-throughput screening rates for ion channels (such as 100,000 data points in 24 h) at comparatively low cost per data point. 17.2.2

Ion Flux Assays

Ion channels are proteins that span the lipid bilayer of the cell membrane and provide an aqueous pathway through which specific ions such as sodium, potassium, calcium and chloride can pass (Choe and Robinson, 1998). The ability of channels to distinguish between different ionic species is a fundamental aspect of their function and is achieved by specific amino acid residues within the pore of the channel (Catterall, 1994; Sather et al., 1994; Bargmann, 1998). Flux assays exploit the fact that the pores of cation channels are permeable to other monovalent cations (Ford et al., 2002; Gill et al., 2003; Parihar et al., 2003), for example, the pore of a potassium channel is permeable to rubidium. By looking at the cellular efflux of potassium channel permeable ions, which are radioisotopic, one can monitor potassium channel activity. For potassium channels, radioactive rubidium 86Rbþ is the preferred isotope due to its high-energy emission characteristics that allows its quantification by Cerenkov counting without the requirement of liquid scintillation fluid addition. This radiometric flux assay principle has also been used to look at other ion channel types: 14C-guanidinium or 22Naþ for sodium channels, 45 Ca2þ for calcium channels, and 125I for chloride channels. Flux assays require a steady-state level of channel activity to carry significant ion flow to provide a robust assay. If the ion channel of interest is not active at resting membrane potential, the channel can be opened by various biochemical techniques. For example, calcium-activated channels can be activated by the addition of calcium ionophores, while voltage-gated channels can be activated using gating modifiers or elevated external Kþ concentrations (typically, 50 mM Kþ) to depolarize the cell. However, modifying the gating mode of the channel can have dramatic effects on pharmacology related to state-dependent interaction of the compound. For example, a high level of external Kþ reduces the affinity of dofetilde and cisapride binding to hERG, possibly by destabilizing C-type inactivation. Furthermore, the permeant tracer itself can affect channel function; for example, Rbþ reduces affinity of compounds for hERG by interacting with inactivation mechanisms (Rezazadeh et al., 2004). Safety and disposal concerns for high-energy radioisotopes are major limitations to configuring a screen for a large number of compounds. Atomic adsorption spectroscopy is an alternative nonradiometric assay system that is more amenable to automation and high-throughput screening. For example, radioactive 86 Rbþ can be substituted with nonradioactive 85 Rbþ and the amount of 85 Rbþ present in a given solution accurately quantified by atomic adsorption spectroscopy (Terstappen, 1999). The correlation between potency determinations between radiometric and these nonradiometric assays are virtually identical. Furthermore, atomic adsorption spectroscopy gives better sensitivity (increased signal-to-background ratio) compared

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to conventional radiometric detection methods. Although the majority of highthroughput nonradiometric ion flux screens have been developed for potassium channels using Rbþ efflux (Parihar et al., 2003; Wang et al., 2004; Gill et al., 2007), the technology is amenable to other cation channels such as sodium channels using lithium as the permeant tracer ion (see Figs. 17.1–17.3) and could be applied to calcium channels using strontium as the permeant ion. An indirect method of measuring Cl channel activity has been reported using measurement of Agþ in solution after precipitation of insoluble AgCl following Cl efflux (Gill et al., 2006). The throughput of nonradiometric flux assays is limited by the rate of measurement using atomic spectrometry. As current technology can measure only up to a dozen wells at once, miniaturization from 96 to 394-well format compromises assay quality without providing any advantage in throughput. A high-throughput reader for ion channel flux assays, ICR12000, has been designed by Aurora Biomed. The system comprises 12 atomic absorption spectrometers in parallel with a built-in plate stacker

FIGURE 17.1 Principles of Liþ influx assay for measuring Naþ channel activity using atomic emission spectrometry. (a) Cooperative effects of veratridine and scorpion venom on Nav1.5 channel kinetics measured using whole patch-clamp technique. Nav1.5 currents recorded in control conditions, 100 mM veratridine, 100 mM veratridine, and 5 mg mL1Leiurus quinquestriatus hebraeus (Lqh) scorpion venom. Whole-cell patch-clamp recordings were performed on a HEK293–Nav1.5 cell line using an EPC9 amplifier with pulse v8.5. Currents were evoked using 10 mV increments from 100 to 40 mV for 50 ms from a holding potential of 100 mV. External solution contained 150 mM NaCl, 10 mM KCl, 3 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and pH 7.4. Internal patch solution contained 160 mM KCl, 1 mM EGTA, 0.5 mM MgCl2, 10 mM HEPES, and pH 7.4. (b) Diagram illustrating basis of the Liþ influx assay. HEK293–Nav1.5 cells are incubated with or without compound for 10 min and then stimulated with 200 mM veratridine and 10 mg1 mL Lqh scorpion venom for 60 min. Incubation buffer contains 139 mM LiCl, 10 mM HEPES, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and pH 7.4. Following washout of Liþ and Naþ channel activators, the cells are permeabilized using 0.1% SDS and the cell contents measured using the atomic emission mode of a ThermoElemental Solaar M series spectrometer connected to Gilson 222XL autosampler.

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FIGURE 17.2 Development of an Nav1.5 Liþ influx assay using atomic absorption spectrometry. (a) Veratridine/scorpion venom-induced Liþ influx is absent in HEK wild-type cells in comparison to Nav1.5 stably expressed in HEK cells (mean þ/ SD, n ¼ 12). (b) Liþ counts for positive (30 mM prenylamine) and negative (0.1% DMSO) control wells for a 45-plate screening run. (c) Z0 values calculated for each screening plate. (d) Correlation between duplicate IC50 values for compounds identified as hits from the LOPAC library. S. Liness, A. Cook and E. Stevens unpublished data.

and liquid handling system and claims measurements of up to 60,000 data points per day. An alternative method to read high numbers of samples is to integrate conventional atomic absorption spectometers with a robot arm, plate hotel, and liquid handling systems, for example, BioFocus DPI’s reader system integrated by Process Analysis and Automation Ltd (see ion channel studies by atomic absorption application note, www.paa.co.uk). The reader system can be further simplified by measuring atomic emission rather than atomic absorption (which is a more sensitive technique for measuring alkali metals); this removes the need for hollow cathode lamps. Atomic emission spectrometers have the added advantage of multielement analysis allowing measurement of ion flux from multiple ion channel types in the same screening plate. 17.2.3

Fluorescent Dye Assays

For ion channels, fluorescent dyes function in one of the two ways. First, dyes may be directly affected by the ion of interest, for example, calcium, halide, and thallium dyes. Second, dyes may indirectly monitor ion channel activity. For example, dyes that are

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FIGURE 17.3 Comparison of concentration–response data for prenylamine, quinidine, and lidocaine using whole-cell patch-clamp and Liþ influx assay. (a–c) Concentration–response data for prenylamine, quinidine, and lidocaine using whole-cell patch-clamp recordings of HEK293–Nav1.5 cells (mean þ/ SEM, n ¼ 3). Currents were evoked using a 10 ms pulse to 40 mV from a holding potential of 100 mV at 1 Hz. (d–f) Concentration–response data for prenylamine, quinidine, and lidocaine using Liþ flux of HEK293–Nav1.5 cells with atomic emission spectrometry (mean þ/ SEM, n ¼ 8). A. Cook, and E. Stevens unpublished data.

