Current Topics in Membranes and Transport VOLUME 37
Channels and Noise in Epithelial Tissues
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Current Topics in Membranes and Transport Edited by Felix Bronner Department of BioStructure and Function The Universio of Connecticut Health Center School of Dental Medicine Farmington, Connecticut
VOLUME 37
Channels and Noise in Epithelial Tissues Guest Editors Sandy I. Helman
Willy Van Driessche
Department of Physiology and Biophysics Universiw of Illinois at UrbanaChampaign Urbana, Illinois
Luboratorium voor Fysiologie Katholieke Universiteit Leuven Campus Gusthuisberg B-3000 Leuven, Belgium
ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers San Diego New York Boston London Sydney Tokyo Toronto
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Copyright 0 1990 hy Academic Press, Inc. All Rights Reserved. No part 0 1 this publication may he reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the puhlisher.
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1
Contributors, ix Foreword, xi Preface, xiii Yale Membrane Transport Processes Volumes, xv
PART I.
THEORETICAL PERSPECTIVES
Chapter 1. Electrical Noise in Physics and Biology H. M. FISHMAN AND H . R . LEUCHTAG
I. Early Observations and Description of Noisc-Producing Phenomena, 4 11. Characterization of Stochastic Processes, 4 111. Application of Fourier Analysis to Noise Problems, 9 IV. Physical Noise in Conduction, 14
V. Noise Measurements and Analysis Techniques, 19 VI. Ion Conductance Fluctuations in Biomembranes, 29 Referenccs. 33
Chapter 2. Analysis of TransepithelialNoise Signals from Ion Channels: Advantages and Limitationsof the Method WILLY VAN DRIESSCHE A N D NOEL VAN DEYNSE
I. Introduction, 37 11. Low-Noise Instrumentation, 38 111. Spontaneous Noise Components, 41
IV. V. VI. VII.
Blocker-Induced Noise, 44 Limitations of the Method, 5 I Problems Related to the Analysis of Blocker-Induced Noise, 55 Conclusion, 57 References, 58
V
vi
CONTENTS
Chapter 3. Ion Channel Fluctuations: “Noise” and Single-Channel Measurements DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
I. Introduction, 61 11. Power Spectral (“Noise”) Analysis, 62 111. Single-Channel Analysis, 77 1V. Comparison of Single-Channel Measurcments with Fluctuation Measurements: A Specific Experimental Example, 92 V. Summary, 112 References, 1 12
PART 11. NOISE ANALYSIS OF EPITHELIAL CHANNELS Chapter 4. Apical Sodium Ion Channels of Tight Epithelia as Viewed from the Perspective of Noise Analysis SANDY I. HELMAN AND NEIL L. KlZER
I. Introduction, 117 11. Theoretical Perspectives, 118 111. IV. V. VI.
VII. VIII.
IX. X. XI.
XII.
Absence of Spontaneous Noise, 120 Blocker-lnduccd Noise: A Simple Three-State Model, 122 Single-Channel Currents and Channel Densities, I26 Noise Analysis with Electroneutral and Charged Sodium Ion Channel Blockers, 130 Choice of Blocker: Criteria, 132 Results from Noise Analysis with 6-Chloro-3,5-diaminopyrazine-2-carboxamide (CDPC) and CGS 4270, 134 Results from Amiloride-Induced Noise Analysis, 136 Amiloride-Sensitive Macroscopic Currents, 142 Results from Triamtcrene-Induced Noise Analysis: A Double Blocker Problem, 144 Dependence of Blockcr and Spontaneous Rate Coeflicients on Apical Sodium Ion Concentration. 149 Refcrcnces, 152
Chapter 5. Noise from Apical Potassium Ion Channels WOLFGANG ZEISKE
I. About the Beginning of Apical Potassium Ion Channel Noise Analysis: A Historical Account, 158 11. Methods for Evaluation of Apical Potassium Ion Channels, 160 I l l . Potassium Ion Channels with Two Putative Roles: Helping Potassium Ions or Other Ions on Their Way across the Epithclium, 162
vii
CONTENTS
IV. Lorentzian Noise from Transepithelial Current: A Fingerprint of “Spontaneous” Apical Potassium Ion Channel Fluctuations, 163 V. Blockers of Apical Potassium Ion Channels, 170 VI. About the Concept of Potassium Ion Channel Selectivity, 177 VII. Microscopic Channel Parameters, 180 VI11. Potassium Ion Channel Chemistry, 181 IX. Influencing Apical Potassium Ion Permeability, 182 X. Family of “Potassium Ion-Specific” and Other Cation Channels, 185 XI. Summary, 186 References, 187
Chapter 6. Basolateral Potassium Channel Noise: Signals from the Dark Side DAVID C. DAWSON, DANIEL J. WILKINSON, AND NEIL W. RICHARDS
I. Basolateral Membrane: Dark Side of the Epithelial Cell, 191 11. Permeabilized Cell Layers: Techniques and Limitations, 194
111. Basolateral Membrane Noise, 201 IV. Is the Noise Worth Listening tom?, 209 References, 209
PART 111. SINGLE-CHANNEL EVENTS IN EPITHELIAL TISSUE Chapter 7. Patch Clamp of Cation Channels SIMON A . LEWIS AND PAUL J. DONALDSON
I. Introduction, 215 11. Technical Aspects of Patch Clamping Epithelial Cells, 216 111. Search for Cation Channels in Epithelia, 233
IV. Use and Physiological Rclevance of Single-Cation Channel Data, 237 V. Conclusions, 242 References. 242
Chapter 8. Chloride Channels in Epithelial Cells RAYMOND A. FRIZZELL AND DAN R. HALM
1. Chloride Channels in Absorptive Epithelia, 248 11. Chloride Channels in Secretory Epithelial Cells, 255
Ill. Volume-Sensitive Chloride Channels, 269 IV. Comparisons among Anion Channels, 271 References 276 Note Added in Proof, 282
viii
CONTENTS
Chapter 9. Reconstitution of Epithelial Ion Channels ROBERT J. BRIDGES AND DALE J. BENOS
I. Introduction, 283 11. Isolation and Purification of Specific Membrane Vesicle Populations, 285 111. Bilayer Techniques Used in Reconstitution, 288
IV. Ion Channel Incorporation into Planar Lipid Bilayers, 291 V. Reconstitution of Ion Channels from Epithelia into Planar
Phospholipid Bilayers, 297 VI. Conclusions, 306
References. 307
Index, 313
Contributors
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
Dale J. Benos (283), Department of Physiology and Biophysics, The University of Alabama at Birmingham, Birmingham, Alabama 35294 Robert J. Bridges (283), Department of Physiology and Biophysics, The University of Alabama at Birmingham, Birmingham, Alabama 35294 David C. Dawson (191), Department of Physiology, The University of Michigan Medical School, Ann Arbor, Michigan 48109 Paul J. Donaldson (215), Department of Physiology and Biophysics, The University of Texas Medical Branch at Galveston, Galveston, Texas 77550 Douglas C. Eaton (61), Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322
H. M. Fishman (3), Department of Physiology and Biophysics, The University of Texas Medical Branch at Galveston, Galveston, Texas 77550 Raymond A. Frizzell (247), Department of Physiology and Biophysics, The University of Alabama at Birmingham, Birmingham, Alabama 35294 Dan R. Halm (247), Department of Physiology and Biophysics, The University of Alabama at Birmingham, Birmingham, Alabama 35294 Sandy I. Helman ( 1 17), Department of Physiology and Biophysics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Neil L. Kizer ( 1 17), Department of Physiology and Biophysics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 H. R. Leuchtag ( 3 ) , Department of Biology, Texas Southern University, Houston, Texas 77004 Simon A. Lewis (215), Department of Physiology and Biophysics, The University of Texas Medical Branch at Galveston, Galveston, Texas 77550 ix
X
CONTRIBUTORS
Yoshinori Marunaka (61), Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322 Neil W. Richards (191), Department of Physiology, The University of Michigan Medical School, Ann Arbor, Michigan 48109 Noel Van Deynse (37), Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, 8-3000Leuven, Belgium Willy Van Driessche (37), Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium Daniel J. Wilkinson (191), Department of Physiology, The University of Michigan Medical School, Ann Arbor, Michigan 48109 Wolfgang Zeiske ( 157), Institut fur Tierphysiologie und Angewandte Zoologie der Freien Universitat Berlin, D-1000 Berlin 41, Federal Republic of Germany
Foreword
This is the last volume of Current Topics in Membranes und Transport for which I have served as editor. In looking back to 1969 when I invited Arnost Kleinzeller to co-edit the series with me, 1 wonder if any of us could have foreseen the enormous leap forward made by biological research in the last two decades. Hans Krebs’ prophetic statement in his foreword to the first volume, “. . . membranes provide a framework for almost every functional activity of cells . . . ,” has come true to the point where there is hardly a field of cellular research that does not need to meet the challenge of membranes, their structure, identity, and specificity. Moreover, increasingly there is concern not only with the plasma membrane and its characteristics, but with the many intracellular membranes and their functional role. In 1970, in our preface to Volume I, we called attention to the need for “the elucidation of the underlying molecular mechanisms . . .” and the relative paucity of the knowledge of structure of transport molecules. Although knowledge of these large molecules is still far from complete, certain basic structural features are now established and their intricacies are becoming known. Just as in 1970 we were able to point to the three major steps of biological transport, i.e., “recognition, translocation, and release,” so today it seems that transport molecules like the ATPases do indeed recognize, translocate, and release their freight. We now know that these molecules contain cytosolic sensors that permit binding and phosphorylation and lead to conformational changes in the transmembrane elements so as to effect transcellular movement. Channels and receptors seem to have a comparable structure, except that sensors may be located on the outside of the plasma membrane. Cycles are common in nature and in human affairs. When the series was started, functional knowledge of transport was advanced, at least in kinetic terms, whereas molecular knowledge was more rudimentary. In the last twentyodd years, this situation has been reversed. We know many molecular details in terms of structure, but knowledge concerning precise function, although emerging, is not yet well advanced. It is perhaps appropriate, therefore, that my last volume as editor of this series deals with channels and noise, a method for studying ion translocation, therefore a functional analysis that complements and at the xi
xii
FOREWORD
same time challenges the molecular knowledge of channels. It does so because ultimately it will be necessary to understand how a given structure leads to the experimentally determined channel current and its density. In some ways the analysis of transepithelial noise signals from ion channels preceded the molecular analysis of channels, but the powerful tools that are permitting the structural dissection of ion channels have allowed structural understanding to catch up to functional analysis. It is likely that the next cycle of biological research will bring further functional understanding to our structural insights. It has been an intellectual feat to have been associated with so many outstanding colleagues and to have had some hand in shaping the knowledge of membranes and transport. 1 look with curiosity and anticipation to the future.
FELIXBRONNER Furmington, Connecticut
Preface
Revolutions in science are often brought about when new methods have been brought to bear on time-honored questions. Epithelial ion transport studies, pioneered by the work of Hans Ussing and colleagues, are now carried on by noise and patch clamp analyses that provide details unimaginable in the 1960s and 1970s. The decade of the 1980s has brought these methods into full bloom and made them into important tools for addressing the ways in which epithelial cells carry out and modulate ion transport. This volume is organized into three parts. Part I provides the theoretical basis and brings out the intimate relationship that exists between noise and singlechannel events, both in theory and in terms of practical implementation. Part I1 provides a discussion of epithelial Na' and K + channels that have been studied by noise analysis. Part 111 discusses single-channel events as revealed by patch clamp and reconstituted ion channel approaches. Excellent reviews of fundamental theory have appeared in the literature in recent years. To the uninitiated, however, these have often been difficult to understand and put in perspective. But even the serious student of patch clamp and noise analysis has had to deal with theoretical and mathematical concepts not normally encountered in the training of biological scientists. Although study of the chapters of this book cannot take the place of a thorough grounding in theory, we hope this volume will contribute to the necessary selfreeducation of researchers as well as alert them to the educational needs of a future generation. At the same time much of the material will be of interest not only to experts but to students of ionic channels in general. We express our sincere appreciation to the contributors and to all concerned with the preparation of this book.
S. I. HELMAN W. VAN DRIESSCHE
xiii
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Yale Membrane Transport Processes Volumes
Emile L. Boulpaep (ed.). (1980). “Cellular Mechanisms of Renal Tubular Ion Transport”: Volume 13 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. William H. Miller (ed.). (198 I). “Molecular Mechanisms of Photoreceptor Transduction”: Volume 15 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. Clifford L. Slayman (ed.). (1982). “Electrogenic Ion Pumps”: Volume 16 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Joseph F. Hoffman and Bliss Forbush ILI (eds.). (1983). “Structure, Mechanism, and Function of the Na/K Pump”: Volume 19 of Current Topics in Membranes und Trunsporr (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. James B. Wade and Simon A. Lewis (eds.). (1984). “Molecular Approaches to Epithelial Transport”: Volume 20 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Edward A. Adelberg and Carolyn W. Slayman (eds.). (1985). “Genes and Membranes: Transport Proteins and Receptors”: Volume 23 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, Orlando. Peter S . Aronson and Walter F. Boron (eds.). (1986). ‘“a+-H+ Exchange, Intracellular pH, and Cell Function”: Volume 26 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, Orlando. Gerhard Giebisch (ed.). (1987). “Potassium Transport: Physiology and Pathophysiology”: Volume 28 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, Orlando. William S . Agnew, Toni Claudio, and Frederick J. Sigworth (eds.). (1988). “Molecular Biology of Ionic Channels”: Volume 33 of Current Topics in Membranes and Transport (J. F. Hoffman and G. Giebisch, eds.). Academic Press, San Diego. xv
xvi
YALE MEMBRANE TRANSPORT PROCESSES VOLUMES
Stanley G. Schultz (ed.). (1989). “Cellular and Molecular Biology of Sodium Transport”: Volume 34 of Current Topics in Membranes and Transport (J. F. Hoffman and G. Giebisch, eds.). Academic Press, San Diego. Toni Claudio (ed.). (1990). “Protein-Membrane Interactions”: Volume 36 of Current Topics in Membranes and Transport ( J . F. Hoffman and G. Giebisch, eds.). Academic Press, San Diego,
Part I
Theoretical Perspectives
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 37
Chapter I
Electrical Noise in Physics and Biology H . M. FiSHMM Deparfmenfof Physiology and Biophysics The University of Texus Medical Branch at Gulvesron Galveston, Texas 77550
H . R . LEUCHTAG Department of Biology Texas Southern Universiiy Houston. TexuJ 77004 I , Early Observations and Description of Noise-Producing Phenomena Observations of Fluctuations 11. Characterization of Stochastic Processes A. Expectation Value, Moments, Variance, and Correlations B . Probabilities and Distributions C. Stationarity and Ergodicity D. Markov Processes 111. Application of Fourier Analysis to Noise Problems A. Fourier Series and Transforms B. Linear Driving-Point Analysis: Impedance C. Convolution D. Spectral Density and Autocorrelation Function IV. Physical Noise in Conduction A. Fluctuation-Dissipation Theorem B . Johnson-Nyquist Description C. Shot Noise D. Fractal Noise E. Diffusion Noise V. Noise Measurements and Analysis Techniques A. Signal-to-Noise Ratio B. Signal Amplitude Statistics C. Spectral Analysis V1. Ion Conductancc Fluctuations in Biomembranes A. Stationary Noise by Spectral Type B . Nonstationary Noise Analysis References
3 Copyright 0 1990 by Academic Press. Inc All rights of reproduction in any form reserved.
4
H. M. FISHMAN AND H. R. LEUCHTAG
1. EARLY OBSERVATIONS AND DESCRIPTION OF NOISE-PRODUCING PHENOMENA Observations of Fluctuations The history of' the science of spontaneous fluctuations-briefly, noise-may be taken to begin in 1827 with an observation by the English botanist Robert Brown. Pollen grains in aqueous suspension, and later mineral and smoke particles, played out a ceaseless dance under Brown's microscope. It was not until the development of the kinetic theory of gases in the latter part of the nineteenth century that the cause of Brownian movement was correctly determined to be thermal molecular motions in the liquid environment of the particles. However, problems in the quantitative description of the behavior of a Brownian particle remained until the 1905 analysis by Einstein. Many other instances of fluctuations and stochastic processes exist in physics, chemistry, and biology (Beck, 1976; Bennett, 1960; DeFelice, 1981). A sensitive galvanometer may be constructed by suspending a lightweight coil in a magnetic field by means of a fine quartz fiber on which a small mirror is cemented. Even with no current passing through the coil, the light beam reflected from the mirror will exhibit some broadening due to random bombardment by air molecules. In a circuit containing a potential barrier such as a rectifying junction in a transistor, the current is limited to those electrons that have sufficient thermal energy to surmount the barrier. As a result, the current fluctuates in a way that is determined by the thermal fluctuations in the position and energy distribution of the electrons, producing a typc of noise known as shot noise. These and similar problems are analyzed by the methods of statistical physics, some of which will be briefly described in this chapter. After an overview of the formalisms by which stochastic processes are characterized (Section II), we review the way Fourier analysis is applied to noise problems (Section 111). Section 1V describes the classical types of noise that are observed in electrical conduction. One of these types, fractal noise, involves nonequilibrium processes and is currently of great interest because it has been shown to encompass the more traditional concept of Ilfnoise. Section V describes the techniques employed in the measurement and analysis of noise. The chapter concludes with a discussion (Section VI) of the fluctuations in ion conductance observed in biological membranes.
II. CHARACTERIZATION OF STOCHASTIC PROCESSES This section is concerned with the formal framework within which stochastic processes are discussed (van Kampen, 1981). The definition of a stochastic vari-
5
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
able is quite different from that of a deterministic variable. A stochastic variable, X, is defined by specifying the set of possible values it can take (its range or state space), along with the probability distribution over this set. The set of states may be discrete or continuous or may be a mixture of the two.
A. Expectation Value, Moments, Variance, and Correlations For a continuous one-dimensional set, the probability distribution is given by the probability density function, P ( x ) , which is nonnegative
and normalized by the condition
where the integral extends over the entire range of the stochastic variable. The average or expectation vulue of any function f(X) defined on the state space of the variable is
In particular (X) =
j x P ( x ) dx
=
p,
(4)
p2
(5)
is the first moment, or mean, and ( X Z ) = Jf x2P(x) dr =
is the second moment, or mean square, of X. The mth moment p, is ( X m ) . The variance of a distribution is given by cr2 =
((X - (X))*) = p2 - p:
(6)
The variance is the square of the standard deviation, u,also known as the rootmean-square (rms) deviation.
6
H. M. FISHMANAND H. R. LEUCHTAG
A function of a stochastic variable is also a stochastic variable; it may be a function of an ordinary variable such as time as well. Such a function, representing a stochustic process, may be written
This function may be regarded as an ensemble of samplefunctions in which the stochastic variable X is replaced by one of its possible values x . As before, the average is obtained by integrating over the probability density of X,
Another average of particular interest is the autocorrdation function
The form on the second line, obtained algebraically from the definition on the first line, is more convenient for computation. For t , = f 2 the autocorrelation function reduces to the time-dependent variance, u2(t)= ( Y 2 ( t ) ) - (Y(r))2.
B. Probabilities and Distributions An alternative way to specify a stochastic process Y , proceeds by constructing a hierarchy of distribution functions. Let P I(y, t) represent the probability for Yx(t)to take the value y at time t. The joint probability that Y has the value yl at time t l and also the value yz at time r, is designated P2(yI,tl ;y,, f2). Proceeding in this way, an infinite hierarchy of probability densities P,,, with n = 1, 2 , . . . , is defined. From these, all averages of the stochastic process, such as ( Y ( l l ) Y ( t 2 .) . . Y(t")) =
I
y1y2, .
. y,,P,,(y1,t , ; y 2 , t,; . . . ; Y,, t,,) d y , , dyr . . . dyn (10)
can be defined. The P , obey the usual probability conditions: they are nonnegative and PI satisfies
7
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
For the joint probability densities the corresponding condition is
I
PAYl, t , ; .
.
. ;y,,-,, L l ; y n ,
f,,)
dy,, = P,,-l(Yl, t , ; .
..
; y " - I * t,$?l)
(12)
Furthermore, any P, is left unchanged by the interchange of any two of the pairs of variables in its argument. Specification of a hierarchy of probability densities obeying these consistency conditions is an alternative way to define a stochastic process. It is useful to define the conditional probabiliry; this function P,1,(y2, t21yl, t , ) is the probability density for Y to take the value y, at t2provided that its value at t , is yI.
C. Stationarity and Ergodicity If all the moments of a stochastic process are unaffected by an arbitrary shift in time, the process is called stationary. The mean of a stationary process is independent of time. This mean may be subtracted from Y ( t ) to yield a zeromean process. The autocorrelation function of a stationary process depends on the absolute time difference It, - tZI only and is not affected by the subtraction of the constant mean. For many processes a constant time difference, 7,, exists such that K ( t l , t,) is zero or negligible for It, - t21>7,. The constant 7cis called the autocorrelation time. It is a remarkable fact of nature that processes that vary with time in an extremely complicated way often lead to observable averages that obey simple laws: the rapid irregular bombardment of a piston by gas molecules, when integrated by the inertia of the piston, causes the piston to move smoothly in a motion determined by Boyle's law. Similarly, the instantaneous current fluctuations in a resistive circuit average out to Ohm's law. Physicists have developed special techniques for dealing with the way in which these macro o ic regularities arise from microscopic systems. A monatomic gas in a cyl' der is completely determined if the three position coordinates and the three omentum coordinates are specified for each of the N molecules at some instqht called the initial time. Any physical quantity such as the instantaneous force on the piston may be computed (at least in principle) from the microstate of the system, a specification of the 6N position and momentum coordinates of the system. The force may be represented by the function Y,(t), where x stands for the 6N-dimensional microstate. The basic technique of statistical mechanics is to replace the evolving system by an appropriate ensemble of systems, all obeying the same equations of motion but having different initial microstates x. The structure of this ensemble is then specified by a density function in the 6N-dimensional hyperspace (called phase
JP
8
H. M. FISHMAN AND H. R. LEUCHTAG
space) in which one microstate, x , is represented as one point. Replacing a single system with such an ensemble converts x into a stochastic variable X. From an appropriate probability distribution P, one can then compute averages, which are interpreted as macroscopic physical quantities. The ensemble therefore serves to visualize the probability distribution in phase space. In this way the force on the piston, for example, is identified with an ensemble average rather than a time average. This is a reasonable procedure if the function Y,(r) for each system of the ensemble will, in the course of a sufficiently long time, pass through essentially all values accessible to it. Justifying this so-called ergodic assumption is the fundamental problem of statistical mechanics. In practice it is not possible to solve the microscopic equations of motion, and an additional assumption is necessary. The “random phase approximation” consists in repeatedly averaging over the irrelevant rapidly varying variables so as to retain only the slowly varying ones in the differential equations describing the evolution of the system. This process yields macroscopic equations, such as rate equations for chemical reactions and the Nernst-Planck electrodiffusion equation. Because of the way they were derived, they are approximate, and the observed quantities will exhibit small deviations from them, the fluctuations or noise with which this chapter is concerned. Unable as we are to determine the solutions of the microscopic equations, we are also unable to solve exactly for the fluctuations. However, their stochastic properties can be determined by the same repeated randomness assumption that went into the macroscopic laws.
D. Markov Processes The subclass of stochastic processes that possess the Markov property play an important role in physics and chemistry. In a Markov process, the conditional probability density at a time, r, , is uniquely determined by the value y,- I at time t,-l and is unaffected by knowledge of the values at earlier times. Examples of Markov processes in physics are Brownian motion and the number of neutrons in a nuclear reactor. In chemistry, the dissociation of a gas of binary molecules and the components of a reacting mixture are Markovian. Formally, a Markov process is defined as a stochastic process with the property that, for any set of successive times,
From Pl(yl, rl) and the rrunsirion probability Plll(y2, tz(yI, t l ) , the entire hierarchy of probability densities can be constructed (van Kampen, 1981). An identity that must be obeyed by the transition probability of any Markov
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
9
process is the Chapman-Kolmogorov equation
That is, the transition from state 1 to state 3 may be determined by integrating over all possible paths from state 1 to state 3 via state 2. A necessary relation between PI and Plrl is
Any two nonnegative functions, P , and P,I I , that obey these two equations uniquely define a Markov process. Of special interest are stochastic processes that are both Markovian and stationary. When a closed, isolated system with a quantity describable as a Markov process is in equilibrium, it is a stationary Markov process. For example, when a resistor in contact with a bath at fixed temperature is in parallel with a capacitor, the voltage Y ( t ) is a stationary Markov process. Markov chains are stationary Markov processes in which the range of Y is a discrete set of states and the time variable is likewise discrete, taking only integer values. The analysis now becomes simpler, in that the transition probabilities for a finite Markov chain of N states can be described in terms of N X N matrices. [For a discussion of the validity of assuming that the kinetics of ion channel conductances are Markovian processes, see Fishman (1985).J
111. APPLICATION OF FOURIER ANALYSIS TO NOISE PROBLEMS In the analysis of stochastic processes, the method of expansion in a Fourier series is a valuable tool. It provides the advantage of shifting our attention from the complex random function Y ( t )to a series of simple sinusoidal functions, of which Y(t) is composed (waveform synthesis) or decomposed (spectral analysis). It is particularly useful in linear systems, in which the principle of superposition (see Section III,B,l) applies and each frequency component can be traced from input to output.
A. Fourier Series and Transforms We begin by expanding one sample function, Y,(t), of the stochastic process Y ( t ) in a fixed time interval 0 < t < T (van Kampen, 1981). Because the sample
10
H. M. FISHMAN AND H. R. LEUCHTAG
function is an ordinary function of waves (assuming that ( Y ( t ) ) = 0)
t,
it may be written as a Fourier series of sine
Here the nth Fourier coefficient is given by An, , = T I O ' Y X ( t sin(? )
1)
dt
The coefficients obey the Parseval identity
From these expressions the equations for the stochastic variable Y(r) may be determined by averaging over all possible values of n with probability density P x ( x ) . In this process the Fourier coefficients A,,,I become stochastic variables A, with average
The Parseval identity averages to
B. Linear Driving-Point Analysis: Impedance Processes that are adequately characterized by linear differential equations can be analyzed rigorously and thoroughly due to the property of superposition. Superposition is the additive property of perturbation-response pairs. The response to more than one perturbation applied simultaneously is equivalent to the sum of the individual responses to each perturbation applied separately. Most natural processes, including ion conduction in biological membranes, are nonlinear. But because of the completeness and conceptual simplicity of linear theory, nonlinear processes often are studied as linearized processes by restricting the perturbation to small amplitudes for which the process remains close to equilibrium or close to a stationary state. This approach is particularly
11
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
well developed and useful in chemical kinetics (Eigen and de Maeyer, 1963), wherein rate processes become first order (linear) near equilibrium, and in modeling ion channel kinetics in membranes (FitzHugh, 1965; Armstrong, 1969), wherein transitions between channel states are assumed to be Markovian. For a linearizable process, once the range of perturbation amplitude that yields a linear response has been determined, linear methods can be applied to obtain a linear description of the process at various equilibrium or stationary states (e.g., membrane potentials). There are many methods for linear analysis (Oliver and Cage, 1971). Here we focus on the concept of impedance because of its fundamental role in characterizing linear processes and its relationship to fluctuating equilibrium processes. Any linear differential equation of the form of Eq. (21), hlv
a,-
dmi
Cl"-lv
dm-li
+ a , __ + . . . + a,, = b,-dt + b, + ..dtn dt"-' dr-'
+
bm
(21)
where i ( t ) is a current-driving function of time and v(t) is the voltage response function, and a and b are constant coefficients, can be transformed into an algebraic equation by means of the Laplace transformation (Cheng, 1959). Thus Eq. (21) becomes
where V(s)and I ( $ ) are the transformed functions of complex frequency s = j w and w is angular frequency). Rearrangement of Eq. (22) to obtain the complex ratio V ( s ) l I ( s )defines the driving-point impedance, Z ( s ) , as (j =
m)
V ( s ) (bos'n Z(s) = -= I(s) (a,,"
+ b,s'"-l + . - . + b,") + a,s"-' + . . + a,>
(23)
*
That is, impedance is the ratio of polynomials of complex frequency, s, obtained by transformation of the temporal differential equation into the frequency domain. A complex impedance can be described in rectangular form Z ( j w ) = R(w)
+ jX(w>
(24)
in which the ratio of complex polynomials in Eq. (23) can be manipulated into a real part, R ( w ) , and an imaginary part, X(w) (terms that have a j in common), which are functions of w. An alternative polar form of the impedance function is expressed as
12
H. M. FISHMAN AND H. R. LEUCHTAG
where (21is the magnitude or modulus of the impedance formed by the real and imaginary components at each w in Eq. (24), i.e., (21 = [RZ(w)
+ X2(w)]”*
The phase angle associated with the impedance, L Z , is the angle whose tangent (tan-’) is the ratio of the imaginary part to the real part in Eq. (24), i.e., L Z = tan-‘[X(w)/R(w)]
Thus complex impedance functions of frequency are most commonly graphed as either a locus of frequency points in the complex plane [plot of X(w) versus R(w)J or as magnitude and phase functions of frequency. An impedance function contains all of the information about a conducting process necessary for a kinetic description. Thus, in addition to aiding in the characterization of electricalstructural properties of tissues (Eisenberg and Mathias, 1980), impedance measurements in membranes have provided an alternative way in which to characterize the kinetics of ion channel processes (Fishman et d., 1983; Warncke and Lindemann, 1985; Hayashi and Fishman, 1988). Furthermore, a membrane impedance function provides a way to assess background noise in membrane fluctuation measurements (Fishman rt d., 1983, 1984) and a basis for predicting fluctuations due to equilibrium processes (thermal noise) (Frehland, 1982; Fishman, 1985). Impedance analysis of membranes has undergone revolutionary changes since the early work by Cole (1972) and colleagues. Digital signal processing techniques and advances in calculating Fourier transforms (Brigham and Morrow, 1967) have produced several new methods for rapidly acquiring high-resolution driving-point functions in membranes. These techniques involve use of synchronized pseudorandom binary signals (Husimi and Wada, 1976; Poussart el al., 1977) and use of Fourier-synthesized signals (Fishman rt al., 198 1; Fishman and Law, 1987; Kottra and Fromter, 1984; Nakamura et al., 1977, 1981).
C. Convolution Driving-point functions (impedance or admittance) are fundamentally used to characterize a linear or linearized (nonlinear) process because they enable analytical determination of the response to any arbitrary stimulus applied at the same set of terminals. The transfer function provides the analogous stimulus-response relationship when the response is obtained at a location different from the site where the stimulus is applied. The inverse Fourier transform of a transfer function is the impulse response, which provides a way to calculate the response to an arbitrary driving function. By use of the superposition principle, a driving
13
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
function can be represented as a series of impulses that have the same value at each time of occurrence as the original function it replaces. The response, R(T), to an arbitrary driving function, D ( t ) , is obtained by a convolution, defined as R(T) =
i
D ( t ) h ( ~- t ) dt
where T is a time-shift variable that depends on the relative shift in time imposed between the function D ( t ) and h ( t ) , the latter representing the impulse response. In words, convolution involves folding the impulse response function about the origin, displacing the function in time by 7,multiplying by D ( t ) and integrating in the region of overlap of the two functions.
D. Spectral Density and Autocorrelation Function Let us now suppose that the process Y ( t ) is stationary, with a zero average and a finite correlation time 7,. Then the second moment ( Y 2 ( f ) )is independent of time and may be taken out of the integral in Eq. (20) to give
This equation states that the mean square of the fluctuations is equal to half the sum of the mean squares of the Fourier coefficients, each referring to a single sine wave with angular frequency w . It remains to be determined how ( Y 2 ) is distributed over the frequencies. To do this it is necessary to compute the spectral density of fluctuations s ( ~ )which , is defined by S(W)Aw
=
1 w~:~,,rrl7Ic:w+Aw 2
C
-(A:)
The period T must be chosen large enough that many values of n fall within the interval Aw, even though Aw must be small. The formula for S(w) is given by the Wiener-Khinchine theorem, which states that the spectral density is the cosine transform of the autocorrelation function ~ ( 7 ) :
The proof (van Kampen, 1981) depends on the requirement, mentioned earlier, that K ( T ) decreases rapidly for time intervals greater than the autocorrelation time, r,.
14
H. M. FISHMAN AND H. R. LEUCHTAG
IV. PHYSICAL NOISE IN CONDUCTION We will begin this section with a discussion of equilibrium processes, including the relation between fluctuations and dissipative processes, and Nyquist’s analysis of Johnson noise. Thereafter we will discuss nonequilibrium processes, including shot noise, the currently very active field of fractal noise, and diffusion noise.
A. Fluctuation-Dissipation Theorem The problem of Brownian motion can serve as a prototype example to illustrate the way in which frictional forces arise in a macroscopic description. These forces are irreversible, even though the laws of mechanics that govern the underlying microscopic mechanics are reversible, that is, are equally valid whether time goes forward or backward (Reif, 1965). Consider a small macroscopic (colloidal) particle immersed in a liquid of absolute temperature T. For simplicity we treat the problem in one dimension. It would be a hopelessly complex task to describe the motion of the center of mass, x, of the particle in terms of the other degrees of freedom in the system. These, including both the motions of the atoms making up the colloidal particle and those of the molecules of the liquid surrounding it, are lumped into a heat bath at temperature T, while their interactions with x are lumped into a net force, F ( t ) , acting on the particle. If there is an external force on the particle as well, say F , ( t ) , the velocity of the particle can be determined from Newton’s second law of motion, rn(dvldt)
=
F,(t)
+
F(t)
(30)
Using the ergodic assumption, we visualize F ( t ) as a stochastic function of t , and the system as a sample of a very large number of similarly prepared systems. We recall that F ( t ) can be characterized by a correlation time T,, which roughly measures the mean time between maxima of F( t ) . The correlation time is quite small, about l o - ” sec. Because F ( t ) is a rapidly fluctuating function of time, v must also fluctuate, although less rapidly, due to the inertia of the particle. We can write the velocity, v , as the sum of the ensemble average velocity ( v ) and a rapidly varying part v ’ , v = (v)
+
V‘
(31)
The external force F, (gravity or electromagnetic interactions) can be considered slowly varying, so that it remains effectively constant during brief time, T. The variation of ( v ) is, however, not simply dependent on F,: even if F, = 0,
15
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
the interaction force F will gradually restore the particle to its mean equilibrium velocity, ( v ) = 0. Thus we must also write
F = (F)
+ F’
(32)
where F‘ is the rapidly varying part of the interaction, with zero mean. If ( v ) is not too large, ( F ( ( v ) ) )can be expressed as a power series in ( v ) , the first nonvanishing term of which is linear in (v). Because the higher terms are negligible, ( F ) has the general form
(F)
= --CY
(v)
(33)
where a is a positive friction constant. The minus sign indicates that force ( F ) tends to reduce (v) to zero as time increases. The equation of motion of the Brownian particle can therefore be described by the Langevin equation, m(dv/dt) = F, - a v
+ F’(t)
(34)
Interestingly, this equation contains the dissipative term - a v , which makes the equation irreversible, even though the microscopic equations do not involve frictional forces and are reversible in time. An electrical problem analogous to that of the Brownian particle is an electrical conductor of self-inductance L carrying current i , with an applied voltage v ( t ) . The current is carried by electrons that interact with the atoms of the conductor, resulting in an effective ff uctuating v(t). This voltage can be decomposed into a slowly varying part ( v ) = - Ri, where R is a positive constant, and a rapidly fluctuating part v’(t), whose mean value vanishes. The analog of the Langevin equation therefore is L ( d i / d t ) = v - Ri
+ v’(t)
(35)
where the friction constant, R , is the electrical resistance of the conductor. Returning to the problem of the colloidal particle, we might suspect that the frictional constant a is also derivable from the interaction force F ( t ) , since it arises from the interaction. This is in fact the case, and further analysis (Reif, 1965) shows that a is related to the correlation function K ( T ) = ( F ( t ) F ( t + T ) ) ~ , , introduced earlier. The subscript zero refers to the absence of external forces, F, = 0. The constant a is given by
16
H. M. FISHMANAND H. R. LEUCHTAG
where k is Boltzmann's constant. This relation, the Pucfuafion-dissipution theorem, is an explicit expression for the friction constant in terms of the correlation function of the fluctuating force F ( t ) in the equilibrium situation. Returning to the analogous electrical problem, we see that, if the rapidly fluctuating voltage, v'(t), is neglected, we have L(dildt) = v - Ri
(37)
Following the analogy, we obtain the specific expression for R in terms of the fluctuating voltage, v(t), 1
R
=
2kT
fa
J
(v(0) ~ ( 7 ) dT )~ -m
B. Johnson-Nyquist Description Einstein predicted in 1906 that Brownian motion of the charge carriers would lead to a fluctuating voltage across the ends of any resistance in thermal equilibrium. This effect was first observed by Johnson in 1926, and in 1928 Nyquist calculated its power spectrum (Robinson, 1974). Consider two equal resistances at the same absolute temperature, T, connected in parallel. The fluctuating thermal voltage that appears across one of these resistors, R, must equal that across the other, or else they would not be in thermal equilibrium. Furthermore, this equality must be true of each frequency component, because a filter containing only inductances and capacitances should not destroy thermal equilibrium. The power spectrum of the voltage fluctuation therefore must be a universal function of R, T, and the frequency, f. A thermodynamic argument due to Nyquist shows that the power spectrum of open-circuit voltage fluctuation is S ( f ) = 4kTR
(39)
The spectrum of Johnson noise is white, that is, independent of frequency, so the mean-square value of the noise voltage in a band Afis (v:)
=
4kTR hf
(40)
For complex impedance, Z ( j o ) [see Eq. (24)], the noise voltage depends only on its real part (Re) as pure reactances do not generate noise:
Nyquist's results apply only to thermal equilibrium.
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
17
C. Shot Noise Current flow in conducting materials is the result of the movement of charge carriers (for example, electrons in semiconductors and ions in nerve membranes). Because of the discreteness of charge carriers and the randomness of their production, current flow is not smooth; instead it is accompanied by a noise of the same character as the sound produced by rain falling on a metal roof. Schottky (1918) first investigated this phenomenon and called it shot noise. The term “shot noise” has been generalized to include different processes, such as fluctuations in plate current of a vacuum diode that arise from the random emission of electrons from the cathode and conduction noise in semiconductor devices due to the random generation and recombination of carriers (random transitions of carrier from bound to conduction states). The classical example of shot noise in membrane conduction is that of conductance fluctuations in the end plate of the neuromuscular junction due to the random release of quanta1 amounts of acetylcholine from presynaptic stores (del Castillo and Katz, 1954; Boyd and Martin, 1956). The characteristic relationship between the mean-square of the current fluctuation, (i*), and the mean current, ( i ) , in a shot process is ( i z ) = 2 q ( i ) Af
where q is the charge of the carriers and Af is the noise bandwidth over which the measurement is made. The utility of shot noise as a model for a variety of physical and biological processes has led to the definition of shot noise as a random process:
,= - m
which is formed by superposition of a pulse, h ( t ) , occurring at random times, t , . Furthermore, the occurrence of a pulse at any time is usually assumed to be equally likely and the number of pulses that occur in a given time per observation time interval approaches a limit as the observation time becomes large. Under these conditions, the number of pulses in a given time is distributed according to the Poisson distribution. When the average number of pulses is large, the fluctuations approach a Gaussian distribution [see Eq. (50)). The power spectrum of shot noise, like thermal noise, is usually flat, so that the power density per unit bandwidth is independent of frequency. However, when t, in Eq. (43) becomes comparable to the observation time, the power spectrum will vary with frequency and depend on the shape of the event defined as h( t ) .
18
H. M. FISHMAN AND H. R. LEUCHTAG
D. Fractal Noise Another well-known conduction noise that is observed during current flow in a wide variety of conducting media is flicker, or “one-over-f” (Ilf) noise (named for the spectral distribution, which falls linearly with approximately unit slope on a log-log plot with frequency). There is a vast physical literature on I/f noise, dating from the first use of vacuum tubes as amplifiers to the era of explosive development of semiconductor devices (see Gupta, 1977). Flicker noise arises in many different physical processes. The classical example is current flow through a carbon resistor, which consists of a large number of conducting grains that make poor contact with each other. The macroscopic resistance consists of all the local contact resistances between grains, which become evident during current flow as a resistance fluctuation. tiowever, flicker noises in semiconductors are generated by completely different mechanisms, as is the 1 /f noise produced during current flow through porous membranes separating ionic solutions (Dorset and Fishman, 1975). In biological preparations (Neumcke, I978), the carliest spectral determinations of nerve membrane voltage fluctuations (Derksen and Verveen, 1966) and current fluctuations (Poussart, 1971; Fishman, 1973) showed a strong I /fcornponent associated with conduction in K channels. Curiously, spectra of Na current fluctuations showed low intensity or negligible Ilfnoise (Conti et d . , 1975, 1976; Fishman rf u l . , 1983). Sauve and Szabo (1985) argue that I l f noise in biological membranes may simply be an artifact of limited spectral range. They show that two relaxation (Imentzian) spectral components can approximate a 1(f’ function to 90% confidence limits over 3 decades of frequency. All of these observations can be reconciled by a new branch of mathematics called fractals. In 1975 Mandelbrot ( 1983) initiated a new scientific discipline based upon a geometry that describes many of the irregular and fragmented patterns of nature, such as coastlines, mountains, and trees. He called this family of shapes “fractals.” With respect to fluctuation phenomena, Iractal processes involve both regularities and irregularities that are statistical but in which the degree of irregularity and/or fragmentation is identical at all scales. A fractal process is defined as one in which a physiciil property, L . depends on the scale, E , at which it is measured such that L ( E ) = Kc’-”
where K is a constant and D is the fractal dimension. Flicker noise behaves with a change of time scale in the same way as the coast of England behaves with a change of spatial scale: both remain essentially the same. If a tape of I lf noise is played, it sounds the same at all speeds (Gardner, 1978). More specifically, as Brophy ( 1969) found, the probability density distribution of interval spacings
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
19
between zero crossings of Ilf noise is independent of sample length; it is statistically stationary. Thus, flicker noise, which is defined by the power spectrum
where a has values from 0.5 to 1.5, is a fractal noise. As mentioned earlier, power spectra of current fluctuations from a large population of potassium channels show strong I ljcomponents. Analysis of patch-clamp recordings from single-ion channels in rabbit corneal endothelium showed the effective kinetic rate constant to be time scale dependent. The frequency histograms of measured nonconducting times of the channel were fitted well by a fractal ion channel model over three orders of magnitude in time (Liebovitch et d., 1987). The interpretation of this result is that Markov chain models of channel kinetic states may be only approximations to a continuum of intermediate states and rate constants corresponding to variations in channel protein conformations. Furthermore, each time a channel returns to a specific state, the channel protein may have a different conformation.
E. Diffusion Noise Because diffusion is often involved in ion transport through membranes, it can be an important source of conduction noise. The power spectrum of fluctuations arising from diffusion was derived by MacFarIane ( 1950) and by Burgess (1953). They showed that the high-frequency asymptotic behavior declined as ,f’- W , whereas the low-frequency behavior leveled off. A transition region between low and high frequency closely approximates a I !f spectrum and often is mistakenly identified as I!fnoise. As Lax and Mengert (1960) have shown, t h e f - ” ? highfrequency spectral behavior is generally characteristic of diffusion processes. An extensive review by van Vliet and Fassett ( 1965) covers fluctuations due to transport in solids. Diffusion noise associated with ion transport through multipore membranes was studied by Green (1976), who concluded that such noise could approximate a I l j spectrum if the distribution of pore lengths is inversely proportional to length.
V. NOISE MEASUREMENTS AND ANALYSIS TECHNIQUES
A. Signal-to-Noise Ratio
In quantifying the performance of a measurement system that operates on small-amplitude signals, the ratio of noise power to power due to the signal
20
H. M. FISHMAN AND H. R. LEUCHTAG
source defines the noise factor, F, which is a useful quantity for comparison purposes. The noise figure, NF. is then a logarithmic expression of F (in units of decibels) given by
NF = 10 log F Noise figure thus conveys the degree of degradation in signal-to-noise performance by a signal processor, with N F = 0 dB indicating that the processor adds no noise to the thermal noise of the source. The extent to which a signal can be amplified and useful information extracted from it is limited by the accompanying background noise produced by extraneous sources. The extraneous noise comes from sensors (electrodes) and subsequent amplifying stages, which are in cascade with the desired source. If the extraneous noise generated by electrodes can be made low, relative to the noise of the process under study (membrane conductance fluctuations), then the remaining extraneous noise will be due to that introduced by the input stage. Furthermore, if the input stage provides signal amplification, noise contributed by subsequent stages and instrumentation will be negligible relative to that contributed by the headstage. Thus, discussion of noise performance (Poussart, 1973) can be focused on the headstage, in which signal-to-noise ratio can be increased by xerting control over: ( I ) the input device type and/or impedance of feedback elements and (2) the magnitude of the source impedance (by implementation of electrical isolation and measurement over a small membrane area). As an example of how signal-to-noise performance depends on factors ( 1) and ( 2 ) above, consider the measurement of current fluctuations through an electrically isolated patch of membrane, as depicted in Fig. 1 . We wish to examine the effect on the signal-to-noise ratio of increasing the isolation, Rlh, and the effect of different device values of en and i,,. The signal-to-noise ratio is defined as the ratio of pipet current, i,, to undesired current noise, i,. The relevant relations are:
(44) i,, = in
where, assuming R P >> Rsh,
+ i, + i,
(45)
21
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
FIG. I . Simplified diagram of a patch clamp used [or calculation of i,/id(ratio of input current to desired current) in Table I and SIN (signal-to-noise ratio) in Table 11. e . Fluctuating voltage source; R,, patch resistance; R,,,patch isolation resistancc; R,,pipet resistance; R,, feedback resistance; A . operational amplitier; r,,, intrinsic voltage noise of A ; in. intrinsic current noise of A .
and, assuming R f >> (R,
+ R,h), I, =
4kT
Rc
+
(47)
R,h
so that the signal-to-noise ratio is
S/N
=
i,li,
R,h
=
R,
+ R,, i,,
-
I ld (R
From Eq. (48), the isolation resistance, R,,, that maximizes S / N is large relative to the pipet resistance, R,. Increasing id (by increasing the patch area, thereby increasing the number of channels contributing to the current fluctuations) provides a useful way to improve SIN and overcome i,,. In fact, the first patch clamp from which fluctuations were recorded and analyzed achieved isolations of small areas (10 to cmz) of squid axon of only a few megohms, but the patch area was large enough to contain a sufficient number of channels to yield a good iJid ratio (see Table 1) (Fishman, 1985; Fishman rt al., 1975). This ratio is comparable to those obtained today with gigohm isolations. In Table 11, S/N is calculated from Eq. (48) for an operational amplifier, A (Fig. I), selected for low input-current noise density (in = 0 . 2 f A i f i ) and for
22
H. M. FISHMAN AND H. R. LEUCHTAG TABLE I
( i , ) WITH
P l P E r T I P DIAMFTFR
ANI) P A I ( H R F S l T r A N < I .
(Rn).A N D IS01 ATION
VAKlArlON IN PIPET CURRENT
I13 S S
I
1 x lo-* 2 % 10 2 % 10 8 x lo-"
((1). RESISTANCF ( R , ) (RLh)
I0
2
0 0s
0 97
5000
I0
0.67
sono
I00
5.0 5.0 SO
I 3 x lo7
I000
0 95 O 995
variation in electrical isolation, R,,,, from 10 Mi1 to 10 Gf1. The voltage and current noise densities were converted i n t o rms voltage and current, assuming a measurement bandwidth of 1 kHz. The best S/N (21.4) is achieved when the patch isolation is maximum (I0 Gi1). Notice, however, that at all levels of isolation the thermal current noise, i,, is the dominant source of undesirable noise.
6. Signal Amplitude Statistics As described in Section II,A, the moments of a stochastic signal provide a way to charactcrixc thc amplitude statistics. The first moment (mean value) and second moment (mean-square value) are the most commonly determined of all the moments of a random signal bccausc of their intuitive physical interpretation. In a Fourier representation of a waveform, the mean value corresponds to the xcro-frcqucncy (dc) value relative to which thc fluctuations are measured. The mean-square value provides a measure of how much the fluctuations deviate from thc mean. In clcctrical terms, the mean-square value is the constant (dc) value that produces the same power dissipation (amount of heat) in a resistor as that produced by thc fluctuations. The Fourier components in a waveform derived from a stationary process, expressed as mean-square function of frequency, convey how the power density is distributed among the frequencies in the wavcforrn, known as the power densic spectrum or power spectrum. Mean values can bc mcasurcd vcry simply with a dc voltmeter. The mean TARIX II SIGNAL-TO-NOISE CALCULATIONS'< K , (Mil) I0 10 I0 10
R,,, ( M O 1
i , (fA)
t.,,
ipV)
i,, ( f A )
I .sx I.S8 I.SR
6.3
I ono
50 91 99
10000
009
I.SX
6.3
I0
I00
6.3 6.3
i,,, ( f A )
79.0 14.4 I .57 0. I 6
i , (tA)
SIN ( i , / i x , )
900 380 I30
0.5
40
21.4
7 7 -._
7:;
"Based on Fig. I and Eqs. (44). (45). and (48). and operational amplifier A with input characteristics: 5 0 " V i a , i,, = 0.2 t A / V % ~ a n d~ m ~ ~ u r ~ n i at c nat handwidth h of 1 kHr.
(I,,
=
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
23
square can be obtained by instruments (known as true rms voltmeters) that use “square law” detectors such as thermocouples. Alternatively, mean squares can be obtained by digital or analog implementation of the separate mathematical operations of squaring, integrating, and dividing by the integration time. The moments of a random process, such as the mean and mean square, are important quantitative attributes of noise, but many different sources of noise can have the same moments. Thus, moments do not provide sufficient information to distinguish among noises arising from different physical processes. To provide a more complete characterization, the distribution function is defined as the fraction of time or probability that the noise amplitude is less than a specified value. If CP(x) is the distribution function for a random process for which x is the amplitude, 9 ( x 2 ) - P(x,) is the fraction of time that the amplitude lies between values x, and x?. The slope of the distribution function is the probability densityfunction, P ( x ) , defined in Section I1,A. The probability density function and distribution function allow one to deal with problems in physics and biology in which the probability of amplitude x. P ( x ) , is a continuous function of x rather than a collection of discrete numbers whose sum is unity. The area under the P ( x ) curve in the interval from x = x, to x = x2 is equal to the fraction of time x spends in that interval because
There are many distribution functions that arise from certain classes of physical noises. We mention a few that have relevance to noise in biological membranes. In the Guussian distribution of amplitudes, the probability density function is given by
with distribution function
where the error function, erf(x), is defined by
The Gaussian distribution is the form the distribution function takes for the sum of a large number of independent quantities, which individually may have
24
H.M. FISHMAN AND H. R. LEUCHTAG
distribution functions that are quite different from a Gaussian function. This result is known as the "central limit theorem." A noise source is often composed of the sum of a large number of independent processes (e.g., a population of independent membrane ion channels) and yields a Gaussian distribution. Thermal and shot noise are two different physical processes that produce Gaussian amplitude distributions because of the superposition of a very large number of random, independent contributions to the fluctuations. Another important distribution for ion channel conductance fluctuations in membranes is the binomial distribution. This distribution describes the expected results of a series of trials in which the outcome of a single trial has only two possibilities. Thus, given a population of N independent channels with a single conducting state (with probability p), the probability of having n channels in the conducting state is N P ( n ) = n ! ( N - n)! P"qN
(53)
-
where q = 1 - p is the probability of a channel being in a nonconducting state. If n is allowed to vary from zero to N , Eq. (53) has the form of the binomial expansion (p + q)", from which its name is taken. That is,
' N
n
o
N
P(n)
N!
?:on!(N
-
n)!
P"9N-"= ( p
+
4)"
=
1
so that the P ( n ) are defined as probabilities. The mean number of channels, n, in the conducting state is defined as
The n = 0 term is zero, and substitution of Eq. (54) yields N
(4= Let k = n
-
c
n=i
N! P"P" (a - 1)!(N - n ) !
1 and, because N ! N- I
(n> =
=
N(N
-
l)!, the above becomes
N ( N - l)! p'+lqN - 1 - k)!
C k!(N
k=o
N- I
-
Np
( N - I)! k!(N - 1 - k)! pkqN-"
70
(54)
25
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
From Eq. (54) the above can be written in terms of the binomial expansion (p + 4Y-I as (n) = Np(p
+ q)”-’
=
Np
(56)
A similar procedure shows that the mean-square value is ( n Z )=
Npy
+ (NP)~
(57)
By Eq. (6), the variance, v2,of the distribution is given by
From the previous discussion, it becomes easy to obtain the mean, mean square, and variance of a conductance fluctuation, g ( t ) , for a single channel that conducts with conductance A, = A and probability P( 1) = p or is in a nonconducting state (Ao = 0) with probability P(0) = q. From Eq. ( 5 3 , the mean of the conductance fluctuation is
and the mean square is
The variance, vi,of the conductance fluctuation is then
Rearrangement yields the square of the single-channel conductance as
Extension to the case of N similar, single conducting-state channels in an isolated membrane area is made by defining the mean membrane conductance as (g> = NAP or
(63)
26
H. M. FISHMAN AND H. R. LEUCHTAG
Squaring Eq. (63) and equating to Eq. (62) yields A -(x) NP
=
CT;
P(1 - P )
From this, the single-channel conductance of N identical channels is obtained as
C. Spectral Analysis From a practical point of view, the most useful approach to characterizing fluctuations is decomposition of the waveform into its Fourier components and graphical presentation of the components in an amplitude-versus-frequency spectrum (Blackman and Tukey, 1958). When the amplitudes are plotted as meansquare values, the graph is known as a power spectrum. For fluctuations derived from a stationary process, the power spectrum provides a “signature” that is indicative of the process kinetics. The power densities relate to the thermodynamics of the process. Thus, fundamental information about unknown mechanisnis can be obtained from spectral analysis. The main disadvantages of this method come from the general mathematical nature of the analysis. As in any spectroscopic method, spectral components are identified only by comparison with known simple processes and by changes in the power spectrum that follow alteration, in some way, of the process that produces the fluctuations. In principlc, the autocorrelation function defined in Section III,C contains the Same information as that in a power spectrum. Why, then, is spectral analysis the preferred way of characterizing a random process‘! The answer rests on the problems associated with implementing an autocorrelation function. First is the presence of periodic extraneous signals, such as those induced by power lines and those produced by current flow between instruments having different voltage reference points (grounds). When these extraneous inputs are summed with the desired inputs, the autocorrelation function is distorted by the superposed periodic components. To prevent this distortion, any extraneous, periodic signals must have amplitudes an order or more below the amplitudes of the desired fluctuations. Achievement of this condition usually requires extraordinary efforts (in shielding, isolation, grounding, etc.). By comparison, in a high-frequency resolution spectral analysis, the periodic signals appear as discrete lines, which are easily identified in the spectra. They can be either ignored or removed in fitting a model to the spectral form. Spectral analysis can even be performed without significant degradation on fluctuations that have superposed, periodic, extraneous signals with amplitudes larger than any of the Fourier components in the fluctuation waveform. The second significant limitation in implementing an autocorrelation function
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
27
determination is that the calculation involves a squaring operation (the timedomain function is multiplied by a shifted version of itself and an integration performed). Given instrumentation in which a fixed-amplitude (dynamic) range is a real limitation on signal processing, implementation of a squaring operation effectively reduces the dynamic range of the input in order that saturation of the output not occur. Furthermore, when extraneous periodic signals have significant amplitudes relative to those of the fluctuations, saturation and nonlinearity produced by the extraneous signals can preclude calculation of an autocorrelation function. Filtering of the extraneous signals usually is not a viable solution, because rejection filters are not ideal: they reject desired components along with the extraneous signals, and they add noise to the measurement. Finally, spectral determinations from a data array are achieved much more efficiently and therefore more rapidly via the fast Fourier transform algorithm (Cooley and Tukey, 1965; Brigham and Morrow, 1967) than via a computation of an autocorrelation function. Consequently, for stationary fluctuations, many power spectral determinations, produced continuously from a single time record, can be averaged to yield an accurate power spectrum much more rapidly than could be obtained from calculation of an autocorrelation function. In fact, an autocorrelation function is usually best determined by inverse Fourier transformation of an averaged power spectrum. In order to extract useful information from a power spectrum, manipulations such as the subtraction of one spectrum from another to obtain a difference spectrum are often carried out. However, these operations may produce spectral distortion if the effect of the conditions under which the measurements were made is not taken into account. A common procedure for producing a conductance fluctuation spectrum for a uniform channel population is to subtract the spectrum of fluctuations after channel blockage from the spectrum obtained prior to blockage. The assumption underlying this procedure is that the “residual” spectrum of fluctuations obtained during the blockaded state is the same spectrum as that which existed during the fuctuation measurement before channel blockage. In most instances this assumption is not valid because the membrane impedance is changed after channel blockage (Fishman ct ul., 1983, 1984). The background noise of all voltage-clamp systems depends upon the membrane impedance, which is part of the feedback-determining factor and which transduces the intrinsic input noise sources from instrumentation into an output background noise (Fishman, 1982). Because membrane impedance is voltage sensitive, the proper spectral correction for background noise requires measurement of the membrane impedance under the same unblocked channel conditions in which the fluctuations are measured and at cvery value of membrane voltage (Fishman et ul., 1983). Another example of improper manipulation of spectral data occurs when there are multiple sources of noise in the spectrum that obscure the spectral form of the noise source of interest. The most frequently encountered noise that compli-
28
H. M. FISHMAN AND H. R. LEUCHTAG
cates spectral analysis is “llf” noise (see Section IV,C). For many years the origin of Ilf noise was not understood because it seemed to arise from many diffcrent phenomena. As the branch of mathematics called “fractals” evolved, identification of 1 (f noise as characteristic of fractal processes explained why so many different processes produced this type of noise. The spectra of potassium channel fluctuations in nerve membranes always exhibit a strong llf component (Derksen and Verveen, 1966; Poussart, 1971; Fishman, 1973; Conti rt ul., 1976). Among the investigators modeling the spectra of these fluctuations, the assumption of udditive spectru of noises of different form became prevalent. A complicated spectrum was fitted by the function
which assumes the spectrum to be composed of “white” noise (first term), l/f noise (second term), and a Lorentzian (third term). In adding these terms, the implicit assumption is made that the sources of each of the noises are uncorrelated. This assumption may not be valid: current flow through isolated cytoskeletal material obtained from the inner surface of squid axon showed llfnoise (Fishman, 1981). Thus the Ilfnoise produced in the cytoskeleton and cytoplasm during current flow through conducting channels could modulate (a multiplicative operation in the time domain) the stochastic channel currents. The resulting spectrum represents the convolution (see Section 111) of the frequency-domain Fourier transforms of the fluctuations produced by the two sources. The power spectrum would contain not only separate additive terms as in Eq. ( 6 3 , but also cross-terms, which could actually dominate the spectrum. With cross-terms, spectral intensity rolloffs with noninteger slopes are possible. Thus, estimation of important parameters such as corner frequency, variance, and low-frequency asymptote from spectra that reflect multiple sources of noise should not be limited to an assumption of uncorrelated sources. Model fits of power spectra should include convolved spectra as alternative models. With evolution of the patch-clamp technique, competition to spectral analysis as a means of characterizing fluctuations has arisen in the analysis of conducting and nonconducting time-interval probability distributions. The argument in favor of the time-interval distribution approach is that rate constants for transitions between conducting and nonconducting states, and the number of such states, can be obtaincd directly from these distributions, if one assumes a Markovian model to fit a sum of exponential functions to the interval distributions. Analysis of power spectra does not provide a direct estimate of the forward and backward rates, but instead yields the relaxation times that depend on a function of the forward and backward rate constants. However, production of dwell-timeinterval distributions is not a trivial process because dwell times in a particular conducting state may be so short that the recording system is unable to follow momentary transitions to or from a state. Thus short dwell times will either
1 . ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
29
reflect the limited patch-clamp bandwidth or be missed entirely. The missed dwell times will bias the overall estimate of the time-interval distribution toward longer dwell times. This problem becomes more severe when the signal-to-noise performance is poor, so that further reduction in system bandwidth by use of a low-pass filter is necessary. The effect of time-interval omissions on distributions has been examined (Roux and SauvC, 1985). Nevertheless, the problems arising from the process of deriving estimates of time-interval distribution come from the attempt to separate a particular noise signal, the stochastic binary waveform, from other sources of noise. The criteria established for accomplishing this objective (Dionne and Leibowitz, 1982; Methfessel and Boheim, 1982; Sachs et al., 1982; Moczydlowski and Latorre, 1983; Bechem et al., 1983; Sakmann and Trube, 1984) are arbitrary and produce aberrations because of bandwidth limitations. Power spectral analysis, on the other hand, yields an unbiased description of all sources of noise in the fluctuation waveform. Therefore, spectral analysis provides an unbiased measure of all the significant kinetic components of the waveform without producing additional distortion.
VI. ION CONDUCTANCE FLUCTUATIONS IN BIOMEMBRANES A. Stationary Noise by Spectral Type One way to classify or characterize sources of noise is by their spectral distribution or form. As in the time domain, the response of a system in the frequency domain is describable in terms of an nth order linear differential equation,
which has a solution in terms of sums of exponentials,
An exponential correlation function transforms via the Wiener-Khintchine integral [Eq. (29)) to a frequency function (power density spectrum) that is known as a Lorentzian function, i.e.,
transforms into
30
H. M. FISHMAN AND H. R. LEUCHTAG
where the “corner frequency” A. = ( 2 m ) I . Sums of exponentials yield sums of Lorentzian terms in the power spectrum. The pragmatic question of how many Lorentzians can be distinguished in a power spectrum is an important issue. Generally, in the time domain more than two relaxation times are difficult to resolve because of limitations in real measurements, such as the finite dynamic range of signal-processing instrumentation and the intensity of background noise, which determine the lowest discernible signal amplitude. The same handicaps hamper spectral analysis: a power spectrum relating to a process of interest usually is limited to about 3 decades in frequency. This restricted frequency range can lcad to nonunique determinations of corner frequencies, ,f; (see Fig. 2) (Neher and Stevens, 1977). Furthermore, if non-Lorentzian sources of noise are also present, curve fits of a power spectrum with a singlc Lorentzian function can yield inaccurate estimates of model quantities. What other non-Lorentzian sources of noise are likely to be encountered? The most common type is I lfnoise. As discussed earlier (see Section IV,D), l/f noise is not uniquely associated with a single physical process (electrodes, diffusion, etc.), but rather constitutes a class of noise produced by fractal processes, which yield power spectra that fall logarithmically with frequency, with slopes ranging from -0.5 to - I With the possibility of this variation in the slopes of spectra, in which thc intensity of this component is equal to or greater than the intensity of Lorentzian components, fits of functions such as
can yield inaccurate values for K , , K , , and ,t.if the exponent cy on f in the first tcrm of Eq. (70) is not exactly 1 .O. Another difficulty with assumption of a sum of spectral components [Eq. (70)] is that this form also implicitly assumes that the sources of I/J’and Lorentzian noise are independent (i.e., not correlated). An example of a typical ion channel noise spectrum, based on correlation between these sources, was given by Fishman (1981). Cytoskeleton and axoplasm, in series with squid axon ion channels, were found to produce excess noise when isolated in a pipet and during current llow. If this noise is produced in situ. the channel conductance fluctuations would be modulated (multiplied in the time domain) by the I / f fluctuation in current through thc axoplasm. The power spectrum of this process would thus contain cross-terms that could dominate the spectrum and produce a spectral form that could be fitted by Eq. (70), but which would yield erroneous values. Furthermore, the possibility always exists that the kinetics of the system or process that produces the fluctuations is nonlinear. In such cases, a time-domain solution of the system response will not be described by the sums of exponentials of Eq. (67), although, with enough of them (more than two), a good approxi-
31
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY A
OL................................................................
-5
1
_-.
7
---
_---
/--
.i
B
.r
2.4
N D
0.8
c
0
7.2 10.8 (nA) FIG.2. Nonstationary noisc analysis. Variancc and mean sodium current in a frog nodc of Ranvier from 32 groups of four 20-msec depolarizations to IS mV after 50-msec prepulscs to - 105 mV. (A) Variancc calculated at each sample point. (B) Temporal variation of mcan current from four responses. (C) Plot of the variance of conductance versus mean current. Continuous curve is drawn from Eq. (74) with i = -0.55 pA and N = 20.400. [From Sigworth (1980).]
3.6
f
~
mation may be possible. Similarly, power spectra of fluctuations from a nonlinear process generally will not yield Lorentzian functions, although several Lorentzians may yield a good fit over a limited frequency range; such fits are only approximations to the actual spectral form.
6. Nonstationary Noise Analysis Random processes are characterized by their statistical properties (moments and distributions; see Section V,B). In order that the statistical properties be interpretable and relate to theory, they must be time invariant. As mentioned in
32
H. M. FISHMAN AND H. R. LEUGHTAG
Section II,C, the attribute of time invariance of statistical properties of a noisy process is called stationarity. The statistical properties of a stationary stochastic process can be determined from a single, finite time record of the fluctuation waveform. Such processes are called ergodic because averages across an ensemble of records are equivalent to averages over time along a single record of infinite extent (Papoulis, 1965). Stationarity allows extraction of useful information from spectral analysis of a single record of a fluctuation waveform. That is, the power spectrum of fluctuations is a useful tool for identifying and characterizing sources of noise according to their spectral content and for distinguishing between extraneous and substantive noisy processes. Spectral analysis also provides a definitive test of the stationarity of fluctuations. Sufficiently long time records can be subdivided into various segments that can be analyzed separately to determine whether later segments of the record yield the same spectrum as early ones. This method was used to show stationarity in K current fluctuations (Fishman et al., 1981) and in Na current fluctuations under Cs perfusion in squid axons (Fishman et al., 1983). However, in most instances, membrane currents, produced by populations of ion channels in response to membrane voltage changes, are transient events. If the current is transient, the underlying process is clearly nonstationary because the current fluctuations have a time-varying mean value, a time-varying mean-square value, and a time-varying frequency structure. Interpretation of the analysis of fluctuations under these conditions is not straightforward. In fact, analysis of nonstationary noise requires assumptions about the process and a methodology that exploits the assumed properties. For example. nonstationary noise during Na current transients in frog nodal membrane was processed by Sigworth (1977, 1980, 1981) on the basis of the assumption of a homogeneous and statistically independent population of channels having only one conducting state for each channel. Then the mean current, ( I ) , is given by (I)
=
Nip
(71)
where N is the number of channels, i is the current through a single channel, and p is the probability that a channel is in the conducting state [cf. Eq. (%)I. The variance of conductance fluctuations, ui, is then [cf. Eq.(61)]
u,2 = Ni2(l - p ) p Division of Eq. (72) by Eq. (71) yields u ; / ( l )= i ( l - p )
(73)
Thus, from a determination of ( I ) , a:, and p , the single-channel current, i, and
1. ELECTRICAL NOISE IN PHYSICS AND BIOLOGY
33
the number of channels, N , can be estimated. Elimination of p from Eqs. (71) and (72) results in the quadratic relation between u: and ( I ) : cr: = i ( / ) - ( I ) 2 / N
(74)
Eq. (74) allows determinations of N and i by measurement of the temporal variation of cr: and ( I ) from nonstationary currents, as was introduced by Sigworth (Fig. 2). The advantage of this method is that p need not be evaluated separately, and from determination of N and i, by application of Eq. (74) to data and by use of Eq. (71), p ( t ) can be determined without assumption of a specific gating model. The main disadvantages of the method come from the loss of spectral analysis as an interpretive tool. Specifically, contributions from extraneous noise sources (l/fand others) are difficult to assess and remove from estimates of cr;. Furthermore, preparation stability is essential in order that the procedure for extracting fluctuations in I from the temporal variation in ( I ) not produce artifactual fluctuations (Fishman, 1985). Finally, this method only yields interpretable information about nonstationary processes that conform to the set of assumptions stated above and is therefore not generally applicable, for example, to multiple conducting state or nonohmic channels. ACKNOWLEDGMENTS We thank Ms. Catheryne Randall for typing the manuscript. This work was supported in part by ONR Contract N00014-87-K-0055 and NIH Grant NSI 1764. REFERENCES Armstrong, C. M. (1969). Inactivation of the potassium conductance and related phenomena caused by quaternary ammonium ion injection in squid axons. J . Gen. Physiol. 54, 553-575. Bechem, M . , Glitsch, H. G., and Poot, L. (1983). Properties of inward rectifying K channel in the membrane of guinea-pig atrial cardioballs. PJuegers Arch. 399, 186- 193. Beck, A. H. W. (1976). “Statistical Mechanics, Fluctuations, and Noise.” HalstedlWiley, New York . Bennett, W. R. (1960). “Electrical Noise.” McGraw-Hill, New York. Blackman, R. B., and Tukey, J. W. (1958). “The Measurement of Power Spectra.” Dover, New York. Boyd, 1. A,, and Martin, A. R. (1956). The end-plate potential in mammalian muscle. J. Physiol. (London) 132,74-91. Brigham, E. 0.. and Morrow, R. E. (1967). The fast Fourier transform. IEEE Spectrum 4, 63-70. Brophy, J. J. (1969). Zero-crossing statistics of Ilfnoise. J. Appl. Phys. 40,567-569. Burgess, R. E. (1953). Contact noise in semiconductors. Proc. Phys. Snc. London B 66, 334-335. Cheng, D. K . (1959). “Analysis of Linear Systems.” Addison-Wesley, Reading, Massachusetts. Cole, K. S. (1972). “Membranes, Ions and Impulses,” 2nd ed. Univ. of Calif. Press, Berkeley. Conti, F., De Felice, L. J., and Wanke, E. (1975). Potassium and sodium ion current noise in the membrane of the squid axon. J . Physiol. (London) 248,45-82. Conti, F., Hille, B., Neumcke, B., Nonner, W., and Stampfli, R. (1976). Measurement of the conductance of the sodium channel from current fluctuation at the node of Ranvier. J . Physiol. (London) 262,699-727.
34
H. M. FISHMAN AND H. R. LEUCHTAG
W. ( I 965). An algorithm for the machine calculation of complex Fourier Cooky. J. W., and Tukey, .I. series. Muth. Cumput. 19, 297-301. De Felice, L. J. (1981). “Introduction to Membrane Noise.” Plenum. New York. del Castillo, 1.. and Katz, €3. (1954). Quanta1 components of the end-plate potential. J . Physiol. (London) 124, 370-384. Derksen, H. E . . and Verveen, A. A . (1966). Fluctuations of resting neural membrane potential. Science 151, 1388-1389. Dionne, V. E., and Leibowitz, M. D. (1982). Acctylcholine receptor kinetics. A description from single-channel currents at snake neuromuscular junctions. Biophys. J . 39, 253-261. Dorset, D. L . , and Fishman, H . M. (1975). Excess electrical noise during current flow through porous membrane separating ionic solutions. J . Membr. B i d . 21, 291 -009. Eigen, M., and de Macycr. L (1963). Relaxation methods. Tech. Org. Chrm. 8, X95- 1054. Eisenberg, R . S., and Mathias, R. T. (1980). Structural analysis of electrical properties. CKC Crit. Rev. Biorng. 4, 203-232. Fishman, H. M. (1973). Relaxation spectra of potassium channel noise from squid axon membranes. Proc. N o t / . Acud. Sci. 1J.S.A. 10, 876-879. Fishman. H. M. (1981). Matcrial from the internal sufiace of squid axon exhibits excess noise: Implications in modelling membrane noise. Biuphvs. J . 35, 249-255. Fishman, H. M. (1982). Current and voltage clamp techniques. In “Tcchniqucs in Cellular Physiology” (P. F. Baker, ed.), Part 11, pp. 1-42. Elsevier. Amsterdam. Fishman, H. M. (19x5). Relaxations. fluctuations and ion transfer acruss membranes. f r o g . Biophvs. Mot‘. B i d . 46, 127- 162. Fishman, H. M.. and Law, W. C . , Jr. (1987). Rapid acquisition and analysis of driving-point functions in nerve membrane. Proc. Annu. Conf. /EEE E n g . M f d . Biol. Soc.. 9th 2, 460-461. Fishrnan, H. M.. Poussart, D. J. M., and Moore. L. E. (1975). Noise measurements in squid axon membrane. J. Memhr. Biol. 24, 2x1 -304. Fishman, H. M., Moore, I.. E., and Poussart, D. (1981). Squid axon K conduction: Admittance and noise during short- versus long-duration step clamps. In “Thc Biophysical Approach to Excitable Systems“ (W. J. Adelman and D. E. Goldman, eds.), pp. 65-95. Plenuni. New York. &.hnran, H. M., Leuchtag, H. R., and Moore, L. E. (1983). Fluctuation and linear analysis of Na current kinetics in squid axon. Biophvs. J . 43, 290-307. Fishman, H . M., I.euchtag, ti. R., and Pousaart. D. ( 19x4). Nonlinear single-channel sodiumconductance in squid axon. Biophys. J . 45, 4 6 49. FitzHugh, R. ( 1965). A kinetic model of the conductance changes in nerve membrane. J . C d / . Cump. Ph~VSiCJ/.66, I 1 I - 1 18. Frehland, E. (1982). “Stochastic ‘Transport Processes in Discrete Biological Systems.” SprrngerVcrlag, Berlin. Gardner, M. (IY78). Mathematical games. Sci. A m . 238, 16--32. Green, M . E. (1976). Diffusion and Iif’noise. J . Mrmhr. B i d . 28, 181- 186. Gupta. M. S . (1977). “Electrical Noise: Fundamentals and Sources.” IEEE Press, New York. Husimi, Y . . and Wada, A. (1976). Time-domain measurement of dielectric dispersion as a response to pseudorandom noise. Rev. Sci. /ns/rum. 47, 213-219. Kottra, G.,and Fromter, E. ( 1984). Rapid determination of intraepitheiial resistance barriers by alternating current spectroscopy. I. Experimental procedures. f ‘ / / r r c y c m Arch. 402, 40%420. Lax, M., and Mengert, P. (1960). lnlluence of trapping. diffusion and recombination on carrier concentration Iluctuations. J . Phys. Chem. Solids 14, 248-267. Liebovitch, L., Fischbarg, J., and Koniarek. J. P. (1987). Ion channel kinetics: A model based on fractal scaling rather than multistate Markov proccsses. Muth. Biosci. 84, 37-68. MacFdrlanc. G . G . (1950). A theory of contact noise in semiconductors. Pruc. Phvs. Soc. London r$63.xo7-8~.
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35
Mandelbrot, B . (1983). “The Fractal Geometry of Nature.” Freeman, New York. Methfcsscl, C., and Boheim. G . ( 1982).The gating of single calcium-dependent potassium channels is described by an activationihlockade mechanism. Biophys. Srruct. Mech. 9, 35-60. Moczydlowski, E., and Latorre, R. (1983). Gating kinetics of Ca?+-activated K’ channels from rat muscle incorporated into planar lipid bilayers. Evidence of two voltage-dependent Ca2+binding reactions. J . Grn. Physi1~l.82, 51 1-54?. Nakamura, H., Husimi, Y.. and Wada. A. (1977). An application of Fourier synthesis to pseudorandom noise dielectric spectronietcr. J p n . J . Appl. Phvs. 16, 2301 -2302. Nakamura, H . , Husimi. Y . , and Wada. A . (1981). Time domain measurement of dielectric spectra of aqueous polyelectrolyte solutions at low frequencies. J . Appl. Phys. 52, 3053-3061, Ncher, E., and Stevens. C . F. (1977). Conductance fluctuations and ionic pores in membranes. Annu. Rev. Biophvs. Bioeng. 6, 345-381 . Neumcke. B. (1978). IifNoisc in nicmbranes. Biophys. Srruct. Mech. 4, 179- 199. Oliver, B. M., and Cage, J. M. (1971). “Electronic Measureinents and Instrumentation,” InterUniv. Electron. Ser. 12. McGraw-Hill, New York. Papoulis. A. (1965). “Probability. Random Variables, and Stochastic Processes.” McCraw-Hill. New York. Poussart, D. J. M . (1971 ). Mcnibrane current noise in lobster axon under voltage clamp. Biophy.~. J . 11, 21 1-234. Poussart. D. J. M. (1973). Low-level average power measurements: Noise figure improvements through parallel or seriea connection of noiay amplifiers. Rev. Sci. Insirurn. 44, 1049- 1052. Pousaart. D., Moore. L. E., and Fishman, H. M. (1977). Ion movements and kinetics in squid axon. I . Complex admittance. Ann. N.Y. Acad. Sci. 303, 355-379. Reif, F. (1965). “Fundarnentals of Statistical and Thermal Physics.” McCraw-Hill, New York. Robinson, F. N. H. (1974). “Noise and Fluctuations in Electronic Devices and Circuits.” Oxford Univ. Press (Clarendon), London and New York. Roux, B., and Sauvb, R. (1985). A gencral solution to the time interval omission problem applied to single channel analysis. Biophvs. J . 48, 149- 158. Sacha, F., Neil, J . , and Barkakati. N . (1982). The automated analysis of data from single ionic channels. F‘flurprs Arch. 395, 331 -340. Sakinann, R . , and Trubc, G . (1984). Voltage-dependent inactivation of inward-rectifying singlechanncl currents in the guinea-pig heart cell membrane. J . Physiol. (London) 347, 659-683. Sauvb, R . , and Szabo, G . (1985). lnterprctation of Iif’fluctuations in ion conducting membranes. J . Theor. Biol. 113, 501-516. Schottky. W. (1918). Spontaneous current fluctuations in various conductors. Ann. Phys. 57, 54 I -567. Sigworth, F. J. (1977). Sodium channels in nerve apparently have two conductance states. Nuture (London) 270, 265-267. Sigworth. F J . (1980). The viiriancc of sodium current Huctuations at the node of Ranvicr. 1. Phwiol. (London) 307, 97- 129. Sigworth. F. J. (1981). Covariance ot nonstationary sodium current fluctuation at the node of Ranvier. Biophvs. J . 34, I I I - 133. nd Chemistry.” Northvan Kampen, N. G (1981). “Stochastic Transport Processes in Physi Holland, Amstcrdam. van Vliet, K . M., and Faasett. 1. R. (1965). Fluctuations due to electronic transitions and transport in solids. I n “Fluctuation Phenoincna in Solids” (R. E. Burgess, ed.), pp. 267-354. Academic Press, New York. Warncke, J . , and Lindemann, B. (1985). Voltage dependence of Na channel blockage by amiloride: Relaxation effects in admittance spectra. J . Mernhr. B i d . 86, 255-265.
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CURRENT TOPICS IN MEMBRANES AND 'TRANSPORT, VOLUME 37
Chapter 2
Analysis of Transepithelial Noise Signals from Ion Channels: Advantages and Limitations of the Method WILLY VAN DRIESSCHE AND NOEL. VAN DEYNSE Lahorutorium voor Fysiologie Katholieke Universiteit Leuven, Gasthuisberg B-3000 Leuvm, Belgium
1. Introduction 11. Low-Noise Instrumentation 111. Spontaneous Noise Components A. Calcium Ion-Sensitive Channels in Amphibian Epithelia B. Chloridc Ion Channels in Frog Skin IV. Blocker-Induced Noise A. Noise from One Single Blocker B. Interaction between Blockers V. Limitations of the Method A. Model Dependency of the Analysis B. Driving Force C. Peaking, Frequency-Dependent Attenuation VI. Problenis Related to the Analysis of Blocker-Induced Noise A . Activity of the Blocker B. Dependence of the Block on Voltage VII. Conclusion References
1. INTRODUCTION Most studies of ion channels with noise analysis were done by recording the fluctuation in transepithelial current under voltage-clamp conditions. The reason for studying the fluctuation in current instead of the voltage noise is that, with ideal voltage-clamp conditions, the power densities of current and conductance are directly proportional, whereas the relation between voltage and conductance 37 Copyright (0 1990 by Academic Press. Inc. All right, of reproduction in any form rcservcd.
38
WlLLY VAN DRIESSCHE AND NOEL VAN DEYNSE
fluctuations is more complicated and depends on the mcmbrane impedancc. As the impedancc changes with frequency, the shape of the voltage noise spectra will be distorted by the frequency dependence of the impedance. This issue was extensively discussed in the early days of noise analysis (Fishman ef a/. , 1975), and since then the study of voltage noise was abandoned. Therefore, we confine ourselves in this chapter to the discussion of the analysis of the fluctuation in current. Noise analysis has been a very valuable method for the study of several ion translocation mechanisms in epithelia. The history of current noise analysis started with the discovery of the blocker-induced noise resulting from the interaction olamiloride with the Na' channel (Lindemann and Van Driessche, 1977). The analysis of these fluctuations allowed the calculation of the current through the single Na' channel and of the Na' channel density. This method has been applied in several studies of the apical Na' permeability, such as its hormonal activation (Helman et d . , 1983; Li er a / ., 1982); the effect of K ' dcpolarization of thc basolatcral membrane (Tang ct ul., 1985); and the effect of aldosterone (Palmer et ul., 19821, quinine, and quinidine (Abramcheck ef u l . , 1985). Sirnilarly, single-channel currents and channel densities were determined for the basolateral K + permeability (Van Driessche, 1986; Dawson et d . . 1988) and the K + conductance in the apical membrane of frog skin (Van Driessche and Zeiske, I980b). In addition to these studies of blocker-induced noise, fluctuation analysis methods contributed to the discovery or identification of several ion-transporting pathways such as the apical K' channels in the amphibian gallbladder (Giigelein and Van Driessche, 19Xlb); the apical K' channels in frog skin (Van Driessche and Zeiske, 1980a); CI channels in frog skin (De Wolf et ul., 1989);and cationselective Ca?+-sensitive channels in the apical mcmbrane of frog skin (Van Driessche and Zeiske, 198S), toad urinary bladder (Van Driessche el ul., 1987), and tadpole skin (Hillyard et u l . , 1982). In these studies noise analysis provided additional evidence for the transcellular localization of these pathways and for the fact that the translocation occurred through a channel-type structure. The first part of this chapter will deal with a brief overview of the experimental requirenients related to noise analysis. In the subsequent sections we will discuss the benetits of noise analysis i n studies of the spontaneous as well as blocker-induced fluctuations. Finally, the possible pitfalls and shortcomings of the method will be reviewed. ~
II. LOW-NOISE INSTRUMENTATION In studies of the transepithelial current noise, the tissue is clamped to 0 mV with a low-noise voltage clamp. Instrumentation noise, which limits the recording of transepithelial noise, originates from different components in the equip-
2. NOISE ANALYSIS IN EPITHELIA
39
Rrn Em
FIG. 1 . Noise sources in the voltage-clamp circuit. The membrane is represented by a simple resistor-capacitor (RC) circuit and an electromotivc force E,,,. Noise originates from fluctuation in R,,,.Ahbrcviations: DA, differential amplifier; FRA. feedhack amplifier; CA, current amplifier. Noisc sources: e,"and c,,, thermal n o w of voltage and current clectrodcs, respectively; e,,,, thermal noise of membrane; en and P , , equivalent voltage noise at the input of DA and CA; in and i, , equivalent current noise at the input of DA and CA. The instrumentation noise mainly originates from clamping the resulting voltage noise at thc input stage to zero.
ment (Fig. 1): ( I ) noise from the current-recording amplifier (CA) ( e , and i'), (2) thermal noise from the voltage-recording electrodes (e,"),and (3) noise from the input stage ofthe voltage amplifier (DA) of the feedback circuit (e,,and i n ) . As far as the current-recording amplifier is concerned, a low-noise operational amplifier can be used. The contribution of the noise of this amplifier is normally far below the noise originating from the two other noise sources. For example, for an Analog Devices AD707 with 100-kfl feedback resistor, the equivalent transepithelial current noise is 0.32 p A / a at 10 Hz. The thermal noise voltage, or Nyquist noise, of the voltage-sensing electrodes is proportional to the square root of its resistance. Careful design of low-resistance electrodes is therefore a prerequisite far this electronic circuitry. With relatively short, thick 1 M KCl agar bridges, combined with Ag-AgC1 wires, the resistance per electrode can be kept below 2.5 kfZ. The thermal noise for such an electrode is 6.3 n V i a . Voltage clamping this noise to zero will result in a transepithelial current noise that depends on the impedance of the preparation. For a 3-kR preparation, the resulting current noise is 2.1 p A / f i , which is an order of magnitude larger than the equivalent current noise of the current amplifier. The third noise source constitutes the major contribution to the instrumentation noise in the feedback circuit. The design of this input stage must therefore focus on the reduction of the noise levels in this part of the circuit. Therefore it is highly recommended to utilize low-noise discrete field-effect transistors (FETs) (e.g.,
40
WlLLY VAN DRIESSCHE AND NOEL VAN DEYNSE
SK146, Toshiba), which have better noise characteristics than do low-noise operational amplifiers. These FETs have low equivalent input current noise (in), which will generate voltage noise at the input stage that is proportional to the input impedance of the circuit ( e , = inZJ. Typically, i,, is 0.01 PA/at 10 Hz; Z, consists of the impedance of the epithelial preparation (2,) and voltage electrodes (2").As Z, and Z , are usually small (maximally lo5 the equivalent voltage ( e , ) will be smaller than the equivalent voltage noise of the FETs (en) and the thermal noise of the voltage electrodes. Typically, en is of the order of 3-7 n V / a at 10 Hz, which is of the same order of magnitude as e,".The latter will be kept as small as possible by designing low-resistance electrodes to measure the transepithelial potential. With input resistances of epithelial tissues of less then 10 k f l and carefully designed recording electrodes, the equivalent voltage noise source of the input FETs (e,,)constitutes the major instrumental limitation for noise recordings. As the current noise of the input FETs accounts for a contribution that is several orders of magnitude less, the input stage can be improved by utilizing a parallel arrangement of FETs. This method will increase the equivalent current noise and lower the equivalent voltage noise. Current noise signals are analyzed after removing the dc component of the transepithelial current. The remaining fluctuation current noise is then sufficiently amplified with standard low-noise operational amplifiers to reach levels in the voltage range that can be accurately digitized with standard analog-todigital converter boards. The power density spectrum of the digitized signal is then calculated by fast Fourier transform methods. In order to reduce statistical scatter, spectra of subsequently recorded time sweeps are averaged. With presently available personal computer systems, the calculations require less than 1 sec per sweep for a sequence length of 1024 points. The time required to collect the data depends on the fundamental frequency of the Fourier analysis (e.g., 5 sec/sweep for a fundamental frequency of 0.2 Hz). Current noise spectra recorded from voltage-clamped epithelia may contain three different noise components (see Fig. 2): ( I ) low-frequency noise or Ilf noise, ( 2 ) Lorentzian or relaxation noise components, and (3) amplifier noise. In most current noise spectra recorded from epithelia, the three noise components are present. The general theoretical background underlying the low-frequency and Lorentzian noise is discussed by Fishman and Leuchtag in Chapter 1 of this volume. The low-frequency noise dominates at the lower frequency end of the spectrum. Its origin is in most studies not clear, although it seems to be correlated to the rate of transepithelial transport. Most interest was given to the Lorentzian noise components that originate from interruptions of currents passing through ion channels that open and close randomly. The fluctuations can be either spontaneous, or caused by a compound that randomly interacts with the channel structure and causes random flickering of the channel, i.e., blockerinduced noise. We will discuss the spontaneous and blocker-induced noise com-
a>,
41
2.NOISE ANALYSIS IN EPITHELIA
I
I
1
10
100
1000
f Hz FIG. 2. Noise components in a typical power density spectrum. The low-frequency (LF) noise dominates at the low-frequency end and is generally described by the following equation: S,,(f) = Alp, where a (usually between 1 and 1.7) is the slope of the line in the double-logarithmic plot. The Lorentzian component S,.[see Eq. (2)] originates from interruptions of the current through channels that open and close randomly. The amplificr noise is caused by clamping the equivalent voltage noise at the input of the differential amplifier to zero. The increase in amplifier noise results from the decrease of the membrane impedance at higher frequencies because of the membrane capacitance.
ponent in the next two sections. Finally, the amplifier noise surpasses the Lorentzian as well as low-frequency noise at the higher frequency end. The increase in instrumentation noise is a result of the decrease of the transepithelial impedance caused by the capacitive shunt of the membrane. Because of the capacitive reactance, the currents, needed to clamp the noise voltage at the input stage of the voltage amplifier to zero, will increase at higher frequencies. For epithelial preparations the instrumentation noise usually surpasses the membrane noise at frequencies above 500 Hz.