sensitive to changes in the cell resting potential can be used to assay the function of channels that control the membrane potential of a cell. Measurement of intracellular calcium concentration has mainly been developed to support screening of GPCRs (Monteith and Bird, 2005) but is amenable to Ca2þ channel and TRP channel screening (Witte et al., 2002). Single-wavelength dye measuring fluorescence intensity has been developed, for example, Fluo-4 (Invitrogen) or no-wash kits (comprising calcium dye with fluorescence quenchers) that increase throughput, for example, FLIPR Ca2þ assay kits (Molecular Devices). Single-wavelength dyes suffer from uneven dye loading, photobleaching, and dye leaking, which can be minimized using dualwavelength ratiometric dyes such as Fura-2 (Monteith and Bird, 2005). However, the majority of HTS labs use single-wavelength dye no-wash kits for simplicity and to increase throughput (Comely, 2006). Chloride channel assays have been configured by measuring halide quenching of quinolinium compounds (e.g., 6-methoxy-Nethylquinolinium iodide, MEQ), and pyrido[2,1-h]-pteridin-related compounds or the more successful (but patented) halide-sensing green fluorescent proteins (Galietta et al., 2001). On the contrary, Kþ channel assays have been developed by measuring thallium influx using the thallium-sensitive fluorescent dye benzothiazole coumarin

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acetoxymethyl ester (BTC-AM). This technology has been developed further by Molecular Devices to generate a no-wash dye kit using a novel dye ThalKal. Dyes that are sensitive to changes in the cell resting potential can be used to assay the function of channels that control the membrane potential of a cell. However, these conventional single-dye systems are susceptible to variation in dye loading, cell density, and cell volume. The use of a second dye during the assay allows ratiometric analysis and reduces artefacts, such as dye loading and cell density variation. Fluorescent cell-based assays that monitor ion channel activity often rely on membrane potential-sensitive dyes such as dis-(1, 2-dibutylbarbituric acid) trimethine oxonol (Di-BAC4(3)). Membrane potential-sensitive dyes are charged molecules that migrate across a lipid bilayer in a manner depending on the cell membrane potential. For example, a stable cell line expressing a voltage-gated potassium channel will be hyperpolarized and the negatively charged bis-oxonol dye DiBAC4(3) will be unable to migrate across the cell membrane into the cell. Blocking the voltage-gated potassium channel will set the resting membrane potential to a less hyperpolarized state, allowing DiBAC4(3) to migrate into the cell. Fluorescence produced from dye present in the cell can then be quantified by bottom plate reading on a conventional fluorescent plate reader in the presence of a cell impermeable dye quencher. Open potassium channels give low fluorescent counts, and blocked potassium channels give high fluorescent counts. Improved homogenous (by removing the wash steps) membrane-potential-sensitive dye systems developed by Molecular Devices using proprietary fluorescence masking or quenching compounds (Baxter et al., 2002) are faster, responding in less than 2 min, and are much less sensitive to temperature changes than DiBAC4(3). Fluorescence resonance energy transfer (FRET)-based dyes (commercially available from Invitrogen) are also used for looking at changes in membrane potential (Gonzalez et al., 2005). Briefly, an external voltage-insensitive donor dye (which is a coumarinlinked phospholipid) is FRET coupled to fast voltage-sensitive acceptor dye, such as DiSBAC2(3). When cells are in a hyperpolarized state, DiSBAC2(3) migrates out of the cell and FRET couples with the coumarin-linked phospholipid. For example, blocking potassium channels that control resting membrane potential causes the cells to become less hyperpolarized. DiSBAC2(3) migrates back into the cell, so that FRET (between donor and acceptor dyes) is unable to occur across the cell membrane. These FRETcoupled dye systems offer subsecond responses, ratiometric analysis, and reduced background fluorescence (since measurements are confined to the cell membrane). New brighter FRET donor dyes have been developed that unlike coumarin dyes are pH insensitive and have been used for screening ASIC channels (Maher and Wu, 2007). Modified cell-based fluorescent plate readers such as Molecular Device’s fluorometric imaging plate reader, FLIPRTETRA, Hamamatsu’s FDSS 6000 and PerkinElmer’s CellLux have been specifically developed for ion channel drug discovery including dual excitation for ratiometric measurements. These systems use a chargecoupled device (CCD) imaging system for simultaneous optical measurement on all wells of a 384- or 96-well microtiter plates and a simultaneous dispensing device for compound addition. High-energy dye excitation is from a water-cooled argon ion laser or xenon lamp. The FDSS 6000 system has been used for multiplexed assays where

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both membrane potential and Ca2þ mobilization are monitored in parallel, which could generate information-rich data for Ca2þ channel and TRP screening. Newer generation plate readers have now introduced built-in field stimulation capabilities for activating ion channels, which avoids having to simulate the channels using biochemical techniques, such as increasing extracellular potassium (Bugianesi et al., 2006; Huang et al., 2006). For example, FRET dye measurements of compounds acting at Nav1.3 using the modified VIPR system with integrated field stimulation, e-VIPR (Vertex Pharmaceuticals) shows a closer correlation with patch-clamp data than a VIPR FRET assay configured using sodium channel openers. In particular, FRET dye measurements of Nav1.3 using e-VIPR have been tested at different electrical stimulation frequencies and can identify use-dependent blockers such as lidocaine (Huang et al., 2006). 17.2.4

Flash Luminescence

Following the development of suitable readers and integrated HTS platforms, flash luminescence has been adopted for measuring Ca2þ mobilization assays for screening GPCRs (Comely, 2006). However, the technology can be extended to voltage-gated Ca2þ channels and ligand-gated channels (Dupriez et al., 2002; Di, 2007). The technique uses a bioluminescent Ca2þ-sensitive photoprotein such as aequorin as a reporter protein that is cotransfected with the target ion channel into a host cell line. Apoaequorin is the apoenzyme that is converted to the active enzyme aequorin in presence of its prosthetic group coelenterazine. Following Ca2þ binding, aeqourin oxidizes coelenterazine to coelenteramide, which results in emission of light measured as luminescence (Dupriez et al., 2002). The technique offers certain advantages over fluorescent assays such as lower cost, higher assay performance (low background and high signal-to-background ratio), requirement for lower cell numbers, and application to cells in suspension. For most ion channel assays, a cytoplasmic phosphoprotein is sufficient for detecting Ca2þ flux; however, rapidly inactivating Ca2þ channels or transient ligand-gated responses have Ca2þ flux kinetics that are too rapid to be detected by available instrumentation. Therefore, the commercially available photoprotein MitoPhotina (which has 70% similarity to aequorin) can be selectively targeted to mitochondria, which delays the detection of calcium waves by the photoprotein and slows the recording kinetics (Bovolenta et al., 2002). The MitoPhotina technology has been applied to P2X1 screening assays (Di, 2007). 17.2.5

Ion Channel Protein–Protein Interactions

Another approach to ion channel screening is to target compounds at sites of protein– protein interaction between or within subunits (Arkin and Wells, 2004), for example, reducing N-type inactivation by inhibiting docking of the inactivation ball or disturbing the interaction between a and b subunits. Wyeth has applied this approach to interaction of Kv4.2 with accessory subunit KChIP and interaction of Cav2.2 with Cavb3 subunits using a high-throughput yeast two-hybrid screen (Young et al., 1998; Bowlby et al., 2005). For example, the Cav2.2 screen used the Cavb3 coding sequence

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fused to the Gal4 DNA binding domain, while Cav2.2 intracellular loop domain I–II was fused to the Gal4 activation domain. Compound inhibition of binding of Cav2.2 to Cavb3 prevents reconstitution of Gal4 protein activity, preventing reporter gene expression (Young et al., 1998). A fluorescent protein–protein interaction screening assay has been developed by Leptus for Kv1.1-Kvb1 by immobilizing biotin-tagged Kvb1 core domains to streptavidin-coated plates and applying the T1 interaction domain of Kv1.1 labeled with Cy3 dye to the plates (Stafford et al., 2005). This technology has been extended to a screen for compounds that inhibit interaction of Cavb3 with Cav2.2 channels (Kanumilli et al., 2005). The emergence of label-free sensors, for example, Biacore (GE Healthcare) using surface plasmon resonance has provided another technique for monitoring affinity of protein–protein interactions and has been used to validate compounds identified from a yeast two-hybrid screen of compounds against Kv4.2–KChIP interactions (Bowlby et al., 2005). 17.2.6