111. SPONTANEOUS NOISE COMPONENTS The term “spontaneous” noise components is used to refer to Lorentzian noise components caused by fluctuating ion channels, for which the gating mechanism is unknown, in contrast with the opening and closing caused by the random interaction of a known, usually externally administered, blocker molecule with a receptor site of the channel. The latter, blocker-induced noise, will be discussed in Section IV. In several studies the occurrence of a spontaneous noise component has been favorably exploited for the study of ion pathways in the cell membrane of epithelia. The presence of this noise type was considered as evidence for a channel-type pathway for the macroscopic current. The fluctuation of the macroscopic current would result then from the random opening and closing of the channels, a phenomenon observed in patch-clamp studies (see
42
WlLLY VAN DRIESSCHE AND NOEL VAN DEYNSE
Chapters 3, 7, and 8). The spectrum of the current passing through one single or an ensemble of fluctuating channels has a Lorentzian shape (Verveen and DeFelice, 1974). However, it is conceivable that Lorentzian noise arises from other mechanisms that modulate the current flow across cell membranes in a stochastic way. For example, a random fluctuation of the driving force between two discrete values could modulate the macroscopic current. The random steps of the driving force would then result in discrete jumps of the total current across the cell membrane of at least one ccll. Such a stepwise random change in driving force is unlikely because of the extremely high degree of cooperativity required. This is very unlikely, as cells in epithelial tissues have an excellent electrical as wcll as chemical coupling. So the random interruption of currents through fluctuating channels is the only mechanism for which electrophysiological evidence exists to explain the presence of a Lorentzian component in the power density spectrum. Moreover, until now such fluctuating channels have only been demonstrated in cellular membranes. Presently, no evidence has been provided for a localization of such channels in the paracellular pathway. However, uncertainty still exists about the localization of the CI- channels that are activated by voltage (Willumscn and Hviid Larsen, 1986), forskolin (De Wolf rt d., 1989), and isoproterenol (Thompson and Mills, 1981). Lorentzian noise associated with the forskolinactivated (De Wolf rt a/.. 1989) as well as thc isoproterenol-activated (M. Zizi, unpublished observations) Cl- currents has been reported recently, Most cvidence points toward a localization in the mitochondria-rich cells, but a paracellular localization of the fluctuating CI - channels cannot be excluded completely. Noise analysis is especially advantageous in conditions wherein spontaneous Lorentzian noise is detected with macroscopic currents that are small and therefore difficult to analyze. Such situations occurred in noise analysis studies of K + channels in the amphibian gallbladder (Van Driessche and Gogelein, 1978) and Ca' -sensitive cation-selective channels in the apical membrane of frog skin (Van Driessche and Zeiske, 1985). Changes in transport rate can then be monitored by comparing the magnitude of the Lorentzian plateau. A remarkable case is the amphibian gallbladder, in which the transepithelial open-circuit potential and thus also the short-circuit current are very small. Even with short-circuit currents close to zero, a Lorentzian noise component originating from K + efflux through the apical membrane could be demonstrated. This is discussed in detail by Zeiske in Chapter 5 of this volume. An overview of the channels for which a Lorentzian noise component has been demonstrated is given in Table 1. For a detailed discussion of the characteristics of the K Lorentzians, see Chapter 5 in this volume. In the following two sections we will briefly discuss the spontaneous noise associated with currents passing through Ca?+-sensitive cation-selective channels and through CI- channels. +
43
2. NOISE ANALYSIS IN EPITHELIA TABLE I CHARACTEKN ICS or SPONTANEOUS LOKENTZIAN NOISECOMPONENT
so Channel type
Tissue
Membrane (A'.sec/cm2)
F,
(Hz)
Reference
Frog skin
Apical
15.0
81.0
Amphibian gallbladder
Apical
69.0
4.3
Frog skin
Apical
44.9
160.8
Todd bladdcr
Apical
5.7
563.4
Tadpole hkin
Apical
I .o
33.4
Hillyard ef nl ( 1982)
-
4.1
61.1
De Wolf ~f crl (1989)
Frog skin
Van Driessche and Zeiske (1980a) Gogelein and Van Driessche (1981b) Van Driessche and Zeiske (1985) Van Driessche et a / . (1987)
For both transport systems the presence of spontaneous Lorentzian noise provided evidence for a channel type of pathway for ion translocation and presumably for its cellular localization.
A. Calcium Ion-Sensitive Channels in Amphibian Epithelia Calcium ion-sensitive channels were found in the apical membrane of several amphibian epithclia: frog skin [Rana temporaria (Van Driessche et ul., 1989) and R a m catesbeiuna (Van Driessche and Zeiske, 1985)], toad skin (Bufo marinus), toad urinary bladder ( B . marinus), and tadpole skin ( R . catrsbeiunu). Under many experimental conditions, the macroscopic currents were very small (between I and 2 pA/cm2, depending on the ion species) and therefore were difficult to study. However, the Lorentzian components were clearly above the background noise and changes in Lorentzian plateau values were easy to study. The Lorentzian parameters for these channels are summarized in Table 1. Except for the tadpole skin, these channels are closed when the apical surface is exposed to normal Ca' -containing Ringer solutions. On complete removal of Ca?' and other divalent cations froni the mucosal bathing media, this channel allows the passage of several monovalent cations (Na+, K + , Rb', Li', CS+, and NH2). In frog skin we found two types of cation-selective channels. Type 1 is highly sensitive to Ca'+, with a Michaelis-Menten constant K,, = 30-50 nmol/liter; for type I I , K,,, = 30-60 pmollliter. The K,,, for inhibition of the cation-selective channels in the toad bladder resembles the K, of the type 11 channels in the skin.
44
WlLLY VAN DRIESSCHE AND NOEL VAN DEYNSE
f
Hz
FIG. 3 .
Spontaneous Lorentzian noise originating from the opening and closing of Ca'tsensitive channels in the apical membrane of frog skin. The serosal bathing medium was NaC1Ringcr's, and the mucosal side was exposed to a solution of either NaCl or KCI-Ringer's. Thc and a Lorentzian function (S,.), continuous line represents a sum of the low-frcquency noise (S,,,.) which was fitted to the data points.
Typical spontaneous Lorentzian components recorded in an experiment with frog skin exposed to Ca2+-frecmucosal Na+ and K + solutions are shown in Fig. 3 . Recently we found that Ca2+ induces additional fluctuation in the cation current through the type l channels in frog skin. This results in a blocker-induced Lorentzian component (Van Driessche et al., 1989). As a detailed description of this pathway is beyond the scope of this chapter, we refer the interested reader to the original papers and reports on this subject (Aelvoet et al., 1988; Desmedt et ul., 1989; Van Driessche et uf., 1989).
B. Chloride Ion Channels in Frog Skin Chloride ion currents through the amphibian skin were recorded after activation of the pathway using voltage, theophylline, procaine, or forskolin. Spontaneous Lorentzian noise was recorded from the forskolin-activated pathway (De Wolf et ul., 1989). The changes in transepithelial current and Lorentzian plateau were correlated. The currents as well as the Lorentzian component were depressed by CI- channel blockers and an elevation of the driving force for Claugmented both parameters. This suggested that the currents and Lorentzian originated from CI - movements through fluctuating CI- channels. The presence of the Lorentzian was taken as evidence for a transcellular localization of the C1- pathway.
IV. BLOCKER-INDUCED NOISE The first study in which blocker-induced noise in epithelia was demonstrated was published in 1977 by Lindemann and Van Driessche. The authors demonstrated for the Na channel that a reversible blocker interrupts the current
2. NOISE ANALYSIS IN EPITHELIA
45
through the channel randomly, which causes fluctuation in the macroscopic current. The analysis of this current noise revealed a Lorentzian component that corner frequency linearly increased with blocker concentration. Similarly, blockerinduced noise was demonstrated for the interaction of Ba2+with the K + channel in the apical membrane of frog skin (Van Driessche and Zeiske, 1980b) and for lidocaine, quinine, and quinidine with the basolateral K + channel in the toad bladder (Van Driessche, 1986) and the tadpole skin (Van Driessche and Hillyard, 1985). The analysis of blocker-induced fluctuations is based on theories dealing with the kinetics of chemical systems (Colquhoun and Hawkes, 1981). To illustrate the application of this theory we will first describe the simple two-state model based on first-order kinetics and the interaction of two blockers with the receptor of the channel. However, more complicated reaction schemes for the blocker-receptor reaction are conceivable. For example, the occlusion of the channel could require the binding of two blocker molecules to the channel receptor, or the channel has, in addition to the open and blocked state, a spontaneously closed state. The latter model was used to analyze the interaction of Ba?+ with the apical K' channel in frog skin (Van Driessche and Zeiske, 1980b) and was proposed for the interaction of amiloride with the Na+ channel (Lindemann and Van Driessche, 1978). Evidence for the three-state K + channel model comes from the recorded power density spectra: spontaneous Lorentzian noise is recorded in the absence of a blocker molecule, whereas double Lorentzians are recorded in the presence of small blocker concentrations. The justification of the three-state model for the Na' channel came from the concept of Na' selfinhibition, which suggested a spontaneous open-close transition for the Na' channel. More recently this hypothesis has been confirmed with patch-clamp recordings (Hamilton and Eaton, 1985), which showed spontaneous channel gating. A detailed analysis and application of the three-state model to channel density and single-channel current calculation has been done recently by Helman and Baxendale (1988).
A. Noise from One Single Blocker The simplest model to describe random interruptions of the current through a single channel is the two-state model, having one open and one blocked state. In the conducting state the blocker does not occupy the channel, whereas the channel is occluded when the blocker made a complex with the channel receptor:
Chemical rate theory (Colquhoun and Hawkes. 1981) predicts that the number of Lorentzian components equals the number of states of the channel minus one.
46
WlLLY VAN DRIESSCHE AND NOEL VAN DEYNSE
So, with the assumption of the above simple two-state model, the power density spectrum of the fluctuation in current consists of a single Lorentzian function:
Assuming that density of receptors ([RI) is negligible to the concentration of blocker (IB I), the Lorentzian plateau (S,,) and the corner frequency (f;), or chemical rate (2rrf,),can be calculated as:
where M represents the channel density, i is the single-channel current, P,>is the probability for the channel being in the open state, and PI is the probability to find the channel in the closed state:
Equation ( 3 ) allows the calculation of the chemical rate constants in a very elegant way from the linear shift of the corner frequency with the blocker concentration. This has been applied for the interactions of amiloride (Lindemann and Van Driessche, 1Y77), CGS 4270 (Abramcheck p t c r l . , 198S), and 6-chloro-3,5diaminopyrazine-2-carboxamide (CDPC) (Helman and Baxendale, 1988) with the Na' channel in frog skin; of Ba?+ with the apical K + channel in frog skin (Van Driessche and Zeiske, 1980b); and of lidocaine (Van Driessche, 1986), quinine, and quinidine (Van Driessche and Hillyard, 1985) with the basolateral K + channel in the urinary bladder and tadpole skin. Except for amiloride, k,,, as well as k , , ,can be determined with sufficient accuracy. The error on the estimate of k , , , for amiloride is considerable because amiloride-induced noise can only be recorded at amiloride concentrations far above the Michaelis-Menten constant ( K , = / ~ , ~ / k , ,For , ) . these concentrations, k,,, becomes much smaller than ko,[B] in Eq. (3), and its estimate obtained from linear regression analysis of the 2rrfc and [B] relation becomes erroneous. Therefore, in several studies k,,, was calculated from the K,,, value obtained from the dose-dependent inhibition of the macroscopic current, whereas k,,, was obtained from linear regression of Eq. (3). Once k,,and k , , , are determined, the single-channel current ( i ) and the channel density (M),can be calculated from Eq. (4) and the expression for the macro-
47
2. NOISE ANALYSIS IN EPITHELIA
scopic current ( I ) : I = MiP,,
(7)
By rearranging Eqs. (4)-(7), we obtain:
and M
= 2~rAll(ik,,,)
It is clear from Eqs. (8) and (9) that the uncertainty on k,,, in the experiments with amiloride does not affect the results for i, but causes large errors in the channel density ( M ) . The advantage of this analysis is that all parameters used are recorded in the same electrophysiological experiment and that because of the simplicity of the model, no further assumptions of other model parameters are required. A prerequisite for its application is the assumption of pseudo-first-order kinetics for the blocker-receptor interaction. This assumption is not valid if the parameters of the reaction scheme depicted in Eq. ( 1 ) are influenced by other transitions of the channel. Indeed, if the channel fluctuates spontaneously between an open and closed state, these transitions will influence the occupation probabilities and thus the number of channels available for blockage by the blocker molecule. In the latter situation a more detailed three-state model analysis might be required. Such an analysis was applied by Helman and Baxendale (1988) to the amilorideNa' channel interaction in frog skin. The coupling of the blocker-receptor interaction to another transition of the channel between the open and closed state will not only influence the amount of available channels for blockage, but also the kinetics of the blocker-receptor reaction. As pointed out by Van Driessche and Lindemann ( l 9 7 9 ) , the amiloride kinetics are not significantly altered by the spontaneous transitions of the Nai channel between its open and closed state. This can be explained by the low rate of the spontaneous open-close reaction of the Na' channel. The analysis of the interaction of two blockers with the channel receptor described in the following section will be illustrated by calculating the corner frequencies of two coupled chemical reactions.
B. Interaction between Blockers To illustrate the influence of the coupling of two first-order reactions as depicted in Eq. ( I ) , we will consider the interaction of two competitive blockers
48
WlLLY VAN DRIESSCHE AND NOEL VAN DEYNSE
1000 150
2TLf,CGS 2lTfpM'
500 I5
5-1
5 -1
L
OO
0 lAM11,
polll
160
80
[CGSI,
pmolll
FIG.4. Corner frequency versus blockcr concentration for amiloride (A) and CGS 4270 (B). The on- and off-rates determined by linear regression were for CGS 4270 [kal = (3.18 f 0.37) pmol I sec I * liter and k20 = (272 ? 36) sec-'1. The on-rate for amiloride was k,, = (22.3 ? 0.9) prno1-l sec- liter.
for the Na+ channel: amiloride and CGS 4270. Both compounds inhibit the Na' current in a reversible way and induce additional fluctuation in the Na+ current, CGS 4270 was used in studies of the effect of quinine and quinidine on apical N a t permeability (Abramcheck et a l . , 1985). The corner frequencies for both blockers defer markedly, thefc values for CGS 4270 being an order of magnitude larger than those for amiloride. This makes the analysis of the noise induced by CGS 4270 much easier than the amiloride noise: (1) the Fourier analysis can be made in a higher frequency range, which reduces the recording time considerably and (2) the interference with low-frequency noise (Ilf noise) is smaller for CGS 4270 than for amiloride. Figure 4 compares the relation between fc and the concentration of amiloride and CGS 4270 for the blockage of Na' channels in R . temporaria. The rate constants obtained from linear regression analysis differ markedly. Whereas the off-rate for amiloride is small and difficult to derive, this parameter can be determined accurately for CGS 4270. The Michaelis-Menten constants calculated from the inhibition of the macroscopic current were K k = (0.12 rfr 0.01) pmoll liter and K$ = (23.8 ? 6.4) pmol/liter for amiloride and CGS 4270, respectively. In order to study the interference between amiloride and CGS 4270 we calcu-
49
2. NOISE ANALYSIS IN EPITHELIA
lated the corner frequencies of the fluctuation in current resulting from the simultaneous blockage of the channel assembly by both compounds, the kinetic equation for this interaction being
where, in state 0 the channel is in its open state, in state 1 (AR) the Na' channel is occupied by amiloride, and in state 2 CGS 4270 is blocking the channel. According to chemical rate theory, the corner frequencies of this system can be calculated as eigenvalues of the A-matrix (Chen and Hill, 1973) of transition probabilities, which is for this system:
The chemical rates thus calculated are
where r , and r2 represent the rates (27rL) of the individual reactions without competition
and p is the shift of the rates caused by the coupling of the two first-order reactions p = 0.5(r,
-
r 2 ) + d{(ri
+ rd2
-
4k,,k2,(l
+
[AUK,
+
[C]/K,)}
(16)
where K A = ki,,/koiand Kc = kzolk(,2.Equations (12) and (13) show that the coupling causes an equal but opposite directed shift of the corner frequencies. Therefore, it is easier to monitor the shift, p , of the smallest corner frequency. To test the above theory we determined the shift of the amiloride-induced Lorentzian, recorded with 2 pmol/liter amiloride, in experiments with varying doses of CGS 4270. Figure 5 shows the arniloride-induced noise spectra recorded in the absence and in the presence of CGS 4270. In the control (without
50
WlLLY VAN DRIESSCHE AND NOEL VAN DEYNSE
. ,CGS
4270
10-2' *.
1
10
f
100
1000
HZ
FIG. 5 . Interaction between amiloride and CGS 4270. The skin of Runu femporcrriu was incubaled with NaCI-Ringer solution on both sides. The control spectrum, which was recorded with 2 pmolilitcr rnucosal amiloride. consisted of a single Lorentzian. The spectrum labeled with CGS 4270 was recorded after addition of 100 fimoliliter CGS 4270 to the amiloride-containing solution. This yxctrum consisted of two Lorentzian components, induced by airdoride (low frequency) and CGS 4270 (high frequency).
CGS 4270), one single Lorentzian induced by amiloride is present in the power density spectrum. In the presence of 100 prnol/liter CGS 4270, an additional Lorentzian appears and the amiloride-induced Lorentzian is shifted toward lower frequencies. Figure 6 shows mean values of the shift of the corner frequency of the amiloride-induced Lorentzian recorded from five experiments. The on- and off-rates for CGS 4270 (k,,, and k?,,) in Ey. (13) can be determined accurately from the analysis as shown in Fig. 4B. Also, the on-rate for amiloride can be determined with sufficient accuracy. However, the uncertainty on the off-rate of
0
50
[CGSI,
100
150
pmolll
FIG. 6 . Shift of the comcr fretliicncy o f the niltiloride-induced 1.orentriaii by addition of diffcrent doses of CGS 4270 to the iriucosal medium. The solid line was obtained by fitting Eq. (16) to the data points. In the lit procedure we assumed the I;,,!, A,,). and I;,,, values of the analysia in Fig. 4 and determined the unknown off-rate for amiloride. k , ( , , by nonlinear regression.
2. NOISE ANALYSIS IN EPITHELIA
51
amiloride is considerable (see Fig. 4A). We therefore used the data in Fig. 6 to determine k,, by nonlinear curve fitting. We assumed the values for ko,, kzo, and k,,? from Fig. 4, so that k,,, remained the only unknown in Eq. (16). We found the best fit for k , , , = 4 sec-’ so that the Michaelis-Menten constant K , = k,,/k,,, was 0.179 p.mol/liter. The latter value agrees rather well with the Michaelis-Menten constant obtained from the macroscopic current inhibition, which was in this series of experiments 0.1 12 pmol/liter.
V. LIMITATIONS OF THE METHOD The application of noise analysis has several limitations that will be discussed in the following sections.
A. Model Dependency of the Analysis Studies aimed at the calculation of the single-channel current and the channel density use chemical reaction schemes as previously illustrated by the two-state model and the interaction between two blockers. The assumption of such models is based on the knowledge of the kinetics of the channel system or on the appearance of the Lorentzian components in the power density spectrum. For example, initially the blockage of the apical Na+ channel was modeled with pseudo-first-order kinetics (Lindemann and Van Driessche, 1977). More recently Helman and Baxendale (1988) used a three-state model for the analysis of their noise data. The justification of the three-state model comes from the apparent changes in channel density with blocker concentration observed when the analysis is based on the two-state model and from the Na’ self-inhibition hypothesis proposed by Lindemann and Van Driessche (1978). Another system wherein a three-state model was used to describe the channel kinetics is the interaction of Ba” with the apical K + channel in frog skin. Here the assumption of the three-state model is based on the appearance of two Lorentzian functions in the power density spectrum. One of the Lorentzians was already observed in the absence of the blocker and was therefore described as “spontaneous.” This spontaneous Lorentzian could be seen together with the blocker-induced component at small Ba? concentrations. It is conceivable that for some systems the kinetic interaction is more complicated than the simple two- or three-state model. As chemical rate theory predicts that the number of Lorentzian components is equal to the number of states minus one, the spectral analysis could show the presence of additional steps in the reaction scheme. However, Lorentzian components caused by some of the steps could be too small to be resolved in the spectral analysis, or the corner frequency could be too small or too large to be found in the frequency range of the analysis. The underestimation of the complexity of the system will cause then errors in the calculations of channel densities and single-channel currents.
52
WlLLY VAN DRIESSCHE AND NOEL VAN DEYNSE
8. Driving Force Noise analysis allows the calculation of channel density and single-channel current, as illustratcd previously with thc assumption of a two-state model, without necessity of knowing the driving force for ion movement. However, an estimate of the latter parameter is required to determine the single-channel conductance. In most studies, values for cell potential as wcll as intraccllular ion concentrations from independent experirncnts were assumed in the calculations. This could lead to considcrable errors because of differences in the experimental conditions used. Moreover, the driving force could change during thc experiment in which the blocker concentration was increased. For example, in studics of the apical Na+ channel, thc intracellular potential will hypcrpolarize as the amiloride conccntration is increased. This will also cause a change of the singlc-channel current with blocker concentration. Similar effects can be expectcd in studics of other pathways. e.g., the apical K ' channels with Ba?' . Indeed, Ba2+ added to rriucosal K + Ringer's causes a dose-dependent hypcrpolarization of the intracellular voltage and thus a varying driving force for K + movcments.
C. Peaking, Frequency-DependentAttenuation As pointed out in Section 1, noisc analysis studies are done under voltageclamp conditions to monitor the fluctuation in currcnt. This rcquires that the resistance in scries with the investigatcd membrane is small compared to the cell mernbranc resistance. The serics resistancc will decrease the current requircd to clamp the membrane at the imposed potential and also thc amplitude of the current fluctuations. To illustrate the effect of a series resistance on the noise amplitudes at the lower frequcncy limit, we calculated the current fluctuations ( A l ) caused hy the fluctuation in conductance of the apical membrane in a membrane model (Fig. 7) clarnped at 0 niV.
Here Ag;, is the fluctuation in apical membrane conductance, R , rcpresents thc resistance of the bathing solution in series with thc epithelium, R, is thc shunt resistance, and K,,and R, are thc apical and basolateral membranc resistances, respcctively. For tight epithelia R , is smaller than R,,. so that Eq. (17) simplifies to
If the basolatcral membrane resistance is much smaller than R , ,
Af, heconies
53
2. NOISE ANALYSIS IN EPITHELIA
Signal from
PRNG
FIG. 7. Equivalent circuit used to simulate the attenuation of the noise signals. R, and C',, represent the passive clcctrical equivalcnt components of the basolatcral membrane. R,, is the paracelMar shun1 resistance and R, is the resistance of the bathing solution between the voltage clcctrodes. R,,, C,, and E,,represent thc cquivalent electrical parameters of the apical membrane. The fluctuation in conductance of thc apical memhrane is similatcd by randomly switching the FET from its conducting to its nonconducting statc. The signal imposed on the FET consisted of rectangular pulses derived from a pseudorandom n o i x generator (PRNG). The spectrum of this pulse train had a Lorcntzian shape.
equal to the fluation A/,,, expected under ideal voltage-clamp conditions:
The square of the ratio of Al,,, by A/, represents the attenuation factor of the power density of the current noise signal:
In epithelia, noise analysis aims at the study of conductance fluctuation of one of the cell membranes, e.g., fluctuation in conductance of the apical menibrane caused by the interaction of amiloride with the Na channel. As the basolateral membrane resides in series with the apical membrane, the attenuation of the noise signal will increase with decreasing fractional resistances [jRo = R,/(R;, R,)] (Gogelein and Van Driessche, 1981b). For studies of the amiloride-induced noise, ,@,, approaches I at higher amiloride concentrations. As the studies of amiloride-induced noise utilize amiloride concentration far above the K,,,, ,@,, will be close to 1 and as consequence the attenuation of the noise signals will be negligible (A = I ) . On the other hand, spontaneous noise signals from channels in cells with comparable apical and basolateral resistance will be considerably attenuated. Such a situation could arise when the basolateral membrane +
+
54
WlLLY VAN DRIESSCHE AND NOEL VAN DEYNSE
resistance was increased, for example, by removal of C1- from the basolateral solution. So far we have considered only a frequency-independent attenuation of the noise, which will cause a constant downward shift of the noise amplitudes over the whole frequency range. However, if the noise source and/or the series resistance are shunted by a capacitor, the attenuation will become frequency dependent. This is thc case in epithelia in which the apical and basolateral impedance have a reactive, capacitive component. Thus the impedance of the noise source (apical membrane) as well as of the series element (the basolateral membrane) will change with frequency. Therefore, the attenuation of the noise signals will be frequency dependent, which results in a change of the shape of the power density spectrum. We simulated the effect of the capacitive shunting of the membrane and its series resistance with a simple electronic circuit based on the model in Fig. 7. The conductance changes of the apical membrane are simulated by randomly switching a field-effect transistor between its conducting and nonconducting state. In this way, a conductive pathway with resistance R( (100 kiZ), parallel to R,,,,is opened and closed randomly. The command signal for the FET was derived from a pseudorandom binary noise generator with a sequence length of 2” I . The spectrum of the pseudorandom noise sequence had a Lorentzian shape (curve 1 in Fig. 8). The circuit in Fig. 7 was connected to a low-noise voltage clamp and power density spectra were recorded. Figure 8 comparcs the shape of the spectrum of the unattenuated pseudorandom noise sequence with those recorded with two different sets of equivalent membrane parameters. In set A (curve 2 in Fig. 8), we used R, = 4 kf2, R , = 1.33 kR, C , = 4.7 p F , C, = 13.2 pF, R, = 200 R, and R,, = 2 k i l ; in set B (curve 3 in Fig. 8) we used the same parameters as in A with exception of R,, = 8 kfi. In the lowfrequency limit. the attenuation (square 0 1 A I J A I , ) calculated with Eq. (17) was 2.3 for model A and 1 I . I for model B, which results in a downward shift of both spectra over the cntirc frequency range. Note that the curve I spectrum was recorded by direct analysis of the signal from the pseudorandom noise generator and its absolute amplitudes are therefore not comparable with curve 2 and curve 3 spectra recorded under voltagc-clamp conditions. With Fig. 8 we do not intend to show the dc attenuation, which can be calculated with Eq. (17), but we illustrate the frequency dependence of the attenuation, which might cause, under soirie conditions, distortions of the power density spectrum. No signiticant distortion of the power spcctrum was observed with model A, up to frequencies of 150 Hz. At higher frequencies the attenuation of the spectral values became larger than the dc attenuation, which results in an additional downward shift of the power densities. This is caused by the decrease of the impedance of the epithelium to values below or comparable to R,. A larger distortion of the power density spectrum was observed with model B. Here, we observed “peaking” in the power density spectrum. This is caused by the fre~
2. NOISE ANALYSIS IN EPITHELIA
f
Hz
FIG.8.
Power density spectra recorded by voltage clamping the equivalent circuit in Fig. 7. Curvc 1 is the power density spectrum of the signal from the pseudorandom noise generator and is shown to compare the shapes of this original powcr density spectrum with spectra recorded by voltage clamping the equivalent circuit and applying the noise signal to the FET (curves 2 and 3). The absolute noise amplitudes of curve I . however, arc not directly comparable with those of curves 2 and 3.
quency-dependent change in the impedance of apical and basolateral membranes. In model B the decrease in basolateral membrane impedance will occur at lower frequencies than does the decrease in apical membrane impedance. Indeed, the time constants of the apical and basolateral membranes are comparable in model A (apical, T , = 18.8 msec; basolateral, T , = 17.6 msec), whereas in model B 7, is much larger than T, (7, = 105.6 msec). The decrease in basolateral membrane impedance will reduce the attenuation and elevate the spectral densitics. The "peak" results from the subsequent drop of the spectral density at frequencies above the corner frequency of the Lorentzian. Such spectra were recorded by Giigelein and Van Driessche ( 1981b) in experiments with Necturus gallbladders. The results were correlated with the changes in impedance of this preparation (Giigelein and Van Driessche, 1981 a).
VI. PROBLEMS RELATED TO THE ANALYSIS OF BLOCKER-INDUCED NOISE A. Activity of the Blocker The chemical rate of the blocker-receptor interaction is, according to Eq. (3), proportional to the blocker concentration. When in solution, many substances have different chemical forms. For example, amiloride has a pK of 8.9 and at pH 7.5 is predominantly protonated. It is assumed that this form of the drug is interacting with the Na+ channel (Lindemann and Van Driessche, 1978). Con-
56
WlLLY VAN DRIESSCHEAND NOEL VAN DEYNSE
sequently, the concentration of the active form of amiloride is approximately equal to the dissolved amount. For other blockers, the dissolved amount and conccntration of the active form might, however, differ considerably. For example, lidocaine, which blocks basolateral K ’ channels (Van Driessche, 1986), is a weak base with a pK of 7.9 (Dawson et al., 1988). It is presumed that the nonprotonated form crosses cell membranes rapidly so that, when administered rnucosally, the drug can reach the basolateral membrane rapidly, where its protonated form occludes the K channels. Consequently, the determination of the on-rate from the shift in corner frequency by linear regression analysis of Eq. (3) has to take the differences in the active and dissolved concentrations into account. These differences will, according to Eq. ( 3 ) , only affect the estimate of the on-rate ( k 0 , )and leave the offrate ( k , , , ) unaltered. Based on the Henderson-Hasselbalch equation, Dawson et ul. (1988) calculated the effect of the pH on the on-rate and the MichaelisMenten constant for lidocaine: +
Similar problems are encountered when using triamterene to induce fluctuation in Na + current (Zeiske and Van Driessche, 1986). This compound is a weak base with a pK of 6.2. The concentration of the protonated form of this compound will therefore change considerably in the physiological pH range. Also, changes in the local pH in the solution layer adjacent to the membrane will affect the concentration of the active form and thus the corner frequency. This was observed in experiments when using bicarbonate, which has only a small buffering capacity. Corner frequencies changed irregularly as the concentration of the blocker was raised, probably due to effects of the inhibition of Na’ transport on the proton transport and therefore on the local pH at the membrane surface. The irregular changes in corner frequency were not observed when buffers such as HEPES were utilized, which are able to clamp the pH in the range around 7.5.
6. Dependence of the Block on Voltage If a blocker needs to be charged to interact with the channel receptor, a dependence on voltage of its inhibitory effect can be expected. The effects of voltage on the chemical rate have been most clearly shown for the interaction of Ba2+ with the apical K’ channel in frog skin (De Wolf and Van Driessche, 1986) (see also Chapter 5 by Zeiske). A larger affinity of mucosal Ba2+for the apical K + channel is expected when the mucosal side is made more positive, whereas its inhibitory effect will be reduced at negative rnucosal potentials. The analysis of the voltage dependence of the Ba2+block was based on Eyring rate theory, which
57
2. NOISE ANALYSIS IN EPITHELIA
describes ion translocation in terms of elementary jumps over energy barriers. Assuming one binding site for Ba?’ in the apical membrane, the dependence of the rate constants on apical membrane voltage ( V z )fulfills
k,,, = b exp
[
(6
I)zFV, 2RT
-
]
where F, R , and T have their usual meaning, 6 is the fraction of the potential difference (V,) across the apical membrane experienced at the binding site, z is the valence of the blocking ion, and a and b are voltage-independent components of the rate constants. The latter parameters are related to Gibbs free activation energy. Equations (23) and (24) clearly predict the voltage dependence of the rate constants and consequently of the corner frequency. This problem becomes crucial for epithelia in which the transmembranal potential changes as the permeability of the apical membrane is decreased by the blocker. Indeed, in experiments wherein the mucosal solution consists of K+-Ringer’s, to investigate apical K + channels, the intracellular potential is hyperpolarized by increasing the mucosal Ba2+.This will complicate the analysis of the 2~JCc-blockerconcentration plots, which could become nonlinear. However, over the range of Ba2+ concentrations used, the changes in intracellular potential are rather small (2030 mV) compared to the control values of the intracellular potential (80 mV).
VII. CONCLUSION This overview illustrates that the analysis of spontaneous relaxation noise can be exploited favorably for the identification of ion transport pathways, as discussed in Section 111. Moreover, the analysis of blocker-induced fluctuation is a powerful application of noise analysis that allows the study of changes in singlechannel parameters as well as in channel densities. We also pointed out a number of restrictions and pitfalls of the method, such as the model dependency and the attenuation of the noise signal. One of the major advantages of noise analysis compared to patch-clamp studies is that the experiments are done with the intact tissue. This allows the study of regulation of channel properties, which may be of benefit in many situations, for example, during hormonal treatments. ACKNOWLEDGMENT This work was financed by Grants OT/86/62 and OT/88/22 of the “Onderzoeksfonds KULeuven” and by support of the Belgian Science Foundation.
58
WlLLY VAN DRIESSCHE AND NOEL VAN DEYNSE REFERENCES
Ahramcheck, F. J., Van Driessche. W.. and Hclman. S. I. (1985). Autoregulation of apical mcmbranc Na+ permeability of‘tight epithelia. Noise analysis with aniiloride and CGS 4270. J. Cen. P h v s i d . 85, 555 - 582. Aelvoet, I . . Erlij, D.. and Van Driesschc, W. (1988). Activation and blockage of a calcium-sensitive cation-aelectivc pathway in the apical membrane of toad urinary bladder. J . Phvsiol. (London) 398,555-574. Chen, Y.. and Hill. T. (1973). Fluctuations and noise in kinetic systcms. Application to K channels in the squid axon. Riophys. J . 13, 1276- 1295. Colquhoun. D., and Hawkes. A. G. (1981). On the stochastic properties of single ion channels. Proc. R . Soc. London B 211, 205-235. Dawsctn. D. C . . Vnn [>ricssche. W.. and Hchilan, S. I. (lY88). Osmotically induced hasolateral K ’ conductance in turtle colon: Lidocaine-induced K + channel noisc. A m . J . Phvsiol. 254, C16.5 C 174. Desmedt. L... Van Driesschc, W., and Simaels, J. (1989). Microelectrode study of a Ca”-sensitive pathway in the apical membrane of frog skin (Runu k w q x m x i u ) . Arch. Int. Physiol. Biochirn. 97, 55. De Wolf. I., and Van Driessche, W. (1986). Voltage-depcndcnt Ba” block of K’-channels in the apical membrme of frog skin. Am. J . Phvsiol. 251, C696-C706. De Wolf. I., Van I)riessche, W.. and Nagel, W. (1989). Forskolin activatcs gated CI channels in frog skin. Am. J . P hvsiol. 256, C 1239-C I24Y. Fishman, H. M . . Moore. L. E . , and Puussart, D. J. M . (1975). Potassium-ion conductance noise in squid axon membrane. J. Membr. Riol. 24, 305-3718. Giigelein. H.. and Van Driesschc. W. (1981a). Capacitive and inductive low frequency inipcdancc of Necwrus gallbladder epithelium. P f l i q P n Arch. 389, 105- 113. GBgclcin. H., and Van Driesschc. W. (1981h). Noisc analysis ofthe K ’ current through thc apical memhrane of Nectrrrus gallbladder. J . Membr. Biol. 60, 187- 198. Hamilton, K. L.. and Eaton. D. C. ( 1985). Single-channel recordings from amiloride-sensitive epithelial sodium channel. Am. J . Phvsiol. 249, C200-C207. Helman. S. I . , and Baxendale, L. M. (1988). Open channel probability of apical Na channels in epithelia of frog skin. FASEB J . 2, A750. Helman. S. I.. Cox. T. C., and Van Driessche, W. (1983). Hormonal control of apical membrane Na transport in epithelia: Studies with fluctuation analysis. J . Gcn. Phvsiol. 82, 201 -220. Hillyard, S . D.. Zeiske, W., and Viin Driessche. W. (1982). Poorly selective cation channels in the skin of the larval frog (stage < XX). ~ f l u e ~ Awwsh . 394, 287-293. Li, J., Palmer. I,.. Edclman. I..and Lindcmann, B. (1982). The role of sodium-channel density in the natriferic response of the toad urinary hladder to antidiuretic hormone. J . Memhr. Bin/. 64, 77-89. Lindemann, B.. and Van Driessche, W. (1977). Sodium-specific membrane channels of frog skin are purea: Current fluctuations reveal high turnover. Scirnw 195, 292-294. Lindemann, €3.. and Van Driessche, W. (fY78). The mechanism of Na-uptake through Na-selective channcls in the epithelium of frog skin. I n “Membrane Transport Processes” (J. F. Hoffman, ed.). Vol. I. pp. 155- 178. Raven, New York. Palmer, L., Li. J., Lindemann, B . , and Edelman. I. (1982). Aldosterone control of the density of sodium channels in the toad urinary hladdcr. J. Membr. B i d . 64,91- 102. Tang. J.. Abramcheck, I-‘. J . , Van Driessche, W.. and Helman, S. I. (1985). Electrophysiology and noise analysis of K ’&polarized epithelia of frog skin. Am. J. Phvsiol. 249, C421-C425. Thompson. I . G.. and Mills. J . W. ( I Y X I ) . laoprutercnol-induced currenl changes in glands of frog skin. Am. J. Phvsiol. 241, C25O-C257. Van Driessche. W. ( 1986). Lidocaine blockage of hasolatcral potassium channels in the amphihian urinary hladdcr. J . Phvsiol. ( L u n c h ) 381, 575-593.
2. NOISE ANALYSIS IN EPITHELIA
59
Van Dries\chc, W., and Giigelein, H. (1978). Potassium channels in the apical membrane of the toad gallbladder. Nulrrre (London) 275, 665-667 Van Driesxhc. W.. and Hillyard. S . D. (1985). Quinidine hlockagc of K ’ channels in the basolateral membrane of larval bullfrog skin q f / i w p w A r d i . 405 (Suppl. I ), S77-S87. Van Driessche. W., and Lindemann. B . ( 1979). Concentration dependence of currents through single sodium-selective pores in lrog skin. Nofitre ( h i d o n ) 282, 5 19-520. Van Driessche, W.. and Zeiske. W. (1980a). Spontaneous fluctuations of potassium channels in the apical membrane of frog skin. J. f/7wio/. (Londot7) 299, 101- I 16. Van Driessche, W., and Zciskc, W. (198Ob). 6a”-induced conductance fluctuations of spontaneously fluctuating K ’ channels in the apical membrane of frog skin (Rnnu rcvnporarin). J . Mrmhr. Biol. 56, 3 1-42. Van Driessche, W.. and Zeiske. W. (1985). Ca” -sensitive, spontaneously fluctuating, cation channels in the apical membrane of the adult frog skin epithelium. PJluegers Arch. 40.5, 250-259. Van Dricssche, W., Aclvoet, I . , and Erlij, D. (1987). Oxytocin and CAMP stimulate monovalent cation movements through a Ca?+-sensitive, amiloride-insensitive channel in the apical membrane of toad urinary bladder. f r o c . Nut/. A i d Sci. U.S.A. 84, 313-317. Van Driessche. W., Desmedt, L.. and Sirnaels. J. (1989). Forskolin and scrosal hypotonicity activate a calcium-sensitive pathway in the apical membrane of frog skin. FASEB J . 3, A983. Verveen, A. A., and DcPelice. L. J. (1974). Memhrane noise. P r o g . Biophys. M o / . B i d . 28, 189265. Willurnsen, N. J . , and Hviid Larsen, E. (1986). Membrane potentials and intracellular CI activity of toad skin epithelium in relation to activation and deactivation of the transapical CI conductance. J . M m h r . R i d . 94, 173- 190. Zeiskc, W., and Van Driessche, W. (1986). Impairment of Na’ transport across frog skin by TI’: Effects on turnover, area density and saturation kinetics of apical Na channels. Pjhr,qers Arch. 407. 145- 152.
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CURRENT ‘TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 37
Chapter 3 Ion Channel Fluctuations: “Noise” and Single-Channel Measurements DOUGLAS C. EATON AND YOSHINORI MARUNAKA Depurtment ojPhy.~iology Emory University School of Medicine Atluntu. Georgiu 30322
I . Introduction 11. Power Spectral (“Noise”) Analysis A. Determination of Channel Properties from Fluctuation Measurements B. Advantages and Disadvantages of Power Spectral Analysis 111. Single-Channel Analysis A . Distributions of Intervals B. Advantages and Disadvantages of Single-Channel Measurements I v. Comparison of Single-Channel Measurements with Fluctuation Measurements A Specific Experimental Example A . Background B . Problems Measuring Sodium Channel Fluctuations C. Power Spectra from Singlc-Channel Data D . Single-Channel Analysis E. Implications of a Four-State Model V. Summary References
1. INTRODUCTION Ion “channels” form an interesting class of membrane proteins that have specifically evolved to provide an essentially aqueous pathway through the otherwise highly hydrophobic barrier of the cell membrane. As such, they are capable of carrying relatively large numbers of ions per unit time. In general, to regulate the number of ions that cross the cell membrane, a channel protein undergoes conformational changes, producing states of the molecule that either afford an
61 Copyright Cl 19W hy Acudeniiu Press, Inc. All rightr d rcpruduclivii i n any furin reserved.
62
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
aqueous pathway across the protein or that have no such pathway for ions. Typically, there may be several “open” states that allow ion movement as well as several “closed” states in which there is no ionic current across the protein. Because the primary function of channel proteins is transport of ions across the membrane, the conformational changes that lead to alterations in current Row have been the subject of significant investigation. Examination of the transitions between open and closed states of channel proteins is also motivated by the fact that regulation of ion transport is often mediated by controlling the rates of transitions between open and closed states and, thus, controlling the relative amount of time the channel is open, conducting ions, compared to the amount of time it is closed. There are currently two methods for examining the fluctuations in current flow through channel proteins: power spectral (or “noise”) analysis and singlechannel analysis using patch-clamp methods. The purpose of this chapter is to present the fundamentals of these two techniques and to compare their strengths and weaknesses. with particular emphasis on their application to epithelial tissues. The chapter will emphasize epithelial tissues primarily for two reasons. First, the use of both “noise” and single-channel analysis has been extensively reviewed in excitable tissue (Neher and Stevens, 1977; Lauger, 1980; Fishman and Leuchtag, Chapter I , this volume) and, second, epithelial tissues present a special situation that makes either analysis method very attractive, but that also poses special problems for interpretation of both approaches.
II. POWER SPECTRAL (“NOISE”) ANALYSIS Historically, power spectral analysis, which is also known as noise or fluctuation analysis, antedates single-channel measurements. The initial description of its use in epithelial tissues dates from 1977 (Lindeniann and Van Driessche, 1977). To provide a basis for comparison of fluctuation analysis with singlechannel measurements, we will first provide a brief description of the method and then give a short history of its use to study channel proteins in epithelial tissues.
A. Determination of Channel Properties from Fluctuation Measurements A much more detailed treatment of methods and theoretical Considerations relevant to noise analysis is given in Part 11 of this volume; however, a simple description of the method is appropriate here to provide a basis for comparison of single-channel and fluctuation measurements.
3. ION CHANNEL FLUCTUATIONS
63
1. PROBABILISTIC CHARACTER OF SINGLE-CHANNEL FLUCTUATIONS
Ordinarily, fluctuations of any physically measurable property are not apparent. The reason for this is intuitively obvious; most measurable quantities represent a very large number of individual events temporally superimposed to produce a mean value that is large compared to the magnitude of the individual events. This relatively large mean value, compared to the small magnitude of individual events, implies that any fluctuation from the mean must also be small. Thus, if we are to examine the fluctuations of a system as well as the mean behavior, then the recording situation must be arranged to accentuate the magnitude of the fluctuations relative to the mean value. For a more specific example of this concept, consider the case of flipping a coin. If the coin is a “fair” coin (i.e., symmetric), there are equal chances of obtaining either a head or a tail. If one side is represented as a 1 and the other as a 0, then the mean or expected value, ( e ) . is
and the mean-square value is
where c, is the value of any state, i, and p , is the probability of occurrence of the ith state. In this case, thcre are only two states with values of 0 and 1 and the probability of occurrence of each state is 0.5. A quantity of more importance to this discussion is the mean-square deviation or variance, u2:
The square root of the variance is the standard deviation, which for a single coin flip would be equal to 0.5, as large as the mean value, implying that the fluctuation of a single flip is as large as the average value. The standard deviation is an experimentally relevant parameter because the actual size of measurable fluctuations is described by the standard deviation of the mean signal rather than the variance. This distinction is important, because, in practice, numerous events (coin flips) would be “superimposed” to produce a signal. For a large number of flips, say 20, the expectation would be
64
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
with a variance of
In this case our intuition as well as Eq. (4) imply that the expected value of 20 coin tosses should be 10, whcrcas the calculated standard deviation is 2.24 (square root of 5). Thus, by increasing the number of events from 1 to 20, the ratio of signal fluctuation size to signal mean value has changed from a ratio of 1 to 0.224. Obviously, for much larger numbers of coin tosses the ratio would become even smaller. For the example of the coin (and in many cases for channels also), the elementary events underlying the mean value and the Huctuations are identical and independent, which leads to a simplification of the expressions above as
where n is the number of events (coin flips or conducting channels). These equations lead to the interesting conclusion that the ratio of the standard deviation to the mean value is proportional only to the number of superimposed events, because
For the coin flip experiment (or a channel that spends equal probability of being either closed or open), the proportionality constant r (the ratio of the standard deviation to the expected value for a single trial) is 1 . But regardless of the specific value of r, as the number of independent events increases, the size of the measurable fluctuations decreases. For loh channels (or coin flips, for that matter), the size of the individual fluctuations would be only I / 10 of 1% of the magnitude of the average signal. This result is consistent with the observation that “noise,” that is, fluctuations, in a signal can be reduced by averaging several signal records together, thereby increasing the apparent number of superimposed fluctuating signal sourceb (in our case the openings and closing of channels). On the other hand, if we are primarily interested in the fluctuations, then to obtain large signals, the number of fluctuating units from which we are recording must be limited to a relatively small number. In the most extreme case, the membrane surface area can be so restricted that fluctuations can be obtained from a single channel and thc singlechannel analysis methods described below may be used to analyze the tluctua-
65
3. ION CHANNEL FLUCTUATIONS
tions. But in the more usual case, the fluctuations of tens to thousands of channels are examined to determine the fluctuation properties of an entire population of channels. In the example of the coin tosses that was used above to give an intuitive feeling for the characteristics of probabilistic fluctuations, the expectation for either of the two states of the coin was 0.5. For membrane channels with only two states, the expectation for either state may be quite different from 0.5; some channels may be open much more often than they are closed, or vice versa. Nonetheless, based on the discussion of the coin tosses, it is easy to develop similar arguments for a channel with an arbitrary probability of being open. Given that a channel can be only open (a value of I ) or closed (a value of 0), then the expectation or average value for the channel is
(4= p
(9)
and
where p is the probability that the channel is open. The variance of the channel is
That is, the variance is equal to the product of the open probability and the closed probability. For n independent channels, the expected mean value and variance are
Mean membrane current, I , (at constant voltage) will be the unit current of a single channel, i, times the average number of open channels (e,,) (I)
=
i(e,,) = inp
(13
and the variance in mean current will be
The ratio of variance to mean value gives an expression for unit current and is u f / ( I )= i(I - p )
(15)
66
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
If the open probability is known to be very small, then this expression gives a direct estimate of unit current. If the open probability is comparable to the closed probability, then the ratio of variance to mean current will underestimate unit current.
2. AUTOCOVARIANCE FUNCTION A S A TEMPORAL MEASURE OF FLUCTUATIONS So far we have described the properties of channel fluctuations at a specific point in time. In general, though, membrane current records are made continuously for long periods of time compared to the rate of the fluctuations due to single-channel contributions. The information that describes the variation of fluctuations with time can provide additional information about channel propcrties over and above the static information provided by the mean current and the variance. In particular, it may provide information about the rates of transition between the open and closed states of the channel. The tcmporal pattern of the fluctuations can be characterized in two ways-either by generating the autocovariance function from the data or by constructing the power spectral density function. In effect, these two approaches are not different, because, as pointed out in Chapter 1 of this volume, the two functions are simply related by a Fourier transform. For Some function of time, fcl), the autocovariance function CoV(T)is:
where 1, is the length of the observation period which must be long coinpared to the value of 1'. CoV(T) is a measure of the correlation betwcenflt) and,f(t)at an interval T later. By definition, the value of the autocovariance function at T = 0 is the variance of the function , f ( i ) . This must be a positive quantity. As T increases to values larger than the average duration of a fluctuation, the value of CoV(T) decreases toward zero. Thus, the autocovariance function is a measure of the average time course of a fluctuation. 3. FUNCII O N A L FORMOF AUTOCOVARIANCE FUNCTION OF CHANNEL. FLUCTUATIONS The form of the autocovariance function for channel fluctuations is not intuitively obvious, although an explicit, rigorous derivation of its form can be found in several places (Neher and Stevens, 1977). However, by considering the singlechannel properties that underlie fluctuations, it is possible to gain a nonrigorous understanding of the function. To do this, it is necessary to understand how the durations of intervals during which a single channel is open or closed are distrib-
3. ION CHANNEL FLUCTUATIONS
67
uted. Let us return for the moment to our coin-tossing analogy. Previously, we determined the statistical variables related to the appearance of one side of the coin or the other, with the tacit assumption that the only outcome of a coin toss was either a head or a tail. In point of fact, for real coins with a finite thickness, every once in a rare while the tossed coin will land on edge. Our intuition tells us that landing on edge is a “rare” event. Practically, the definition of a “rare” event is one in which the probability of success (landing on edge) approaches zero while the probability of failure (either a head or a tail) approaches 1 . The probability of success for such rare events is given by a limiting case of the binomial expansion and was first described by Poisson, who gave his name to the statistic. The formulation that is most useful for application to channel fluctuations is given by (Colquhoun, 1971)
where p(n, t ) is the probability of n events within the interval t and T is the mean value of the distribution. As with the coin landing on edge, the probability that a channel will make a transition between one conformational state and another is also a “rare” event. The trials are the random thermal fluctuations of the channel molecule, which occur on a picosecond time scale. Most of these fail to produce any change in the conformational state of the molecule, but, for every 10“’ or 10” fluctuations, there will be one that is large enough to lead to a state change. This is indeed a rare event and is described by Poisson statistics. In the continuous case, the distribution becomes an exponential distribution whose mean value is equal to the average lifetime of a given conformation or the time the protein remains in a given state before it undergoes a transition to a different state. The mean value is also related to the chemical kinetics of the transitions between states; the frequency of leaving a particular state (the reciprocal of the lifetime) is equal to the sum of all the transition rates for leaving that state (Colquhoun and Hawkes, 1983). The reason that the distribution of lifetimes for any given channel state should have an exponential form may not be immediately obvious. The exponential distribution implies that very short-duration lifetimes are more probable than long durations; if transitions are such “rare” events, one might anticipate that it would require a long time for a transition to occur. A careful consideration of the transition process belies this notion. The probability of a transition (a success) for any given trial is a very small, but finite value, say E ; the probability of failure for the same trial is very large, but, nonetheless, less than one ( 1 - E ) . Therefore, the probability of the first trial being a failure and the second trial being a success is E( I - E ) , which is smaller than the probability of success on the first trial. In general, the probability of success on the nth trial is
68
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
so that the probability of success on the nth trial must always be greater than the probability of success on the n + 1 trial. In other words, the probability of the shortest intervals (relatively small number of trials) must be higher than that of longer intervals (with more trials). In fact, the entire distribution is just the sum over all trials as the number of trials becomes very large:
Of course the sum of all the probabilities must equal I . The sum in Eq. (19) is the power series representation of an exponcntial function with a time constant at the mean value of the distribution. Therefore, it is again clear that the distribution of lifetimes of any state of a channel must be an exponential distribution whose mcan is the sum of the rates of leaving the state. Knowing the distribution of open lifetimes allows us to determine the form of the autocovariance function. The problem reduces to the question of calculating the probability that a channel is still open at some time, t T, given that the channel was open at time, 1. This implies for a channel with one open and one closed state, that
+
CoV(7‘) = A exp( - t h )
(20)
where T is the reciprocal of the sum of the rates of entering and leaving the open state. In Fig. 1, simulated single-channel data obtained from the model given in Eq. (21) CLOSED
=OPEN kl
k.1
(with k, and k k , = 20 sec-I) are shown along with thc autocovariance function obtained from similar data simulating approximately 9 niin of channel transitions. For comparison with the autocovariance function, the distribution of open intervals for the same data is also plotted to demonstrate that the mean values (or characteristic time) for the two functions are not the same. The mean of the open interval distribution (the mean open time) is the reciprocal of k - , , the rate of leaving the open state, and the mean of the autocovariance function is the reciprocal of the sum of k , and k - , . Nonetheless, the autocovariance function is a measure of the fluctuations of a single channel and in the case of this very simple model the properties of the function provide specific information about the transition rates between the two conformational states of the channel. Even when multiple channels contribute to fluctuations, the autocovariance function still
3. ION CHANNEL FLUCTUATIONS
- 0
- c
A
- 0
- c - 0
- c - 0
loo lnsac
- c
20
0
%
100
1%
DURATION (rnsec)
I . Single-channel events and their autocovariance function. (A) Simulated single-channel records obtained by generating the random transitions expected for a simple two-state model described in the text. To simulate normal single-channel events, Gaussian noise has been superimposed on the single-channel currents. The records in A represent approximately 4 sec of data. (B) The distribution of open intervals for approximately 8.7 min of single-channel data of the same type as that shown in A. (C) The same 8.7 min of data has been used to generate the autocovariance function. For both functions the solid line through the data points is the best least-squares fit to an exponential function of the form A exp( - I / T ) . Both the open-interval distribution and the autocovariance function are single exponential functions. The characteristic time of the open-interval histogram is equal to the mean lifetime of the open state in the model, and the characteristic time of the autocovariance function is the reciprocal of the sum of the rates of entering and leaving the open state.
70
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
- 5 y 3 - 0
150
g I
A
B
I
z
Y
AMPLllUDE (orbitrory units)
0
50
1w
150
2w
250
DURATION (msac)
Pic;. 2. Multiplc-channel records and their autocovariance function. (A) Channcl lluctuations generated from thc same model as those of Fig. 1 except that there are five channels present, as demonstrated in B. ( B ) Thc distribution of amplitudes of the digitized points within the record. Each peak represents a preferred amplitudc of the data, and the area under each peak is the relative probability of occupying the level represented by the peak. Because of the multiple channels. the openinterval distribution i s nut specifically cornparahle to the autocovariance function and therefore is not presented. However, the autocovariance function in C is, except for y-axis scaling, very similar to the autocovariance function for a single-channel presented in Fig. I .
71
3. ION CHANNEL FLUCTUATIONS
provides similar information for this simple model. Figure 2 shows simulated channel transitions for the same model as in Fig. 1 , but from a patch that contains five channels. Except for a change in the scale of the amplitude and minor differences due to inaccuracies in simulation, the autocovariance function for multiple channels is essentially the same as that derived from the single channel shown in Fig. 1 . In general, the autocovariance function contains one term for each set of transitions from a closed state to an open state. For example, both of the following two kinetic schemes contain two sets of transitions from closed (zero current) to open (finite current) states.
CLOSED,
LI
======= I,
k2
(23)
CLOSED, LOPEN k-2
The simulated single-channel data for the two kinetic schemes and the autocovariance functions for the data are shown in Fig. 3. The autocovariance functions have two distinct exponential components corresponding to transitions from either of the two closed states, directly to the open state for the kinetic scheme in Eq. (22), and for the scheme in Eq. ( 2 3 ) directly from the open state to the state labeled “CLOSED,” and back to open, or from the state labeled “CLOSED,” through the state labeled “CLOSED,” to the open state. Moreover, the characteristic times for the two exponential components do not correspond to the mean lifetime of the open state, but rather are the characteristic times for the individual reactions; i.e., for the scheme in Eq. (22), 7 l = l / ( k , k - , ) and T? = l / ( k 2 k J. In general, for n such sets of transitions between closed and open states, the autocovariance function can be represented by
+
+
where A, is the magnitude of each exponential component at t = 0. 4. POWER SPECTRAL DENSITY FUNCTION IS THE FOURIER TRANSFORM OF T H E AUTOCOVARIANCE FUNCTION
Although the autocovariance function can give us an intuitive feeling for the relationship between chemical kinetic events and the current fluctuations pro-
A -c -c -c
(-
25
s
l
M M
-
10 mow
100
M M
-
108 msec
10
1
0
im
50
200
I50
250
DURATION OF CLOSED INTERVALS (rnrce)
0
YI
I00
150
DUf7AllON (mscc)
FIG. 3 . Single-channel events and their autocovariance function for a three-state model. (A) Simulated channel fluctuations obtained by generating the random transitions expected for two simple three-state modcls described in the text IEqs. (22) and (23)]. To simulate normal single-channel events, Gaussian noise has been superimposed on the single-channel currents. The records in A represent approximately 4 sec of data. These records demonstrate that for both modcls, there appear to be two classes of closed intervals, short and long. (B)The two classes are demonstrated by plotting the distrihution of closed intervals for approximately 8.7 min of single-channel data of the same type as that shown in A. To emphasize that the distribution contains two exponential components, the data have been plotted with a logarithmic frequency axis. (C) The same 8.7 min of data has been used to gencrate the autocovariance function. Because there are two transitions to the open state from closed states, both the closed-interval histogram and the autocovariance function have two expunential components. For both functions the solid lines through the data points arc the best least-squares tit to the sun1 of two exponential functions of the form Al exp( - 1/71) + A, exp( - 1 / 7 2 ) . The characteristic times of the two exponential components of the closed-interval histogram are equal to the mean lifetimes of the two closed states of the model. and the characteristic times of the autocovariance function are the reciprocal of the sum of the rates for entering and leaving the two closed states.
73
3. ION CHANNEL FLUCTUATIONS
duced by kinetic transitions, it is often easier to consider temporal events in the frequency domain rather than the time domain. This is true when examining chemical kinetic systems because chemical kinetic rates are directly related to the frequency of transitions, whereas in the time domain, rates must be extracted from the characteristic times obtained from autocovariance functions. But more importantly, as pointed out by Fishman and Leuchtag (Chapter 1, this volume), handling data in the time domain is significantly more difficult than handling data in the frequency domain; calculation of autocovariance (or autocorrelation) functions is time consuming compared to the generation of power spectra using any of several fast Fourier transform methods. Recognizing this, we can produce the general power spectral density function by taking the Fourier transform of Eq. (24):
If Eq. (25) represents the power spectrum of a single channel, the power spectrum of M independent channels, S,(f), is M S ( f ) . As with the autocovariance function, the power spectral density function contains one component, a Lorentzian, for each separate set of transition rates leading from a closed channel to an open channel. Moreover the half-power point, or corner frequency, f c , is directly related to the transition rates by the relationship 2irfc = k ,
+ k-
(26)
Unfortunately, k , and k - are not, in general, directly related to the rate constants of kinetic schemes such as those in Eqs. (22) and (23). The difficulty is best illustrated by examination of the model given by Eq. (23). Here, transitions between the OPEN state and the CLOSED, state are simply represented by a corner frequency proportional to the sum of k, and k - 2 , However, besides these simple transitions between a single zero current (CLOSED,) and a single finite current (OPEN) state, there are also transitions in which the channel, after a transition to CLOSED,, rather than returning to the OPEN state, makes a transition to CLOSED, [with a probability, p , of k - , / ( k - , k,) and a rate k , = of ~ k - ~This ] . implies a second class of closed intervals with a sojourn consisting of at least one set of CLOSED, to CLOSED, to CLOSED, transitions. Obviously, the rate of returning to the OPEN state from such a sojourn is slower than that in which the channel immediately returns to the OPEN state from CLOSED, and the rate of return (the reciprocal of the lifetime in this long closed interval) from sojourns that include transitions to CLOSED, includes contributions from all of the rate constants, k , , k - , , and k2. Thus all possible combinations of transitions from closed states to open states can lead to measurable Lorentzian com-
+
74
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
00.00
10.00 L
z LL
0,
1 .00
0.10
0.01 0.1
1.o
10.0
100.0
1000.0
100.0
1000.0
Frequency (Hz) 100.00 I
2.41 Hz
10.00
0.1
1 .o
10.0
Frequency (HA FIG. 4. Power spectra of single-channel data. An X I Wpoint fast Fourier transforni algorithm was used to transform thc sanie single-channel data that were used to construct the autocovariance function in Figs. I and 3 . The data from Fig. I are in the upper panel and the data from Fig. 3 are in the lower panel. The power spectra were fit with either one (top panel) or two (lower panel) Lorentzian functions. The corncr frequency of the spectra in the upper panel is 12.7 Hz and the corner frequencies of the spectra in the lower panel are 4.35 and 69.2 Hz. These corner frequencies correspond to characteristic times of 12.5 msec for the spectra in thc upper panel and 36 and 2.3 msec for thc spectra in the lower panel. Thesc values for characteristic times arc equal (within the crror of the lilting proccdurc) to the characteristic times ohtained from the autocovariance functions in Figs. I and 3 .
75
3. ION CHANNEL FLUCTUATIONS
ponents in the power spectrum. In general, for N states in a kinetic scheme, there will be N - 1 Lorentzian components to the power spectrum (although as we will see later, often, not all of them are experimentally resolvable). Figure 4 illustrates this point by taking the data of Figs. I and 3 and transforming them via an 8192-point fast Fourier transform to produce the power spectra of the single-channel data. The data have been tit with one Lorentzian function in the upper panel and two in the lower panel. The tits provide estimates for the plateau levels, S(O), and the corner frequencies,,L.. Within the error of the fitting procedure, l / 2 ~ j iis. equal to the characteristic times obtained from the autocovariance functions of Figs. 1 and 3. For the simple two-state model, additional information can be obtained from the power spectrum. The integral of the spectral density function is equal to the variance of the fluctuations. That is,
but, from Eq. (1% (r' = (I)i(1 - p,,,,,,); if total macroscopic current, ( I ) ,is known and the reverse rate constant, k - , , is known or can be derived from other experimental measurements, then, unit current, i , can be calculated from the p(,wn = pLlrrrcd = k , / ( k , + k- ,) = following expression (remembering that I ~
k - I/27rf,):
Once the unit current is calculated, the total number of channels, M , contributing to the power spectrum can also be estimated, because
then
Similar methods may be used to derive corresponding expressions for more complicated kinetic schemes (Frehland, 1979; Colquhoun and Hawkes, 1977; Neher and Stevens, 1977; Baxendale and Helman, 1986; Lauger, 1980). In general, for a kinetic scheme with 4 states, r of which are open, the plateau level of the nth Lorentzian component is given as 'I
I
I,
I
S,(O) = 4i'N
b,,l(k;
+ k;)
76
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
where k,: and k, are the n possible rates for transitions from combinations of closed states to open states and
where p , and pJare the probability of being in the open state i and j , respectively, and is the delta function, which is 1 for i = j and 0 otherwise. An example of one such derivation will also be given in Section IV.
B. Advantages and Disadvantages of Power Spectral Analysis The advantage of current fluctuation analysis lies in its ability to provide inforniation about the properties of entire populations of channels. Unfortunately, the price for obtaining information about large numbers of channels is the necessity for assuming the properties of individual channels. In particular, it requires the assumption that all the channels contributing to the fluctuations are identical and behave independently. Moreover, to estimate the unit current of a channel [from Eq. (14)] requires an assumption about the open probability of the individual channels. Neither of these assumptions are excessively restrictive, as evidenced by the application of fluctuation analysis in the experiments described in Section IV. A more serious problem arises when fluctuation measurements are used as a basis to provide kinetic information. As is obvious from Figs. 2 and 4 and Eqs. (21)--(23),the form of the fluctuation functions can give information about the minimum number of closed and open states if the frequency resolution is adequate; however, even with appropriate frequency resolution, fluctuation analysis cannot provide information about how the states communicate and can provide only limited information about closed states that do not communicate directly with an open state. Therefore, to interpret thc fluctuation data, some kinetic model that contains the minimum number of states must be assumed. In addition, the corner frequencies of power spectra do not give information about individual rate constants, but, rather, provide the sum of at least two rates. Unless one of these rates can be experimentally manipulated, determination of individual rate constants is usually not possible. In the abscnce of a frequency resolution that includes the time constants for all of the channel transitions, the situation becomes more problematic because unresolved transitions may contaminate thc otherwise wcll-resolved fluctuations (sce example below). Thus, the ideal situation in which to use fluctuation analysis would be one in which the true kinetic scheme has been independently verified by some other experimental method, but one in which large populations of channels must be monitored. As we shall demonstrate in Section 111, a close to ideal situation may be provided by the combination of fluctuation rncasurements and singlechannel measurements.
3. ION CHANNEL FLUCTUATIONS
77
111. SINGLE-CHANNEL ANALYSIS Recently, the ability to measure the currents associated with single-membrane channels has dramatically changed the manner in which current fluctuations can be analyzed. Because single-channel analysis methods have been dealt with extensively by others (Colquhoun and Sigworth, 1983; Sachs et al., 1982) and also within this text (Lewis and Donaldson, Chapter 7), we will only deal here with methods that are directly relevant to comparison of “noise” and singlechannel methods. Although all of the statistical considerations that were developed in conjunction with power spectral density functions and autocovariance functions are applicable to the fluctuations of a single channel, there are other methods that can only be applied to the fluctuations of a single channel. These methods are particularly useful in providing estimates of minimum kinetic schemes that represent the conformational transitions of channel proteins. There are two types of information available from a single-channel record that cannot be obtained from the fluctuations produced by multiple channels. First, the duration of individual open and closed intervals can be directly measured; and, second, the sequence of intervals can be determined. In the first case, histograms of the duration of open and closed intervals can be used to estimate the number of different classes of intervals. In the second case, once classes of intervals are established, then the temporal relationship between classes can be determined; that is, does an interval of one identified class always follow an interval of another identified class‘?
A. Distributionsof Intervals In Section II,B,3, we demonstrated that the distribution of durations for any state of a channel can be described by an exponential function. Experimentally, the underlying distribution is estimated by generating a histogram of all open intervals and a similar histogram of all closed intervals. The form of these discrete distributions would, in the limiting case of a very large number of measured intervals and a large number of histogram bins, each representing a very narrow range of durations, simply be an exponential distribution. The number of exponential components in the distribution of open intervals represents the minimum number of open states of the channel, whereas the number of exponential components in the closed-interval histogram gives an estimate of the minimum number of closed states. In the simplest case, the mean of each exponential component is just the mean lifetime of one of the states of the channel, and the reciprocal of the mean duration is the sum of all the rate constants for leaving that state. There are several practical considerations involved in constructing the interval histograms (Colquhoun and Sigworth, 1983). As with all temporal measurements, the data have a limited bandwidth; therefore, exponential components whose mean duration is short compared to the bandwidth of the system will be
78
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
underrepresented, that is, short-duration intervals associated with the distributions will be missed. Also, because the data are sampled at a finite rate, the temporal resolution of the raw data leads to estimates of interval durations with a certain "graininess" and some inaccuracy in placing intervals into histogram bins. Both of these problems can, to a certain extent, be corrected to yield a more accurate description of the interval data within the bandwidth constraints of the recording system (Wilson and Brown, 1985).
I . RESOI.IJTION OF EXPONENTIAL COMPONENTS Because a determination of the minimum number of states in a kinetic model of channel transitions involves a determination of the number of components in interval histograms, significant attention has been given to methods that would help to resolve these components. Some of the problems are similar to welldescribed problems of resolving the exponential components of a function that represents sums of exponentials; fitting such functions often yields poor cstimates for the parameters of individual cxponentials if the time constants of the exponential components arc separated by less than a Factor of 10. There is no general solution to this problem and each experimental situation must be examined individually. On the other hand, there is with single-channel records often a problem of an opposite sort-the time constants for two exponential components are often different by several orders of magnitude, Because the histogram bins represent finite intervals, it is often difficult to pick a bin width that can resolve very short-duration events and yet still provide enough events in the bins that represent long-duration events to adequately resolve the long time-constant components. An example of this problem is shown in Fig. 5 . When shortduration intervals arc chosen for the size of the histogram bins (in A), there are only a few events in the bins that represent long-duration events, and many events fall outside the range of the histogram. If an attempt is made to fit two exponential functions to this distribution, the distribution consisting of short intervals is fit well, but the mean time for long-duration intervals is very poorly estimated. In Fig. 5B, long-duration intervals adequately resolve the long timeFIG.5. Interval histograms with exponential components w h o s time constants diflfer by inore than an ordcr of magnitude. Resolving exponential components of interval histograms can be difficult with widely aeparatcd time constants. For simulated data consisting of the sum of two exponential distributions with time constants of 30 and 1000 msec, an attempt to determine the value of the shortduration component by using narrow bins (A) lcads to an accurate deterniination of the short-duration component but to an overestimate of the long-duration component hy 85%. An accurate determination of the long-duration component ( B ) overestimates the short-duration component. A poaaiblc solution to this problem, which tits all of the data only one time, is shown in C, where the histogram bin sizes arc logarithmically distributed in a fashion similar to that described hy Sigworth and Sine (1987). This method has the interesting property that each distribution rcachcs a maximum at the mean duration for the distribution.
79
3. ION CHANNEL FLUCTUATIONS 500 400
300 Y M = 30.6 marc
200
100
0 0
200
100
400
300
DURATION (rnsec)
3000
(--
t,
YEAN = 52 rnae
2000
W z
3
s K
1000
0 lo00
0
2000
00
DURATION (msec)
MEAN = 991 mmc
t, z
C
400
W
3
s L
200
0 1
10
100
1000
DURATION (rnsec)
1E4
1 :5
80
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
constant component of the histogram, but, because essentially all of the events associated with the fast time-constant component fall within the first or second bin, the histogram gives essentially no information about the fast component other than its existence. In this case, the mean time of the long-duration events is estimated well but the mean of the short-duration events is very poorly estimated. Although it is possible to successively determine the parameters of different distributions by multiple fits at different temporal resolution, a more satisfying resolution of this problem has been the development of methods for assigning logarithmically distributed intervals to the histogram bins; that is, bins representing fast events also represent relatively short intervals, and bins including long-duration events are temporally much wider. There are several possible ways of accomplishing logarithmic binning (McManus er al., 1987; Sigworth and Sine, 1987). One such method (Sigworth and Sine, 1987) is illustrated in Fig. 5C in which the data originally presented in Fig. 5A and B are reanalyzed with logarithmic bin sizes. The transformation that produces the logarithmic bin sizes has the interesting additional feature in that a transformed exponential function reaches a maximum at the mean value of the function, thus, making it visually somewhat easier to discern different exponential components. The new functional form with a maximum at the mean is due to the temporally very narrow bins for short-duration events. Even though there are large numbers of short-duration events, there are also large numbers of bins, so that the number of events per bin is smaller than for bins that contain longer duration events. The function decreases from its maximum because of the scarcity of very longduration events.
A MINIMUM KINETICMODEL 2. DETERMINING
As mentioned above, noise analysis can only give information about the minimum number of states present in a kinetic model. Without additional experimental manipulation, the rate constants associated with these states are often difticult to determine individually, and the relationship between states, that is, which states communicate with one another, is often impossible to determine unequivocally. A better determination of a correct kinetic model can often be made by analysis of a single-channel record. There are two reasons for this. First, the mean duration of measurable states is determined only by the rate constants leaving the state; that is, half as many rate constants contribute to the mean durations compared to corner frequencies. Therefore, experimental manipulations can more easily isolate individual rate constants. In addition to simpler expressions to determine rate constants, there is also specific information about communication between states in the single-channel record. By observing which classes of events occur temporally together, it is possible to determine which states are “adjacent” or communicate in a kinetic model.
81
3. ION CHANNEL FLUCTUATIONS
3. THEORETICAL EXAMPLE As an example of such methods consider the kinetic schemes given in Table I . a. Power Spectra. Each of these schemes involves four conformational states, two open and two closed; each has one long-lived open and one longlived closed state and one short-lived open and one short-lived closed state; each has two distinct transitions from closed to open states and one transition from multiple closed states to an open state. Thus, there are three sources of measurTABLE I COKNER FREQUENCIES FOR THREEFOUR-STATE MODELS" Model
Model data
Kinetic schemes k l = 0.01
#I
k2 = 0.2
k., = 0.02
k.2 =
0.01
k.3 = 0.2
0 01
OPEN, / CLOSED, 4 CLOSED,
CLOSED,
-.
w OPEN,
0 01
02
#3
F====== CLOSED,
02
02
#2
kl = 0.01
CLOSED, / OPEN, 4 OPEN,
0 01
0 01
02
0 01
0 01
. OPEN, 0 01
CLOSED,
OPEN,
02
Characteristic rates
#I #2 #3
r
t:
27Tf
(Hz)
(rnw ) 0.10 0. I I 0.20
16
17 32
f?
27Tj:.
f2
(Hz) 32 21 32
(msec-') 0.20 0.13 0 20
(Hz) 78 92 53
2Tf
;
-'
(msec ) 0.49 0.58 0.33
Probability of occupancy #I #2 #3
CLOSED, 0.48 0.02 0.02
CLOSED, 0.02 0.48 0.48
OPEN, 0.02 0.02 0.48
OPEN 0.48 0.48 0.02
"The probability of occupancy of the different states in the models is given. The corner frequencies of any kinetic model can be calculated using standard methods (Colquhoun and Hawkes, 1977). The probability of occupancy for each state (which is necessary to calculate the plateau levels of the Lorentzians) can also be derived using documented methods (Runyan and Gunn, 1989). For the three models given here, there are always two states of equal, but low, occupancy and two of equal, but high. occupancy. Whether these are open or closed states depends upon the specific model. In addition, for the rate constants chosen as examples, the corner frequencies are often similar enough to be difficult to resolve in power spectra. Rate constants are in units of msec -I. The value 27rfc is equal to the sum of the forward and reverse transition rates, k + + k - .
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
82
MODEL 1
1000.0
.-2!
fi
5
100.0
i?
3
10.0
f
1.0
v
0.1
0.01
0.1
1
10
100
FREOUENCY (Hz)
0.01
0.1
1
10
100
FREOUENCY (Hz) 1000.0
0.1
FK;.6 . Power spectra of several four-state models. The three models described in the text were each used to generate 16 min of single-channel data. An 8192-point fast Fourier transform was then used to transform the single-channel data tu produce the power spectra in this figure. Each model has two transitions hetween open and closed states with two corresponding curncrs in each power hpcctra .
3. ION CHANNEL FLUCTUATIONS
a3
able current fluctuations. Therefore, the power spectra of the single-channel fluctuations, theoretically, contain three corner frequencies (Fig. 6), but in practice the closeness of the corner frequencies or the relative power associated with some transitions in the models makes resolution of all three corners and plateau levels difficult. Table I gives the approximate calculated corner frequencies and the probability of occupancy of the different states in the three models. With knowledge of each model, the power spectra can be predicted. The reverse, however, is not true; the power spectra d o not uniquely determine the models or even the number of states, and, because of poor resolution of at least one corner frequency in each model, even a two-state model fits the data fairly well. Even with a more prominent corner, several three-state models and many models with four or more states will produce similar power spectra. Determination of the individual rates is, in general, not possible unless one can be manipulated independently. The necessity for additional information about at least one of the rate constants arises because the power spectra in Fig. 6 potentially contain six measurable quantities (the corner frequencies and plateau levels) that describe six variables (the rate constants). But the expressions for the corner frequencies and the plateau levels both contain the sum of a forward and reverse rate constant and are, therefore, not independent; and, as mentioned above, the rates of transitions from zero current states to finite current states (the basis for the fluctuations) are generally not simply related to individual rate constants in a kinetic scheme.
b. Single-Channel Datu. On the other hand, the single-channel records for each of these kinetic schemes (Fig. 7) are more distinctive, with characteristics that allow identification of the number and also the order of the states. In Figs. 8 and 9, the open- and closed-interval histograms each have two components, which immediately demonstrates that there are at least two open and two closed states. The mean duration of each of the histograms provides some information about the rates of leaving the different kinetic states. Unfortunately, for the fourstate models given in Table 1, the mean durations of two of the four components in the interval histograms uniquely determine the rates for leaving both end states, but the mean durations associated with the interior states consist of contributions from at least two rate constants and, therefore, as in the case of power spectral analysis, thcy can only be determined if there is some additional experimental evidence from which one can be determined individually. The situation for the first and second models in which two states of similar current magnitude directly communicate is even more difficult, because transitions between two open states and two closed states are not resolvable in the single-channel record. For example, in kinetic model # 1, the channel can make a transition from either a closed state to an open state; once the channel is open, it may make repeated transitions between the two open states with no indication of the transitions in the current record. Thus, the mean duration that the channel is open depends
a4
DOUGLAS C. EATON AND YOSHINOR1 MARUNAKA
MODEL # l
A -0
- c
MODEL #2
B
MODEL #3
C
Fic;. 7. Single-channel data from several four-state models. This figure depicts typical records from the single-channel data that wcrc used to generate the power spectra in Fig. 6 . Each set of 5ingle-channel data has a characteristic distribution of open and closed intervals, which could be used to dctine the number of states in the undcrlying kinetic model.
3. ION CHANNEL FLUCTUATIONS
MODEL # 1
1o00,
800
600 400
200
0 0.01
0.1 0
1 .00
10.00
101 1.00
100.00
MODEL #2
lo00
B
400 200
0
C 800600
-
400 200 0 0.01
0.10
1.00
10.00
100.00
101 1.00
OPEN DURATION (ma)
FIG. 8. Open-interval histograms for the three niodels described in the text. Although each distribution has quantitative features that distinguish it from the others, qualitatively, all of the distributions are similar: each distribution has two components, which implies two open states in each model.
86
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
MODEL #1 800 '"--
4w
i
A
600
3
400
200 0 0.01
0.10
1
.oo
10.00
1000.00
100.00
MODEL P2
I B
jooO
$ W
600
3 0
F
i
100.00
10
100.00
1000.00
MODEL 93
4
600
3 W
E 0.01
0.10
1
.oo
10.00
CLOSED DURATION (ms)
FK;. 9. Clohed-interval histograms for the thrcc inodels describcd in the text. As with the openintcrval distributions, all of thc distributions arc qualitatively similar. Each distribution ha.\ two coniponents. which irnplics two closed statcs in each modcl.
3. ION CHANNEL FLUCTUATIONS
a7
upon the mean duration for each open state (and, therefore, the rate constants for leaving the state), as well as the probability of repeated transitions between the open states. Methods for dealing with this problem have been described (Colquhoun and Hawkes, 1983). Although interval histograms can generally define the number of different kinetic states, except for very simple schemes, the order of the states may not be obvious. Determination of the minimum kinetic model requires a variety of experimental approaches. One class of approaches, which was mentioned previously, involves the examination of “adjacent” states; that is, states that directly communicate. There are various methods for performing such “adjacent state” analysis (McManus et a / . , 1985; Colquhoun and Hawkes, 1988; Ball et uf., 1988). We will present two here. The first method provides only a qualitative method of looking for adjacent states. Nonetheless, this topographic approach can be a relatively simple method for initially screening a new preparation, or the plots may be used as a “fingerprint,” which is characteristic of certain classes of kinetic schemes. For brevity, we present only the results from applying the approach to data from one of the schemes we have presented in Table I. For the model #2 mentioned previously, Fig. 10 shows a topological representation of the relationship between open (or closed) states and their immediately “adjacent” state (in a temporal sense). In the upper panel of Fig. 10, we have constructed a three-dimensional histogram of all pairs of open and subsequent closed events (or closed and subsequent open events, depicted in the lower panel). One horizontal axis represents the durations of one class of events (either open or closed) and the other axis represents the duration of the event immediately following the events chosen for the first axis. As an example consider all open events. One horizontal axis is divided into intervals, a t , with the ith interval beginning at i6t and ending at ( i + 1)6t. The other horizontal axis is divided similarly. The two axes define a histograni H,, whose elements are defined by the frequency that an open interval, which falls in the ith interval, is immediately followed by a closed event whose duration falls within the.jth interval. Thus, the height of the histogram bins represents the number of open events, which fall in a specific temporal class, that are followed by closed events, which also fall in a specific temporal class. In the simplest case, the temporal classes are defined by the components of the interval histograms and correspond to individual states in the kinetic model. For practical reasons, in Fig. 10, the intervals along both horizontal axes are logarithmically distributed. In the upper panel, the duration of events that are “adjacent” to open intervals is plotted; in the bottom panel those intervals that are temporally adjacent to closed intervals are plotted. The two peaks in the top panel indicate that there is an open state of short duration that directly communicates with a closed state of short duration, and another open state of long duration that directly communicates with a closed state of long duration.
08
>
2
3
s L
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
20
1s 10
S 0 0.1
1
10
OPEN WFATKIN (me)
.-. >-
u
M
a w
20
63 (L
I 10
0 0.1
1
10
100
CLOSE0 WRATlON (me)
FIG. 10. Temporally adjacent states for one four-state model. To determine possihle communication hetween different states in model #2 (top), the duration of all open events plotted on the ncar axis arc comparcd to the duration of the closed event (plotted on the far axis), which immediately follows the open event. The height of the function ( z axis) is proportional to the iiumbcr of open events, in a specific duration range, that are immediately followed by a closed interval within sume range of closed intervals. The intervals on both the near and far axes are the log of the interval duration; i.e., every 10 intervals represents a decade change in the open or closed intervals. In the lower panel, a similar plot compares the duration of open intervals that immediately follow closed intervals.
Another method for examining “adjacent states” is, in theory, more quantitative, but is in many senses philosophically similar to the topographic approach described above. By examining the correlation between temporally adjacent open and closed intervals, it is possible to determine the minimum number of open-closed transition pathways and, also, in cases such as the four-state models discussed previously, whether states of similar duration (e.g., short open with
89
3. ION CHANNEL FLUCTUATIONS
short closed) communicate or if states of dissimilar duration are kinetically adjacent. The correlations are calculated from the expression (Ball et a l ., 1988) given in Eq. (36),
where 0,is the duration of the ith opening and c, is the j t h closing after this opening up to some maximum, LAG. T,,, T , , u,,, and u<are the mean open and closed times and the standard deviations of the mean open and closed times, respectively. Strictly speaking, this function is not the cross-correlation of individual points in the digitized single-channel data, but rather is the correlation of individual pairs of open and closed events when compared with the mean open and closed times. Specifically, the sign of the function distinguishes two categories of events. These are summarized in Table 11, which demonstrates that the sign of the function is negative if open and closed states of dissimilar duration communicate and it is positive for kinetic schemes in which states of similar duration communicate. The cross-correlation function of intervals associated with the three four-state models in Table I are shown in Fig. 1 1 . Kinetic model # I produces a negative correlation and models #2 and #3 are both positive correlations. On the basis of our foreknowledge of the underlying kinetics, we know that in the first model long-duration openings often lead to short-duration closures whereas long-duration closures often lead to short-duration openings. For the other two models, the long-duration open states communicate with longduration closed states, and the short-duration open states communicate with short-duration closed states, leading to a positive correlation. In the absence of our foreknowledge of the underlying kinetic models, the correlation information would be particularly useful in deciding upon the correct kinetic model.
TABLE I I SIGNOF INTERVAI CROSS-CORRELATION FUNCTION" Duration of closed and open intervals
"When open and closed intervals are paired, the relative duration compared to the mean duration of either the open interval or the closed interval can determine the sign of the correlation function.
90
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
0.15
V
A
0.10
-
0.05
-
0
I
0.00
1
0
0
0
0
~
~
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0 -0.05
-
0
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5
10
15
LAG Fit;. I I . Cross-correlation of pairs of open and closed intervals from three four-state models. A table of opcn intervals is paired with the closed interval that inmediately followed. The duration of thc ith open interval is correlated with the duration of the i nth closed interval for values of n (lag) varying from 0 to 15. Communication betwcen open and closed intervals of similar duration (compared to the mean open or closed duration) produces a positive correlation (B and C) and communication between states of dissimilar duration produce a negative correlation (A).
+
3. ION CHANNEL FLUCTUATIONS
91
B. Advantages and Disadvantages of Single-Channel Measurements As the name implies, the primary advantage and, curiously, the primary disadvantage of single-channel measurements is that they represent the properties of individual channels. The disadvantage of this is that it is difficult to guarantee that the properties of one channel are representative of the entire population of channels. To gain any insight from single-channel measurements into the average properties of a large group of channels requires large numbers of experiments that sample a relatively large number of channels. Nonetheless, it is generally not reasonable to sample anywhere near the number of channels that can be sampled in a single fluctuation experiment. On the other hand, in patches with only one channel, significantly more information can be had about the one channel than can be obtained unequivocally about a population of channels in a fluctuation measurement. Specifically, single-channel measurements are more capable than fluctuation measurements at suggesting appropriate kinetic models that describe channel transitions. There are several reasons for this. First, examination of the relationship of different classes of events often restricts possible kinetic schemes. The qualitative examination can be quantitated by so-called burst analysis, which examines subsets of kinetic states (Colquhoun and Hawkes, 1983). or “adjacent state” analysis, which identifies which states communicate with one another (McManus t’t al.. 1985; Colquhoun and Hawkes, 1988; Ball et a / ., 1988). Besides these analysis methods, which strongly restrict possible kinetic models, there is a technical aspect of single-channel measurement that also contributes to a more accurate definition of channel kinetic states. Singlechannel methods. in general, have better temporal resolution (with new integrating patch-clamp headstages) at both high and low frequencies than do conventional fluctuation measurements. Therefore, not only can the relationship between states be more easily determined from single-channel measurements, but states that are not resolvable in fluctuation measurements can be observed in single-channel records. In addition, once a likely kinetic scheme has been identified, analysis of kinetic parameters is usually easier with single-channel data than with fluctuation data. This is because interval histograms can provide information about individual rate constants (compare Figs. 1B and C and 3B and C) or, if multiple rate constants are involved, the expressions are usually simpler than those derived from fuctuation data. Although these analysis methods make single-channel recording very attractive to examine channel properties, there are disadvantages other than the issue of population representation. Most of these are technical problems of implementation. In general, fluctuation experiments require attention to detail (Van Driessche and Zeiske, 1985; Gogelein and Van Driessche, 1981; Lindemann, 1984b), but can usually be implemented in most epithelial tissues. For patchclamp experiments, there is no N priori reason to expect that it will be possible
92
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
to form high-resistance seals on a new tissue. It appears to be somewhat more difficult to form seals on epithelial cells than on many excitable tissues. Renal collecting-duct cells, in isolated tubules, have been a notoriously difficult preparation on which to form seals. One part of this problem is access; problems of forming gigaseals are particularly acute for measurements of basolateral channels, although tissue culture systems may allow access to the basolateral surface of cells. In addition, to form seals on many cells from native epithelial tissues requires pretreatment with enzymes. Because some epithelial channels may be modified or degraded by proteolytic enzymes, it will always be necessary to demonstrate the normalcy of channels observed after enzyme treatment.
IV. COMPARISON OF SINGLE-CHANNEL MEASUREMENTS WITH FLUCTUATION MEASUREMENTS: A SPECIFIC EXPERIMENTAL EXAMPLE The examination of potassium channels using both single-channel and fluctuation measurements has been dealt with in another chapter of this volume; we will therefore concentrate our comparison on the properties of the amilorideblockable sodium channel of tight epithelial tissues. Amiloride-blockablesodium channels represent a unique ion permeation mechanism found in “tight,” i.c., high-resistance, epithelial tissues. The channel represents the principal site for the discretionary control of sodium reabsorption in the kidney. As such, sodium channels play a critical role in total body sodium balance and are the target of numerous modulatory factors, including the hormones aldosterone and vasopressin or antidiuretic hormone (ADH). Because of its importance to salt balance, the channel has been investigated extensively both in renal tubules (where it is found primarily in the distal nephron) as well as in various model tissues such as frog skin or toad urinary bladder, which contain high densities of the channel. In previous sections, we have examined the use of single-channel measurements and fluctuation measurements individually. It is appropriate to compare the relative usefulness of the two methods. To do this, we will consider the fluctuation and single-channel measurements that have been made on the epithelial sodium channel.
A. Background Some of the first fluctuation measurements on any epithelial tissue were performed to investigate the properties of the sodium channel in frog skin (Lindemann and Van Driessche, 1977). The hope was that, as with channels of excitable tissue, it would be possible to measure the spontaneous openings and
93
3. ION CHANNEL FLUCTUATIONS
closings of the sodium channel. However, the conclusion from these original measurements was that there was no obvious spontaneous Lorentzian in the range 0.2- 100 Hz. Only after application of channel blockers such as amiloride was it possible to observe a Lorentzian component of the power spectra. Because of the lack of any observable spontaneous Lorentzian, Lindemann and colleagues (Lindemann and Van Driessche, 1977; Lindemann, 1980; Lindemann, 1984a) argued that, at least as a good approximation, the sodium channel block by amiloride could be modeled as a two-state process; that is amiloridr
OPEN A OPEN BLOCKED
(37)
This notion effectively sidestepped the issue of the underlying kinetic scheme for the sodium channel in the absence of blocking agents. Lindemann and colleagues were always careful to point out that there might be spontaneous fluctuations of the sodium channel, but that they had no experimental basis for concluding whether they existed. Unfortunately, this presentation also tended to cloud the fact that there was also no a priori basis for assuming that the kinetic scheme above was a completely correct description of amiloride’s interaction with the sodium channel. I t is true that the corner in the power spectra implies that one of the mechanisms of amiloride’s block is an interaction with open channels, but such an interaction does not preclude other types of interaction. Nonetheless, the simple model of amiloride block was useful for a variety of studies involving the nature of the amiloride-binding site at the external surface of the channel (Li and Lindeniann, 1983b; Li ~t d . , 1985, 1987) and has been used as a qualitative estimate of the number of amiloride-blockable sodium channels present in tight epithelia (Lewis et ui., 1984; Loo et a/., 1983; Sandle ct ul., 1986; Zeiske t’r ul., 1982; Halm and Frizzell, 1986). However, when more quantitative estimates of the sodium channel density were required, the two-state model of Eq. (37) was not generally adequate. The problems developed because of a variety of interesting features of the epithelial sodium channel. As might be expected in considering the channels’ role in maintaining homeostatic balance, the sodium flux through the apical membrane via these channels is strongly modulated by a variety of physiological factors. One of the mechanisms by which this modulation takes place is to alter the density of conducting channels in the apical membrane; the hormones aldosterone and ADH appear to regulate the apical sodium entry in this manner. In addition, there appears to be at least one mechanism, which has been termed “homocellular” (Schultz, I98 I ) , to maintain a balance between apical sodium entry and total sodium transport. This modulation of sodium entry has been termed “sodium self-inhibition” (Fuchs et a/., 1977; Van Driessche and Erlij, 1983; Li and Lindemann, 1983a; Frehland P t ul., 1983). In one version of “self-inhibition,” sodium ions are capable of directly or indirectly closing open channels. Of course, this concept of self-inhibition requires that there must be a closed state of the channel and, there-
94
DOUGLAS C. EATON AND YOSHlNORi MARUNAKA
h e , a( lcast a three-state kinetic scheme ( i f aniiloride block is included), which is usually written as CLOSED
=OPEN
iiiiiloride
OPEN R1.OCKED
(38)
Such a three-state niodel has been used to describe the effect of a variety of blocking agents (Li rt ( J / . , 1985; Abramcheck et a / . , 1985) as well as several hormones (Helman PZ al., 1983) and drugs (Baxendale and Helman, 1986). Nonetheless, despite the apparent necessity for a closed state of the channel in the absence of blocking agents, there was no direct evidence for such a state. To summarize these points, examination of the microscopic properties of individual sodium channels using power spectrum analysis methods has been complicated by the fact that there is no discernible corner to the spectra from intact tissues, at least down to frequencies of about 0.5 Hz (Abramcheck et a / . , 1985; Lindemann and Van Driessche, 1977; Lewis et d.,1984). This implies that either channels are open all of the time (Lindemann and Van Driessche, 1977) or that their rate of transitions between open and closed states is less than I sec-’ (Abramcheck r t u l . , 1985).