Automated Single-Cell Electrophysiology

With the exception of FRET technology, the temporal response for the majority of the HTS assays is greater than a second. In contrast with these biochemical assays, electrophysiology is considered the “gold standard” for measuring modulatory effects of drugs on ion channels. Voltage-clamp recordings are direct measurements of channel activity and have a rapid temporal response (in milliseconds). Furthermore, voltage-clamp recordings are independent of the cell resting membrane potential, do not require modified external potassium concentrations to alter membrane potential and have exceptional signal-to-noise characteristics. Therefore, when using electrophysiology to study the action of compounds on ion channels, analysis can be performed under physiologically relevant timescales, voltages, and ionic conditions. Although the quality and reproducibility of electrophysiological data is substantially higher than data derived from biochemical assays, throughput by conventional electrophysiology is markedly lower. Therefore, considerable attention has been directed toward developing higher throughput electrophysiological assays and integrating these techniques earlier into the drug discovery process. However, the throughput of the systems that are currently available does not yet match the throughput of the biochemical assays that previously been described. One electrophysiological approach to increasing throughput has been to use two-electrode voltage clamp of the Xenopus oocyte heterologous expression system (Shih et al., 1998; Shieh et al., 2003). Some commercially available systems allow cDNA or cRNA injection and voltage-clamp recording from Xenopus oocytes in a 96-well plate, as well as a measuring head containing glass electrodes, reference electrodes, and perfusion ports. Some systems record from eight oocytes in parallel and have the potential of generating hundreds of data points per day and are suitable for higher throughput assays than are possible with conventional electrophysiology using mammalian cells. Such systems can be useful for quickly evaluating the function of a novel gene, but expression of mammalian ion channels in an amphibian expression system can often result in an abnormal pharmacology. Consequently, these systems are not often used for intensive screening or supporting medicinal chemistry.

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The electrophysiological technique used most commonly for more rigorous drug discovery is the whole-cell patch-clamp technique of mammalian cell lines transiently or stably expressing recombinant ion channels (Hamill et al., 2005). Under voltage-clamp conditions, measuring currents associated with the flow of charged ions monitors channel activity. The voltage-clamp technique is rapid and precise, allowing channel activity to be recorded over a millisecond timescale at different voltages. During the discovery process, electrophysiology has traditionally been used to validate HTS data and generate accurate IC50 or EC50 values later in lead optimization. Also, electrophysiology has been essential for determining mechanism of action of channel blockers by examining voltage dependence, state dependence, and frequency dependence. However, recently electrophysiology has been considered a potential platform for HTS by integrating it earlier into the drug discovery process where it can be used to explore hits discovered by other screening methods. Several automated electrophysiology systems have used a modification of conventional whole-cell patch-clamp technique where the entire patch-clamp system is miniaturized by removing the need for cell visualization, for example, Xention’s in-house “interface patching” (Treherne, 2002) and Wyeth’s RoboPatch (Vasilyev et al. 2006). Wyeth’s technology relies on blind patching of sedimented cells. In contrast, interface patching uses an inverted configuration where cells are loaded into a glass capillary and allowed to sediment to the air–solution interface, while a patch pipette is positioned beneath the capillary. A commercially available electrode-based automated electrophysiology system is Flyion’s “flip-the-tip” technology in which cells are placed within the electrode and subsequently flushed toward the tip where they form gigaseals (Lepple-Wienhues et al., 2003). The automated screening platform Flyion 8500 is capable of up to six recordings at once and has been adapted to recordings from intracellular organelles with diameters less than 1 mm, such as mitchondria. In contrast to conventional patch-clamp electrophysiology; however, these devices are higher throughput and largely controlled through software that controls the entire clamping process and allows the application of compounds and the analysis of electrophysiological data obtained from such drug applications. Chip-based planar patch-clamp systems dispense with the need for borosilicate patch pipettes (Bruggemann et al., 2003; Kutchinsky et al., 2003; Wang and Li, 2003; Wood et al., 2004) and instead use a substrate that has both optimal electrical insulation and membrane sealing behavior with machined apertures into which individual cells are positioned. Membrane currents are recorded across the aperture containing the cell using a current-to-voltage converter. Systems have been developed based on a planar patch-clamp system using, silicon, quartz, glass, polymer, or polydimethylsiloxilane (PDMS) chips (Sigworth and Klemic, 2005). There are currently a variety of commercially available systems for planar patch clamping. For example, Molecular Devices’ IonWorks currently provides the highest throughput using a 384-well recording chip (Schroeder et al., 2003). This level of throughput is achieved by compromising the quality and continuity of the patch-clamp recording. The system uses a perforated patch-clamp configuration with a low seal resistance (typically 50–100 MW) and no

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compensation for capacitance artefacts or series resistance compensation. During compound addition, the recording head is removed to allow pipettor access; therefore, voltage clamp is interrupted during recordings that results in cell depolarization and change in channel state, The system is designed for single-compound applications, therefore, data are analyzed in a similar way to conventional high-throughput screening using positive/negative controls and Z0 values to determine assay performance statistically. IonWorks HT compensates for recording failure rate by applying one compound to four recording sites; however, the system has recently been modified by using a “population patch-clamp” technique (IonWorks Quattro), with 64 apertures in each recording site resulting in a current recorded from multiple cells. This has a dramatic effect on success rate as the technique accommodates a low percentage of apertures unoccupied by cells. Therefore, only one recording site is required for each compound resulting in a fourfold greater throughput than IonWorks HT (Finkel et al., 2006). As each recording is constructing from multiple cells, stable cell lines with a high percentage of nonexpressors can be successfully screened. In addition, multiplexing with a mixture of different ion channel cell lines on the same recording site is possible (Dale et al., 2007). Planar patch-clamp systems based on high-quality recordings using both “gigaseal” formation and whole-cell recording configuration are available, for example, Sophion’s QPatch, Molecular Device’s PatchXpress, and Nanion’s Patchliner. These systems use uninterrupted recordings and are configured for sequential additions of compounds with intervening washout periods or addition of cumulative concentrations of compounds to single cells. PatchXpress uses Aviva Biosciences’ Sealpatch planar patch-clamp technology that consists of 50–100 mL wells with 1–2 mm diameter planar patch-clamp holes using PatchXpress wash probes (consisting of combined inlet and outlet channels) to washout compounds., where 90% solution replacement takes several hundred milliseconds (Xu et al., 2003; Tao et al., 2004). The newer generation of planar patch-clamp plates such as Sophion’s QPlate and Nanion’s NPC-chips uses integrated microfluidics that provide faster rates of solution exchange (<100 ms), making the systems more suitable for screening fast ligand-gated channels (Farre et al., 2007). The data quality of the gigaseal systems can be exploited by using sophisticated voltage-pulse regimes to test multiple parameters for each compound on each recording site (e.g., testing both use dependence and state dependence). Throughput of these systems is significantly lower than that of IonWorks (e.g., 8, 16, and 48 recordings in parallel using Patchliner, QPatch16/PatchXpress, and QPatch HT, respectively). However, strategies have been employed to increase throughput, for example, Cytocentrics’ Cytopatch, which is scalable from 1 to 20 recording sites (but is not yet commercially available) uses an asynchronous recording mode allowing maximum usage of available recording sites by replacing failed recordings with new chips without interrupting neighboring recordings (Stett et al., 2003). Sophion have tackled throughput constraints by integrating a cell stirrer on the QPatch16 which allows screening to continue for up to 4 h without interruption, allowing screening to continue unattended. In addition, the QPatch16 has four pipettors controlled by scheduling software that maximizes the use of available whole-cell recordings (Kutchinsky et al., 2003). Recently, Sophion released the QPatch HT that records