6. Problems Measuring Sodium Channel Fluctuations Since these fuctuation measurements were made, single-channel measurements (Palmer and Frindt, 1986; Marunaka and Eaton, 1988) have demonstrated in several renal preparations that epithelial sodium channels do have both an open and closed state in the absence of blockers, and that, as expected, the spontaneous transition rates between the closed and open states are in the range 0.4-0.7 sec-I. This corresponds to a corner frequency of about 0.1 Hz. To resolve such a spontaneous corner would require a frequency rcsolution down to approximately 0.01 Hz. Although this is theoretically possible, practically frequencies as low as this are generally not accessible to investigators measuring fluctuations from whole tissues for two reasons. First, resolution of low frequencies rcquires preparations that are stable for very long periods of time, and, second, even if preparations were stable, there is always a significant component of background noise that is very large at low frequencies and makes resolution of Lorentzian components difficult. Single-channel recording suffers less from thew problems. Maintaining stable recordings of one or a few single channels with constant open probability for relatively long periods of time is possible (Palmer and Frindt, 1986; Marunaka and Eaton, 1988). Moreover, the background noise of the random fluctuations in either open or closed channels has much lower power than that of transitions between open and closed channels. At the resolution of the power spectra presented in this discussion and for the frequency range examined (<1 KHz), the background noise from channels appears essentially “white” (see Fig. 14 and Sigworth, 1985).
95
3. ION CHANNEL FLUCTUATIONS
1
0.1
-- plus arniloride 0.01
0.001
0.0001
difference
I
--
I u 1
10
100
FREQUENCY FIG. 12. Power spectrum typical of arniloride-induced noise from various tight epithelia (see, e.g., Zeiske PI ul.. 1982; Van Driessche and Erlij, 1983; Lewis e t a / . , 1984). The two records are obtained in the absence of blocker (labeled background) and in the presence of 1 pM amiloride (plus arniloride). The difference is best tit by a single Lorentzian function representing the arnilorideinduced noise, although there is excess noise at low frequencies (which may be due to spontaneous fluctuations).
For these reasons, measurements of macroscopic fluctuations from epithelial sodium channels have generally been confined to blocker-induced fluctuations of the channel. One example of such a spectrum obtained from rabbit colon, an aldosterone-sensitive tight epithelium (Zeiske et al., 1982), is illustrated in Fig. 12, and other examples are given in Chapter 4 of this volume, by Helman and Kizer. There are several difficulties making measurements with blocking agents. First, the induced fluctuations often represent a relatively small component to the overall power, with the background noise contributing large amounts of power at the corner frequencies of the blocker-induced noise. The background noise consists of both llfcomponents of unclear origin (see Fishman and Leuchtag, Chapter 1 ) as well as l / p noise from the spontaneous fluctuations of the unblocked channel. The contributions of the background noise have, therefore, been modeled as Ilf” noise, where n is observed to vary between 1 and 2 (Van Driessche and Zeiske, 1980). Second, because the spontaneous fluctuations cannot normally be measured, much of the information that might suggest possible kinetic models is lost. In particular, the plateau level of the low-frequency fluctuations could help to suggest potential minimum kinetic models.
96
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
1
n
0.1
V
x
(--
Amiloride
0.01
ru'
5
0.001
E
g 0
0.0001
a
0.0000 1
0.000001 0.01
0.1
1
10
100
FREQUENCY (Hz)
B
1
c 'V
g
0.1 0.01
N'
%
v
0.001
E
0.0001
a
0.00001
0.000001 0.01
0.1
1
10
100
FREQUENCY (Hz)
FIG. 13. Power spectra of single-channel data from cultured (A6) rcnal cells. Single-channel records of 15-20 min duration from gigaseal cell-attached patches on A6 cells wcre filtered at 100 H7 and then sampled at S-nisec intervals. The amount of data was usually derived by appending several 2- to 8-min records. Most of the records were chosen to contain three to five channels to increase the magnitude of the fluctuations, although records that contain only single channels also produce similar power spectra for the same number of total open-to-closed transitions. To produce the spectra in this figure, a 16,384-point fast Fourier transform was applied to thc digitized data. (A) In the control power spectrum, the square symbols represcnt the power spectra of a patch in the absence of channel-blocking agents. This power spectrum demonstrates that spontaneous channel fluctuations do produce a corner in thc power spectra, but at the relatively low corner frequency of 0.155 Hz. The open triangles represent the power spectrum of a patch with no channel activity. This
3. ION CHANNEL FLUCTUATIONS
97
C. Power Spectra from Single-Channel Data One way to get around these problems is to generate the power spectra directly from single-channel data. There are two benefits of this approach: first, the background noise of a channel-free patch (or a patch during the periods when all channels are closed) is very low and is also “white,” i.e., with no dependence on frequency, at least over the range of frequencies used by us (Sigworth, 1985); second, the frequency resolution is increased because of the very long recording times possible with stable gigohm-seal patches. Even the fluctuations of an open channel have very low power compared to the power of open-to-closed transitions of the channel, so that open channel noise is also essentially “white” (cf. Sigworth, 1985). In Fig. 13, power spectra have been produced from fluctuation data measured in isolated membrane patches from A6 epithelia, which contain anywhere from one to several sodium channels. The first immediately obvious feature of the spectra is that there is an easily discernible low-frequency corner (0.15 Hz) in the untreated control tissue, at a frequency that would be expected from single-channel measurements on the same tissue. The second clear feature is that both 1 pLM amiloride and a 3 pM concentration of an amiloride analog, 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), induce a second corner at much higher frequency than that of the spontaneous corner (2.4 and 12 Hz, respectively). The effect is particularly clear in the presence of CDPC. The two corners are consistent with the simple three-state model described by Eq. (38) (two distinct open-closed transitions), but the effect of amiloride is interesting because of the large decrease in the low-frequency power caused by application of amiloride: it is such a large decrease that the low-frequency corner is obscured. Some reduction in power would be expected, but the decrease seen here seems somewhat inconsistent with a three-state model. The decrease in power cannot be easily attributed to other factors. When amiloride is applied to frog skin epithelial tissue, there is a different “up-regulatory” effect on the total number of functional sodium channels, an effect that has been described by Helman and co-workers (Abramcheck et al., 1985; Helman and Baxendale, 1988). The phenomenon is apparently related to sodium entry across the apical membrane of the entire tissue. In the patch experiments, however, amiloride only blocks a demonstrates that, at least at these low frequencies, the background power does not contribute significantly to the power spectra of channel fluctuations and that the power of a bare patch is “white” rather than proportional to f I . The effect of the channel blocking agent, amiloride ( I pM),is represented by the power spectrum with open squares. Amiloride signiticantly reduces the lowfrequency power, making it difficult to resolve the low-frequency corner, but it docs induce a corner at higher frequency (2.4 Hz). The large reduction in low-frequency power suggests that the mechanism of amiloride block may involve more than only the block of open channels. ( B ) Reproduction of the control data of the upper panel, but also showing the power spectrum of a channel after treatment with 3 pM CDPC. This blocker produces a pronounced corner at 12.4 Hz, with less reduction of the plateau value for the spontaneous Lorentzian.
98
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
:;\\-'= -
n
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0.11 -
u
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-
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1 .
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FREQUENCY (Hz) FIG. 14. Power spectra of sodium channels with restricted frequency range. If the singlechannel data of Fig. 13 are sampled at 0.5-msec intervals and an 8,192-point fast Fourier transform is applied to the digili7ed dala, the frequency range of the data is more comparable to that of power apcctra obtained from Iluctuitions in the shoit-circuit current of whole tissues.
single sodium channel on one cell. Therefore, regulatory phenomena dependent upon whole tissue entry of sodium seem an unlikely explanation of the effects of amiloride on single channels. In addition, the effects of amiloride on single sodium channels are the same in cell-attached patches as well as inside-out patches, which have no intracellular components present on the inner surface of the patch.
3. ION CHANNEL FLUCTUATIONS
99
Also, because the power spectra can be generated from the fluctuations of a single channel, loss of channel activity within the patch is easy to detect. To appreciate the difficulty in obtaining fluctuation information from whole tissues, the single-channel data were sampled at a higher rate and new power spectra were produced that covered a frequency range more consistent with those that have been reported in the literature for whole tissue measurements. As can be seen in Fig. 14A and B, the control spectrum contains no discernible corner, but application of either amiloride or CDPC induces a measurable Lorentzian. Fortunately, the background noise of the patch is of very low power relative to the power of the open-closed transitions of the channels, so that over the frequency range sampled in this experiment, for both open and closed states, channel noise is very close to being “white” (Sigworth, 1985). Thus, the Lorentzian components attributable to the fluctuations produced by the two channel blockers are relatively easy to see; in a whole tissue experiment, the spectra of Fig. 14 would be superimposed on a large 1 (f component of noise, which would further mask any low-frequency Components and even confound the determination of the blocker-induced corner frequencies. Therefore, although theoretically possible, practically speaking, measurement of the spontaneous fluctuations of single sodium channels is experimentally difficult because, first, resolution of low frequencies requires preparations that are stable for very long periods of time, and, second, even if preparations were stable, there is always a significant component of background noise that is very large at low frequencies and makes resolution of Lorentzian components difficult. Thus, it is likely to be difficult in whole tissues to measure low-frequency corners of power spectra from sodium channel fluctuations and, therefore, to unequivocally determine that there are spontaneous fluctuations of the channel in the absence of blocking agents. Even if the low-frequency corner were observable, the rate constants for the transitions could not be determined from the corner frequency, although the rate constants for block and unblock by amiloride or CDPC could be determined by measuring the corner frequencies at several different blocker concentrations (see Helman and Kizer, Chapter 4; Abramcheck et ul., 1985).
D. Single-Channel Analysis To gain additional insight into channel properties requires examination of the single-channel data. Figure 15 shows single-channel data from three cellattached patches formed on A6 cells: one in the absence of sodium channel blockers (Fig. 15A) and the other two in the presence of either I pM amiloride or 3 pM CDPC (Fig. 15B and C, respectively). The channel in the absence of blockers clearly has regular transitions that occur on a time scale of the order of seconds.’ Both blockers produce a profound change in the character of single‘Marunaka and Eaton (1988) have suggested that, in addition to the long-duration openings and closings that are obvious in Fig. 15. there are two other classes of events, short-duration openings
100
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
channel events. The effect of CDPC is primarily to add many fast closures to the long open periods of the unblocked channel. The effect of amiloride is more interesting because it apparently does not produce such rapid closures as does CDPC, and amiloride also produces longer closures than does the control.
I . INTERVAL HISTOGRAMS
To obtain a more quantitative description of the single-channel events. the interval histogram for open and closed events are shown in Figs. 16 and 17. For these cells the mean open time, T,,!,~,,,in the absence of blockers is 1.20 sec and the mean closed time, T , , ~ , , ~is~ 2.55 , sec. In the presence of 1 pM amiloride or 3 FM CDPC, the open time is reduced to 83 and 116 msec, respectively. The closed-interval histograms in the presence of blockers both have at least two components, one of relatively short duration and one of much longer duration. The two histograms in Figs. 16A and 17A imply that, for these cells, the unblocked channel has two stable states, one long-duration open state and one longduration closed state (but see footnote I). These observations imply the following kinetic scheme for the channel in the absence of blocking drugs: CLOSED
kl
OPEN
(35))
k~i
Unlike the case for the power spectra in Fig. 12, the rate constants, k, and k I , in this kinetic scheme can be easily calculated from the interval histograms and and I / T , , ~ " , respectively. Because the most obvious are just equal to I/T',,,,,.,, eft'ect of amiloride and CDPC in Fig. 168 and C was to reduce mean open time, it seems clear that one effect of the blockers must be to block open channels (Palmer and Frindt, 1986; Hamilton and Eaton, 1985). This view is also supported by noise measurements on whole tissues (Abramcheck et a / ., 1985; Heland short-duration closures. Some examples of these are visible in Fig. 15A. In the lirst opening of the top record, there is a fast closure, whereas in the first long closure of the second record from the top, thcrc is a fast opening. These ohscrvations imply that the channel in the absence of blockers has at least four statcs. a long-duration closed stare and a long-duration open state, as well as ahortduration open and closcd states. Despite the apparent prcsence of these additional short-duration states, wc will consider only a two-state kinetic scheme to describe the unblocked channel, for several reasons. First, the rclative number of the short-duration states (particularly short closures) seems to he variable from one batch of cells and even from one patch to the next. The data wc have chosen for this chapter contain relatively few rapid open or closed events. Second, the duration of the fast events is such that they do contribute in a signilicant way to the overall open probahility of the channel, compared to the long-duration events, and, therefore, whilc being of theoretical interest, do not seem to he of major physiological interest. Finally, inclusion of the short-duration open and closed events so complicates the discussion of the relationship between power spectra and singlechannel analysis that it serves only to confound the comparison without shedding any significant light on the subject of this chapter.
101
3. ION CHANNEL FLUCTUATIONS
CONTROL
1pM AMlLORlDE
B
3pM CDPC
C
FIG. 15. Currents from single sodium channels in the presence and absence of blocking molecules. When patches of A6 cell membranes containing only a single amiloride-blockable sodium channel are isolated (A), the channels display a characteristic pattern of transitions between open and closed states, which occur on thc order of seconds (but see footnote I on p. 99). When similar patches are formed, except that 1 pM amiloride (B) or 3 pM CDPC (C) is included in the patch pipet filling solution, then the open time of the channel is significantly reduced. For CDPC, in particular, many fast closures are induced.
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DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
CONTROL 1000
A
0.1
1.o
10.0
100.0
1pM AMlLORlDE
21 k
lwo.o
1. lE4
1000
B
600
400 200
0 0.1
1.o
10.0
100.0
lwo.o
1 lE4
3uM CDPC
C 600 400 200 0 0.1
1.o
10.0
100.0
1m.o
1.OE4
OPEN DURATION (ms)
FIG. 16. Open-interval distributions of epithelial sodium channels in the prcscnce and absence of blocking agents. The open-interval distributions confirm the qualitative impression given by thc data in Fig. 15, that amiloride and CDPC reduce mean opcn time. The mean duration in the absence of blockers is I .20 sec. The mcan open times arc 83 and I16 msec after 1 or 3 W M amiloride or CDPC. respectively.
103
3. ION CHANNEL FLUCTUATIONS
800
-
A
800
-
B
0.1
8W
1.o
10.0
100.0
1ooo.o
1.OE4
C
-
0.1
1.OE5
1.o
10.0
100.0
1m . 0
1.OE4
1.OE5
CLOSED DURATION (ms)
FIG. 17. Closed-interval distributions of epithelial sodium channels in the presence and absence of blocking agents. In the absence of blocking agents, there is essentially only one class of closed intervals, represented by the single exponential distribution in A, with a mean duration of 2.55 sec. After application of 1 pM amiloride (B) or 3 p M CDPC (C), there are at least two components to the interval histograms, with mean durations of 201 msec and 5 .4 2 sec for amiloride and 20.5 msec and 2.88 sec for CDPC.
104
DOUGLAS C . EATON AND YOSHlNORl MARUNAKA
man and Kizer, Chapter 4). This implies that the kinetic model of Eq. (39) should be altered to include this block as follows: CLOSED
ki
I/
OPEN
k.1 k2
k.2
OPEN OCCLUDED
This model is essentially the same as three-state models that have been previously suggested (Baxendale and Helman, 1986); it requires that the long open time should be reduced in the presence of blocker (as observed) and that the mean duration of the time spent in the open state should be dependent upon the blocker concentration according to the relationship:
Although with the simple three-state model, it is possible to determine all of the rate constants from the values for mean duration, the relationship in Eq. (41) allows an additional method for determining the validity of the blocking rate. In addition to the effect on the mean duration of open intervals, the model given in Eq. (40) also implies that, in the presence of blockers, a second class of closed events should appear (OPEN OCCLUDED channels). The interval histogram in Fig. 17 is consistent with there being at least two classes of closed events.
2.
IS A
THREESTATE MODELTHE MINIMUMKINETICSCHEME?
Because there appear to be two exponential contributions to the closed-interval histogram and one exponential contribution to the open-interval histogram, the single-channel interval data appear superficially consistent with a three-state kinetic scheme. However, the interaction of blockers with open channels does not preclude the possibility that the blockers might also interact with closed channels to produce an occluded form of the closed channel. Additionally, if such an occluded form is possible, then open occluded channels may be capable of closing with the blocking molecule in place, thus entering the closed occluded conformation. That is, rather than the three-state model of Eq. (40), the channel might best be described by the kinetic model of Eq. (42), in which both long closed and long open states of the channel can interact with blocking molecules, and occluded channels can make transitions between the OPEN OCCLUDED state and the CLOSED OCCLUDED state: CLOSED
.
ki
k.4
. OPEN
3. ION CHANNEL FLUCTUATIONS
105
3 . SINGLE-CHANNEL METHODS THATSUGGEST A N ADDJTIONAL CLOSED STATE
In Section 111, we presented a variety of methods that can help determine the minimum kinetic model consistent with the kinetic data and that may provide support for or against the kinetic scheme in Eq. (42). Close examination of the interval histograms provides the first suggestion of a departure from a simple three-state model. If there are two closed states (CLOSED and OPEN OCCLUDED) that do not communicate except through the open state according to the kinetic scheme shown in Eq. (40), then there should be two components to the closed-interval histogram, as observed, but the mean duration of one component should correspond to the mean time in the OPEN OCCLUDED state and the other mean duration must be equal to the mean time of the CLOSED state, in the absence of blocker. That is, there should be one component of the twocomponent closed-interval histogram in the presence of blocker that has the same mean duration (albeit with lower amplitude) as that of the single component of the closed-interval histogram obtained in the absence of blocker. For both amiloride and CDPC this is not the case; however, the difference for amiloride is most striking: the mean of the long-duration component in the presence of amiloride is 5.42 sec and the mean of the long-duration component in the absence of blockers is 2.55 sec. Such a result is difficult to reconcile with any linear kinetic scheme with three or more states. An examination of the adjacent-state map (Fig. 18) shows open states are shortened, as expected, but that, particularly for amiloride, one class of closed events appears to be too long and too broadly distributed to consist of only a single exponential component, as the three-state model implies. Correlation analysis would seem well suited to the elucidation of cyclic reaction schemes [as hypothesized by Eq. (42)l; however, because there is only one open state, correlation functions generated in a manner similar to those of Fig. 1 I are flat and very close to zero. Unfortunately, a null correlation does not restrict the class of models, which might explain the effects of amiloride (Ball et al., 1988). On the other hand, if amiloride or CDPC can interact with closed as well as open channels, there should be one very characteristic change in channel kinetics. If there are transitions between the occluded states [Eq. (42)], then the most straightforward demonstration involves examining the total duration of open “bursts.” The definition of a burst is the period of time after a channel opens following a long closed interval (CLOSED state or CLOSED OCCLUDED state) until that time when the channel closes and enters another long closed interval (i.e., the open time, when brief closures are ignored). For CDPC in Fig. 15, the bursts of activity after opening are particularly obvious. A burst of activity can, therefore, only end by a transition from the OPEN state to the CLOSED state, or, hypothetically, in the presence of blocker, by a transition from the OPEN state to the OPEN OCCLUDED state to the CLOSED OCCLUDED state
DOUGLAS C . EATON AND YOSHlNORl' MARUNAKA
106
No BLOCKER
0 001
0.01
......................... 0.1
1
..........,,.-.10
CLOSED WRITlW (SEC)
MILORIDE
W W WRIllON (ssc)
FIG. 18. Adjacent-state map for open states in the presence and absence of blockers. To determine possible comniunication between different states of the sodium channel, in the absence of blocking agent5 (upper panel), the duration of all open events plotted on the near axis is compared to the duration of thc closed event (plottcd on the far axis) that immediately follows the open cvcnt. The height of the function ( 2 axis) is proportional to the number of open events in a specific duration range that are immediately followed hy a closed interval within some range of closed intervals. Thc intervals on both thc near and far axes are the log of the interval duration: i.e., every 10 intervals represents a decade change in the open or closed intervals. In the middle and lower panels, similar plots examine the effcct of 1 FLM amiloride and 3 p.M CDPC. respectively. The spread of the closed intervals for similar opcn times appears too large to be consistent with the single exponential distributions cxpectcd for linear sequcntial kinetic schemes.
(and possibly then to the CLOSED state). Thus, there should be a relationship between the burst duration, or the rate of leaving a burst, and blocker concentration. If an OPEN OCCLUDED channel cannot become a CLOSED OCCLUDED channel, burst duration should increase with increasing blocker concentration (Colquhoun and Hawkes, 1983). If an OPEN OCCLUDED channel
107
3. ION CHANNEL FLUCTUATIONS
can become a CLOSED OCCLUDED channel, burst duration should not increase as much or may even decrease. Following the approach of Colquhoun and Hawkes (1983) for calculating burst durations, using the probabilities for transitions between various states given a specific starting state (OPEN), we calculate the rate of leaving a burst (the reciprocal of the burst duration) as: k-,
I lTbur*,
=
I +
+ k - ?k&,+ k - , k, k
+ k-,
[BLOCKER] (43) [BLOCKER]
where k - I is the rate of leaving a long opening in the absence of blockers. This expression is a hyperbolic relationship with a nonzero intercept ( k - I ) and a value the transition rate from OPEN OCat large blocker concentrations equal to k CLUDED to CLOSED OCCLUDED. Figure 19 shows data for burst durations in the presence of various concentrations of amiloride or CDPC with the best nonlinear least-squares fit to the function in Eq. (43). Even in the absence of any quantitative measurements, it is qualitatively clear from the data of Fig. 19 that there must, in the presence of blockers, be a new path from the OPEN to the CLOSED state, because the rate of leaving a burst increases as blocker concentration increases. If there were no such alternative pathway, the rate of leaving a burst should decrease (Colquhoun and Hawkes, 1983). Thus the simplest explanation of the properties of sodium channels (including the effects of blockers) can be represented by the kinetic model given by Eq. (42) and reproduced in Table 111 with the values for the various rate constants.
2
0.51:
‘2. 0.0 BLOCKER CONCENTRATION (UM) FIG. 19. Rate of leaving a “burst” versus the blocker concentration. The smooth curvcs are the best nonlinear, Icast-squares lit to Eq. (43).
108
DOUGLAS C.EATON AND YOSHlNORl MARUNAKA
TABLE, 111 KINETIC SCHFMr TOR OPrN ANDCI O5FD T K A N $ I I I O A NN I )SFOR BI OC K 13Y A M I ORII)F I CLOSED
.
ki
CLOSED OCCLUDED
AND
CDPC''
OPEN
OPEN 0CCI.UDED k-4
Unblocked
Amiloride
CDPC
"Note: all values are for no potential applied lo the patch pipet.
E. implications of a Four-State Model Because of the kinetic scheme implied by the single-channel data, one question naturally arises: How will this kinetic scheme affect the interpretation of fluctuation data that assumes either a two- or three-state linear model? The answer depends upon the nature of the experiment and also on the blocking agent used to induce fluctuations. In particular, the action of CDPC can be very well approximated by a threc-state model in which CDPC blocks only open channels. Because of the very rapid off-rate from the channel, a CDPC-blocked channel rarely closes with the blocking molecule in place. Thus, the transitions reflected in the power spectra do not reflect a significant contribution of states other than the OPEN and OPEN OCCLUDED states. Therefore, as pointed out by Helnian and Kizer (Chapter 4). CDPC is a better blocker to use cxperinientally for the induction of noise. Amiloridc, on the other hand, is more coniplicated. At high concentrations of amiloride, a significant fraction of the channel molecules reside in a closed configuration, with an amiloride molecule bound to the channel. The off-rate constant for amiloride is so slow that there is a significant reduction in thc number of channels that are available to open and, therefore, are available for open channel block. In the absence of large numbers of open channelblocking events, the total power produced by amiloride block is less than cxpected for a true open channel blocker of similar affinity. Another potential problem involves the use of fluctuation nicasurenients to "count" channels. As pointed out in Section 11, if the appropriate kinetic scheme is known as well as the macroscopic current and some estimate of the open probability of the channel can be made, then the unit current and total number of channels contributing to the total current can be calculated from the valucs
3. ION CHANNEL FLUCTUATIONS
109
of the plateau and the corner frequency of the power spectrum. Choosing an inappropriate kinetic scheme implies applying the wrong formulas to make the calculations of channel number. To determine the inaccuracy of choosing a twostate model to describe the blocker-induced fluctuations, rather than the fourstate cyclic model implied by single-channel measurements, it is only necessary to compare the formulations for the plateau values implicit in the two different models. The blocker-induced corner frequencies for each model are the same; that is, they are composed of the rate constants that link the open state with the blocked open state. For a two-state model (given in Eq. (37), but repeated here for clarity] amiloride
OPEN W OPEN BLOCKED
(44)
the blocker-induced plateau level is given as
where k, is the rate constant for block, N<, is the total number of channels, pa is the open probability of one channel and,f;. is the corner frequency. For two states the unit current is
where pc is the closed probability of one channel and k - , is the unblocking rate. For the four-state, cyclic model [Eq. (42)J there are three Lorentzian components: two simple ones, corresponding to the spontaneous fluctuations of the unblocked channel (CLOSED to OPEN) and blocker-induced fluctuations (OPEN to OPEN OCCLUDED), and one more complex one, corresponding to transitions from the OPEN state through either the CLOSED or the OPEN OCCLUDED states to the CLOSED OCCLUDED state. The latter transitions may involve several transitions among zero-current states before finally returning to the OPEN state. The expressions for the plateau values of these Lorentzians can be derived following standard methods (Colquhoun and Hawkes, 1977). Following the notation of Eq. (42):
where j ; , f ? , and .fi are the corner frequencies of the three Lorentzian components, respectively; k - , is the spontaneous closing rate of the unblocked channel;
110
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
k? is the blocking rate in the presence of blocker; and k , is the rate for entering the compound closed states involving the CLOSED OCCLUDED channcl. The rate, k , , depends upon the rates for entering and leaving the CLOSED and the OPEN OCCLUDED states and is given as
The unit current can in thcory be derived from any of the plateau values and is given by
(50)
i = S,,(0)7Tf,12pc(I)
where S,,(O)and A, are the nth plateau value and corner frequency, respectively, and pL is the overall closed probability of one channel. The calculated values for the plateau levels are shown in Fig. 20 for different concentrations of ainiloride and CDPC. Expressions for the blocker-induced plateau values and unit current appear similar. The difference between the expressions is hidden in thc open and closed probability terms, which for the four-state model are quite different than those for the two-state model. Specifically, the open probability of the two-state model is
and the corresponding open probability for the four-state cyclic scheme [following the notation of Eq. (42)j is
where F = k,k , k - ?
+ k,k
,k-,
+ k,k,k-, + k,k,k
Jblockerl
(53)
and
T =
The open probability calculated from Eqs. (52)-(54) is shown in Fig. 21 for a variety of different blocker concentrations. If rate constants from the two-state model are used to calculate open (or closed) probability, but the real tninimum kinetic model is the four-state model, then the unit current will be underesti-
111
3. ION CHANNEL FLUCTUATIONS
1 E-2 1E-3 1E-4
- - - 4
-/
/
amiloride
1E-5 1E-6
1E-7 1E-8
1E-9
- ./ ./"
\ CDPC
\ \
1
\ \
\.
1E-10 I
0.001
0.010
0.100
1.000
10.000
100.000 1000.000
BLOCKER CONCENTRATION (pM)
FIG. 20. Plateau levels lor a four-state cyclic model. Expected plateau levels were calculated from Eq. (44) for both spontaneous and blocker-induced noise at various different blocker concentrations. The solid and dotted lines are thc level of the spontaneous, low-frequency plateau in the presence of amiloride and CDPC. respectively. The dashed and dash-dot lines are the blockerinduced plateaus.
mated and the calculated number of channels will be overestimated, particularly at high blocker concentration. Therefore, it will be important to verify which minimum kinetic model is most appropriate for the epithelial sodium channel if a detailed description of the sodium channel is to be obtained from fluctuation measurements.
\
c
\
2 m a m
\
\
0
\
LT
n
\
2
w
Q
CDPC
\ \
0
\ amiloride
\
112
DOUGLAS C. EATON AND YOSHlNORl MARUNAKA
V. SUMMARY Fluctuation and single-channel analyses represent powerful methods for elucidating the properties of individual membrane channels. Each method has certain restrictions that make them difficult to use except under special circumstances. In particular, fluctuation analysis can be used when recording from multiple channels, but the correct kinetic scheme must be known for fluctuation methods to be successful. On the other hand, single-channel methods are suitable for determination of the correct kinetic scheme, but only if single channels can be isolated. As demonstrated above, a hybrid combination of the two methods is a real possibility that has the potential of combining the best of both methods. This may be particularly important because an examination of the properties of single channels in epithelial tissues is often impeded by the difficulty of consistently finding many single-channel patches. The combination of fluctuation measurements with single-channel measurements would mean that only enough single-channel patches are necessary to establish the correct kinetic scheme, after which multiple channel patches may be used to determine many of the channel properties. An additional bonus is the low level of background noise, which is essentially “white” when power spectra are generated from single-channel data. The combination of the two techniques may allow significant advances in the understanding of a variety of epithelial channels. REFERENCES Abramcheck. F. J . , Van Driesschc, W., and Helman, S . 1. (1985). Autoregulation of apical membrane Na’ permeability of tight epithelia. Noise analysis with arniloride and CGS 4270.1. C m . Phvsiol. 85, 555-582. Ball, F. G . , Kerry, C. J., Ramsey, K. L., Sansoin, M. S. P., and Usherwood, P. N. R. (1988). The use of dwell timc cross-correlation functions to study single ion channel gating kinetics. Bioplrys. J. 54, 309.- 320. Baxendalc, L. M., and Helman, S. 1. (1986). A three state model for regulation of apical membrane Na’ transport of epithelial cells. Fed. Pror.. Fed. Am. SOC. Exp. B i d . 45, 516. Colquhoun, D.(1971). “Lecturcs on Biostatistics,” pp. 374-395. Oxford Univ. Press (Clarendon), London and New York. Colquhoun, D., and Hawkes, A . G. (1977). Relaxation and fluctuations of membrane currents that How through drug-operated ion channels. Pruc. R. Soc. Londun B 111, 231-262. Colquhoun, D., and Hawkes, A. G . (1983). The principles of the stochastic interpretation of ion channcl mechanisms. In “Single-Channel Recording” (B. Sakrnann and E. Neher, eds.), pp. 135- 175. Plenum, New York. Colquhoun, D.,and Hawkes, A. G. (1988). A note on correlations in single ion channel records. Proc. R. Sol-. London B 230, 15-52. Colquhoun, D . , and Sigworth, F. J. (1983). Fitting and statistical analysis of single channel records. In “Single-Channel Recording” (B. Sakmdnn and E. Neher, eds.), pp. 191-263. Plenum, New York. Ikhland, E. (1979). Theory 0 1 transport noise in nicmbrane channels with open-closed kinetics. Biophys. Struct. Merh. 5, 91 - 106.
3. ION CHANNEL FLUCTUATIONS
113
Frehland, E., Hoshiko, T., and Machlup, S. (1983). Competitive blocking of apical sodium channels in epithelia. Eiochim. Biophvs. Acra 732, 636-646. Fuchs, W., Hviid Larsen, E., and Lindemann, B. (1977). Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin. J . Physiol. (London) 267, 137- 166. Gogelein, H . , and Van Driessche, W. (198 I ). The effect of electrical gradients on current fluctuations and impedance recorded from Necturus gallbladder. J . Membr. B i d . 60, 199-209. Halm, D. R . , and Frizzell, R. A. (1986). Active K transport across rabbit distal colon: Relation to Na absorption and CI secretion. Am. J . Physiol. 251, C252-C267. Hamilton, K. L., and Eaton, D. C. (1985). Single-channel recordings from amiloride-sensitive epithelial sodium channel. Am. J . Physiol. 249, C200-C207. Helman, S. I . , and Baxendale, L. M. (1988). Open channel probability of apical Na channels in epithelia of frog skin. FASEB J . 2, ,4750. Helman, S . I . , Cox, T. C., and Van Driessche, W. (1983). Hormonal control of apical membrane J . Gen. Physiol. 82, 201-220. Na transport in epithelia: Studies with fluctuation analy Lauger, P. (1980). Kinetic properties of ion carriers and channels. J . Membr. B i d . 57, 163- 178. Lewis, S. A., Ifshin, M. S., Loo, D. D., and Diamond, J. M. (1984). Studies of sodium channels in rabbit urinary bladder by noise analysis. 1. Membr. B i d . 80, 135- 151. Li, J., and Lindemann, B. (1983a). Chemical stimulation of Na transport through amiloridehlockable channels of frog skin epithelium. J . Membr. B i d . 75, 179- 192. Li, J., and Lindemann, B . (1983b). Competitive blocking of epithelial sodium channels by organic cations: The relationship between macroscopic and microscopic inhibition constants. J . Membr. B i d . 76, 235-251. Li, 1. H.-Y., Cragoe, E. J . , Jr., and Lindemann, B . (1985). Structure-activity relationship of amiloride analogs as blockers of epithelial Na channels: 1. Pyrazine-ring modifications. J . Memhr. B i d . 83, 45-56. Li, J. H.-Y., Cragoe, E. J . , Jr., and Lindemann, B . (1987). Structure-activity relationship of amiloride analogs as blockers of epithelial Na channels: 11. Side-chain modifications. J . Membr. B i d . 95, 171-185. Lindemann, B . (1980). The beginning of fluctuation analysis of epithelial ion transport. J . Membr. B i d . 54, I - I I . Lindemann, B . (1984a). Fluctuation analysis of sodium channels in epithelia. Annu. Rev. Physiol. 46,497-515. Lindemann, B . (1984b). Analysis of additively contaminated Lorentzians by integration. Biophys. J . 46,409-41 1 . Lindemann. B.. and Van Driessche, W. (1977). Sodium-specific membrane channels of frog skin are pores: Current Iluctuations reveal high turnover. Srience 195, 292-294. Loo, I). D., Lewis, S . A., Ifshin, M. S . , and Diamond, J. M. (1983). Turnover, membrane insertion, and degradation of sodium channels in rabbit urinary bladder. Science 221, 1288- 1290. Marunaka, Y., and Eaton, D. C . (1988). The effect of amiloride and an amiloride analogue on single sodium channels from a renal cell line. FASEB J . 2, ,4750. McManus, 0. B., Blatz, A. L., and Magleby, K . L. (1985). Inverse relationship of the durations of adjacent open and shut intervals for CI and K channels. Nature (London) 317,625-627. McManus, 0. B., Blatz, A . L . , and Magleby, K. L. (1987). Sampling, log binning, fitting and plotting durations of open and shut intervals from single channels and the effects of noise. Pfluegers Arch. 410, 530-553. Neher, E., and Stevens, C. F. (1977). Conductance fluctuations and ionic pores in membranes. Annu. Rrv. Biophvs. Bioeng. 6, 345-381. Palmer, L. C . , and Frindt, G . (1986). Amiloride-sensitive Na channels from the apical membrane of the rat cortical collecting tubule. Proc. Narl. Acad. Sci. U.S.A. 83, 2767-2770.
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Kunyan, K. K.,and Gunn, K. B . (1991). Generation of steady-state rate equations for enzyme and carrier-transport mechanisms: A microcomputer program. In “Methods in Enzymology,” Vol. 171, pp. 164- 190. Acadcmic Press, San Diego. California. Sachs, F.. Neil, J., and Barakati, N. (1982). The automated analysis of data from single ionic channels. k‘//ilrrrRrrsArch. 395, 331-340. Sandlc, G . I., Wills, N. K . , Allcs. W., and Binder. H. J. (1980). Electrophysiology of the human colon: Evidence of segmental heterogeneity. Cur 27, 999 1005. Schultz. S. G . ( I Y X I ). Homocellular regulatory mechanisms in sodium-transporting epithelia: Avoidance of cxtinctiun by “llush-through.“ Am. J . Phvsiol. 241, F57Y-F590. Sigworth. F J. ( 19x5). Open channel noise. I . Noise in acetylcholine receptor currents suggests conformational Iluctuations. Biophvs. J . 47,709- 720. Sigworth. F. I., and Sine, S . M. (1987). Data transformations for improved display fitting of single channel dwell time hiatograma. Riophw. J . 52, 1047- 1054 Van Driessche, W., and Erlij. D. (1983). N o k c analysis of inward and outward N a ’ currents across the apical horder of nuahain-treated frog skin. ~flucgrr-sArch. 398, 179 - 188. Van Dricssche, W., and Zeiskc, W. (1980). Ba! ’ -induced conduccancc Huctuationa of spontancously fluctuating K ’ channels in the apical membrane of frog skin (Runu femporuriu). J . Mmzbr. Biol. 56, 3 1 - 42. Van Driessche, W.. and Zeiske, W. (1985). Ionic channels in epithelial cell membranes. Phvsiol. Rev.65, X31-903. Wilson, D. L., and Brown. A. M. (1985). Effcct of limited intcrval resolution on single channel Truns.Riomcd. Eng. 32, 786-797. measurements with application to Ca channels. I Zciske. W.. Wills. N. K . . and Van Dricsschc. W. (19x2). Na’ channcls and amiloride-induced noise in the m:imm;ilian colon epithelium. Riochirn. Biophys. Acru 688, 201 -2 10.
Part II
Noise Analysis of Epithelial Channels
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CURRENT TOPICS IN MEMBRANES A N D TRANSPORT. VOLUME 37
Chapter 4 Apical Sodium Ion Channels of Tight Epithelia as Viewed from the Perspective of Noise Analysis SANDY 1. HELMAN AND NEIL L . KIZER Department of Physiologv and Biophvsics University of Illinois at Urhuna-Champaign Urhanu, Illinois 61801
I. Introduction II. Theoretical Perspectives 111. Absence of Spontaneous Noise IV. Blocker-Induced Noise: A Simple Three-State Model V. Single-Channel Currents and Channel Densities VI . Noise Analysis with Electroneutrdl and Charged Sodium Ion Channel Blockers VII. Choice of Blocker: Criteria VIII. Results from Noise Analysis with 6-Chloro-3,5-diaminopyrazine-2-carboxamide and CGS 4270 IX . Results from Amiloride-Induced Noise Analysis A . Kate Coefficients B. Single-Channel Currents and Densities X. Amiloride-Sensitive Macroscopic Currents XI. Results from Triamterene-Induced Noise Analysis: A Double Blocker Problem Xli. Dependence of Blocker and Spontaneous Rate Coefficients on Apical Sodium Ion Concentration References
1. INTRODUCTION A vast epithelial tissue literature documents the existence of highly selective, normally nonexcitable sodium ion (Na’) channels that reside within apical membranes of tight epithelia. Under the influence of a physiologically labile electrochemical potential difference, Na’ normally enters cells, necessitating extrusion 117
118
SANDY 1. HELMAN AND NEIL L. KIZER
of Na' via basolateral membranes at the steady state of Nat absorption. Owing to the lability of the electrochemical potential differences and regulation of apical membrane permeability to Na ' by hormones and other agents, the rates of apical menibrane Na entry vary widely reflecting altered physiological capability of the tissues to absorb Na' . It is now widely acknowledged that moment-to-moment mechanisms of regulation of Na' absorption reside at apical mcmbranes of the cells and must therefore involve regulation of Na' channels, especially so in tissues such as frog skin and toad urinary bladder, whose apical membranes are functionally and predominantly selective for Na' . Whereas various drugs, hormones, and other substances are known to cause changes of apical membrane permeability to Na' and hence Na' channel density and/or its single-channel conductance, an ultimate description of the physiology of apical membranes will require additionally knowledge of endocytic-exocytic processes whereby channels are shuttled between cytosol and apical membrane, the status of the vesiculated channels, the status of electrically quiescent or dormant channels within apical membranes, and the mechanisms of activation-deactivation of channels from quiescent to electrically active states. The advent of patch-clamp methodology and its intimate relationship to electrical noise arising from channels has provided important new tools to address some of the issues noted above. This chapter focuses attention on analysis of electrical noise arising from cpithelial Na' channels with emphasis on the use o f chargcd and electroneutral Na' channel blockers that have been used to determine Na+ channel densities and single-channel Na+ currents.
II. THEORETICAL PERSPECTIVES Patch clamp of apical membranes of rabbit (Helman et al., 1985) and rat cortical collecting tubules (Palmer and Frindt, 1986a, 1986b, 1987, 1988), of A6 epithelia grown in culture (Eaton and Hamilton, 1988; Hamilton and Eaton, 1985; Ling and Eaton, 1989; Marunaka and Eaton, submitted), of toad urinary bladder (Frings et d.,1988), and of Na' channels reconstituted into planar lipid bilayers (Olans et al., 1984; Sariban-Sohraby et al., 1984), has provided unequivocal evidence that Na' channels fluctuate spontaneously between open and closed states. In the absence of demonstrable subconductance states, the transition rates between closed (c) and open (0)states are defined by the rate coefficients a and p .
c - 0
P a
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
119
The channel open probability is
where NT represents the total pool of electrically active channels or the mean time averaged open (N,,)plus closed ( N , ) states of the channel. Under voltage-clamp conditions with N , independent channels and with single-channel current, iNa, the spontaneous fluctuations between open and closed states give rise to a variance (aL,)of the mean macroscopic rate of Na' transport, INr(Hille, 1984).
Since
Thus, electrical noise arising from fluctuations of channels is reflected as the variance around the mean current and in principle leads to one approach for estimating the single-channel current from the quotient of the variance, and macroscopic current if p' is close to zero. This approach suffers practically in two ways. According to recent reports (Helman and Baxendale, 1988, 1990; Palmer and Frindt, 1988; D. C. Eaton, personal communication), P' of epithelial Na' channels is highly variable among tissues and averages near 0.4-0.5, requiring, therefore, for Eq. ( 5 ) a separate method of determination of P ' . Second, the variance contains noise arising from sources other than fluctuations of the Na+ channels, leading thus to uncertainty in estimating iNawith Eq. (5). In this regard, the particular noise of interest (arising from fluctuations of the Na' channels) can be determined by measurement of the spectral density or power density spectra (PDS) of current noise. Because open- and closed-state lifetimes of a channel are exponentially distributed with a mean open lifetime of I/aand a mean closed lifetime of l l p (Colquhoun and Hawkes, 1983) it follows that Fourier transform of current noise arising from fluctuations of channels between open and closed states will give a Lorentzian spectral density different from a spectral density arising from other nonexponentially distributed sources of current noise. In this way, the particular noise contained in the variance associated directly with fluctuations of channels can be separated from all other sources of noise. For noise following exponential kinetics, Lorentzians are of the form
120
SANDY I. HELMAN AND NEIL L. KIZER
where S,,and f c are the low-frequency plateau and corner frequency of the Lorentzian PDS, and the radian corner frequency of the spontaneous fluctuations is
2Tfr
=
a
+p
(7)
Accordingly, for a two-state scheme of open and closed channels, the radian corner frequency is simply the sum of the rate coefficients. In this regard and in comparison with patch-clamp experiments, wherein mean lifetimes of open and closed channels can be estimated separately, noise analysis provides corner frequencies that can be used as an upper bound for either a or p.