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FIGURE 17.4 QPatch HT automated electrophysiology platform. (a) QPatch HT system with two integrated plate stackers, liquid handling system using 8 pipettes, 48 individual patchclamp amplifiers and touch screen monitor for basic assay configuration. (b) Close-up of the workstation. The on-board cell hotel ensures that cells remain healthy and in suspension. A QPlate 48 is loaded onto the recording site and covered with a mini-Faraday cage to screen electrical noise and seal suction lines. Three compound plates can be accessed by the liquid handling system. Compounds are applied to QPlate to the microfluidic channels through apertures in the Faraday cage. (c) QPlate 48 containing 48 individual planar patch-clamp sites, each with an integrated microfluidic system.

from 48 channels in parallel, has eight pipettors, and provides both QPatch and compound plate stackers (Mathes, 2006) (Fig. 17.4). The present barrier to adoption of automated electrophysiology for highthroughput screening of large libraries is the high price of recording chip consumables. Groups in Yale and Berkley are currently developing planar patch-clamp chips including integrated microfluidics with reduced fabrication costs using PDMS, which is a low-cost material that is easily manipulated using air moulding (Ionescu-Zanetti et al., 2005; Li et al., 2006). However, this material did not give rise to a commercial recording chip during the collaboration between Axon Instruments and Yale in the late 1990s, and in addition the success rates of gigaseal formation on PDMS are low (Ionescu-Zanetti et al., 2005; Li et al., 2006). In general, there are a number of advantages of automated electrophysiology systems over equivalent biochemical assays. For example, they often generate more physiologically relevant data, allowing ion channels to be assayed in the physiological state most relevant to the disease or disorder of interest. In particular, these systems also allow the screening of any recombinant ion channel target that can be stably expressed in a suitable cell line.

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Automated Tissue-Based Electrophysiology

There are, however, a number of disadvantages with the use of automated single-cell electrophysiology for high-throughput screening. For example, the systems described above still rely on using recombinant ion channels expressed in mammalian cell lines, and these channels do not always have the same pharmacology as the native channel. Furthermore, the cells are usually in suspension in automated systems and may respond differently to adherent cells in tissue culture that can produce processes and electrophysiological currents closer to those recorded in native systems. Therefore, it is important to check the pharmacology of any novel chemical “hits” discovered using automated patch clamp as soon as possible on native ion channels in tissues or cells. In any case, this cross-checking should be carried out before committing significant medicinal chemistry resource to the optimization of “hits” discovered by screening against recombinant ion channels. Several commercial systems such as Scientifica Ltd’s Slicemaster and Campden’s Synchroslice have been developed for this purpose. Scientifica’s Slicemaster, developed in collaboration with Merck (see Fig. 17.5), is a semiautomated tissue-based

FIGURE 17.5 Slicemaster tissue-based electrophysiology platform. (a) Scientifica Slicemaster semiautomated recording system that allows one trained operator to run up to 16 concurrent assays simultaneously and independently (only one-half of modular system shown). (b) Online data acquisition and analysis software controlling assay configuration. (c) Integrated brain slice chamber, integrating the tissue recording chamber, control electronics, flow control, and peltier heat control unit within a self-contained modular unit.

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recording system that allows a single trained operator to run up to 16 concurrent independent tissue-based assays at one time, affording the potential of screening up to 50 novel compounds per day. Integrated brain slice chambers (IBSC) underpin the Slicemaster system coupled to a remote user console and integrated acquisition and analysis software, increasing the user’s data-handling productivity to keep pace with the increase in data generation capacity (Stopps et al., 2004).

17.3 USING HTS TECHNOLOGIES IN ION CHANNEL DRUG DISCOVERY Most existing ion channel drugs are derived from chemical series initially discovered by serendipity or using HTS assays that were not specifically configured to detect ion channel modulators. In the future, the screening technologies described above can now be used to accelerate the discovery of novel small-molecule drugs that modify ion channel function. This objective can be achieved by screening a range of carefully selected targets and using validated assays and by applying medicinal chemistry expertise to these targets to identify lead drug candidates. Many of these new drugs will have been optimized from novel “chemical hits” discovered using ion channel HTS assays, so that these hit compounds can open up whole new avenues of research for medicinal chemistry to exploit. As long as medicinal chemistry can develop adequately potent and selective molecules for the primary ion channel target over other ion channels, the pipeline of ion channel drugs entering the clinic is likely to increase (Minkel, 2003). A key objective of any ion channel drug discovery program, then, is to integrate the ion channel screening technologies that produce the most relevant data earlier into the drug discovery process so that medicinal chemists no longer have to rely just on serendipity to initiate the optimization of novel hit compounds. The success of such an approach relies heavily on the quality of the ion channel libraries being screened and the skill of medicinal chemists in optimizing the hits discovered from those libraries. 17.3.1

Chemical Libraries for Ion Channel Screening

Assays are only as good as the compounds that are screened and ion channel targeted libraries can be assembled that use training software to build in diversity space while maintaining certain ion channel modulating parameters. Further, chemical diversity can then be built into a core library through ongoing screening activities through the use of the appropriate software tools. Libraries for screening against ion channels can be compiled from “virtual libraries” on the basis of key physicochemical principles and computational approaches to ensure the maximum occupation of the relevant chemical diversity space (as determined by suitable molecular modeling approaches). As knowledge of the pharmacology within this screening library develops through the integration of key structure–activity relationship (SAR) data, it becomes possible to apply further computational approaches to identify areas of the library for development. A variety

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of computational approaches can allow the virtual screening of large library sets based on 3D structure, and pharmacophore mapping and quantitative structure– activity relationship (QSAR) methodology can then be integrated into the initial computational capability. Depending on the range of ion channels being targeted, the size of the resulting library would usually range from 10,000–100,000 individual compounds. The screening of high-quality ion channel libraries will usually result in finding novel hit compounds, but it is highly unusual to discover a potential drug from such a primary screen. Consequently, medicinal chemistry is required to develop “hit-tolead” programs for the individual chemical series identified in the screening library. A good example of the optimization of several chemical series targeting voltage-gated sodium channels has been described in a review by Anger et al. (2001) and for potassium channels in a more general review by Coghlan et al. (2001). A more specific example of hit-to-lead optimization for a particular potassium channel is described in a further review by Peukert et al. (2003), which describes the optimization of novel blockers of the voltage-gated potassium channel Kv1.5. This potassium channel underlies the IKUR current in the human atrium and is an attractive target for the treatment of atrial fibrillation. The reviews all provide good examples that demonstrate how relatively weak and nonselective hits can be optimized into development candidates for further progression toward the clinic. 17.3.2

Hit Detection, Hit Validation, and Lead Optimization

Screening of compound libraries constituting hundreds of thousands of compounds is limited to ligand binding assays, ion flux assays, or fluorometric assays due to cost and throughput constraints (see Sections 17.2.1–17.2.3). However, such screening assays generate hit data with limited mechanistic information (e.g., state-dependence or voltage-dependence). Therefore, these primary screening assays are ideal for generating libraries of hits suitable for further screening by an automated electrophysiology assay. However, the pharmacological relevance of the generated hits may be compromised by the nature of the high-throughput assay; for example, blockers of particular channel state may not be detected by the assay. Smaller ion channelfocused compound libraries, consisting of thousands of compounds, lend themselves to screening directly using automated electrophysiology, especially IonWorks Quattro, without an initial high-throughput screening phase. The success of such a screening strategy relies on the combination of novel, high-quality ion channel relevant compounds and a high-quality screening assay. High-throughput technologies such as fluorometric membrane potential dye assays generate useful hit compounds as a basis for drug discovery programs; however, their poor pharmacological correlation with manual patch-clamp assays have limited their usefulness as platforms for supporting medicinal chemistry efforts. Automated electrophysiology systems have provided ideal platforms for determining accurate potencies against the primary target and selectivity over other channels, and for supporting establishment of structure–activity relationships (SAR). Indeed, the high-quality voltage clamp necessary for testing important parameters such as state-dependence and

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use-dependence achieved using gigaseal-based automated electrophysiology technologies, such as PatchXpress, QPatch 16, or higher throughput QPatch 48, provides the best available ion channel platforms for supporting hit-to-lead development. 17.3.3

Safety Pharmacology

Inhibition of the myocardial hERG potassium channel can cause a prolonged QT interval in humans, the possibility of ventricular arrhythmia, and, in some cases, sudden death. Due to these potentially serious side effects, the early testing of compounds for hERG inhibition is now a standard component of cardiac safety profiling in drug discovery. According to ICH guideline S7B, in vitro electrophysiological testing of compounds against hERG should be used as part of an integrated risk assessment of QT prolongation. This recommendation was introduced after the FDA withdrew terfenadine, astemizole, terodiline, grepafloxacin, cisapride, prenylamine, sertindole, and droperidole from the U.S. market following reports of ventricular arrhythmia associated with these drugs.

FIGURE 17.6 Biophysical characterization of hERG currents from a CHO-hERG stable cell line using QPatch 16. (a) Family of hERG currents evoked by 2 s depolarizing pulses between 60 and 50 mV in 10 mV increments, from a holding potential of 80 mV, followed by a 2 s tail current pulse to 50 mV. (b) Current–voltage relationship demonstrating inward rectification, determined from peak activating currents evoked by depolarizing pulses (n ¼ 6). (c) Conductance–voltage relationship determined from peak tail currents. V1/2 ¼ 1.0 0.2, k ¼ 7.1 0.2 (n ¼ 6). The extracellular solution contained 145 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, and pH 7.2, while the intracellular solution contained 120 mM KCl, 5.4 mM CaCl2, 1.75 mM MgCl2, 10 HEPES, 10 EGTA, 4 mM Na2-ATP, and pH 7.2. E. Stevens unpublished data.

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FIGURE 17.7 hERG pharmacology measured using QPatch 16. (a and b) Reproducible hERG IC50s generated for both hydrophilic and hydrophobic compounds using QPatch 16. Four-point concentration–response curves for the (a) hydrophobic pimozide and (b) hydrophilic verapamil were determined from by QPatch 16 recordings from a CHO-hERG stable cell line. Currents were evoked using a 20 ms prepulse to 50 mV (to determine linear leak) preceding a 2 s depolarizing pulse to þ20 mV, followed by a 2 s tail current pulse to 50 mV, from a holding potential of 80 mV. The pulse regime is applied at a frequency of 0.067 Hz. For pimozide mean IC50 ¼ 1.98 nM, range ¼ 1.44–3.02 nM (n ¼ 5), while for verapamil mean IC50 ¼ 217, range ¼ 188–247 nM (n ¼ 5). (c) Correlation between hERG patch-clamp data generated using QPatch 16 (n ¼ 3–7) and manual patch-clamp data. Bold line is y ¼ x, while dashed lines show half-log deviations from y ¼ x. Manual patch-clamp data was sourced from Guo and Guthrie, 2005. E. Stevens unpublished data.

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Rbþ flux, dofetilide binding, and fluorescent membrane potential dye assays have all been used as hERG safety assays. However, the assays show limited predictive capability due to poor correlation with manual patch-clamp data. In contrast, automated electrophysiology platforms, such as IonWorks, PatchXpress, and QPatch, provide more predictive hERG screening assays. In particular, Sophion’s QPatch technology is an ideal hERG screening platform with the advantages of glass-coated microfluidics that prevent false negatives with “sticky” hydrophobic compounds and 100% series resistance compensation which minimizes the chance of false-positive effects due to an increase in series resistance over the course of the experiment (Figs. 17.6 and 17.7). Multichannel systems have developed a multielectrode array (MEA)-based predictive hERG screening assay platform. The QT screen is an assay based upon extracellular recording of the composite cardiac action potentials of spontaneously beating cardiomyocytes, cultured directly onto specifically designed 96-well plates comprising integrated gold electrodes (both recording and reference). This system claims recording of up to 6000 data points per day at a relatively low cost per data point compared to patch-clamp platforms (Meyer et al., 2004). Results obtained from the platform are in agreement with those obtained by classic repolarization and patchclamp assays.

17.4 CONCLUSIONS AND FUTURE PROSPECTS In summary, integrated ion channel drug discovery is an area of research with great therapeutic relevance to a number of important diseases. Historically, discovery of novel inhibitors and modulators of ion channels has been hampered by the lack of functionally relevant high-throughput screens. Consequently, a number of assay platforms have been developed to allow the discovery of novel drugs acting on ion channels. Although biochemical assays have sufficient throughput to support drug discovery they have a number of limitations compared to biophysical techniques. Although conventional electrophysiology is an excellent technique, it is relatively slow in comparison to biochemical assays and is too slow to be effective in the discovery of novel chemistry. Automated ion channel screening systems capable of generating large volumes of functional electrophysiological data from mammalian cells is likely to be the most promising way forward. Exploiting these technologies, where they are effectively integrated into drug discovery programmes, will allow the exploration and development of novel chemistry in this exciting and growing area of medical research.

ACKNOWLEDGMENTS We would like to thank BioFocus-DPI for giving permission to include atomic emission data.

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INDEX

Acceptor sites, 235, 236 Acetic acid-induced urinary bladder pain, 82 Adenine nucleotide transporter (ANT), 159 Adenosine diphosphate (ADP), 156 Adrenal gland, 325 AHP, 197, 198 amplitude, 198 A-kinase anchoring proteins (AKAPs), 283 Aldo-keto reductase enzymes, 133 Alzheimer’s disease, 15, 160 Amino acid residues, 136 Amino acid sequences, 129, 157 Amino-terminal cytoplasmic domain, 345 AMP, 429 dependent protein kinase, 281 Amphibian neurons, 402 Amphipathic helices, 163 Amyloid precursor protein (APP), 15 Angiotensin stimulation, 305 Anomalous rectifier, 262 Anoxia-sensitive neurons, 254 ANT, 175 Antiapoptotic proteins, 166 BCL-2, 166

BCL-xL, 166 Anticonvulsant compounds, 375 zonisamide, 375 Antiepileptic compounds, 209 Antiepileptic drug, see Ethosuximide Antiepileptic drugs, 44, 397 Antihyperalgesic effects, 43 Antisense oligonucleotides, 43 AP firing, 292 Apoptosis, 160 Apoptosis channel, 161 Apoptotic pathways, 258 Apoptotic stimuli, 182 growth factor deprivation, 182 Arginine residues, 6 Arterial smooth muscle cells, 259 Aspartic acid, 332 ATP, 155, 179, 183, 258, 267 binding motif, 204 injection, 181 production, 170 regulated Kþ channels, 265 Automated electrophysiology systems, 454

Structure, Function, and Modulation of Neuronal Voltage-Gated Ion Channels, Edited by Valentin K. Gribkoff and Leonard K. Kaczmarek Copyright Ó 2009 John Wiley & Sons, Inc.