111. ABSENCE OF SPONTANEOUS NOISE In principle, i t is possible to determine the spectral density with any frequency bandwidth of interest. In practice, however, it is most unusual experimentally to encounter a tissue with sufficient stability of the INato determine the spectral density at frequencies less than 0.1 Hz. When PDS of short-circuited epithelia are measured at 0. I < f < 1000 Hz, it has so far been impossible (under a wide variety of experimental conditions) to observe a spontaneous Lorentzian, leading to the conclusion that if channels fluctuate between open and closed states, they must do so at f : < 0.1 Hz. Put in perspective of patch-clamp experiments, wherein mean open and closed times are in the range of several seconds, the INa would be required to remain absolutely stable for about 40 min to allow data acquisition with suitable signal-to-noise ratio so as to be able to observe reliably fr of about 0.01 Hz. This instability of the fNataken with the normally present '' llf" noise arising from other sources will remain problematic in assessing very low-frequency spontaneous fluctuations in noise experiments. Accordingly, from the vantage point of noise analysis, the so-called llf noise at low frequencies is most likely a combination of background llf noise and noise arising from spontaneous fluctuations of the channels at f > f: appearing as 1 lf" noise, where a defined here should not be confused with the a rate coefficient defined above. If it is assumed thatf: has an upper bound of 0.1 Hz, it can be inferred for any combination of a and /3 that a + /3 < 27-r (0. I ) , so that a andlor p must be less than 0.63 sec-I. Assuming for sake of discussion that a = p, with channel open probability therefore equal to 0.5, the mean open- and CloSed-Stdte lifetimes would be in the range of about 3 sec. Indeed, in agreement with the results from noise analysis, epithelial Na+ channels with high selectivity for Na' and with mean openlclosed times of several seconds have been observed in patch-clamp records (as noted above). In the absence of any hint of existence of a spontaneous Lorentzian at 0. I < f: < 1000 Hz, it would be reasonable to infer that the
121
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
10 p M CDPC
7
0.01
1.0
100
Frequency, Hz FIG. 1. Spontaneous and blocker-induced Lorentzians. (A) Spontaneous Lorentzian calculated assuming a = p = 0.33 radlsec, giving a corner frequency of 0.105 Hz. All calculations assumed an N , = 60 million channelslcm? and a single-channel current iNr of 0.5 PA. Introduction of blocker causes appearance of a second Lorentzian as shown in B for the low-rate blocker amiloridc and in C for the high-rate blocker CDPC. (B) &P = 16 rad/sec pM; kg” = 2 rad/sec, giving an unshifted amiloride. Note the symmetrical shifts offr to 0.055 Hz andf: to 2.9 1 blockerf, of 2.86 Hz at 1 ~ L M Hz. Note also the large decrease of S:, so that S:, < Stm”. (C) With CDPC-induced noise (kob = 7 radlsec pM; k, = 200 rad/sec; unshiftedf, = 42.99 Hz at a low concentration of 10 pM CDPC), the shiftedf? = 43.0 Hz andf; = 0.09 Hz. A t f > 0.1 Hz, and at lower frequencies, noise power originating from the spontaneous Lorentzian varies as a function of f-2and adds to the usual lowfrequency “ l l f ” noise, which is not shown in these figures. The practical lower limit for observing for amiloride and 5 p M for CDPC. blocker-induced noise is 0.5 ~ L M
physiological channel subserving Na’ transport is a rather sluggish channel as regards its gating kinetics. Neglecting other nonfluctuating sources of current noise, a single spontaneous Lorentzian would be present in current noise power density spectra as illustrated in Fig. lA, with f;. at 0.105 Hz if a = p = 0.33 sec-I. At f > 0. I Hz, the S ( f ) of the spontaneous Lorentzian would appear to add to the background ‘‘ 1 ( f ” noise appearing as 1If noise. Our own experience, like that of others, has been to observe on very rare occasions the appearance of what in fact may be a Lorentzian at f < 0.2 Hz. To the extent that spontaneous f i have never been observed at higher frequencies, noise analysis rules out existence of appreciable higher rate fluctuations of channels, such as those of poorly selective Na’ channels that have been studied after Naf channel reconstitution in planar lipid bilayers (Olans ef al., 1984; SaribanSohraby et al., 1984) and of Na+ channels in apical membranes of A6 epithelia grown on impermeable supports (Hamilton and Eaton, 1985, 1988). If such deviations in gating kinetics and of selectivity for Na’ over other cations are physiological, they would, according to noise analysis, represent an immeasurably small contribution to the density of apical membrane channels responsible for subserving Na’ transport.
122
SANDY I. HELMAN AND NEIL L. KlZER
IV. BLOCKER-INDUCEDNOISE: A SIMPLE THREE-STATE MODEL A wide variety of compounds are known to act as blockers of epithelial Na' channels (Kleyman and Cragoe. 1988; Li Pt [ i i . , 1985, 1987). When added to the apical solution in graded concentrations, a single Lorentzian appears in current noise PDS, where corner frequency ,fF varies linearly with increasing blocker concentration [B]. Such observations have been described for cationic amiloride and a wide variety of its analogs (Li et al., 1985), for triainterene (Hoshiko and Van Driessche, 1981; Zeiske and Van Driessche, 1984, 1986), and for the electroneutral cornpounds CGS 4270 (Abramcheck et d.,1985) and, more recently, CDPC (6-chloro-3,5-diaminopyrazine-2-carboxamide) (Baxendale and Helman, 1986; Helman and Baxendale, 1990; see also summaries in Tables I-VII). Although no (I priori reason exists to exclude blocker interactions with closed states of the channel, arguments have been presented that favor the view that channel blocker interacts primarily if not solely with the open state of the channel (Helman and Baxendale, 1990; Li and Lindemann, 1983b). Indeed, when channels are exposed to a blocker, a single Lorentzian appears in the PDS whose corner frequency f'! at any blocker concentration ([B]) varies linearly as a function of [BI. Notably, the absence of a second blocker Lorentzian associated with block of closed channels could he interpreted to indicate either inability of the blocker to interact with closed states of the channels, a corner frequency beyond resolution by noise analysis ( 1 kHz < f: < 0.1 Hz), or similar values of f? for block of open and closed states of the channels. As the latter possibility is inconsistent with blocker concentration-dependent changes of channel densities (Helman and Baxendale, 1990), such observations taken with those of others open G blocked argue in favor of a simple three-state scheme of closed state kinetics. The simplest scheme that accounts for such observations (and those given below) is a three-state model in which the blocker interacts primarily if not solely with the open state of the channel (0) causing fluctuations between open and blocked (b) states of the channel.
In the absence of background noise, the current noise PDS would contain two Lorentzians due to the transition rates a and p of the spontaneous reaction and due to the blocker-related transition rates k,,,[B] and kh,, of the blocker reaction. In such a linear scheme, the mean lifetime of the open state will depend upon the sum of the transition rates, a + ~k,,~lB], and hence the corner frequencies of the spontaneous ( , f : )and blocker reactions ( f ? ) will be shifted from their non-
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
123
interacting relaxation rates (Van Driessche and Zeiske, 1980, and see Van Driessche and Van Deynse, Chapter 2). Noninteracting or unshifted spontaneous and blocker relaxation rates are defined as
r'=a+p
(9)
The shifted relaxation rates are
where
Accordingly, because p depends upon all transition rates of spontaneous and blocker reactions, the spontaneous 271-f:.will be shifted by an amount p to lower frequency and the blocker 2rrfB will be shifted to higher frequency by the same amount. In this regard, it is perhaps fortuitous, but important practically, to note that if the spontaneous cf':) S 0.1 Hz, then p d 0.1 Hz. Thus for any blocker with f : >> f ;, the f p will for all practical purposes be essentially equal to rR= 27rj':. Consequently, it should not be surprising that rate-concentration plots according to Eq. (10) or (12) have consistently been observed to be linear, thereby providing a relatively simple method for determination of the koband kh,, of the blocker reaction [Eq. (lo)]. The low-frequency plateau values of the spontaneous (S:,) and blocker (St) Lorentzians will also depend upon the [B]. For the three-state scheme (see Van Driessche and Van Deynse, Chapter 2 )
s: where
4NTikeP
= ___
Q"27i-f
F
124
SANDY 1. HELMAN AND NEIL L. KlZER
and where K , =: kk,lk,,, is the blocker concentration at which open- and blockedstate channel densities are the same. Assuming values of NT of 60 million channels/cn? and iNiiof 0.5 PA. the IB1-dependent changes of S:,and St were calculated for amiloride and CDPC and are illustrated in Figs. 2 and 3 .
]A
0.00
R 0 c
X
@vI Amiloride 2. Amiloridc concentration-dependeni changes of comer frequencies and low-frequency platcaus of spontaneous and blocker-induced Lorcntzians at amiloridc concentrations 5 3 pM. u = 16 rad/sec pLM; kb, = 2 rad/scc; K , = 0.125 p M ;N , = 60 million/cm*; 3!, = 0.33 radlsec; X,,, iNa = 0.5 pA (compare with Fig. 3). (A) Shift of/: with increasing [B]. Note scales of,f: and fy'. (C and D)Changes of S:, and S p l with increasing [BI. Note scales of g, and SB~'. At [B] > 0.5 pM and as shown in Fig. IH. S: S;,. Because amiloride-induced noise is limited to [B] > 0.5 pM, the biphasic dcpendencc of S! on IS] cannot be demonstrated. Fici.
125
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
A
0.00
C
0 0
B
a 0
25
s o
50
0 0
25
50
pLM CDPC FIG. 3. CDPC concentration-dcpendent changcs of corncr frcquencies and low-frequency plateaus of spontaneous and blocker-induced Lorentzians at CDPC concentrations < 50 p M . Noise analysis is practical at [CDPC] < 300 p M . a = /3 = 0.33 radisec; kta = 7 radisec pM; k , = 200 radisec; K, = 28.6 pM: N r = 60 niillionicrn': iN., = 0.5 PA. Panels are arranged for comparison with Fig. 2. Note scales of,f: andf'?'". S;,> S : F ' at all [ B ] (see also Fig. 1C).
For amiloride with a K , of 0.125 pM (kh= 16 rad/sec pM;khaassumed to be 2 radlsec; see below), S:,falls toward values near zero as IB] increases toward I pM and s::"l' exhibits a biphasic dependence on [B] so that at [B] greater than about 0.5 pM 2,"'" > S:, (see also Fig. 1B). Because of this and becausef? >>f;, the amiloride-induced Lorentzian is due virtually alone to fluctuations arising between open and blocked states of the channel. Experience has indicated that amiloride-induced Lorentzians can only be observed at amiloride concentrations between 0.5 and 10 pM, thus limiting study using this blocker to a range of concentrations far exceeding its K , and forcing additionally study of tissues at markedly inhibited rates of macroscopic Na' transport. For the wcaker channel blocker CDPC, for which the K , is near 29 pM (kgtf'(' - 7 rad/sec pM; kX!IlT - 200 rad/sec) and thef'! are considerably higher
126
SANDY I. HELMAN AND NEIL L. KIZER
in value than those ofamiloride at comparable [BIIK,, $,decreases with increasing IB] while S:D1’c‘varies biphasically with increasing IS], as with amiloride (see Fig. 3 ) . As illustrated in Fig. IC, 10 pM CDPC causes the appearance of an ,fr at 43.0 Hz that is immeasurably shifted by interaction with f: (Aj;. = 0.015 Hz at 10 pM CDPC), but is readily distinguishable from the Lorentzian of the spontaneous fluctuations. For the case here of a high-rate blocker, $, > St,but becausef’; >>f:,the blocker-induced Lorentzian is easily distinguishable from the spontaneous Lorentzian, although at the lower frcquencies the S ( f ) of the spontaneous Lorentzian combines with background noise so that, as has becn done repeatedly, PDS can be fit at .f’ > 0.1 Hz to an equation of the form
It is of particular interest to emphasize two points: ( I f High-rate blockers such as CDPC permit noise analysis to be done at [BI << K,3,and thus at l a , (macroscopic rate of Na transport in the presence of blocker) near the resting values of I,, (absence of blocker). In this regard, high-rate blockers act as probes of the channels and thus permit design of experiments at or near normal rates of Na’ absorption. ( 2 ) Noise analysis with high-rate blockers can be done at KB < [B] < K,, thereby allowing measurement of the biphasic dependency of Sa on [BI, which allows determination of the dependence on [BI of the single-channel current and channel density over a wide range of (B] on either side of the K , . From the results of such studics it was concluded for the high-rate CDPC blocker that CDPC interacts principally if not solely with open channels (Helman and Baxendale, 1990). Whether this is the case for all blockers remains to be determined. +
V. SINGLE-CHANNEL CURRENTS AND CHANNEL DENSITIES Because the St and f ! arise from fluctuations of the channels between open and blocked srates, the i;:, at any blocker concentration is calculated directly with Eq. (20) (Lindemann and Van Driessche, 1977).
Hence the open-channel density at any [ B ] is
127
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
The distribution of channels between open ( N : ) and blocked (N,,) states will, in accordance with the law of mass action, depend upon [BJ and the blocker rate coefficients:
If N,a is defined as the sum N : blocked states at any [B], then
+ Nhor as the pool of channels in open and
It is obvious according to the three-state scheme of blocker interaction with open channels that closed channels are "recruited" from closed into open and blocked states (N<,,,)as [ B ] is increased, and in part such an expectation serves as a test of the idea that the blocker interacts preferentially with open channels. In the limit where [ B ] >> K,, Noh = N , = N ! . NF is the total channel density (closed open + blocked) at any [B]. If the sum of channels distributed between closed, open, and blocked states remains constant, then the channel open probability could in principle be calculated as N J N , = NJN p. The [B]-dependent relationships among N ! , N,,, and Nh at constant N , are illustrated in Fig. 4 for amiloride and for CDPC, where p' was assumed to be
+
1
'"1
80
t
z c 0
8
40K 8' ,= 0.5
20
,
K, = 0.061
,
pM
0 0
1 2 pM Arniloride
I
3
0
10
20
30
40
50
pM CDPC
FIG. 4. Blocker concentration-dependent changes of channcl densitiea according to mass law action for the three-statc scheme of Eq. (8)for amiloride ( A ) and CDPC (8).Nt! is the open channel density at any blocker concentration. Nhis the channel density of blocked states, and N,,, is the sum of the channel densities of open and blocked states. N r = N ? is assumed to be constant. When [B] >> K , , N o h + N , . At [amiloride] > 0.5 pM. N,,, approximates the NT within about 10%. provided N'? = N , , which assumes that channels distributed between open, closed, and blocked states remain constant at all [BI. At 0.5 pM amiloride, N,H/N,,is 19.6%, indicating an 80.4% inhibition of IN^ due alone to block of Na+ channels. With 5 p M CDPC, N t I N , is 91.7%. indicating an 8.3% inhibition of I,,, due alone to block of Na' channels. Calculations assumed p' = 0.5.
TABLE I
RATE COEFFICIE.NTS. S l h C L E - C H A N N E L Na* CURRENTS.
A N D O P E N CHANNEL.
IN,
Tissue
Species
Anionh
A'
DENSITIES AS MEASURED WITH ELECTRONEUTRAL BLOCKERS" Xh,
Ha
N"
(sec-')
(PA)
(millions/cm!)
oh
(sec . /AM)-'
(pAtm2)
Reference
CDPC-induced noise
* h)
m
Nondepolarized Skin
21
26.0 i .5 (12.3 53.6) 23.5 t- .6
7 5 2 0.1 212 2 6 0.48 f 0.01 (6.08-9.09) (162-308) (0.32-0.62) 1.4 2 0.2 192 f 5 0.32 2 0.02
56.5 t- 4.1 (25.7- 167) 82.3 t- 8.3
Rana pipiens Xenopus taevis
46 72
20.6 t- I 3.95 f 0.22
6.4 7.9
Xennpirs luevis Rana pipiens
15 15
14.3 t- 0.9 92.9 2 8.1
I1
7.3 i 2.0
Rana pipiens
43
Rana pipiens
A6
A6. aldos terone pretreated Colon
Rana esculenta: temporaria Pseudemu scrrpta
II
220 f 5 254 f 5
0 61 0.51
0.02 0.01
36.5 -t 3 . 2 8.1 f 0.5
80201 6.7 i 0.1
23524 300 i 10
0.44 2 0.02 0.79 2 0.11
31.6 i 3.7 I17.6<
3.8
265
1.1
f 0.1 f
k
0.1
0.1
11.lt-0.8
-C
3
744f53
2 2
2 0.2
-
6.6p
Helman and Baxendale (19901 Baxendale and Helman" Kizer and Helman' Baxendale. Duncan. Sariban-Sohraby. and Helman
'
Baxendale. Duncan. and Helman' Thompson ei a t . (1987) Krattenmacher et al (1988) Wilkinson and Dawsond
Urinary bladder Skin
Pseudemw scripta Rana pipiens
C1
CI
8
7
14.9 2 0 . 6
-
9.53 2 31
CGS-4270-induced noise 18.951.9 3.050.1 3042s
-
-
0.36-CO.05
60.6tll.O
Helman and Stetson' Abramchecketal. (1985)
CDPC-induced noise K
A
N (D
depolarized Skin
+
Rana pipiens
SO,
Rana pipiens
CI
18
8
2.4
7.4 t 0.2
221
-t
6
0.12
0.01
2 9 2 t 29
21.4-C 3.7
6 . 4 2 0.5
236
5
5
0.24 5 0.02
105 -C 26
32.8
-C
5
"Values are means i- SEM for N experiments and range where given. All experiments done at ambient temperature. 'Major anion in Ringer solution is either chloride or sulfate. ' Experiments carried out in the author's laboratory. "Personal communication. ' N o calculated as INa/iNa. 'Experiments done with intact skins; scraped skins were used in all other groups of experiments carried out in our laboratory.
Baxendale and Helman< Kizer and Helman"
130
SANDY I. HELMAN AND NEIL L. KIZER
0.5. Because NClh has invariably been observed to increase as a function of [Bl and because increases of N,,hand decreases of N6 conform rather well to mass law expectations of the three-state scheme, it has been suggested that blockers such as amiloride and CDPC interact primarily if not solely with open channels. If so, it would seem rather clear that the blocker binding site exists within the open channel, and such a thesis is compatible with compelling observations of a voltage-dependent block of cationic amiloride (Henrieh and Lindemann, 1984; Palmer, 1984; Warncke and Lindemann, 1985). Although more complicated schemes may be envisioned, the simple threestate scheme is at the moment capable of encompassing current observations. It should be stresscd from the vantage point of noise analysis that the relative simplicity of the analysis is due to the fact that the rate coefficients governing the spontaneous fluctuations are in absolute terms rather low in value, and in relative terms rather low in value compared to the blocker rate coefficients of amiloride and especially higher rate blockers such as CDPC. Experiments by Y. Marunaka and D. C. Eaton (personal communication) on the sluggish Na' channels in aldosterone-pretreated A6 epithelia are in agreement with those of noise analysis in which, for CDPC, blocker interaction occurs with open channels according to Eq. (8). Although it remains impossible with noise analysis to determine absolute values of cy and /3, channel open probability can be determined from analysis of blocker-induced noise (Helman and Baxendale, 1990). By normalizing N : to those of N,, (absence of blocker), NflIN,, = 1 / ( 1
+ ,!3'[B]/K,)
so that p' can be determined from the fractional decrease of blocker-dependent open channel density at K , < [B] < K,,. Using this approach, /3' measured with noise analysis was found to average near 0.4-0.5 in various groups of experiments on frog skin (Els and Helman, 1988, 1990a,b; Helman and Baxendale, 1990) and A6 epithelia (see Table 1). Such values compare well with those determined by patch clamp, as indicated above.
VI. NOISE ANALYSIS WITH ELECTRONEUTRAL AND CHARGED SODIUM ION CHANNEL BLOCKERS Amiloride and numerous analogs exhibit pK, values that span the physiological range of interest from about pH 3.0 to 10.5. Thus for compounds such as amiloride or triamterene, whose pK, values are 8.67 and 6.20, respectively, channel blockers exist in solution in both charged (B') and neutral forms (B").
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
131
At pH 7.5, 93.7% of amiloride and 2% of triamterene exist as cationic blockers. Defining .f’ as the fraction of [B] existing as [ B + J
Such considerations led Lindemann and colleagues to carry out a variety of experiments at an apical solution pH of 5.5, so that f +was close to unity and so Lorentzian noise could be attributed to the interaction solely of charged blocker with the channel. Under these circumstances and assuming moreover that neutral amiloride does not block channels,
where the apparent on-rate coefficient, k:p = ,f+k;b would appear to be pH dependent. It is true in general at any pH and especially at or near the pK, of the blocker that channels will be exposed to potentially competing blockers, with each blocker (charged and neutral) characterized by respective pairs of on- and off-rate coefficients with a possible scheme of
c+
B a
In addition to the spontaneous Lorentzian, two additional Lorentzians would be expected with unshifted corner frequencies of
With pH-dependent changes off”, the analysis of what may be referred to as a “double blocker” problem is especially complicated when the unshifted values off: and,fl’ are similar. Such double blocker conditions are endemic in both noise and patch-clamp experiments and, at present, no concentrated effort has been made to address such issues for amiloride and other analogs. It is indeed possible even at the extremes of pH (relative to pK,) that the low-concentration form of a blocker may interact with the channel with rate coefficients that could lead to rather complex kinetics that exacerbate delineation of the kinetic scheme governing blocker-channel interactions. Abundant evidence exists indicating that both neutral and charged forms of blockers are capable of blocking Na+ chan-
132
SANDY I. HELMAN AND NEIL L. KlZER
nels, so that it becomes important to note the potential pitfalls to be encountered with double blockers such as amiloride and triainterene (see below). It is additionally clear that block of the channels is mildly voltage dependent. It has been demonstrated that both the on- and off-rate coefficients for amiloride block of the channels are voltage dependent, with cationic amiloride sensing about 13% of the apical membrane voltage (Warncke and Lindemann, 1985). On this basis, a plug-type model has been proposed (Li et. al., 1987) in accordance with the previous suggestion by Cuthbert (1976). As apical membrane voltage is highly variable among Na+-transporting tissues, reflecting differences of transepithelial Na+ absorption and other factors, it should also not be surprising to find that koh and kh,will show variations among tissues due to differences of membrane voltage. For this reason, among others, it is more appropriate to refer to rate coefficients rather than rate constants in discussion of blocker kinetics. Indeed, in addition to voltage dependence of cationic blocker, it has been observed that the rate coefficients of electroneutral CDPC can be altered markedly by hormonal activation of Na + transport (Els and Helman, 1990a,b) and by other maneuvers thought to involve changes of intracellular pH and Ca2 that modify Na absorption (Kizer and Helman, manuscripts in preparation). Such observations underscore the need for additional work in resolving the nature of the blocker-"binding site" interaction. In design and interpretation of experiments utilizing charged blockers with pK, values in the physiological range of interest, several important problems, as indicated above, arise and may not be easily circumvented. These problems center on issues related to the existence of neutral and charged forms of a blocker that may compete for binding to the channels and where rate coefficients of the charged form of the blocker are additionally voltage-sensitive. Such issues remain to be addressed in detail for amiloride and its analogs in order to more completely understand the kinetic nature of the blocker reaction with its binding site. Although the voltage dependency of the rate coefficients of cationic amiloride is weak, its existence argues strongly in favor of the view that the binding site is within the open channel. +
+
VII. CHOICE OF BLOCKER: CRITERIA Blocker-induced noise analysis, aside from relative simplicity and its noninvasive nature, offers advantage as an investigative tool in allowing study of tissues ( I ) not amenable to patch clamp, ( 2 ) over the time frame of minutes to many hours, and ( 3 ) in the absence of mineralocorticoid stimulation of Na' transport as has been necessary in patch-clamp studies of A6 epithelia (Ling and Eaton, 1989), rabbit cortical collecting tubules (Palmer and Frindt, 1987), and bretylium-stimulated toad urinary bladder (Frings ez u l . , 1988). Indeed, noise analysis has permitted comparative study of a wide variety of epithelia (see be-
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
133
low) with quantifiable blocker-induced noise arising from channels specifically involved in Na+ transport, even in tissues with rather low rates of Na’ transport and in the absence of prior treatment with hormones, diets, or drugs. At normal rates of Na+ transport in tissues such as frog skin, in which the channel density is less than 1/pm2,it would not be surprising to note that it has been difficult to find channels in patches as would occur especially if channels are clustered within the apical membrane. It may be noted that in pioneering efforts by Cuthbert and colleagues, the density of amiloride binding sites as determined by displaceable isotopically labeled amiloride far exceeds in value (-400/pm2) the density of Na+ channels measured by noise analysis (Cuthbert, 1973b). Accordingly, it is possible that only a small fraction of plasma membrane-bound channels are electrically active and are capable of fluctuating between open and closed states. Although a significant fraction of channels measured by the amiloride binding method of Cuthbert may reflect nonspecific binding or labeling of the apical membrane or uptake of amiloride by the cells (see below), it remains possible that quiescent channels or nonconductive channels fluctuate at the apical face of the channels and allow access of blocker to the apical binding site, but because of either a “capping” of the channel at its cytosolic face or other mechanisms that maintain the channel shut in an electrically quiescent state, such channels do not contribute to the pool of electrically conductive channels. If such a view is tenable, then it remains of interest to question what mechanisms are involved in determining how and when membrane-bound quiescent channels are activated-deactivated or exchanged between plasma membrane-bound pools of electrically active and quiescent channels. To the extent that channels originate within the cytosol and reach apical plasma membranes by a process of vesicle fusion, it would be reasonable to question whether vesicles contain channels that are electrically active and/or quiescent. Answers to such questions are presently not available. Thus the focus of attention of noise analysis has been on determination of the plasma membrane-bound pool of electrically active channels, and in this regard, the choice of blocker should not in principle be important in measurement of the N , and iNa. There are, however, practical considerations in the choice of blocker to be used as a probe of the channels. First, the blocker should ideally not change appreciably the rate of Na+ transport, because inhibition of apical Na’ entry causes not only hyperpolarization of apical membrane voltage with changes of intracellular electrolyte composition, but also changes of apical membrane Na+ channel density that seems related to a process of “autoregulation” of Na’ channel density that is presently not well understood (Abramcheck et al., 1985; Helman and Baxendale, 1990). With amiloride and other low-rate blockers, relatively high concentrations of blocker that far exceed their KB are required for noise analysis, thereby causing marked inhibition of the Na’ transport rate and thereby limiting analysis of the tissues to conditions of rather low rates of Na’ absorption (<20% of normal transport rates with amiloride as blocker). Such
134
SANDY I. HELMAN AND NEIL L. KlZER
inhibition of transport triggers a large autoregulatory increase of open channel density due to increase of N T , whereas iNs is overestimated due to hyperpolarization of apical membrane voltage (see below). In these regards, it would be advantageous to choose blockers wherein noise analysis is possible at [B] << KB and wherein macroscopic Na' transport is inhibited minimally. Second, it is clear (see below) that the potency of amiloride as a low-rate channel blocker is due principally to its very low value of kc".Because kk, is determined at the 2rf ," intercept of the rate-concentration plots, the statistical uncertainty in measurement of the absolute value of &'" is large, thereby leading to large uncertainties in estimation of the kg:"l and hcnce all subsequent ealculations that depend upon K , . It is thus advantageous to choose high-rate blockers, where k,, >> 0 and where for similar uncertainties that arise in measurement of 27i-f;, both k , and kc,,,can be measured with reasonable certainty. Third, it is advantageous to choose high-rate blockers, where f ," >> f:.so that the f: of the blocker-induced Lorentzian can be easily distinguished fromft and/ or other sources of noise even at [B] < K,. Fourth, it is of advantage to choose electroneutral blockers that avoid the dual blocker problem noted above that arises with compounds with pK, values in the physiological range of interest and where, in addition, the rate coefficients of the charged form of blocker are voltage sensitive. Fifth, it is advantageous to use water-soluble blockers so as to avoid possible effects of organic solvents such as ethanol and dimethyl sulfoxide (DMSO). Sixth, i t is mandatory to use blockers of high purity, as contaminants may introduce the appearance of spurious Lorentzians in noise experiments or in the case of patch-clamp experiments spurious open-blocked states. In this regard, it has come to our attention that some batches of commercially available amiloride contain significant impurities (e.g., Sigma, Lot #47F-0423). Given the above considerations, we have in our own laboratory chosen to emphasize work with the electroneutral Na + channel blocker CDPC, as it fulfills the criteria specified above.
VIII. RESULTS FROM NOISE ANALYSIS WITH 6-CHLORO-3,5DIAMINOPYRAZINE-2-CARBOXAMIDE (CDPC) AND CGS 4270 Compiled in Table I are summary data obtained with the electroneutral blockers CDPC and CGS 4270 in several Na -transporting epithelia, including frog skin, A6 epithelia cultured on permeable supports, frog colon, and turtle urinary bladder. All experiments were conducted at room temperature with tissues bathed with Ringer solution containing either CI- or SO:- as the predominant anion. In one large group of frog skins (N = 43), the macroscopic rates of Na+ transport, INrraveraged 26.0 pA/cm2, with, however, the usual large range of spontaneous rates of transport between 12.3 and 53.6 pA/em2. In this group, the kob for CDPC ranged from 6.08 to 9.09, averaging 7.5 radisec pM. The k ,
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
135
averaged 212 rad/sec, with tissue-to-tissue variability of the kboranging between 162 and 308 rad/sec. Single-channel currents averaged 0.48 PA, which is in rather good agreement with results from patch clamp. Open channel densities ( N , ) averaged 56.6 niillionicrn? (or about 56.5 channels/cell or about 0.6 channelsipm?) and ranged from 25.7 to 167 million/cm2, with No being correlated directly with the INa. Similar observations have been made with A6 epithelia studied in their nonstimulated and aldosterone-stimulated states (Helman et al., 1986, and see Table I) where INawas increased by aldosterone from means of 3.95 to 14.3 pA/cm2. The koh, k,,,, and i,, are in the same range as determined for frog skin, with stimulation of I,, occurring as a consequence of increase of No from 8.1 to 31.6 million/crn2. The blocker rate coefficients are not only varhble among tissues of the same species as noted above, but as indicated in Table I, also among species of tissue. It is of particular interest to note that the rate coefficients for frog and turtle colon and turtle urinary bladder are markedly different from those encountered with frog skin and A6 epithelia. Whereas the kobof R u m pipiens skin averaged near 7 radisec pM, the k,,, of skins from Runu esculentu and Runu ternporuriu is significantly less, averaging 3.8 radisec. The k,,, of turtle colon and turtle urinary bladder averaged 1 1.1 and 14.9 radisec pM, and the k,,, for these tissues is markedly greater than observed in other tissues. Hence, for CDPC, as with charged blockers (to be indicated below), the rate coefficients are labile, indicating that the blocker binding site may be regulated physiologically and may additionally possess major structural, compositional, or other factors that would account for the differences of rate coefficients. Experiments in our own laboratory have indicated a selective dependence of k,,, on adenosine 3’,5’-cyclic monophosphate (CAMP)(Els and Helman, 1990a,b) and different time rates of change of k,, and k, in skins of R . pipiens treated with ionomycin, exogenous COz, and quinine (Kizer and Helman, manuscripts in preparation). It is premature to speculate on the reason(s) for this, but such observations may in some ways be used to advantage in elucidation of the nature of the blocker binding site. Depolarization of apical membrane voltage of frog skin causes no apparent change of the CDPC blocker rate coefficients (see Table I) as can be ascertained in nonpaired experiments. However, in three groups of experiments with R . pipiens bathed with C1 Ringer solution either untreated or pretreated with either 2.5 or 25.0 pM forskolin, K + depolarization of basolateral membranes (50 mM Na+ substituted with 50 mM K’) caused no significant change of the kf;bL‘” but caused kEt’Pc to increase from its paired control value by means of 30 to 55%. Since CDPC is electroneutral, it would appear that such a selective change of the off-rate coefficient can be attributed to change of the “receptor” for binding of CDPC to the channel. It is of interest to note that elevation of CAMP by forskolin in nondepolarized tissues causes a selective increase of the kLDpDPc
136
SANDY 1. HELMAN AND NEIL L. KlZER
(Els and Helman, 1990a), and yet in the above experiments, K + depolarization caused increases of kLDK despite prior elevation of CAMPby forskolin. Thus, it seems that the increases of kLDK may be due to factors other than CAMP. iNa,as expected, was decreased by membrane depolarization to means of 0.12 and 0.24 pA for the two groups of experiments reported in Table I. The decreases of i N awere accompanied by relatively large increases of N,, measured about 30 min after depolarization of membrane voltage and at a time when the INahad restabilized at or near the original control values of INa.The increase of N , is due presumably to activation of N , by CAMP secondary to K + depolarization of the tissue (Cuthbert and Wilson, 1981).
IX. RESULTS FROM AMILORIDE-INDUCED NOISE ANALYSIS A. Rate Coefficients It is evident by examination of Table 11 that there are considerable differences in rate coefficients for amiloride binding with the Na' channel over and above those expected due to variations of temperature. In nondepolarized tissues of frog skin, colon, and urinary bladder, the kbo ranges from values near zero in skins of R . pipiens to 20.6 rad/sec in tissues of frog colon. Regardless of the source and nature of variability of the k,, it is clear that in large part the potency of amiloride is due to its relatively low value of kbn. For CDPC with kh ranging between 150 and 300 rad/sec the mean lifetime of the closed state is about 3-7 msec; for amiloride, the mean lifetime can be in the range of about 50 msec to several seconds, depending on the tissue and the conditions of study. Patch-clamp experiments of A6 epithelia and of cortical collecting tubules have indicated mean amiloride blocked times of several seconds, being compatible with values of k, near 0.3 radlsec. As was pointed out previously, such very low values of k,, cannot be measured with confidence from rate-concentration plots (Abramcheck et ul., 1985). Variability of the k , has also been observed in K+-depolarized frog skins, urinary bladder, and lung (thereby ruling out tissue-to-tissue variations of apical membrane voltage), with mean khuranging between 2.5 and 13.8 rad/sec at ambient temperature and at apical solution pH between 5.5 and 8.1. Notably, the kboand koh appear pH independent (5.5-7.5) in skins of R a m ridibundu, with kk, in skins of R . esculentu and in epithelia of lung derived from Xrnupus luevis being considerably greater in value than in urinary bladder and other species of frog skin. It is to be expected that the apparent on-rate coefficient for amiloride (k:RP) would appear to be pH dependent if cationic amiloride is a more potent blocker than the neutral form of amiloride. On the assumption that cationic amiloride is the only effective blocker, the k,& can be calculated according to Eq. (26), where k g - f + k & . It should be emphasized here that calculation of k 2 in this way
TABLE II RATECOEFFICIENTS FOR AMILORIDE (PK,= 8.67)"
Tissue
Species
Nondepolarized Skin Runa pipiens
Colon
Urinary bladder
Rana pipiens Rana pipiens Rana pipiens Rana pipiens Rana remporuriu Rabbit Rabbit Human Rana esculenra temporaria Rabbit Rabbit Rabbit Pseudemys scripta
Temperature ("C) Ambient Ambient Ambient Ambient Ambient Ambient 27 37 37
Anion
CI CI C1
so, CI CI CI CI C1
pH,
k :i?
k&
k hu
Reference
8. I 7.4 8. I 8.1 8.1 7.8 7.4 7.4 7.4
16.4 2 1.0 16.0 23.3 2 1.0 17.1 2 1 . 1 18.7 t- 0.6 7.1 t- 0.9 20.4 2 6.1 68.4 2 5.5 20.0 2 2.0
20.8 16.9 29.6 21.7 23.7 8.1 21.5 72.1 21.1
0.7 2 0.5 2.0 2.1 2 0.8 0.9 2 0.1 5.0 I 1.3 2.2 t- 0.8 18.9 2 8.3 7.4 2 6.1 7.0 -t 3.0
Helman et a/. (1983) Zeiske and Van Driessche (1984) Abramcheck et al. ( I 985) Tang er a / . (1985) Helman and Baxendale ( 1990) Hoshiko and Van Driessche ( I 986) Zeiske et a / . (1982) Zeiske et al. (1982) Wills er a / . (1984)
13.5 2 0.2 52.1 2 7.3 46.0 -t 1.5 47.5 4.9 34.1 2 2.4
16.4 54.9 48.5 50.1 36.0
20.6 2 11.6 2 13.2 2 13.1 2 0.6 t-
13.3
13.0
13.1 13.2 13.2 19.3 19.8 18.0 17.1 9.6
4.4 3.9 3.9 5.6 3.1 2.7 2.5 13.8
20-23 37 37 37 Ambient
CI CI C1 C1
8.0 7.4 7.4 7.4 7.4
C1
_f
4.8 1.1 0.4 1.2 1.7
Krattenmacher el a / . (1988) Loo et a / . (1983) Lewis e f a / . (1984) Lewis and Hanrahan (1985) S. I. Helman and D. L. Stetson (personal communication)
K + depolarized Skin
Rana esculenta
Ambient
so4
7.5
12.5
Ambient Ambient Ambient Ambient Ambient Ambient Ambient 22-27
so4
Urinary bladder Lung
Rana ridibunda Rana ridibunda Rana ridibunda Rana pipiens Rana caresbeiunu Bufo marinus Bufo marinus Xenopus luevis
7.5 5.5 5.5 8. I 7.4 7.5 7.5 8.0
12.3 -t 0.7 1.9 13.2 13.2 2 0.3 15.2 0.9 18.8 2 0.7 16.9 2 1.9 16.0 8.0 2 0.2
so4 so4 so4 so4
so,
S04-CI
CI
* _f
1.0 1.5 + 0.2 2 1.3 2 2.3 t- 2.4 -t
2
2 1.7
Van Driessche and Lindemann ( 1979) Li and Lindemann (1983a) Li and Lindemann (1983b) Li ef a / . (1987) Tang e r a / . (1985) Van Driessche and Erlij (1983) Li el a / . (1982) Palmer er al. (1982) Fischer e f a/. (1989)
"Values are means 1- SEM when available; pH, is apical solution pH. Units of k","b"and k i,(see , text) are rad/sec. p M ; k Gb calculated as ka,Pbqf+kboin units of radisec.
138
SANDY I. HELMAN AND NEIL L. KlZER
presumes a negligible block of the channels by neutral amiloride. To the extent that no evidence has yet emerged for existence of a high-rate form of amiloride block in either noise or patch-clamp experiments, block of channels by neutral amiloride, if it exists, must also be characterized as a low-rate blocker with corner frequencies possibly in the range of cationic amiloride (see below for t riam terene) . Keeping such reservations in mind, the values of k i p compiled from the literature are summarized in Table 11. As was done by Li et ul. (1987), the k& were calculated as indicated above, assuming a pK, of 8.67. At room temperature, k:, ranged from 8.1 to 36.0 rad/sec pM at the extremes where it is apparent that, like the khgr,variability exists for k,k among skins of R . pipiens (16.9 to 29.6 radlsec p M ) , R. temporariu (8.1 rad/sec p M ) , and turtle urinary bladder (36.0 rad/sec p M ) . In K+-depolarized tissues (Table II), k& ranges from 9.6 to 19.8 rad/sec pM. and thc value of 9.6 reported for lung is clearly less than for other tissues of frog skin and urinary bladder. Although in part, variability of the magnitude of the amiloride rate coefficients may be associated with differences of apical membrane voltage in nondepolarized epithelia, the existence of variability in depolarized epithelia taken together with similar findings with electroneutral blockers argues in favor of the view that the kinetics of the blocker reaction is not iikely a constant, inviolate feature of the epithelial Na' channel. This raises the interesting question of the nature of the molecular composition and structure of the binding site and the factor(s) that are capable of modifying its architecture.
B. Single-Channel Currents and Densities Experiments using amiloride-induced noise analysis have been carried out in a variety of epithelia in both their nondepolarized and K -depolarized conditions (Table Ill) to determine single-channel currents and Na+ channel densities. At the relatively high amiloride concentrations (>0.5 pLM) required for noise analysis, noise analysis is done under conditions in which the macroscopic I,, (absence of blocker) is inhibited by about 80% or more and where apical membrane voltage of nondepolarized tissues is hyperpolarized. Accordingly, it would be expected that iR > iNilrwhere superscript B indicates measurements made in the presence of blocker. Such an expectation has been confirmed in experiments where, in the same tissues, CDPC-induced noise permitted determination of the control iNar and thereafter iR was measured following inhibition of I,, by 0.5 pM amiloride (Helman and Baxendale, 1990). In agreement with the known hyperpolarization of apical membrane voltage caused by amiloride in skins of R. pipiens, single-channel current was elevated by about 43% immediately following arniloride inhibition of I,,, and this increase of i,;,, like membrane voltage, is sustained for the duration of the 40-min experiments.
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
139
In skins of R . pipiens bathed with CI-Ringer solution, i:, averages near 0.5-0.6 pA (Table 111). When some groups of skins are bathed with a sulfate-Ringer solution, i;, is considerably less, averaging near 0.2 PA. Whether such differences of i:, reflect differences of electrochemical potential difference and/or differences of single-channel conductance remains to be determined. We believe a 2- to 3-fold difference of i:, is most likely not due to differences of electrochemical potential difference as the intracellular voltages and Na+ concentrations of amiloride-inhibited tissues are not sufficiently different in skins bathed with either chloride or sulfate Ringer solution. Thus, in the absence of other large significant but unknown errors, there may be significant differences of single channel conductance in skins bathed with a chloride or sulfate Ringer solution. The i;, of rabbit colon at room temperature is quite low in value, averaging 0.1 PA, but rises to 0.39 pA at 37°C. In comparison, the i& of frog colon averages I .OO pA at room temperature. As a 10-fold difference of i;., between frog and rabbit colon cannot likely be explained by a difference of electrochemical potential difference, it remains possible at least in part that such differences of iEd may be attributed to differences of single-channel conductance. Despite relatively low rates of N a+ absorption by rabbit urinary bladder studied at 3 7 T , i;, averages between 0.64 and 0.72 PA. K +-depolarized tissues have been studied mostly while bathed with SO,Ringer solution. Under these conditions (Table HI), i:, varies among species of skin, ranging between means of 0.08 and about 0.4 pA at roughly the same rates of macroscopic Na' absorption (15.6-26 pAicm'). From measurements made in the same tissues, K ' depolarization causes ii;, to decrease from a mean of 0.19 to 0.08 pA (Tang et al.. 1985). In view of three- to fourfold differences of i;, among tissues and with apical membrane voltage reduced maximally toward zero by K + depolarization, it becomes reasonable to speculate that differences of i;, may at least in part be due to variability of the single-channel conductance. With K +-depolarized epithelia, wherein apical membrane voltage is essentially unchanged by 100 pM amiloride (Tang et al., 1985) and presumably i N a = i:, (which assumes, moreover, that the Na+ chemical potential difference driving Na+ into the cells is unchanged), open channel density N,, can be calculated as /Na/i;a,and such values are summarized in Table 111 for K + depolarized epithelia. It is of particular interest in evaluation of mechanisms of regulation of Na+ transport to be able to measure not only open channel densities, but channel open probabilities ( p ' ) and hence the size or density of the pool of electrically active closed and open channels ( N T ) . With high-rate blockers like CDPC and CGS 4270, the i;, can be determined at values of i;, near the spontaneous rates of IN*,thereby leading to determination of iNaand hence N,>.And because the blocker concentration dependency of the N : and N,,b can be assessed at
SINGLE-CHANNEL CURRENTS A
0 P
Tissue
Species
Nondepolarized Skin Rnna pipiens Ratio pipiens
Colon
Urinary bladder
Temperature ("C) Anion
Ambient Ambient
CI
c1
Ranu pipiens Rana pipiens Rana pipiens Rana escuientut
Ambient Ambient Ambient
CI
remporaria Rabbit Rabbit Human Rabbit Rabbit Rabbit
20-23 27 37 37 37 37 37
C1 CI CI CI CI
SO, SO,
CI
CI
AND CHANNEL
pH,
IN,
8.1 7.4
-
17.2
TABLE 111 DENSITIES MEASURED WITH AMILORIDE-INDUCED NOISE"
iL
No
Nl
0 59 2 0.05 0.47 2 0.03
3 9 ? 12 53 2 6 60 234 350 f 90
23 +- 8' 320 2 160 610 f 80 180 ? 50 2.0 f 0.1
8.1 8.1 7.8
22.9 29.6 -
0.48 2 0.02 0.23 0.23 f 0.23
8.0 1.4 7.4 7.4 7.4 7.4 7.4
26.3
1.00 2 0.10
0.10 158 0.39 0.09 0.72 0.64 5.9 0.71 0.72 -
a;,
t 0.03 f 0.06
t 0.03 2
0.06
t 0.06 2 0.40
-
Reference
Helman er a/. (1983) Zeiske and Van Driessche (1984) Abramcheck er a/. (1985)b Tang el d . ( 1985)b Hoshiko er a / . (1988) Krattenmacher er 01. ( I 988) Zeiske et a/. (1982) Zeiske era/. (1982) Wills er a / . (1984) Loo et a/. (1983) Lewis er a/. (1984) Lewis and Hanrahan (1985)
K
depolarized Skin Rona esculenra
Ambient
so,
-
26
0.30
f
0 50
52-87
70- 200
R a m escrilenta
Ambient
so,
-
20.6
0 28
2
0.02
74
-
Rana ridihitnda Rana ridibitnda Rana pipiens Rana pipiens Ram catesbeiana Buj?~marinus BuJo marinus Xenopits laesis
Ambient Ambient Ambient Ambient
so, so,
7 5 8 7
15.6
195
-
-
-
-
0.08 2 0.01 0.10 i 0.02 0.09 0.22 2 0.07
Ambient Ambient Ambient 22-27
SO,
25.0 -
+
Urlnary bladder Lung
SO,
so, so, so,
c1
5 5 1 4
78 7 5 7.5 80
-
20.7
3.2
-
-
25 1
-
260 i 90
0.15 2 0.04
167
340
0.18
-
0.02 0.15 2 0.02 0.29 f 0.04 f
? 200 -
23
-
-
24 2 4
"Values are means t SEM. I,, is the macroscopic rate of N a + transport (@A/cm'). Single-channel current (PA). i, A
p
> 0.5 pM. N , , Noh. and N , are channel densities in millions/cm2. Values of i:, and N o , calculated at 1 pM amiloride. ' Units are c h a n n e l s i j d . "N = I.