467

468

INDEX

Automated single-cell electrophysiology, 453 Automated tissue-based electrophysiology, 457 Auxiliary calcium channel subunits, 223 Axon Instruments, 455 B-adrenergic receptors, 283 B-cell lymphomas, 161 B interaction domain (BID), 7 BAD, 170 pathological roles, 170 physiological roles, 170 Basolateral amygdala neurons, 130 Bax channels, 167 BCL-2 family proteins, 156, 176, 162, 183, 268 BCL-xL injection, 182 BCL-xL interaction, 168 BCL-xL protein, 181 Bepridil, 206, 207 BFNC, 399 Binaural pathway, 198 Binding partners, 162 Biochemical measurements, 303 Bithionol, 207, 208 BK blockers, 435 BK channel(s), 260, 317, 427, 430, 432, 435 BK channel blockers, 436 BK channel inhibitors, 108, 109, 110 BK channel protein, 118 BK channel regulation, 423 BKCa channels, 200, 202, 203 Blood-brain barrier, 45 BMS-204352, 405–406 Bongkrekic acid, 175 Bradykinin-induced pain, 44 Bristol-Myers Squibb (BMS) compounds, 410 BTX binding, 376 C-terminal fragment, 9 C-terminus isoforms, 230 Ca2þ-activated Kþ currents/channel, 197, 200 Calcium-activated potassium, 423 channels, 220

Calcium-dependent inactivation, 9 Calcium-selective channel, 171 Calcium-selective current, 173 Calcium channel antagonists, 3 nifedipine, 3 Calcium channel-associated transcription regulator (CCAT), 9, 19 Calcium channel binding, 7 Calcium channels, 4, 15 discovery, 4 history, 4 involvement of, 15 Calcium homeostasis, 171 Calcium phosphate complex, 171 Calcium signaling, 21 Canine pulmonary arteries, 260 Capsaicin assay, 45 Cardiac long-QT syndrome, 395 Cardiac muscle disorders, 20 Cardiovascular disorders, 6 Carrageenan-induced inflammation, 82 cDNA library, 199, 395 Cell adhesion molecule, 76 Cell membrane, 67 depolarization of, 67 Cell-specific factors, 223 Cell surface expression levels, 138 Cellular processes, 155 Cellular proteins, 161 Central nervous system (CNS), 8, 39, 196, 251, 395 therapeutics, 426 tumors, 106 Cerebellar basket cell synaptic terminals, 143 Cerebellar granule cells, 132 CFA injection, 83 CGMP-dependent protein kinase, 324 Charge carriers, 196 Chemically induced dimerization (CID), 297 Chemotherapeutic agents, 116 Childhood absence epilepsy (CAE), 50 Chinese hamster ovary (CHO) cells, 201, 206, 295 Chronic cardiopulmonary diseases, 256 Chronic constriction injury (CCI), 78 Chronic hypoxia, 256 Chronic nerve injury model, 378 Chronic pain, 366

INDEX

Chronic pain syndromes, 72 ClC channels, 112 CNS-penetrant compounds, 47 CNS cell differentiation, 262 CNS diseases, 22 CNS distribution, 41 Cognition-enhancing compounds, 407 Conductive ion channel, 163 Congenital stationary night blindness (CSNB2), 21 Constitutive exons, 221 Crayfish neuromuscular junction, 178 CREB phosphorylation, 20 Cryo-electron microscopy, 140 Current-clamped DRG neuron, 90 Cysteine string protein (CSP), 226 Cytoplasmic hydrophobic domains, 319 Cytoplasmic proteins, 140 Cytosolic A-kinase-anchoring protein, 325 Cytosolic channel partners, 68 anchoring of, 68 Cytosolic proteins, 282 DAP amplitudes, 198 Dehydroabietic acid, 424 Dentate gyrus granule cells, 333 Deoxycarnitine ester prodrug, 428 Diabetic neurons, 84 Diabetic neuropathy, 47, 84 Dihydropyridine-sensitive calcium channels, 18 Dinucleotide splicing recognition sequences, 233 Disulfide bond, 7 DNA binding protein, 346 DNA databases, 199 DNA fragmentation, 430 DNA plasmid, 432 DNP, 176 Dopamine receptor-induced inhibition, 304 Dorsal root ganglion (DRG), 38, 68, 69, 304, 367 Double-label immunofluorescense studies, 398 Downstream enzymes, 155 DRG neurons, 42, 45, 46, 69, 70, 71, 73, 74, 75, 76, 78, 80, 81, 82, 85, 88, 89, 90, 375, 376, 398, 408

469

DRG/spinal nerves, 367 Drosophila discs, 143 Drosophila embryonic neurons, 258 Drosophila melanogaster, 133, 344 Drug binding site, 6, 236 Drug interaction, 7 Drug targets, 443 EAG cells, 110 EAG channels, 111 Ectodomain-directed antibodies, 141 EF-hand proteins, 358 Electrical excitability, 127 Electron micrographs, 180 Electron microscopy (EM), 349 Electron transport inhibitor, 176 Electrophysiological defects, 21 EM structure, 349 Endogenous death channels, 165 Endogenous promoter, 9 Endoplasmic retention signal (CVLF), 331 Endoplasmic reticulum (ER) membrane, 220, 328 Energy-dependent processes, 174 Epilepsy, 333 Epilepsy/bipolar disorder, 430 Epileptogenic activity, 17 Episodic ataxia, 144 Epithelial Naþ channels, 258 Erectile dysfunction (ED), 430 ERR motifs, 136, 141 ERR receptor protein, 136 EST database, 225 Estrogen response elements (ERE), 328 sites, 329 Ethosuximide, 44 Excitation-contraction coupling, 7 organization of, 7 Exogenous factors, 73 Experimental autoimmune encephalomyelitis (EAE), 71 Expression systems, 201, 202, 259 Extracellular linker, 231 Flash Luminescence, 452 Fluorescence polarization (FP) technique, 446 Fluorescence resonance energytransfer (FRET)-based dyes, 451

470

INDEX

Fluorescent dye assays, 449 Food and Drug Administration (FDA), 40, 374, 375 Fox binding sequence, 236 FRET technologies, 377 G-proteins, 402 G-protein activation, 229 G-protein-coupled receptor (GPCR), 291, 396 G-protein-coupled receptors, 396 G-protein-mediated inhibition, 230 G-protein mechanisms, 301 GABA transaminase, 51 Gene transcription, 35 Gene transcription activity, 9 Genetically epilepsy-prone rats (GEPR), 52 Genome sequences, 226 GFP, 297 GK domain, 7 Glioma cells, 105, 107, 111, 116 cell lines, 114 cell proliferation, 108 Glucose tolerance, 21 Glutamate-threonine (ET), 232 Glutamic acid residue, 6 Glycolytic pathway, 159 Golgi apparatus, 331 GPCR activation, 401 GYG pore sequence, 200 HEK cells, 46, 206, 208, 424 HERG gene, 283 Heterologous expression systems, 136, 137, 140 Heterologous systems, 325 Heteromeric channel complexes, 133 Hexokinase sites, 160 High-affinity subtype-selective T-type channel blockers, 44 High-dose groups, 432 High-frequency discharge, 199, 209 High-frequency signals, 230 High-throughput screening (HTS), 444 High voltage activated (HVA) Cav channels, 4, 35, 36 biophysical properties, 36 Higher order sensory neurons, 78 Hippocampal neurons, 254 Homozygous lethality, 399

HTS approaches, 445 HUGO Gene Nomenclature Committee (HGNC), 395 Human embryonic kidney (HEK), 424 cell line, 206, 257 Human ether-a-go-go-related (HERG), 282, 283, 446, 460–462 Human genome sequence, 224 Human nervous system, 233 HVA channels, 4, 35, 45 HVA, see High voltage activated Hypoxia-inducible factor (HIF) transcription factor, 255 Hydrophilic pathway, 12 Hydrophilic pores, 279 Hydrophilic segments, 157 Hydrophobic residues, 351 Hyperexcitable mutant gene, 344 Hypoglossal motoneurons, 254 Hypoxia-induced cellular pathways, 268 Idiopathic generalized epilepsies (IGEs), 47 ICA-27243, 410, 412 IK channels, 261 Immunocytochemical analysis, 397 Immunohistochemical analysis, 132 In situ hybridization, 42, 51, 79, 132, 200 In vivo phosphorylation sites, 139 Inflammation-induced pain, 80 symptoms, 80 Inflammatory hyperalgesia, 87 Inflammatory mediators, 83 Inflammatory models, 380 SNL, 380 Inflammatory pain, 36, 38, 39, 82, 377, 409 Influx pathway, 174 Inherited disorders, 20 Inherited erythromelalgia, 89 Inhibitory components, 403 Injury-mediated effects, 73 Inner membrane channel, 181 Inner membrane physiology, 170 Insulin secretion, 21 Integral membrane proteins, 7 Intermediate-conductance channels, 202, 261 Intermembrane space proteins, 161 cytochrome, 161 Intracellular calcium homeostasis, 177 Intracellular messengers, 280

INDEX

Intracellular organelle membranes, 162 Ion channels, 105, 127, 155, 171, 196, 252, 264, 279, 283, 284, 291, 443, 447 activity, 162 auxiliary subunits, 140 drugs, 443 expression, 105 HTS technologies, 445 modulation, 286 modulators, 445 proteins, 447 role of, 105, 252 significance of, 286 Ion conducting pathway, 158 Ion flux assays, 447 Ionic conductance pathway, 133 Ionic exchangers, 251 Isoform-selective drug screening, 237 Isoform-specific antibodies, 74, 235 Isoform-specific interactions, 231 KATP channel, 204, 263, 264 KNa channels, 194, 195, 196, 197, 199, 200, 203, 204, 205, 209, 210 pharmacology of, 205 KNa currents, 195 KNa inhibition, 206 KNa modulators, 208 Konig’s polyanion, 158 KCNQ channel(s), 281, 293, 395 Kþ channel, 47, 105, 107, 108, 193, 196–200, 204, 206, 259–268, 281, 282, 286, 393, 395, 411 involvement of, 108 modulation of, 292–295, 302, 304, 344 types, 291, 292 Kþ channel-associated protein (KChAP), 281 Kþ channel interacting proteins (KChIPs), 281, 344–358, 453 Kþ channel modulators, 206 Kv channels, 345 Kv1 channels, 129, 132, 143 activation of, 132 Kv4 channels, 344–358, 453 Kv7 channels, 206, 292–307, 393–412 L-type antagonists, 21 molecular properties of, 129

471

L-type calcium channels, 7, 10, 12, 18, 19, 22, 220 activity, 8 antagonists, 16 physiological and pharmacological expression, 8 L-type channel genes, 20 diltiazem, 21 nifedipine, 21 L-type VGCC inhibitors, 117 Lidocaine, 374 Lidocaine drug, 366 Ligand binding assays, 445, 446 Ligand-gated ion channels, 195, 280 Ligand-receptor interactions, 396 Lipid membranes, 165 Long-QT syndrome (LQTS), 283 Long-term depression (LTD), 14 Long-term potentiation (LTP), 14, 170 Loss-of-function mutations, see Night blindness Low-frequency impulses, 365 Low-voltage activated (LVA) channels, 116 LTP, see Long-term potentiation LVA channels, 46, 116 LVA, see Low voltage activated Lysine residues, 6 M-channels, 291, 295 activity, 292 subunits, 299 M-current suppression, 307 MAC activity, 165 Madin-Darby canine kidney (MDCK), 330 Magnetic resonance imaging, 411 Malignant transformation, 107 Mammalian genes, 282 Mammalian genomes, 223 Mammalian neurons, 254 Mammalian smooth muscles, 423 MCC, 174, 176 Medial nucleus of the trapezoid body (MNTB), 197, 199 neurons, 209 Membrane-delimited preparations, 260 Membrane-spanning d component, 7 Membrane-spanning domains, 357 Membrane-targeted peptides, 297 Membrane depolarizations, 255

472

INDEX

Membrane proteins, 252 Metabolites, 157 ADP, 157 ATP, 157 Metabolite fluxes, 171 Missense mutations, 21, 50 Mitochondrial ATP, 172 regulation of, 172 Mitochondrial ATP release, 178 Mitochondrial bioenergetics, 178 Mitochondrial conductance, 180 Mitochondrial depolarization, 170 Mitochondrial dysfunction, 267 Mitochondrial ion channel, 177, 180 Mitochondrial membrane, 155, 163, 180, 182 activity, 181 component, 165 inner membrane, 155 outer membrane, 155 potential, 174 Mitochondrial metabolism, 160 Mitochondrial permeability transition pore (mPTP), 173, 174 Mitochondrial respiration, 176 Mitochondrial signal, 177 Mitochondrial targeting proteins, 178 Mitogen-activated protein kinase (MAPK) pathway, 14 Mitogenic signals, 110 Modular adapter proteins, 13 Modulatory proteins, 284 Molecular machine, 220 Motogenic signals, 110 MPTP components, 181 ANT, 181 BCL-2 family, 181 BCL-xL, 181 VDAC, 181 MRNA polyadenylation signals, 320 MRNA processing, 320 Multicellular organisms, 169 Muscarinic depression, 297 Naþ channel-blocking drugs, 366 Naþ channel blockers, 257, 376, 378 Naþ channel density, 258 Naþ channel expression, 116 Naþ current, 256 Naþ dependent DAPs, 209

N-ethylmaleimide (NEM), 403 N-linked glycosy lation (NLG), 138 site, 134 N-terminal domain, 346 N-terminal hydrophobic peptide, 349, 352 N-terminal inactivation peptide, 352 N-terminal residue, 139 N-terminal sequence, 204 N-terminus complex, 349 N-type channels, 13, 41, 42, 53, 300, 302 role of, 41 N-type immunoreactivity, 38 Neonatal neurons, 258 Nerve growth factor (NGF), 116 Nerve transection, 72 Nervous system, 9, 219, 222 Neurodegenerative diseases, 283 Neurodegenerative disorders, 209, 268 stroke, 209, 268 trauma, 268 Neuroendocrine cells, 8 Neurological deficits, 22 Neurological disorder, 144 epilepsy, 144 Neuromodulatio, 286 Neuronal disturbances, 3 Neuronal calcium sensor (NCS), 344 Neuronal disorders, 20 Neuronal excitability, 208 regulation of, 208 Neuronal L-type voltage-gated calcium channel (NLTCC), 4 Neuronal pathway, 14 Neuronal sodium channels, 69 Nav 1.6-1.9, 69 Neuropathic pain, 41, 68, 73, 237, 376, 407, 408 Neuropathic pain model, 46 Neuropathy-induced thermal hyperalgesia, 44, 46 Neuropathy models, 44 Neurotransmitter-containing vesicles, 182 Neurotransmitter systems, 20 Neurotrophic factors, 73 Nicotinamide adenine dinucleotide phosphate, 140 Nicotinic acetylcholine receptors (nAChRs), 18 Night blindness, 8

INDEX

NLTCCs, 13, 15, 17, 18, 19 antagonists of, 15, 18 isoforms of, 13 role of, 13 NMDA receptors, 38, 220, 267, 281 Nonradiometric flux assays, 447, 448 Nonneuronal cells, 132 NP encoding exon, 231 Obsessive-compulsive disorder, 45 Oculomotor nuclei, 201 Oligosaccharide-processing enzymes, 138 Optical techniques, 174 Oscillatory network, 48 Oxidative metabolism, 178 Oxidative phosphorylation, 155, 156 Oxidizing agent DTNB, 46 Oxidoreductase active site, 140 Oxidoreductase-like proteins, 140 Oxygen deprivation, 251 Paralogous genes, 130 Paroxysmal movement disorder, 331, 333 Paw-withdrawal latencies (PWLs), 46 Paw-withdrawal test, 44 PCREB signaling, 9 PDZ domain, 204 PDZ-domain sequence, 200 Peptide toxins, 206 Peripheral nervous systems, 8 Peripheral axons, 76 regenerative response, 76 Peripheral injury, 87 Peripheral nervous system, 3, 38, 407 Peripheral neuropathies, 88 Peripheral tissues, 261 Permeability transition pore, 174 PIP2, 296–299, 302, 306–307, 396, 401–402 Pharmacological agents, 205, 376 PKA activation, 139 PKA phosphorylation, 324 PKC blockers, 256 Planar patch-clamp systems, 455 Plasma membrane, 67 Plasma membrane channels, 180 Plasma membrane exchangers, 252 Plasma membrane subdomains, 143 PLC activation, 295 PNS tissues, 69