Lindemann and Van Driessche ( 1977) Van Driessche and Lindemann (1979) Li and Lindemann ( I983a) Li and Lindemann ( 1983b) Tang er a / . (1985) Hoshiko el a / . ( 1 988) Van Driessche and Erlij (1983) Li e t a l . (1982) Palmer C I a / . ( 1982) Fischer er a / . ( I 989) (absence of blocker), and i:,
at [amiloride]
142
SANDY I. HELMAN AND NEIL L. KlZER
K , < ( B ] < K B , the channel open probability can be estimated according to Eq. (24) from noise analysis alone, thereby providing an independent method to that of patch clamp for determination of p' and N.,. (Helman and Baxendale, 1990). With low-rate blockers such as amiloride, this cannot be done, as evidenced by examination of Fig. 4, where N t and Nohare far removed from N,, over the range of amiloride concentrations suitable for noise analysis ( > O . S pM).Accordingly, it would be difficult at best when working at [B] >> K , to analyze for N,, by extrapolation of the [BI-Nfl relationship to zero [B], and such extrapolations of Nu would likely underestimate the correct value of N , even in K' -depolarized epithelia. It should be pointed out that the majority of papers in thc literature report channel densities calculated with Eq. (23) and so are in tact values of N,,hat the rcspective ( B ] at which I;:, and i,$, were measured. With reference again to Fig. 4A and at 1B1 >> K , , the [B1-NObrelationship is nearly flat, with Nohin the range of 90- 100% of N., as amiloride concentration is increased to about 0.5 p M . Hence, the Nc,breported in Table 111 can as a rough approximation be taken as lower bound estimates of N ; , and it is apparent that N , , , > N,,. In the experiments by Li and 1,indemann (l983b), N:! was estimated by extrapolation of Nubto infinite [B], and here Nohwas 1921 millionlcm' as compared with N,, of 195 million/cm'. To thc extent that N . , is constant, the ratio NJN., provides an estimate of p'. which in this case would be near 0. I . As autoregulatory increase of N., accompanies blocker inhibition of INd,values of 6' so calculated can be taken as a minimal estimate of p' if N ; > N , . In review of N a + channel densities of excitable membranes, Hille has noted that channel densities as determined by STX or TTX binding fall in a range of 35 to S33/pm2in a variety of tissues [see Table I , p. 209, in Hille (1984)j, while channel densities determined from the gating charge rangc between 105 and 3000/pm2 lsce lable 11, p. 210, in Hille (1984)l. When such Na' channel densities are compared with those encountered in Na+-transporting epithelia, it becomes readily evident that N . , of epithelial apical membranes (assuming 200 million/cm' or about 2/ym2) is about two to three orders of magnitude less than the N , of excitable membranes. Taking p' = 0.5 and iNa = 0.5 PA, such epithelial Na' channel densities would support a steady-state rate of Na' absorption of 100 pA/cm?.
X. AMILORIDE-SENSITIVE MACROSCOPIC CURRENTS We have referred above consistently to the macroscopic rates of Na' transport as IN,,,where in particular the current carried by Nat is measured as the shortcircuit current, which in epithelia such as frog skin is due entirely to N a + transport at apical membranes of the cells. It must be recognized that the channel densities and single-channel currents measured by noise analysis are specitic to
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
143
those channels that interact with blocker, thereby creating current noise. In this regard, it is important to know the magnitude of the so-called amiloride-sensitive I,,, or maximal fraction of I,, that is blocker sensitive. In practice, this is done by exposure of tissues to 50- 100 pM amiloride ([B] >>> KB)and the amiloridesensitive IN, estimated as the Al,, some 30-60 sec later when I : appears to stabilize. In frog skin the "amiloride-insensitive current" at this time usually averages near 1 pAlcm2, although much higher values have been observed occasionally in molting frogs and indeed in other tissues in which electrodiffusive Na+ transport is amiloride insensitive. In this regard, it is of obvious advantage to carry out experiments with high-rate blockers, where the I!, is considerably larger than the amiloride-insensitive current. With brief exposure of the tissues to amiloride for sufficient time to overcome diffusion of amiloride through unstirred layers, I N a so determined should provide reliable estimates of Na+ transport via channels that give rise to blocker noise. A unique phenomena not encountered at amiloride concentrations less than 0.5 pM occurs when tissues are exposed to high amiloride concentrations (>5 to 20 pM). As documented by Fisher and Lockard (1988) and as observed repeatedly in our own experiments, the I,, continues to fall toward zero after exposure to 100 pM amiloride. At lower amiloride concentrations, the initial decrease of I,, is followed by a secondary but slower return of the I,, toward the original preamiloride value of the I , (Helman and Baxendale, 1990). At 100 pM amiloride the initial decrease of I,, is followed by a secondary and slower decrease of the I,, toward zero. The magnitude of the initial decrease of I,, is often referred to as the "amiloride-sensitive" current, as the rapid initial decrease of I,, is consistent with a direct blocker interaction with the apical face of the Na' channels. At 100 pM amiloride >> K;"" and despite existence of finite unstirred layers, >99.99% of the blocker-sensitive channels must be blocked within seconds after appearance of amiloride in the apical chamber. The Isc that remains, often referred to as the "amiloride-insensitive" Na' current is in fact inhibitable by amiloride since at 100 pM amiloride, the remaining amiloridesensitive current decays toward zero with a time constant of several minutes. Clearly, a fraction of the Na' channels are not directly inhibitable by blocker from the outer apical face of the channel, and it remains unresolved why the amiloride-insensitive current is inhibitable by prolonged exposure of the tissues to very high concentrations of amiloride. Since amiloride addition to the basolateral solution is without effect on the I,, (Briggman et d., 1983), and presuming no special compartmentalization of amiloride after entry of amiloride into the cells. it would seem unlikely that the secondary long time constant decrease of the I,, is due to sensitivity of intracellular enzymes such as adenylate cyclase or kinases (see Benos et al., 1983) that are involved in regulation of channel densities and/or channel open probabilities. Accordingly, whether amiloride at high concentrations exerts a direct effect on blocker-insensitive channels at apical membranes or whether blocker-insensitive channels are lost from the
144
SANDY 1. HELMAN AND NEIL L. KIZER
apical membrane as a consequence of extreme inhibition of the I , remains to be resolved. Because blocker-insensitive channels exist within apical membranes, it is important, especially for purposes of noise analysis, to know the magnitude of the amiloride-insensitive current so as to allow determination of the I!, associated with the blocker-induced PDS. In our own experiments we have turned to fitting falling biexponentials to the I,, records following apical exposure of tissues to 100 pM amiloride. In a large group of tissues of R . pipiens, the amilorideinsensitive current averaged 15.0 ? 0.8% ( N = 206) of the lSc.The time constant for initial and secondary decline of the I,, averaged 22.5 ? 1.3 and 282 ? 26 sec, respectively (Kizer and Helman, manuscript in preparation).
XI. RESULTS FROM TRIAMTERENE-INDUCED NOISE ANALYSIS: A DOUBLE BLOCKER PROBLEM Experiments with amiloride (pK, = 8.7) are normally carried out with bathing solutions of pH < 8.0 so that cationic amiloride is the majority form of blocker. As the pK, for triamterene (T) is 6.2, the charged form of triamterene (T') ranges between an f+ of 1.6% of T at pH 8.0 and 33.4% of T at pH 6.5 (Table IV). If both T + and neutral forms of triamterene (To) block channels, complex kinetics are expected. The degree of complexity will depend on the magnitudes of the rate coefficients governing block of the channels by To and T + . If as indicated above, the rate coefficients can be assumed to be pH independent, and if membrane voltage is held constant, then the unshifted corner frequencies will be given by Eqs. (28) and (29), and the shifted corner frequencies will be given by equations analogous to Eqs. ( 1 1) and (12), where we assume as before that the spontaneous 271-f: << 27rf and 2nf;. Given the remote possibility that 27rf: = 27rfz, it would be impossible to separate out Lorentzians arising from block by To and T + , being.analogous to resolving relaxation rates in any multiexponential kinetic system. TABLE IV APPARENT KATE COEFFICIENTS FOR TWIAMTERENE AS A FUNCIION OF APICAL SOLUTION pHa
PH a 8.0
'f 0.010
N 5
k p!: 1.19
&
k bo
0.06
( I .02- I .35) 7.5
0.048
21
7.0
0.137
5
6.5
0.334
2
2.20 C 0.07 ( I .49-2.70) 5.03 c 0.22 (4.65-5.89) 11.5 2 1.9 (9.60- 13.4)
"Value? ot k:f: (rad/wc * F M ) and k,,, (radlsec) arc means
?
25.4 2 1.5 (2 I .9-29.9) 26.6 2 1.4 ( 17.3-43.4) 21.2 2 2.6 ( 1 3.7 -27.8) 16.5 ? 0 . 7 ( 15.9- 17.2)
SEM (range) ot N experiments
145
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
With such ideas clearly in mind, we examined the blocker-dependent changes of corner frequency as a function of apical solution pH. Skins of R . pipiens were incubated in an HCO?-free Cl/HEPES-Ringer solution containing 100 mM NaCI, 2.4 mM KCI, 2.0 mM CaCI,, and 8.0 m M HEPES, with basolateral solution maintained constant at pH 8.0 and an apical solution pH, of 8.0, 7.5, 7.0, and 6.5. Triamterene was added to the apical solution at concentrations between 5 and 50 pM using a staircase protocol (Helman and Baxendale, 1990). Current noise power density spectra were determined in the usual way. PDS at all pH, yielded what appeared to be single Lorentzians, in agreement with the results of others (see Table V). As indicated in Fig. 5A, the 27rf,B varied linearly with triamterene concentration [TI. The apparent on-rate coefficient k:gP varied as a function of pH,. At a pH, of 8.0, the k i p averaged 1.19 rad/sec p M and was increased to 11.5 rad/sec pM at pH, of 6.5 (Table
Pnf, ,rod/s
C
0
300
b.5
too
25
MM T or T+
f
f+
FIG. 5 . Double blocker complications in determination of rate coefficients; an example with triarnterene (pK,, = 6.2). (A-C) Results of experiments with skins of Rana pipiens. Apical solution pH, was varied between 8.0 and 6.5 (standard error bars not showing are less than the size of the data points). (A) With acidification of pH,, and increase of the fraction of triarnterene ( f ' ) in its charged form, the apparent on-rate coefficient k g was increased (see also Table IV) with relatively little or no change of the kh,. (B) Dependence of k j onf' indicating linear dependence of k:gp on f" , but with an extrapolated nonzero intercept. (C) The k:,p is normalized for f ' , indicating an apparent pH, dependency of the triamterene k&. (D-F) Theoretical calculations according to double forms of triamterene block channels. blocker hypothesis, where both charged (T' ) and neutral (T") (D)For the rate coefficients given in the text, the solid lines superimpose the empirical observations shown in A. Here k#' (solid lines) > &, (dashed lines) in a pH-dependent manner. yielding as shown in E and F the expected changes of k& as a function off" , if Tt alone blocked channels, and of k:@, if both T" and T+ blocked channels.
TABLE V
RATE COEFFICIENTS FOR ‘I‘RIAMTERENE(pK, = 6 2)”
Tissue
Nondepolarized Skin
Urinary bladder
K
depolarized Skin
Species
Temperature (“C)
Anion
pH,
Rana :emporarru
Ambient
CI
6.0
12.4
20.2
19.2
Rana mnporaria
Ambient
so4
7.0
5.0
36.5
36.0
Rabbit
37
c1
7.4
2.0
33.7
100.0
Lewis e f a/. ( I 984)
Ratiw ridibunda Kana ridibunda Bufo marinus Bufo marinus
Ambient Ambient Ambient Ambient
37.0 16.0
34.0 2 2 . 3 36.2 2 3.0 25.3 t 1.9 30.0
Li and Lindemann ( 1 983b) Li and Lindemann (1983b) Li and Lindemann (1983b) Henrich and Lindemann (1984)
L3
bc;
‘
ho
Reference
Hoshiko and Van Driessche (19811 Zeiske and Van Driessche (1986)
+
Urinary bladdei
“See footnote to Table I1
so4
so
4
so4
so,
7 .O 5.5 7 .O 5.5
5.1 2 0.9 13.4 2 0.7 8 . 8 2 1.2 12.0
64.4
14.4
147
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
IV). The mean k,,, ranged between 16.5 and 25.4 radlsec, with considerable variation among tissues at all pH,. Taken at face value, these data can be interpreted to indicate that channels are blocked by T + , with differences of k:gp attributable to pH,-dependent changes of T'. If indeed this is the case, then k:,gp should vary linearly with ,f', with extrapolation through the origin. As illustrated in Fig. 5B, the j ' + - k : ~ p relationship is shifted upward (nonzero intercept), leading as shown in Fig. 5C to an apparent pH dependence of the k& calculated from the quotient k:[plf . Recalling that the rate coefficients for amiloride as indicated above (and see below for CDPC) are pH independent, it would seem strange but not impossible to believe that the kc: for triamterene is pH dependent and at variance with the behavior of other blockers. It is moreover curious that the apparent pH, dependence of k A occurs at the more alkaline pH,, where IT"] >> IT']. If To and T + compete for the same binding site, then in the presence of double blockers with appropriate rate coefficients, we surmised that it might be possible to explain simply this anomaly without resorting to more complex considerations. To examine this idea, we used the empirical data of Fig. 5A as a guide in choosing rate coefficients fork& and kzc,.For kc, we assumed a value of 24 radlsec (Table IV and Fig. 5A) and for k,; we assumed a value of 34 rad/sec p M that was approximated from Fig. 5C, where the k:,gP(f'' appeared to become pH independent with acidification of pH, < 6.5. On a purely trial basis, rate coefficients for k!, and kg,, were chosen using the criterion that the apparent rateconcentration plots so developed would superimpose the empirical data of Fig. 5A. The values so chosen were k!,, = I rad/sec p M and kg,, = 75 radlsec. +
TABLE VI
DOUBLEBLOCKER I.OKENTZIANS IT1
IT"1
[T '1
5 15 25 30 35 40 45
4.92 14.76 24.60 29.52 34.44 39.36 44.28
0.08 0.24 0.40 0.48 0.56 0.64 0.72
sI(
F O R T R l A M r E R E N E AT
s ,:
j.7
(Shifted'.<') 0.36 0.57 0.58 0.56 0.54 0.52 0.50
2.17 3.22 2.06 1.40 0.86 0.47 0.22
pH
.f:
(Unshifted") 12.88 14.29 15.86 16.64 17.43 18.21 18.9Y
4.25 5.12 5.99 6.42 6.85 7.29 7.72
8.0"
.fP
t:
(Shifted' ) 12.70 13.98 15.08 15.60 16.08 16.54 17.00
4.28 5.40 6.71 7.40 8.13 8.87 9.63
~~
"[TI. Triamterene concentration (pLM); [To] and (T 1. concentrations ( p M ) of neutral and charged forms of triamterene at pH 8.0, wheref" = 0.016. Rate Coefficients used in calculation of S,, ( A ' . secicm?) and f , (Hz) were X S , , = 34; Xi,, = 24; k!,, = I ; klj, = 75. N , = 60 millions/cm' and i, = 0.5 PA. h, f( ,I and .f ,' , Unshiftcd frequencies of neutral and charged forms of triamterene, respectively. +
which would apply if each blocker interacted specifically with mutually exclusive groups of channels. ' Shifted ,f, apply where blockers compete for the same receptor. "Note changing ratio ofSg/S!l with increase of T (see Fig. 6).
148
SANDY I. HELMAN AND NEIL L. KlZER
As indicated in Table V1 for calculations done at pH, = 8.0, the shifted y d and ,f: are similar in value. When taken with the similar values of SI: and S; , it becomes apparent that even with excellent signal-to-noise ratio, it would be difficult to know if the PDS as illustrated in Fig. 6 contained one Lorentzian attributablc to T + or a combination of Lorentzians due to competitive To and T + blocker interactions with the channels. For simplicity, these calculations were done assuming absence of a closed state, which nevertheless leads to a complex interrelationship between SX and S,: , where at low [TI, S! < S,: , but at higher (TI, S! > S,: (see also Fig. 6). Calculations similar to these were carried out at all pH,, and only when pH, approached 6.5 was the apparent Lorentzian attributable alone to T' block of the channels leading to values of k,; near 34 radlsec pLM (Fig. SC) and to values of k a g p = k&f+, as illustrated in Fig. SD at pH 6.5. Also illustrated in Fig. SD are the rite-concentration plots for k:,, (dashed lines) where it is observed that at pH, = 6.5, k : g ~= k&, but with alkalinization of pH,, k g > k&. This leads, as illustrated in Fig. SE and F, to replication of the empirical data of Fig. SB and C. Accordingly, although other hypotheses cannot be excluded, it is possible for cases such as triamterene to invokc a double blocker problem to explain the apparent pH, dependence of kJ,, where, in fact, as assumed throughout our calculations, all rate coefficients are pH, independent. Our bias favors the view that kg,hovcr the range of physiological pH, is pH independent and is supported, moreover, by the additional observation that k,,, and k,,,, for CDPC are unchanged by pH, between 6 and 10 (S. I. Helnian, L. M . Baxendalc, and W. Van Driessche, personal communication). 1
1
1
E
Y 25
0.01
1 .(I
100
0.01
pM Triom.
.-_
~~
1.o
I
-
45 +M Triam.
c . A .o 7
100
0.01
1
-
y
-
100
Frequency, Hz FK;. 6 . Double blocker Lorentzians at 5 , 25, and 4.5 pA4 trianitercne with pH, = 8.0 and where f ' :0.016 (1.6% of T as T + ) .Shifted Imcntzians are ahown for T ' and To blocker interactions with channels. Note that at 5 pA4 T, S,: > S::at ; 45 pM T, S,: < S::. The solid lines are thc sum of the Lorzntzians, which in thc face of usual signal-to-noise ratios could not be distinguished from a single Lorenthn. For the calculations carried out here at pH, of 8.0 and at lower pH, ( n o t shown), 1; was taken at the half-power point of the solid curves and provided the data of Fig. SD and the subsequent calculation of !i:p. For aiinplicity and purpose of illustration of this double hlocker prubIcm, the calculations wcrc donc with a three-state schenic, where channels cxisted in open or either of two blocked (b', b") states (no closed state).
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
149
XII. DEPENDENCE OF BLOCKER AND SPONTANEOUS RATE COEFFICIENTS ON APICAL SODIUM ION CONCENTRATION Based upon interdependence of apical solution Na+ concentration ("a],) and amiloride concentration in altering apical membrane Na+ entry into cells, it has been suggested that Na' and amiloride may act as either competitive, noncompetitive, or mixed inhibitors of the Na+ channel (Benos, 1982). In retrospect, it is not surprising that the interaction between "a], and amiloride in modulating the rates of apical Na' entry has been confused. As should be evident, analysis of changes of macroscopic currents in response to changes of "a], and/or (B] in intact epithelia according to simple Michaelis-Menten kinetics would be inappropriate when (1) channels fluctuate spontaneously between open and closed states and with open probabilities that may depend on "a], and/or IS], ( 2 ) autoregulatory changes of channel density occur as consequence of inhibition of apical Na entry by reduction of "a]. and/or exposure to [B], and (3) blocker rate coefficients are themselves labile and dependent on "a],, either directly or via changes to electrochemical potential differences driving Na+ into the cells. The latter question has been addressed in several studies with equivocal results, as summarized in Table VII. Using K+-depolarized epithelia of frog skin, Van Driessche and Lindemann were the first to report that decreasing [Na],,from 110 to 1 1 mM caused no change of the k,,h for amiloride (Van Driessche and Lindemann, 1979). Using nondepolarized epithelia of frog skin, Hoshiko and Van Driessche ( 1 986) observed that the kc,hfor amiloride increased while the k,,, decreased with reduction of "a],, and similar results were obtained with triamterene. Although it would not be difficult to believe that such disparate observations may be due to differences of the blocker binding site in K+-depolarized and nondepolarized tissues, it is of interest to note that in nondepolarized skins, reduction of "a], causes no significant changes of the kohor kbofor electroneutral CDPC (Els and Helman, 1990a), being similar to the observation for amiloride in K+-depolarized tissues. These latter observations share in common the fact that for K+-depolarized tissues, reduction of "a],, would cause little or no change of apical membrane voltage of short-circuited tissues, while for nondepolarized tissues, the rate coefficients for CDPC are voltage independent [M. Awayda and S. I . Helman (personal communication) by impedance analysis; see also Table 11. Because it is known that reduction of "a], in nondepolarized tissues causes hyperpolarization of apical membrane voltage, the observation of increase of kJ, and decrease of kf (amiloride and triamterene) may at least in part be a reflection of voltage dependence of the rate coefficients. Accordingly, our bias favors the view that blocker rate coefficients are not directly dependent upon "a],. Because the corner frequencies of spontaneous Lorentzians arising from fluc-
TABLE VII OF BLOCKER RATECOEFFICIENTS ON A P I C ~sOI.UTIOU L Na CONCEhTRATIOY" DEPENDENCE ~
Blockeritissue
Species
PH,
"a1
Nondepolarized Amilorideiskin
Rano pipiens
7.8 7.8 7.8 7.8 7.8 7.8 7.8 7.4 7.4 6.0 6.0 6.0 8. I 8.1
120 20
Rana esculenta
Rano rempororia
K
Amiloridelurinary bladder Triamtereneiskin
Rana temporaria
CDPCIskin
Rano pipiens
depolarized Amiloride/skin
Rabbit
10
120 10 I20 10
I34 30 120 40 20 100
5
a
k
7.6 17.2 23.9 5.40 2 1.73 14.5 t 0.45 7.09 t 0.86 17.9 2 2.80 46.0 .+ 1.50 55.5 2 10.5 12.4 15.9 18.9 7.22 t- 0.61 7.57 .+ 0.25
Reference
bo
14.8 10.6 5.61
2.19 t- 0.77 0.73 2 1.84 13.2 2 0.40 9.80 t- 1.90 19.2 12.0 13.2 184 f 4 161 2 7
Hoshiko and Van Driessche ( 1986) Hoshiko and Van Driessche (1986) Hoshiko and Van Driessche ( 1986) Lewis er al. (1984) Hoshiko and Van Driessche (1981) Els and Helman (1990aIh
+
Rana esculenia
-
I10
-
80 55 35 I1
7.0
100 10
12.2 2 0.3 12.5 t 0 . 4 13.0 2 0.6 12.4 2 0.5 13.6 2 0.4 7.46 t 0.30 7.56 2 0.32
-
-
Van Driessche and Lindemann (1979)
-
-
2 1 4 2 12 173 -C 7
Els and Helman. submitted(
"Values are means ? SEM. "a], is apical solution Na' concentration (mM).Note that "a], values are concentrations ind not activities. 'Skins were bathed symmetrically with a CUHCO, Ringer solution. Tetramethylammonium was substituted for Na in the apical solution. 'Apical and basolateral solutions were HC0,-free and buffered with 2 m M HEPES. Basolateral solution pH was 8.0.
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
151
tuation of channels between open and closed states are practically immeasurable with noise analysis, it has been impossible with noise analysis to test directly for Na+ concentration dependence of either a and/or p. Lindemann and colleagues have on the basis of indirect evidence suggested that Na+ self-inhibition hypothesis to explain the dependence of open channel density on "a], (Fuchs et al., 1977). They envision that a = a'[Nal,, leading therefore to a channel open probability that is dependent upon "a], (Li and Lindemann, 1983b). According to this hypothesis, reduction of "a], leads to an increase of open channel density with NTconstant. As an alternative to this, it had been suggested that increase of No may arise by an autoregulatory increase of NT secondary to inhibition of the rates of apical Na+ entry (Abramcheck et al., 1985), and more recent observations support this point of view (Els and Helman, 1990a,b; Fisher et al., 1986; Helman and Baxendale, 1990). As a direct test for "a], dependence of the N,, and p', Ling and Eaton (1989) subjected patches of apical membranes of A6 cells to reduction of bathing solution "a], while maintaining intrapipet Na+ concentration constant. It was observed that new channels appeared in the patch within a few minutes either after reduction of "a], to 3 mM or in response to 10 pM amiloride, thereby ruling out a specific Na+ effect. Such observations are in agreement with the autoregulatory hypothesis whereby increase of open channel density is thought to be mediated by intracellular factors and/or messengers. In patches of apical membrane of rat cortical collecting tubules, Palmer and Frindt (1988) observed no change of channel open probability with large changes of intrapipet Na' concentration. If such observations, taken with those above, can be confirmed, it will be necessary to abandon the Na+ self-inhibition hypothesis. In this regard, experiments by Els and Helman (1990a) using CDPCinduced noise indicated that in both control and forskolin-stimulated conditions of Na+ transport in frog skin, reduction of "a], from 100 mM to either 5 or 10 mM causes a large (three- to fourfold) increase of total channel density ( N T )with little or no significant change of open probability. Indeed, 100 pM amiloride causes the same response in frog skin, namely, an increase of NT with no significant change of p' (Helman and Baxendale, 1990), suggesting no direct effect of INa]., on p' from mean values of p' near 0.44. It is recognized that determination of the true open probability in patch-clamp experiments is exacerbated by mean open/closed times of several seconds, and like noise analysis, stable recordings of about 40 min would be required to accumulate sufficient data (-500 events) to determine the a and p and hence open probability, assuming, moreover, that patches contained a single channel. Hence, it is important to recognize that p' may not be the same in value as P , as estimated from 'patch-clamp records of relatively short duration containing multiple channels. In the face of patches that contain no channels ( P o = 0) and patches with multiple channels, it is difficult, as in the experiments by Ling and Eaton ( I 989), to know whether the appearance of new channels arises by in-
152
SANDY I. HELMAN AND NEIL L. KIZER
crease of NT and/or by change of p’. Such problems with patch clamp would seem intractable, especially under conditions wherein changes of NT and/or p’ occur physiologically both in the short- and long-term time intervals after experimental perturbations of Na’ transport. From the physiological point of view, the picture that seems to be emerging is that regulation of apical membrane Na’ transport occurs by change, not only of channel density (NT)but also by change of open probability. Indeed, open probabilities range spontaneously between values of 0 and 1 as measured both by patch clamp (Palmer and Frindt, 1988) and noise analysis (Helman and Baxendale, 1990), so that caution must be exercised in interpretation of population means from small sample sizes. If in fact p’ is so markedly labile, it will be of particular interest to discover which enzymes and/or intracellular factors are responsible for regulation of a and/or p and hence the channel open probability. Accordingly, from the vantage point of noise analysis, there is (1) promise of this continually developing method, which should provide new insights into the mechanisms underlying Na+ absorption by a variety of epithelia, including those not amenable to study by patch clamp, and (2) an independent noninvasive method of obtaining data that can be compared quantitatively with other methods, such as patch clamp, for analysis of Na+ channels. In this sense, it is now truly remarkable that a consensus opinion may be at hand in identifying the nature of the ubiquitous epithelial Na+ channel. ACKNOWLEDGMENTS We gratefully acknowledge financial support from NIH Grant DK 30824 and the collaborative efforts of our colleagues L. M. Baxendale, W. J. Els, R. L. Duncan, S . Sariban-Sohraby. S. M. Thompsun, D. L. Stetson, M. Awayda, and W. Van Driessche. N. Suarez and J. Waters provided superb technical assistance. REFERENCES Ahramcheck, F. J , Van Driessche, W., and Helman, S. 1. (1985). Autoregulation of apical membrane Na+ permeability of tight epithelia. Noise analysis with amiloride and CGS 4270. J. Gm. Physiol. 85, 55.5-582. Baxendale, L. M., and Helman, S. I. (1986). Sodium concentration dependence of apical membrane singlc channel Na’ current and density of nondepolarized frog skin (three state model). Biophys. J . 49, 160a. Benos, D. I. (1982). Amiloride: A molecular probe of sodium transport in tissues and cells. Am. J. Phvsiol. 242, C131LC145. Benos, D. J . , Reyea, J., and Shoemaker, D. G . (1983). Amiloride fluxes across erythrocyte membranes. Biorlrim. Biophys. Acra 734, 99- 104. Briggman, J. V., Graves, J. S . , Spicer, S . S., and Cragoe, E. J., Jr. (1983). The intracellular localization of amiloride in frog skin. Histochem. J . 15, 239-255. Colquhoun, D., and Hawkes, A. G. (1983). The principles of the stochastic interpretation of ionchannel mechanisms. I n “Single-Channel Recording” (B. Sakmann and E. Neher, eds.), pp. 135-175. Plenum, New York.
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
153
Cuthbert, A. W. (l973b). An upper limit to the number of sodium channels in frog skin epithelium. J . Physiol. (London) 228,681-692. Cuthbert, A. W. (1976). Importance of guanidinium groups for blocking sodium channels in epithelia. Moi. Pharmacol. 12, 945-957. Cuthbert, A. W., and Wilson, S . A. (1981 ). Mechanisms for the effects of acetylcholine on sodium transport in frog skin. J. Mernbr. B i d . 59, 65-75. Eaton, D. C . , and Hamilton, K. L. (1988). The amiloride-blockable sodium channel of epithelial tissue. In “Ion Channels” (T. Narahashi, ed.), Vol. I , pp. 251-282. Plenum, New York. Els, W. J., and Helman, S. I. (1988). Forskolin causes large increases of apical membrane Na channel density in epithelia of frog skin. FASEB 1. 2, A749. Els, W. J., and Helman, S. I . (1990a). Forskolin and vasopressin mediated changes of Na channel density and single channel currents in epithelia of frog skin: CDPC blocker-induced noise analysis. J . Gen. Physiol. (submitted). Els, W. J., and Helman, S. I . (1990b). Responses of apical membranes of scraped skins and isolated epithelia of Rana pipiens to indomethacin, forskolin, and WE,: CDPC blocker-induced noise analysis. J . Gen. Physiol. (submitted). Fischer, H . , Van Driessche, W., and Clauss, W. (1989). Evidence for apical sodium channels in frog lung epithelial cells. Am. J. Physiol. 256, C764-C771. Fisher, R. S . , and Lockard, J. W. (1988). Complex response of epithelial cells to inhibition of Na+ transport by amiloride. Am. J . Physiol. 254, C297-C303. Fisher, R. S . , Baxendale, L. M., and Helman, S. I. (1986). Sustained increases of apical membrane channel density after inhibition of transport in frog skin. Fed. Proc.. Fed. Am. Soc. Exp. B i d . 45, 516. (abstract). Frings, S . , Purves, R . D., and Macknight, A. D. C. (1988). Single-channel recordings from the apical membrane of the toad urinary bladder epithelial cell. J. Mernbr. B i d . 106, 157- 172. Fuchs, W., Hviid Larsen, E., and Lindemann, B. (1977). Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin. J. Physiol. (London) 267, 137- 166. Hamilton, K . L., and Eaton, D. C. (198% Single-channel recordings from amiloride-sensitive epithelial sodium channel. Am. J. Physiol. 249, C200-C207. Hamilton, K . L., and Eaton, D. C. (1988). Single-channel recordings from two types of amiloridesensitive epithelial Na+ channels. Memhr. Biochem. 6 , 149- 171. Helman, S. I . , and Baxendale, L. M. (1988). Open channel probability of apical Na channels in epithelia of frog skin. FASEB J. 2, A750. (abstract). Helman, S. I . , and Baxendale, L. M . (1990). Blocker related changes of channel density: Analysis of a 3-state model for apical Na channels of frog skin. J. Gen. Phvsiol. 95, 647-678. Helman, S. I . , Cox, T. C., and Van Driessche, W. (1983). Hormonal control of apical membrane Na transport in epithelia: Studies with fluctuation analysis. J . Gen. Physiol. 82, 201 -220. Helman, S. I . , Koeppen, B. M., Beyenbach, K . W., and Baxendale, L. M. (1985). Patch clamp studies of apical membranes of renal cortical collecting ducts. Pjuegers Arch. 405 (Suppl. I ) . s 7 I - S76. Helman, S. I . , Baxendale, LA. M., Sariban-Sohraby, S . , and Benos, D. J. (1986). Blocker-induced noise of Na+ channels in cultured A6 epithelia. Fed. Proc., Fed. Am. Soc. Exp. B i d . 45, 516. (abstract). Henrich, M . , and Lindemann, B. (1984). Voltage dependence of channel currents and channel densities in the apical membrane of toad urinary bladder. In “Intestinal Absorption and Secretion” (E. Skadhauge and K . Heintze, eds.), pp. 209-220. MTP Press, Lancaster, England. Hille, B. (1984). “Ionic Channels of Excitable Membranes.” Sinauer, Sunderland, Massachusetts. Hoshiko, T., and Van Driessche, W. (1981). Triamterene-induced sodium current fluctuations in frog skin. Arch. Int. Phvsiol. Biochem. 89, P58-P60.
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Hoshiko, T., and Van Driessche, W. (1986). Effect of sodium on amiloride- and triamterene-induced current fluctuations in frog skin. J . Grn. Physiol. 87, 425-442. Hoshiko, T., Grossman, R. S., and Machlup, S.(1988). Effccts of hasolateral ouabain, amphotericin R . cyanide and potassium on amiloride noise during voltage clanip of Himu pipirr1.s skin support sodium-aniiloride competition. Biothim. Biophvs. Ac/tr 942, 186- 188. Kleyman. T. R., and Cragoe. E. J., Jr. (1988). Amiloride and its analogs as tools in the study of ion transport. J. Mernhr. Biol. 105, 1-2 I. Krattenmacher, R., Fischer, H., Van Driessche, W., and Clauss, W. (1988). Pf(uegers Arch. 412, 568- 573. Lewis, S. A , , and Hanrahan. J. W. (1985). Apical and hasolateral membrane ionic channels in rabbit urinary bladder epithelium. F'flurgers Arch. 405 (Suppl. I), S83-S88. lewis, S.A,, Ifshin, M . S., Loo, D. D. F., and Diamond, J. M. (19x4). Studies of sodium channels in rabbit urinary bladder by noise analysis. J . Mernhr. B i d . 80, 135- I S I. Li, J. H.-Y., and Lindemann, B. (1983a). Chemical stiniulation of Na transport through amiloridehlockable channels of frog skin epithelium. J. Memhr. Biol. 75, 179- 182. Li, J . H.-Y., and Lindemann, B. (1983b). Competitive blocking of epithelial sodium channels by organic cations: The relationship betwccn niacroscopic and microscopic inhibition constant. J . Mrrnhr. R i d . 76, 235- 25 1 . Li. J. H - Y . , Palincr. I.. G..Edelman, 1. S . . and Ihdemann, B. (19x2). The roleofsodiurn-channel density in the natriferic response of the toad urinary bladder to an antidiuretic hormone. J . Mrtnhr. B i d . 64, 77-89. Li. J. H.-Y., Cragoc, E. G , , Jr., and Lindemann. B . (198s). Structure-activity relationship ol'arniloridc analogs as blockers of epithelial Na channcls: I . Pyrazine-ring modilications. J . Mrrnhr. nioi. 83,45-56. Li, J . H.-Y., Cragoe, E. J., Jr., and Lindemann, B. (1987). Structure-activity relationship of arniloride analogs as blockers of epithclial Na channels: 11. Side-chain modifications. J . M e l b r . nioi. 95, 171- 18s. Lindemann, B., and Van Driessche, W. (1977). Sodium-specific membrane channels of frog skin are pores: Current lluctuations reveal high turnover. Scirncr 195, 292-294. Ling, B . N., and Eaton, D. C. (1989).Effect!, of luminal Na+ on single Na' channels in A6 cells. a regulatory rolc for protein kinase C.Am. J . Physiol. 256, F1094-FI 103. Loo, D. D.F., Lewis, S. A . , Ifshin, M. S., and Diamond, J. M. (1983). Turnover, membrane insertion, and degradation of sodium channels in rabbit urinary bladder. Sciffrw 221, 1288- 1290. Mamnaka. Y . , and Eaton, D. C. (1990). The effect of amiloride and an ainiloride analog on single Na' channels from a renal cell line. Submitted. Olans, L., Sariban-Sohraby, S., and Benos, D. J. (1984). Saturation behavior of single, aniiloridesensitive Na' channels in planar lipid hilaycrs. Biophys. J. 46, 831-835. Palmer, L. G . (1984). Voltage dependent block by amiloride and other cations of apical Na+ channels in the toad urinary bladder. J . Mcrnhr. Biol. 80, 153- 165. Palmer, L. G . , and Frindt, G . (1986a). Amiloride-sensitive Na channels from the apical menihrane of the rat cortical collecting tubule. Proc. N u / / . Acnd. Sci. U.S.A. 83, 2767-2770. Palnicr, L. Ci.%and Frindt, G . (1986h). Epithelial sodium channels: Characterization by using the patch-clamp tcchnique. Fed. Proc.. Fed. A m . Soc. Exp. B i d . 45, 2708- 2712. Palmer, L. G . , and Frindt, 6. (1987). Effects of ccll Ca and pH on Na channels from rat cortical collecting tubule. Am. J . Physiol. 253, F333-F339. Palnicr, L. G . , and Frindt, G . (1988). Conductancc and gating of epithelial Na channels from rat cortical collecting tubule. Effects of luminal Na and Li. J . Gen. Physiol. 92, 121- 138. Palmer, 1. C.,Li, J. H.-Y., Lindemann, B., and Edelman, I . S. (1982). Aldosterone control of the density of sodium channels in thc toad urinary bladder. J . Mrrnhr. Bid. 64, 91 - 102.
4. SODIUM ION CHANNELS OF TIGHT EPITHELIA
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Sariban-Sohraby, S . , Latorre, R.. Burg, M., Olans. L.,and Benos. D. (1984). Amiloride-sensitive epithelial Na channels reconstituted into planar lipid bilayer membranes. Nature (London) 308, 80-82. Tang, J . , Abramcheck, F. J . , Van Driessche, W.. and Helman, S . 1. (1985). Electrophysiology and noise analysis of K ' -depolarized epithelia of frog skin. Am. J . Physiol. 249, C421 -C429. Thompson, S . , Baxendale, L. M., and Helman, S . 1. (1987). Fluctuation analysis of ion transport by frog colon. Fed. Proc., Fed. Am. SOC. Exp. Biol. 46, 1269. Van Driessche, W.. and Erlij, D. (1983). Noise analysis of inward and outward Na+ currents across the apical border of ouabain-treated frog skin. fflurgers Arch. 398, 179- 188. Van Driessche, W., and Lindemann, B. (1979). Concentration dependence of currents through single sodium-selective pores in frog skin. Nature (London) 282, 519-520. Van Driessche, W., and Zeiske, W. (1980). Ba2+-inducedconductance fluctuations of spontaneously fluctuating K ' channels in the apical membrane of frog skin (Rana remporaria). J . Memhr. B i d . 56, 3 1-42, Warncke, J . , and Lindcinann, B. (1985). Voltage dependence of Na channel blockage by amilotide: Relaxation effects in admittance spectra. J . Memhr. B i d 86, 255-265. Wills, N. K., Alles, W. P.,Sandle. G . I . , and Binder, H. J. (1984). Apical membrane properties F749and amiloride binding kinetics of the human dcsccnding colon. Am. J . Physiol. 247, G749-G757. Zeiske, W., and Van Driessche, W. (1984). Thc sensitivity of apical Na+ permeability in frog skin to hypertonic stress. Pjfuegers Arch. 400, 130- 139. Zeiske, W., and Van Driesschc, W. (1986). Impairment of Na+ transport across frog skin by TI+: Effects on turnover, area density and saturation kinetics of apical Na' channels. f'puegers Arch. 407, 145- 152. Zeiske, W., Wills, N. K . . and Van Driessche, W. (1982). Na' channels and amiloride-induced noise in the mammalian colon epithelium. Eiochim. Biophvs. A m 688, 201 -210. +
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 37
Chapter 5
Noise from Apical Potassium Ion Channels WOLFGANG ZEISKE Institut fur Tierphysiologie und Angewandte Zoologie der Freien Universitat Berlin
0-1000 Berlin 41, Federal Republic of Germany
I. About the Beginning of Apical Potassium Ion Channel Noise Analysis: A Historical Account A. Para- versus Transcellular Pathway for Potassium Ions B. Ways to Tackle Potassium Ion Channel Noise C. Epithelia under Study II . Methods for Evaluation of Apical Potassium ton Channels A. Approaching Potassium Ion Permeability B. Information Derived from Channel Noise 111. Potassium Ion Channels with Two Putative Roles: Helping Potassium Ions or Other Ions on Their Way across the Epithelium IV. Lorentzian Noise from Transepithelial Current: A Fingerprint of “Spontaneous” Apical Potassium Ion Channel Fluctuations A. lntrinsic or Extrinsic Driving Forces: Ways to Discover Fluctuating Channels B. Influencing the Gating Rate C. Summary: “Spontaneous” Conduction Noise V. Blockers of Apical Potassium Ion Channels A . Comparison of Protons, Cesium, Rubidium, 4-Aminopyridine, Quinidine, and Tetraethylammonium B. Cadmium Ions Can Be Potassium Ion Channel Inhibitors C. Signals of Channel Blockers in the Current Noise D. The Rates of Barium Ion Blockade Depend on the Nature of Permeant Ion Species E. Blockers Act in a Voltage-Dependent Way F. Barium Ion-Induced Conduction Noise Helps to Discover Unexpected Potassium Ion Channels VI. About the Concept of Potassium Ion Channel Selectivity VII. Microscopic Channel Parameters VIII. Potassium Ion Channel Chemistry IX. Influencing Apical Potassium Ion Permeability A . Hormone Action Increases Channel Density B. Long-Term Potassium Ion Exposure Also Increases Channel Number C. Channel Gating as Potassium Ion Current Determinant
157 Copyright 0 19W by Acddcrnic Press, Inc. All rights of repnducrion in any form reserved
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X. Family of “Povassium Ion-Specific” and Other Cation Channels A . Basolatcr;il versus Apical Potassium Ion Channels H . Some Evidcncc for Kinship of Amphibian Apical Cation Channels XI. Summary References
1. ABOUT THE BEGINNING OF APICAL POTASSIUM ION CHANNEL NOISE ANALYSIS: A HISTORICAL ACCOUNT A. Para- versus Transcellular Pathway for Potassium Ions This review will focus on the recent discovery and characterization by noise analysis of currents through potassium ion-selective channels in apical membranes of epithelia. For a long time, transepithelial movements of potassium ions (K + )had been thought to occur mainly along paracellular routes. Driving forces were assumed to be provided by an electrical potential, for example, set up by clectrogenic transcellular transport of other ionic species. For instance, K + secretions by distal colon (Frizell and Schultz, 1978; Fromm and Schultz, 1981) or frog skin (Mandel and Curran, 1972) have been visualized in this manner. The long-standing paradigm for tight epithelia possessing K -impermeable apical membranes (Koefoed-Jnhnsen and Ussing, 1958; Nielsen, I97 I ) turned out to be a heavy burden. Conflicting views arose, supported by a variety of experiments, concerning this issue. A good example for such a scientific debate was the case of the descending colon of the rabbit, for which some investigators presented evidence for a paracellular route (Frizell and Schultz, 1978; Fromm and Schultz, 1981) and others provided evidence in favor of at least one finite transcellular route for K’ secretion (Wills and Biagi, 1982; Wills et al., 1982; Yorio and Bentley, 1977). At the same time, microelectrode studies confirmed an apical permeability for K +,not only for colon but also for other tight epithelia such as distal nephron (O’Neil and Boulpaep, 1982; Koeppen et al., 1983) and frog skin (Nagel and Hirschmann, 1980). Also, several leaky epithelia, for example, proximal kidney tubule (Wright, 1981), loop of Henle (Greger, 1985), and gallbladder (Keuss et uf., 1981), displayed apical K’ permeability when studied with microelectrodes. Once the existence of such apical K’ channels had been accepted it became obvious that they were in fact indispensable for transport of other ions: for example, in the loop of Henle (Greger, 1985), K’ channels parallel to a Na-K-2CI cotransporter provide the latter with K + ions that originate from the cytosol. Another example is the parallel arrangement of a H ,K+-ATPase and a K channel in the apical membrane of oxyntic cells in gastric mucosa (Sachs et ul., 1982; +
+
+
159
5. APICAL POTASSIUM ION CHANNEL NOISE
Wolosin and Forte, 1985; Zeiske et al., 1983). Here again, K channels mediate K + recycling so that, as with the above-described cotransporter in leaky epithelia, they do not show up in the overall transport process. The situation is quite analogous to the ubiquitous Na+,K+-ATPase in parallel with a K + channel in serosal cell membranes (Koefoed-Johnsen and Ussing, 1958; Diamond, 1974). For two “model” tight epithelia, frog skin and distal colon, paracellular K + secretion was shown to occur in response to the transepithelial potential difference originating from transcellular electrogenic Na’ absorption (Mandel and Curran, 1972; Fromm and Schultz, 1981). Thus, the eventually discovered apical K + channels in these epithelia (cf. Van Driessche and Zeiske, 1985a) seemed to be superfluous, at least in the beginning. But what if a tight epithelium is, paracellularly, “really” tight? Would a transcellular pathway, which could even be controlled by cytosolic factors, then not make more sense? And what if Na+ transport is for some reason very small but K + secretion is needed? +
B. Ways to Tackle Potassium Ion Channel Noise
By establishing a transepithelial concentration gradient for K + , an inward or outward K + current can be obtained, depending on the gradient’s direction. If potassium ions would cross membranes via channels, then spontaneous conductance fluctuations of the channels or, otherwise, their reversible occlusion by a blocking molecule would lead to fluctuations (or “noise”) in the current passing these channels. This noise increases when the electrochemical driving force for K + is increased across the membrane in question (Van Driessche and Zeiske, 1985a). If the constellation of resistances in series and parallel to the one with conduction noise is favorable and does not attenuate the noise signal (Van Driessche and Gogelein, 1980), then another unique feature can be observed: even without an obvious transepithelial electrochemical gradient for K +, K + channel noise from the apical membrane can be seen as long as the transapical driving force will move enough K + across that membrane to be recorded. For example, this can be witnessed in the case of a gallbladder bathed mucosally and serosally with NaC1-Ringer’s (Van Driessche and Gogelein, 1978). Favorable resistance constellations [ i.e., series resistances low and parallel resistances high in relation to the fluctuating resistance (Van Driessche and Gogelein,l980)) can be present due to the nature of some preparations, as in tight epithelia. They can also be “constructed,” for instance, when the resistance of the ion channels under study is increased (e.g., using a blocker at submaximal concentrations). Of course, any similar manoeuvre with parallel ion pathways would be equally helpful. The use of large “nonpermeant” anions goes in the same direction. Apart from the above-mentioned structured approaches toward acquiring
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WOLFGANG ZEISKE
“hands on” noisy apical K + channels, pure accidents occasionally helped us on our way in the beginning. One such accident has already been mentioned: the very first encounter with a spontaneously fluctuating K + channel in a toad gallbladder bathed in ambient NaC1-Ringer’s (Van Driessche and Gogelein, 1978). Here, nobody expected a current noise signal from an ion whose extracellular concentration was only a few millimoles per liter. A similar situation arose with R a m temporaria frog skin, for which we wanted to assess amiloride-induced Na channel noise. For kinetic studies, we used K + as a Na+ substitute in dose-response assays and so discovered high-frequency noise from spontaneously fluctuating apical K + channels (Van Driessche and Zeiske, 1980a). +
C. Epithelia under Study Potassium ion current noise analysis, with respect to apical K + channels, has been done successfully with adult frog skin, three vertebrate gastrointestinal epithelia (i.e., frog stomach, gallbladders of several species, and rabbit descending colon), larval frog skin, and larval insect midgut.