Polar drugs, 12 Polarized epithelial cells, 142 Polyclonal antibodies, 51 Pore-forming components, 396 Pore-forming HVA, 36 Pore-forming subunits, 177 Pore-localized ERR signal, 136 Postmitotic cells, 107 Postoperative pain model, 45 Postsynaptic cell, 181 Postsynaptic laminins, 232 Posttranslational modifications, 68, 138, 280, 320, 324 Potassium (Kþ) channels, 430, 443 inhibitors, 444 Pre-mRNA splicing, 219 Preganglionic nerve terminals, 40 Preoptic nucleus, 200 Presynaptic calcium channels, 232 Presynaptic nerve terminals, 233 Presynaptic proteins, 233 Proapoptotic mitochondrial factors, 183 Proapoptotic molecules, 163, 182, 183 Bax, 182 DNBCL-xL, 182 Progenitor cells, 107 Programmed cell death, 155, 160, 167 Proliferating tumors, 106 astrocytomas, 106 glioblastomas, 106 Proline-rich tyrosine kinase, 324 Promoter-regulatory motifs, 326 Protein-conducting channels, 174 Protein-mediated inhibition, 229 Protein-protein interactions, 128, 142 Protein binding domain, 221 Protein expression, 9 Protein interaction, 393 Protein kinases, 138, 200, 281, 303 Protein phosphorylation, 393 Protein translocators, 177 Proteomic analysis, 268 Proximal dendrites, 9 P-type channel, 236 inactivation, 232 PTX-treated SCG neurons, 302 Pulmonary arteries, 264 Purkinje cells, 232

473

474

INDEX

Purkinje neurons, 332 Pyramidal neurons, 255 Quinidine, 206, 207 RCK domains, 204 Reactive oxygen species (ROS), 175 Receptor-mediated inhibition, 402 Receptor-mediated mechanisms, 394 Receptor site organization, 4 Recombinant BCL-xL protein, 168 Regulatory proteins, 222 Repressor proteins, 220 Retinal glial cells, 106 retigabine, 404, 406–412 RIM binding protein, 226 RNA binding proteins, 220, 221, 236 RNA expression, 51 RNA processing, 219 RT-PCR analysis/techniques, 234, 398 Ryanodine receptor, 173 Scaffolding proteins, 283 Sciatic nerve, 78 ligation, 374 neuromas, 77 Second-order neurons, 37 Semichronic pain syndromes, 407 Sensory responses, 408 Serine phosphorylation sites, 139 Serotonin-modulated glutamate, 130 Signaling complexes, 286 Signaling pathways, 36, 162, 292, 299 Single-cell analysis, 222 Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), 222 Single-channel conductance, 202 Single nucleotide polymorphisms (SNPs), 50 Sinusoidal currents, 197 Site-directed mutagenesis, 157 Six transmembrane segments, 68 Size exclusion chromatography, 168 SK channels, 260 Skeletal muscles, 259 Slack channels, 202, 203 Slo1, 194, 196, 203, 206, 318–334, 393 Slo2, 193, 200, 203, 319–320 Slack currents, 208

Slo3, 320 Slick-transfected CHO cells, 202 Slo interating protein 1 (SLIP1), 281 Slo binding protein (Slob), 281–282, 284–285 Small-and intermediate-conductance KCa channels, 196 Small-molecule blockers, 366, 382 Small interfering RNA (siRNA), 41 Smooth muscles, 259 Sodium-activated potassium current, 194 Sodium channel blockers, 87, 91 Sodium channels, 67, 68, 72, 76, 91 dysregulation of, 68, 78, 84, 87 expression of, 68, 69, 78, 91 isoforms, 69, 91 transcript, 84 Sodium ions, 67 Somatosensory pathway, 42 Spared nerve injury (SNI), 72 Spinal cord injury (SCI), 86, 87 Spinal cord neurons, 258 Spinal nerve ligation (SNL), 72 Spliced exons, 220 Splicing enhancers, 220 Splicing factors, 236 Starvation-induced death, 284 State-dependent actions, 12 Superior cervical ganglion (SCG), 292 neurons, 401 Supramolecular protein complexes, 128 Susac’s syndrome, 19 SWD discharges, 49 Swept field confocal (SFC) system, 305 Sympathetic nerve dysfunction, 41 Sympathetic neurons, 304, 306 Synaptic depression, 182, 282 Synaptic machinery, 226 Synaptic transmission, 38, 178 T-type calcium channels, 37, 42, 44, 45, 46, 47, 48, 50, 51, 52 blockers, 42, 44, 47 currents, 49 inhibitor, 117 involvement of, 117 isoform, 48 expression of, 42 role, 42, 51

INDEX

Tail-flick test, 44 TC neuron, 43 Tetramerization domain, 345 Thalamic reticular neurons, 48 Thalamocortical (TC) neurons, 43 Therapeutic agents, 206 Therapeutic index, 237 Thermal hyperalgesia, 45 Time-dependent analgesia, 46 Time-dependent hyperalgesia, 46 Trafficking signal, 135 Trans-membrane domains (TMD), 300 Transient subconductance levels, 195 Transmembrane domains, 325 Transmembrane reporter proteins, 142 Transmembrane voltage, 400 Tricyclic antidepressants, 381 TTX-resistant currents, 74, 79 Turtle neurons, 258 Two-hybrid screens, 281 Tyrosine kinases, 307 Tyrosine phosphatase inhibitors, 139 Tyrosine phosphorylation, 139 Untranslated region (UTR), 320 Variance-mean analysis, 195 Ventilation/perfusion ratios, 252 Ventral posterolateral (VPL), 87 thalamocortical neurons, 43 Ventricular myoctyes, 257 Vesicle pools, 179, 182 Vestibular nuclei, 201 VGCC expression, 117 VGCC inhibitors, 116, 117 VGCCs inhibition, 117 VIPR technologies, 377 Visceral organs, 83 inflammation of, 83 Voltage-activated calcium channels, 427 Voltage-clamp techniques, 403

475

Voltage-dependent anion channel (VDAC) 156, 158, 264 channel, 160, 167 dimerization, 167 genes, 169 inhibitor, 164 interacting proteins, 167 oligomerization, 167 protein, 157 Voltage-dependent channels, 264 Voltage-dependent currents, 114 Voltage-dependent potassium (Kv) channels, 127, 343 Voltage-dependent sodium (Naþ) channels, 365 Voltage-gated calcium (Ca2þ) channel, 5, 6 schematic representation, 5 Voltage-gated calcium channels, 4, 9, 20, 223, 231, 237 HVA, 4 LVA, 4 Voltage-gated ion channels, 4, 155, 157, 193, 195, 291, 412 Voltage-gated sodium currents, 70 Voltage-independent inhibition, 230 Voltage-independent pathways, 229 Voltage-sensing domains, 345 Voltage-sensitive calcium channels, 255 Voltage sensor region, 345 Water-filled channels, 177 Western blot analysis, 200 Whole-cell currents, 113, 202 Whole-cell levels, 303 Whole-cell voltage-clamp, 400 Willing/reluctant model, 300 Women’s Angiographic Vitamin and Estrogen (WAVE) 333 Xenopus oocytes, 202, 203, 208 Zinc pirithione, 405