II. METHODS FOR EVALUATION OF APICAL POTASSIUM ION CHANNELS A. Approaching Potassium Ion Permeability Several methods unambiguously demonstrated the existence of apical K channels. For example, methods included microelectrode studies in a number of epithelia (for a review, see Macknight, 1977; Wright, 1981; Van Driessche and Zeiske, 1985a), on-cell patch clamping in kidney proximal tubule (Gogelein and Greger, 1984) and gallbladder (Maruyama et id., 1986), excised patch clamping of distal tubule cells (Hunter and Giebisch, 1987), and reconstituted channels from choroid plexus (Zeuthen et al., 1987). However, some epithelia (especially those discussed later) implicate considerable structural or methodological complexity in obtaining a suitable patch-clamp or vesicle preparation. Current fluctuation analysis turns out to be of greatest value for such epithelia in obtaining microscopic data from a channel ensemble in equilibrium conditions. +
B. Information Derived from Channel Noise To ensure that the reader can follow the arguments presented, a brief [many times before discussed at length (cf. Van Driessche and Zeiske, 1980a,b, 1985a)l
161
5. APICAL POTASSIUM ION CHANNEL NOISE
sketch of the relationships relevant to current noise analysis of a two- or threestate channel model is given in Eqs. (1)-(7): h
blocked
=======
“blocker induced”
a
channel
open
A closed B
+ relaxation noise 4 “spontaneous”
Open-channel probability
Closed-state probability
Lorentzian corner frequency
Lorentzian plateau value
S:
=
4liP,B(1/2i~f,B), S,,= 41iP,(1/2vfc)
Mean current I
=
P:iM,
I = P,iM
(6)
Single-channel current
In Eq. (7) y represents the single-channel chord conductance, and in Eq. (6) M is the channel area density. The model here is formulated for the simpler case of negligible interference of spontaneous and blocker-induced channel fluctuations. The fluctuations are assumed to occur between one fully open state and one spontaneously or blocker (B)-induced closed state. The parameters a , /3, k,,, and k,ff are then rate constants for pseudo-first-order kinetics. The determination of mean macroscopic ion-specific current, I, can be achieved easily. If only spontaneous (blocker-induced) fluctuations of the channels between the open and closed (blocked) state occur, the above-described model predicts one
162
WOLFGANG ZEISKE
Lorentzian function in the power density spectrum of the current, with corner frequencyf, ( f , " ) and plateau value S,, (S:). When both spontaneous and blockerinduced transitions occur, the superposition of two Lorentzian functions can be observed in the spectrum, depending on the relative magnitude of~fi. andf?, and S, and S:. If the rate constants are known, channel area density ( M )and singlechannel current ( i ) can be calculated. Knowledge of transmembrane electromotive force ( E ) for the ion in question and true transmembrane voltage ( V ) enables estimation of y , the single-channel conductance.
111. POTASSIUM ION CHANNELS WITH TWO PUTATIVE ROLES: HELPING POTASSIUM IONS OR OTHER IONS ON THEIR WAY ACROSS THE EPITHELIUM Potassium ion secretion by tight epithelia has long been thought to follow a paracellular pathway. Some years ago, apical conductive pathways were shown to definitely exist in the skin of the grass frog, R . temporuriu (Nagel and Hirschmann, 1980), rabbit descending colon (Wills and Biagi, 1982), and distal mammalian nephron (Koeppen rt ul,, 1983). These epithelia are not always, perhaps even never (frog skin), confronted with mucosal "a+] large enough to establish the transepithelial voltage for secondary paracellular K i secretion, and will certainly need an alternative secretory pathway for K + , i.e., the transcellular route. As an independent method, fluctuation analysis provided evidence for the transcellular K' pathway. According to results obtained in the last few years (Sachs rt ul., 1982; Wolosin and Forte, 1985), apical K + channels in the HCI-secreting oxyntic cells from stomach epithelium are able to perform their task only then when K + ions are available on either the serosal or the mucosal side. A delayed assessment of apical K + permeability was due to the fact that the concerting systems for HCI generation, i.e., parallel-operating K + , H t -ATPase, K + , and CI-- channels in nonstimulated stomach tissue, are almost exclusively found in vesicles near the apical membrane (Wolosin and Forte, 1985). Only after histamine stimulation do these vesicles fuse with the apical membrane (Clausen rr ul., 1983) and render the incorporated transport mechanisms such as K + channels accessible to electrophysiological measurements. As was the case for frog skin or colon, only nonphysiological conditions such as large transepithelial [ K + ]gradients (occasionally supported by appropriate electrical potentials) were able to reveal the channel nature of the apical K pathway via fluctuation analysis of trdnsepithelial current (Zeiske rt ul., 1983). Toad gallbladder represents the class of leaky epithelia. There, K + channels may be required to maintain a functioning parallel Na-K-CI, symport unit, which is known to be inhibitable by the diuretic furosemide (Greger, 1985). On +
5. APICAL POTASSIUM ION CHANNEL NOISE
163
the other hand, the gallbladder epithelium seems to involve antiporters such as Na’-H’ and CI--HCO; in order to allow NaCl uptake (Reuss et al., 1981). Nevertheless, apical K’ channels could be demonstrated in all leaky epithelia of this type (cf. Van Driessche and Zeiske, 1985a). In the case of the Na-K-Cl? symporter present in apical membranes, a “silent” K t recycling from cytosol (piled up there by the basolateral Na+, K+-ATPase) to the external face of the apical membrane and back to the cytosol, via the cotransporter, will warrant the function of a Na-K-Cl, symport (Greger, 1985). A similar concept would apply to the role of K’ channels in stomach HCI secretion. Furthermore, apical K’ channels could play a mandatory role for K’ secretion in low-resistance epithelia, which lack large transepithelial voltages as paracellular driving forces. For instance, secretion of anions such as of bile acids, or bicarbonate, could accompany the secreted potassium. Thus, bile acid K’ salts would be piled up in the gallbladder lumen, concentrated by the concerting NaCl and H,O absorption. It is also conceivable that the introduction of electrogenicity for the apical membrane, i.e., a K + channel parallel to Na+-H+ and CI--HCO; exchangers, will establish a cytosol-negative, K+-dependent membrane potential that would add to the serosal one and thus would increase the electrical driving force for a basolateral passive C1- exit from the cell. This view would then be similar to the one discussed for other NaC1-absorbing epithelia, e.g., the thick ascending loop of Henle (Greger, 1985).
IV. LORENTZIAN NOISE FROM TRANSEPITHELIAL CURRENT: A FINGERPRINT OF “SPONTANEOUS” APICAL POTASSIUM ION CHANNEL FLUCTUATIONS
A. Intrinsic or Extrinsic Driving Forces: Ways to Discover Fluctuating Channels This discussion in this section refers strictly to “K+-specific” channels, i.e., channels that do not pass ions other than K + , Rb’, NH: and TI+ (Latorre and Miller, 1983; Hille, 1984) and thus are distinct from hybrid channels, which will carry not only K’ but also Na’ (Hillyard et al., 1982; Van Driessche and Zeiske, 198%) and even Ca” (Van Driessche, 1987). The focus of this section is the socalled spontaneous K + channel noise that can be detected ( I ) with transepithelial [ K + ] gradients (i.e., in frog skin, frog stomach, and rabbit colon) or (2) without a [K+1 gradient (i.e., in toad gallbladder). Actually, additional electrical driving forces can elicit the typical K’ channel noise (cf. below) in either case, if only the resulting net transapical driving force is sufficient for monitoring and analyzing even very small currents. Here, as depicted in the following figures, some of those experimental procedures will be emphasized.
1 64
WOLFGANG ZEISKE
n
N
E
N
5 @ (D .d
1o-20
0
C I
Y
100
10'
1 o2
Frequency (Hz) FIG. 1. Power spectrum of trdnsepithctial current noise in spontaneously acid-secreting frog gastric mucosa: estahlishment of an inward-directed [ K ' ] gradient after replacing mucosal sodium gluconatc-Ringer's (Na; ) with potassium gluconatc-Ringer's (K?: ) elicits a Lorentzian noise in the short-circuit current at 0 mV transepithelial potential. Smooth lines from data tit with sum of linear background noise and Imentzian component (cf. inset Fig. 13). Mucosal positive potential ( +35 rnV) enhances Lorentzian plateau by ahout 200% whilc ,/; increases from 15.4 to 24.5 Hz. [From Zeiske e/ ol. (1983).]
Let us consider the frog stomach epithelium. When electrogenic C1- secretion (Machen and Zeuthen, 1982) is prevented using ambient gluconate, and a [K'] gradient is established, directed from mucosa to serosa, the power density spectrum of the fluctuations in the short-circuit current shows a Lorentzian component (Fig. 1). If a mucosally positive voltage is applied, this K+-dependent Lorentzian component rises even more from the background, just as expected for a noise source dominated by passive driving forces. Many arguments (Zeiske et al., 1983) show convincingly that K + channels in the apical membrane of oxyntic cells generate this noise, e.g., its histamine-dependent rise or its sensitivity to mucosal blockers such as Ba?' or tetraethylammonium (TEA).
165
5. APICAL POTASSIUM ION CHANNEL NOISE
n N
E
0
‘ v)
N
5
t 1oo
10’
lo2
lo3
Frequency (Hz) FIG. 2.
Rabbit descending colon: influence of the pore-forming antibiotic, nystatin (NYST,) (40 U h l ) on Kt-dependent channel noise when added to the mucosal (M) K+-Ringer’s. A , Control; + , after nystatin; serosa, Na gluconate-Ringer’s. Lorentzian noise in short-circuit current (shoulder in smooth line from data fit;f, = 14 Hz)is attenuated by nystatin-induced shunt. [From Wills e l a/. (1982).]
As with stomach epithelium, the epithelium from rabbit descending colon also displays apical K + channel noise. These channels respond to [K+] gradients and transepithelial voltages in a manner similar to the response of stomach K’ channels; both are permeable for K + in either direction (Wills et al., 1982). When a high mucosal [K’] is present, the apical colon membrane can be “shortcircuited” via the addition of the pore-forming antibiotic nystatin. Consequently, the K current noise generated by the cytosol-directed K + gradient is abolished (Fig. 2). In toad gallbladder, contrary to the tissues mentioned previously and later, spontaneous K + channel noise from apical membranes can be seen even without any parallel sign in short-circuit current (Van Driessche and Gogelein, 1978). When the gallbladder is bathed in physiological Na+ saline (characterized by very small transepithelial voltages or currents, but comparably large conductances), a Lorentzian current noise can be seen. This noise can be abolished by mucosal Ba2+and TEA, both well-known K + channel blockers. Figure 3 shows that the Lorentzian plateau magnitude was directly proportional to the transapical K’ gradient. Treatment with ouabain depletes cytosolic K’ and also reduces the relaxation noise. Thus, we assume the existence of apical fluctuating K + channels. Two features that appear to be common to all the above-described gastrointestinal epithelia should be further emphasized. (1) Transepithelial “macroscopic” parameters are usually difficult to relate to the events seen through the +
166
WOLFGANG ZEISKE
'O-'I
tlbi,\\,,
(
,
,do
\
\
I \
I
IK1s,m(mM)
FIG.3 . Potassium channel noise in the apical membrane of'toad gallbladder. Lorentzian plateau and mucosal ([K].,) K ' concentration. Gallbladder initially values (S,) as a function of serosal ([K],) bathed in ambient NaC1-Ringer's, where [K'l>,m= 2.5 mmoliliter. Mucosal (0) and serosal (0) substitution of' Na by K ' . At [K]., = 36 mmoliliter, the Lorentzian is snialler than the background noise level. Fully expressed l.orent7,ian noise is obtained in thc absence of, hut also in presence of, a large transepithelial [ K ' 1 gradient in either direction. Corner frcquencics (-5 Hz) do not change. [From Van Driesache and Ghgelein (197X1.1 +
window of noise analysis, and ( 2 ) the corner frequencies are all in the range of 5-25 Hz, which is much lower than is the case for frog skin (cf. Fig. 4).
B. Influencing the Gating Rate Generally accepted is the assumption of spontaneous channel fluctuations between different conductance states. The transition probability between them may be changed, for example, by voltage and/or triggering molecules such as transmitters or Caz+ (Hille, 1984). At least for the gallbladder, patch-clamp experiments (Maruyama et uf., 1986) demonstrated an apical K' permeability associated with cellular negativity as well as cytoplasmic Caz+.Indeed, the Necturus gallbladder (Gogelein and Van Driessche, 1981) showed a voltage-dependent Lorentzian noise, equally K + dependent, in addition to the spontaneous one, which could also be blocked by application of mucosal TEA. A dependence on calcium has not been tested. The situation for the other investigated epithelia (stomach, colon, and frog skin) seems, however, to be different. In fact, for frog skin, intracellular Ca2' seems to induce channel closure rather than opening (Van Driessche, 1984), although voltages might play a role (Zeiske and Van Driessche, 1981): To begin
5. APICAL POTASSIUM ION CHANNEL NOISE
167
B
10-18
oo
10‘ 1o2 103 Frequency (Hz) FIG.4. Potassium channel noise in the apical membrane of frog skin ( R a m temporaria). Lorentzian noise in short-circuit current obtained with rnucosal KCI- and serosal NaC1-Ringer’s (“control”) disappears when serosal Na’ is replaced with choline (“during”). The effect is reversible (“after”) and accompanied by a drastic reduction in Ba”-blockable current; ,h = 70-80 Hz. [From Van Driessche (1984).1 1
with, the addition of quinidine to solutions bathing the skin of R . temporaria led to a reduction of the Ba2+-blockable,transcellular K + current (Van Driessche, 1984; cf. also Fig. 6). Although this agent has been claimed to evoke a cytosolic [Ca2+]rise in toad bladder (Arruda and Sabatini, 1980), direct K + channel blockage is likely, as for excitable membranes (Hille, 1984). Another way to increase intracellular Ca?’ is to shut down the serosal Na+-Ca2+ exchanger (Chase, 1984) by replacing serosal Na+, e.g., with choline. Figure 4 depicts that such a procedure results in a drastic reduction in Ba*+-blockableK + current as well as in a complete suppression of apical K’ channel noise in frog skin (Van Driessche, 1984). Contrary to the inhibition by intracellular Ca2+,interesting effects of mucosal Ca2+and positive potentials on frog skin K’ channel gating have been witnessed: mucosal Ca2+ions, between 0 and 20 mmol/liter, have no significant impact on
168
WOLFGANG ZEISKE
n N
E 0
\ (D
dM
1
N
a
W
0 v) ..-I
0
c I
U
1oo
1o2
10‘
Frequency ( H A
a 20 5,. 10” 15 i A z s/rmzl 10
B
b
5
ti
1
I
I
L
FIG. 5. Apical K ’ channel noise in frog skin. Mucosal Ca?+affects the Lorentzian component in current fluctuations hut not short-circuit current, which is carried by K . (a) Power spectra obtained with mucosal KCI- and serosal NaCILRinger’s, in the absence ( 1 ) or presence (2) of 20 rnrnol/ liter Ca*’ in the inucosal solution. (b) Lorentzian plateau value (S,) and corner frequency ( J ) , as well as short-circuit current (SCC), as functions of mucosal Ca2’ concentration. [Ca2+].[From Zeiske and Van Driessche (1981).1 +
transepithelial, Ba*+-blockableK + currents. However, the current noise spectra change in a dramatic way (Fig. 5): corner frequencies decrease and Lorentzian plateaus increase steadily, whereas the K +-carried short-circuit current remains practically constant during [Caz+]rise (Zeiske and Van Driessche, 1981). Basically the same pattern can be obtained with other external polyvalent cations, and also with highly concentrated choline and protons above pH 4 (Zeiske and Van Driessche, 1981). When varying [H+]and lCaz’] simultaneously, the
169
5. APICAL POTASSIUM ION CHANNEL NOISE
effect is not additive. When mucosal positivity is increased by transepithelial voltage clamping, a similar effect can be obtained that is smaller at higher external Ca2+concentrations. From this behavior it was deduced that frog skin apical K + channel gating responds to electrical fields set up either transepithelially or locally (by surface charge screening). Also, K’ channels in stomach and colon show minor voltage effects on spontaneous K +-dependent Lorentzian corner frequencies. Yet, for all species investigated so far regarding noise analysis, dramatic changes, in the sense of an all-or-nothing behavior within a physiological apical voltage range, have not been observed. The detailed analysis for frog skin revealed, however, that by means of the above-described procedures, only the large and dominating (Van Driessche and Zeiske, 1980a,b) channel-opening rate (/I) is reduced, with increasing mucosal positivity, whereas the very small closing rate (a)remains constant (Zeiske and Van Driessche, 1981). Such an analysis was not attempted for the other channels (or it was, but with reservation), because the openiclosed-state probabilities were not known or were tentatively set to 0.5 (Van Driessche and Zeiske, 1980a; Gogelein and Van Driessche, 1981). Van Driessche and Zeiske (1980a,b) found no dependence of the spontaneous gating corner frequency in frog skin on mucosal K + . The recent reinvestigation by De Wolf (1989), however, showed thatf,, indeed, increased linearly from 60 to 90 sec - I when the mucosal K + concentration was raised from 20 to 1 17 mmol/ liter, whereas the voltage across the apical membrane was held constant. Thus, an involvement of K+ ions in the mechanism of channel gating seems likely.
C. Summary: “Spontaneous” Conduction Noise In summary, spontaneously fluctuating apical K channels could be detected in frog skin, frog stomach epithelium, toad gallbladder, and rabbit descending colon, when a transapical driving force for K + entry or exit existed. This driving force was usually established by appropriate [K’] gradients, at either shortcircuit or at a K + current-enhancing voltage. For the gallbladder, the outwarddirected transapical driving force under short-circuit conditions (Reuss and Finn, 1975) in ambient NaC1-Ringer’s was apparently sufficient, and a K+-dependent Lorentzian was seen with a short-circuit current that was close to zero. In fact, only frog skin produced a current that could be easily related to a transcellular K + movement (Van Driessche and Zeiske, 1980a; Nagel and Hirschmann, 1980) and that was quickly and reversibly blocked by the K’ channel blockers Cs’ (Van Driessche and Zeiske, 1980a; Zeiske and Van Driessche, 1979) or Ba2+ (Van Driessche and Zeiske, 1980b; Nagel and Hirschmann, 1980; Zeiske and Van Driessche, 1983) in the external medium (cf. later). Again, noise analysis detected events in the gating process that could not be seen at all by following macroscopic parameters such as the K -dependent short-circuit current. Hence, apart from establishing the mere existence of apical K + channels from the appearance of K+-dependent Lorentzians in the current noise, an insight into mi+
+
170
WOLFGANG ZEISKE
croscopic processes such as channel gating can be obtained. The window of noise analysis allows for a comprehensive description of complex epithelial systerns that cannot, or only with great difficulty, to date be approached by other microscopic methods such as patch clamping.
V.
BLOCKERS OF APICAL POTASSIUM ION CHANNELS
A. Comparison of Protons, Cesium, Rubidium, 4-Aminopyridine, Quinidine, and Tetraethylammonium It has been a paradigm that channels of a specific kind could be recognized by their specific interaction with certain agents, such as ions, toxins, or drugs. However, this rather strict concept had become blurred for channels of a “mixed” selectivity and also when the drugs, which were considered to be specific, showed cross-reactions with different channel types. So-called typical K + channel blockers are Cs , Ba*+, 4-aminopyridine, quinine/quinidine, or tetraethylammonium derivatives (Hille, 1984; Latorre and Miller, 1983). Protons also block K + channels; however, due to their nature, they interfere with many different types of cation channels and cannot be called typical. Besides the aforementioned H +-induced “slowing down” of the channelopening rate at pH > 4 (Zeiske and Van Driessche, 1981), external protons can completely shut down K’ channel activity below pH 3 , e.g., in frog skin (Zeiske and Van Driessche, 1979, 1981) or gallbladder (Goigelein and Van Driessche, 1981). Although it has not been studied in colon or stomach epithelium, a prediction for stomach can nevertheless be made: Because K channels are thought to supply K + ions for, and are therefore operating parallel to, a H + ,K+ -ATPase, their function is absolutely indispensable even at a very acidic pH level of the gastric gland lumen. Hence, they should be either inaccessible to external H+ or else be protonated much below the typical pH of stomach lumen. Ra2+ ions, which are (due to their radius) potent imitators of potassium ions, seem to be the primary competitive K + channel blocker, reacting with almost all types of K + channels (cf. reviews by Hille, 1984; Van Driessche and Zeiske, 1985a,b). Actually, apical colon K’ channels resisted a block by mucosal Ba2+ applied in the millimolar range (Wills ef al.. 1982), although larger doses had not been tried. Mucosal Cs’ was effective, as a K competitor, in the colon (Wills et a l . , 1982) and in frog skin (Zeiske and Van Driessche, 1979) but not in the gallbladder (Giigelein and Van Driessche, 1981). TEA, on the other hand, showed K ‘ channel inhibition in the gastrointestinal epithelia but did not affect frog skin (Van Driessche and Zeiske, 198Sa). However, it could block larval cation channels, which are not strictly K + specific (Hillyard ef d.,1982). Quinidine was tested in frog skin and was shown to be a probable cytosolic +
+
5. APICAL POTASSIUM ION CHANNEL NOISE
171
inhibitor (Van Driessche, 1984). Finally, 4-aminopyridine cannot block epithelial K + channels. Among monovalent cations that have a naked-ion radius close to that of K + , Rb+ (Zeiske and Van Driessche, 1979; De Wolf, 1989) and T1+ (Zeiske and Van Driessche, 1983) can show voltage- and dose-dependent competitive blockage of K + current. However, in contrast to the above-described blockers, Rb+ and TI+ can pass through the K + channels (see Section V,D). Although inhibitory effects on K + conductance are usually inferred from ionic radius versus charge considerations, they are sometimes accompanied by lipophilic effects, as with quinineiquinidine or TEA and its derivatives (Hille, 1984).
8. Cadmium Ions Can Be Potassium Ion Channel Inhibitors An unexpected and most remarkable finding was the inhibitory action of mucosal Cd" ions (similar in radius not to Ba2+,but to Ca'+ and Na') on apical K + conductance in frog skin (Zeiske and Van Driessche, 1980; Van Driessche and Zeiske, 1985b). This finding is unique and most striking, because it appears to link K + , Na', and Ca2+channels (cf. also Section X), based on the evidence that ( 1 ) Ca?+ channels are usually blocked by Cd2+ions (Hille, 1984; Lansman et al., 1986) and (2) Cd2+ions open frog skin Na+ channels (Hillyard and Gonick, 1976; Scholtz and Zeiske, 1988).
C. Signals of Channel Blockers in the Current Noise Some blockers will interact very slowly or very quickly with K + channels. Due to the limited frequency range of noise analysis, these random interruptions of K current through a (spontaneously) open pore will not be visible as blockerinduced Lorentzian current noise in addition to the spontaneous K* channel noise. But blockers reduce the number of open K' channels (available for spontaneous conductance fluctuations), thus the spontaneous Lorentzian noise will be diminished, e.g., with Cs+ (Van Driessche and Zeiske, 1980a; Wills et a l . , 1982), Ht (Zeiske and Van Driessche, 1979, 1981), or quinidine (Van Driessche, 1984). The latter case is depicted in Fig. 6. If random interruptions of K' channels (during a spontaneous open period) are slow when compared to the spontaneous noise, they can be recognized as a second, low-frequency Lorentzian component in the power spectrum of the short-circuit current. This is the case for frog skin with Ba2+ and Cd2+ as blocking ions (Van Driessche and Zeiske, 1980b, 1985a,b; Zeiske and Van Driessche, 1980). Figure 7 shows that mucosal Cd2+elicits a second, low-frequency relaxation noise for frog skin in addition to the preexisting spontaneous K + channel fluctuations. One would expect, according to the theory of first-order blocking re+
172
WOLFGANG ZEISKE
I
rnin
1o1 102 10’ Frequency (Hz) FIG 6 . Current noise from apical K i channels in frog skin. Serosal quinidine depresses the Lorentzian component and Ba? -blackable short-circuit current (mucosa, KCI-Ringer’s; serosa, NaC1-Ringer’s). Slow but reversible effect of quinidine (0.5 mniol/litcr) indicates intraccllular sitc of drug action. (From Van Driesschc (1984).] 100
+
..
, +Cd
2.
. CTR
€ L
101
loo
Frequency
loz
lo3
Hz
FIG. 7. Double 1,orentzians from apical K + channels in frog skin. Superposition of spontaneous and blacker-induced current fluctuations. Single Lorentzian component for control (CTR) case (smooth line: f; = 75 Hz). Sum of high-frequency, spontaneous noisc and low-frequency, blockerinduced noisc with 0.5 mmol/liter niucosal Cd” (smooth line, +Cd”; low-frcquency,f; 15 Hz). Mucosa. KCI-Ringer’s; serosa, NaC1-Ringer’s. K current block by Cd” is moderate and only partially rcversible. \From Van Dricssche and Zeiskc (1985b).j ;
+
173
5. APICAL POTASSIUM ION CHANNEL NOISE
action (Lindemann and Van Driessche, 1978; Van Driessche and Zeiske, 1980b; Zeiske and Van Driessche, 1983), that the corner frequency of the “blockerinduced” noise would linearly increase with blocker concentration. However, this could not be clearly seen with Cd?+, probably because it is a comparably poor K + channel inhibitor, with (as compared to Ba2+;see below) a small association rate but a large dissociation rate at its blocking site (unpublished observations; Zeiske and Van Driessche, 1980).
D. The Rates of Barium Ion Blockade Depend on the Nature of Permeant Ion Species Ba2+ ions are much better for demonstrating the experimental fulfillment of the channel-blockade theory (cf. Fig. 8). Here, increasing blocker concentration shifts (initially in a linear manner) the Ba2+-induced Lorentzian to higher frequency values, but at large [Ba2+ a saturation of this relationship is found. Concomitantly, the plateau values of the Ba2+-inducedLorentzian noise rise to a maximum (predicted for the point of 50% of maximal current depression), but they decrease again at higher [Ba’+ I. The linear part of the 2qfl.-[Ba] relationship (cf. Section 11) allows us to calculate on-rate (slope) and off-rate (ordinate intercept) constants for the interaction of Ba?+ with the K + channel. and IBaz’Io 400
1
10
I
I
IpMI 100
1000 1
I
300 --b
zr f:“
5~2’x102‘
ZOO
150
(5‘I
(A’ s/cm2)
0
1
0
1000
0
I 6a ’*4,
2000 (p M I
FIG. 8. Blocker (Ba’+)-induced low-frequency K + channel noise in frog skin. Variation of Lorentzian plateau (SJ and blocking rate (27rf;) with mucosal Ba” concentration, [Ba’+],,. Linear initial increase in 27rj supports pseudo-first-order reaction of Ba2+with open channel. Saturation at large (Ba?+1 illustrates the now rate-limiting factor, i.c., delivery of open K + channels from a comparably short-lived spontaneous closed state. The bell-shaped curve for S,, as well as the saturating 27rf; relation are in full agreement with three-state channel model (closed-open-blocked) for slow Be’ + blockade with long-lived spontaneously open channel. [From Van Driessche and Zciskc ( 1 980b).]
174
WOLFGANG ZEISKE
their ratio yields the Michaelis constant of K + current block as seen by macroscopic experiments (Van Driessche and Zeiske, 1980b, 1985a; Zeiske and Van Driessche, 1983). The flattened portion (Fig. 8) of the 2?f;.-[Ba2+] plot at higher [Ba?' J arises because of interference between spontaneous and blocker-induced channel fluctuations. This has been successfully interpreted assuming that the frog skin K ' channel is open most, i.e., 95%, of the time (Van Driessche and Zeiske, 1980b). Hence, the observed saturation limit of the blockcr noise equals the rate for the overall rate-limiting spontaneous fluctuations. As will be shown in Section VI, different permeant ions interact in a very specific and multiple way with the K + channel. The kinetics of the BaZ+ions' interaction turned out, as well, to strongly depend on the nature of the given permeant cation, e.g., K' , Kb+, NH,' , and TI+ (cf. Section VI), as well as on the membrane potential. In the case of either permeant species, Ba' ' induces a Lorentzian channel noise with very different corner frequencies at a given le a2 + ] .Figure 9 shows that a linear relationship between the Ba?'-induced Lorentzian corner frequency and rnucosal [Ba2+1 exists, with either K', Rb', or NH: as permeant ion, in the investigated blocker concentration range. The Ba?+ association rate with the channel (slope) has been found to be about twice as fast with K + as permeant ion when compared to the presence of Rb ' . With NH: as a permeant cation, Ba2+ is 53 times faster than with Rb+. Almost the opposite is true for the Ba2+ dissociation constant, which is largest with Rb , 2.5 times slower with N H 2 , and I 1 times slower with K + (Zeiske and Van Driessche, 1983). +
d
Rb'
K' I
I
1000
500 pM
[Ba2'l,
FIG. 9. Frog skin apical K channel. Kinetics of Ba' ' blockade in the prescncc of three permcant ion species, i.e.. K ' , NH: . and Rb+. Ba?+ induces typical hlocker noise with either of these permeant cations in the mucosal solution (I15 mM: serosa, NaCI-Ringcr's. Betwcen 0 and loo0 pinolilitcr cxtcrnal Ba'+; blocker rate, 27rA, depends linearly on [Ba? I,,, indicating a pseudo-twostate blocking mechanism. On-rates for Ba" assuciation with (slopc) the K + channel and off-rates for dissociation from (ordinate intercept) the K ' channel strongly depend on thc nature of the respcctivc pcrmeant ion species (these include K + . R b + , NH:. and TI+.). [From Zeiske and Van Driessche (1983).] +
5. APICAL POTASSIUM ION CHANNEL NOISE
175
Due to irreversible effects of TI+, a clear relationship between Ba*+-induced TI+ current noise and [Ba”] could not be established, but it became clear that the dissociation rate of Ba’ is again much smaller, i.e., only one-fiftieth of the rate in the presence of Rb+. Ba2+ association in presence of TI+ is extremely slow; a 10-fold increase in [Ba2+]did not result in the expected increase of the corner frequency of the Ba2+-inducednoise at all. This situation is reminiscent of the situation during Cd2+block, when K + carries the current (see above). These results will enable us, in conjunction with the studies of channel selectivity described in Section V1, to understand that K + channels in every cell membrane (Hille, 1984; Van Driessche and Zeiske, 1985a,b) do not obey the Goldman independence principle (Goldman, 1944). Rather, they show strong multiple interactions between channel molecules and ions that are permeant or reversibly “anchoring” in the channel mouth. We shall return to this problem later, when we discuss multisite, single-filing theory.
E. Blockers Act in a Voltage-DependentWay As has become apparent from selectivity studies in frog skin, the abovedescribed variation in Ba2+ block cannot be due to effects arising from simple competition phenomena. In addition, different membrane potentials may be expected in the case of different permeant ions, thus the blockade could vary individually with voltage. The question of a voltage dependence of the rates of the barium block has been addressed by De Wolf and Van Driessche (1986). They measured inward K + currents and conductances by means of current-voltage relationships for a wide range of transepithelial potentials as well as for different mucosal [Ba2+]. It was found that the macroscopic inhibition constant decreased exponentially with mucosal positivity. A similar result was obtained from current noise analysis of Ba2+-inducedLorentzians. Figure 10 illustrates that the on-rate of the Ba2+ block is apparently strongly enhanced by mucosally positive potentials. Conversely, the off-rate decreases somewhat. It was concluded that the site that must be reached by mucosal BaL+during this voltage-dependent channel block is located at about 72% (relative electrical distance) of the apical membrane field as measured from the cytosolic side. De Wolf and Van Driessche (1988) also investigated whether K + channel block by musocal Cs’ was voltage dependent. They came to a similar conclusion by analyzing current-voltage curves; however, contrary to investigations using Ba2+, they could not study blocker-induced noise, because Cs’ is a high-rate blocker, whose (principally existing) Lorentzian blocker noise cannot be recorded in this manner (Van Driessche and Zeiske, 1980a). Its blocking efficiency is also over 100 times less than that of the block by Ba”. Contrary to findings using Ba2+, the site for interaction with the channel seems to be located more on the inside
176
WOLFGANG ZEISKE V , lmVl
r&fm
MI
FIG. 10. Voltagc-dependent Ha2+blockade of frog skin K' channel. Slope of linear relationship between blocker rate, 27rL (from B a l l -induced low-frequency relaxation noise; mucosa, KCIRinger's; scrosa, NaCI-Ringer's) and mucosal Ba' concentration. for submaximal [Baz+ 1, increases with mucosally positive transepithclial potential, V , . The ordinate intercept decreases correspondingly. Thc data favor the concept that the location of Ba2+ interaction is within the depth of the external channel mouth. [From De Wolf and Van Driessche (1986).] +
(at 0.32, relative electrical distance, with reference to the cytosol). Thus, Cs', compared to Ba2+,apparently is more deeply embedded within the channel. In conclusion, the effectiveness of the K' channel block depends not only on the nature of the permeant species and the nature of the blocking ion, but in some cases also on the voltage dependence of the blocker. Higher or lower transmembrane potentials can help a blocking ion to attain a site of interaction within the depth of the channel. Of course, there are a number of parameters influencing transapical voltage even in a short-circuited state. For example, functional elimination of the serosal membrane via serosal depolarization with high [K'], as has been successfully performed to effectively voltage clamp the apical membrane for the study of Na + transport (Fuchs et aL., 1977; Lindemann and Van Driessche, 1978), is of course not applicable here. This procedure would eliminate the driving force for passive, transepithelial K' movement (Van Driessche and Zeiske, 1980a).
5. APICAL POTASSIUM ION CHANNEL NOISE
177
F. Barium Ion-Induced Conduction Noise Helps to Discover Unexpected Potassium Ion Channels The midgut epithelium of the caterpillar of the moth, Manduca sextu, is able to secrete large amounts of K + . First, K + ions cross the serosal membranes containing a “normal,” Ba2+-blockableK + channel (Zeiske et al., 1986) but no Na+,K+-ATPase. In the next step, K + ions are transferred apically by a unique K + -ATPase (Harvey er ul., 1983). A large lumen-to-hemolymph-directed [K+1 gradient is similar to the naturally occurring situation (Dow et al., 1984). Establishing this gradient in vitro generates a small, hemolymph-directed current that depends on luminal K + and can be blocked reversibly by adding Ba2+ to the luminal fluid. The inhibition by Ba2+was manifest through a Ba2+-inducedLorentzian current noise, but no spontaneous K + channel noise could be recorded (unpublished observations; Zeiske and Schroder, 1988). This newly discovered K + channel seems to counteract the epithelium’s task to secrete K + ions, and the significance of this finding is far from being clear. Yet, it again demonstrates that (1) suitable methods allow a successful search, and (2) K + channels, being ancient with respect to evolution (Hille, 1984), are ubiquitous and deserve further attention, although we do not at present know what their function is in this tissue.
VI. ABOUT THE CONCEPT OF POTASSIUM ION CHANNEL SELECTIVITY Some years ago (Zeiske and Van Driessche, 1983), we presented data demonstrating frog skin apical K + channels to be blocked by Ba2+ ions, with a graded apparent “permeability” for so-called K + -like ions in the decreasing order of T1’ > K + > Rb+ > NH: . Of course, the permeability we are referring to here is deduced simply from the magnitude of Ba2+-blockable current for comparable concentrations of the permeant ion species. Unique features of the frog skin K + channel exist (for the ion concentration range between 0 and 120 mmol/liter). 1. Current kinetics, which seem to obey Michaelis-Menten behavior when the solution contained either K + or TI , turned out to be quite complex for Rb+ and NH,‘ , based on an overproportional current decrease that was observed when the outer ion concentration was reduced (see Fig. 11). 2. The dependence of the specific channel current on the continuously increasing [TI+]of Kt-T1+ mixtures shows a minimum at low Tl+/K+ ratios, whereas the current for 100% TI+ is larger than for 100% K + (see Fig. 12); in fact, low (TI+] appeared to block K + current competitively (Zeiske and Van Driessche, 1983). A similar situation was encountered with mixtures of potassium and rubidium (De Wolf, 1989). +
40
r X'I 'x
\
\ 2c
rnM c m 2
I
50
I
100
1
150
mM [X'l FIG. I 1 , Apical ti + channel in frog skin. Current kinetics depend on the nature of the permeant ion. Hanes plot of ion-spccilic ( X ' = K', R b ' , NH:) inward currents. I, (mucosal XCI ~Ringcr's rcplaccs stepwise NaC1-Ringer's containing 50 pmol/liter amiloridc): [X ' ] / I , vs. [X ' 1. Only for ti' (or TI+)is a linear (thus simple) saturation bChdVlOr found. Strong cooperative effects are obtained with Rb' and NII; . (From Zciske and Vdn Driessche (19831.l
15
vol % FIG. 12. Mole fraction relationship for total ion current through frog skin K ' channel exposed to various inucn~alti ' /TI mixtures. Afacrepresents current change when rnucosal NaNO1-Ringerk ( i SO pmol/litcr amiloride) is replaced by the rcspcctivc K +/TI' mixture. Mole fraction relationship shows minimuni (competitive K channel block by TI ) and indicate5 better channel permeability for TI ' alone than for K alonc. This picture is typical for a variety of K ' channels and can be explained on thc basis of bingle-filc diffusion along a multisite narrow pore. [From Zciske and Van +
Driessche (1983).I
179
5. APICAL POTASSIUM ION CHANNEL NOISE
lo0
lo1
10'
103
Frequency (Hz) FIG. 13. Frog skin apical K ' channel. Spontaneous channel gating depends on the nature of the permeant ion. Current noise power spectra and fits (lines) obtained with either mucosal KCI-. NH,CI-, or RbCI-Ringer's, respectively (serosa, NaCI-Ringer's). Note ion-specific height of Lorentzian plateaus and corner frequencies (f; = 59 Hz for K ' , 76 Hz for NH: , 230 Hz for R b + ) . Plateau level order matches that for ionic currents (cf. Fig. I I ) . Inset shows, for K ' . how linear background (B) and Lorentzian (L) component superimpose to resultant fit (R). [From Zeiske and Van Driessche (1983).1
3 . With any of these permeant ions a spontaneous Lorentzian channel noise is obtained (and the Lorentzian plateau heights roughly reflect the above-mentioned apparent permeability sequence). However, there are clear differences in gating, i.e., corner frequency, decreasing in the order Rb+ >> TI+ > NH,'- > K + (see Fig. 13 for a comparison of Rb', NH,' , and K + ) . All of these findings, along with the observation that the nature of the permeant ion influences the on- and off-rate of the Ba2+ blocking kinetics, allowed us (Zeiske and Van Driessche, 1983) to classify the frog skin K + channel as a pathway with multisite, single-file permeability character, a concept that fits the
180
WOLFGANG ZEISKE
current view of K ’ channels (Hille, 1984; Latorre and Miller, 1983). In our case, a minimal model, starting from the mucosal channel mouth, with two energetic wells in front of a selectivity tilter followed by an inner channel gate (Zeiske and Van Driessche, 1983), has been proposed to account for the observed pcculiarities. A mathematical modeling hereof is already extremely complicated, even when based on rather simple assumptions (Hille and Schwarz, 1978). Intuitively, appropriate intimate interactions of more or less hydrated permeant or blocking ions at permeability- or gating-determining sites on the rim, or even within the depth of the channel, could explain all observed deviations from “simple” channel behavior. This would be in terms of electrical interactions between ions and channel moieties, possibly accompanied by slight structural rcarrangements.
VII. MICROSCOPIC CHANNEL PARAMETERS Here we are interested in channel area density, single-channel current, singlechannel conductance, and rates of spontaneous channel gating. The problems confronting the determination of these parameters were discussed in Section 11. Because Lorentzian current noise can be recorded at the observed magnitude of currents and noise power, the apical K + transporters, with a turnover of millions of ionic charges per second, will classify as ion channels in terms of tunnels. Even when a lack of knowledge of transapical net electrical driving force is taken into account, a single-channel conductance of a few picosiemens can be tentatively calculated (Giigelein and Van Driessche, 1981). The correct data, however, bear on the clear determination of ( I ) the apical membrane area, as well as on knowledge of (2) opedclosed channel probabilities, in the case of spontaneous noise, or rates, for channel blockade mechanisms, and (3) on welldefined ion-specific currents. This also should not be hindered by attenuation effects, that is. the apical membrane should be the one with the dominating resistance (Van Driessche and Gogelein, 1980). For frog skin, the following figures were obtained (Van Driessche and Zeiske, IOXOa,b; De Wolf and Van Driessche, 1986; Zeiske and Van Dricssche, 1981): a channel density of 5-20 X IOh per cm’; 1-2 pA for single-channel current at mucosal IK + 1 of I IS mmol/liter; a single-channel conductance of a few picosiemens, depending on the estimated transapical net driving force for K + movement; and a large channel opening rate, inferring a mean open lifetime of 23 msec and a mean closed lifetime of 2 msec. With respect to the movement of K ’ ions, all of these parameters were comparable in magnitude to the ones obtained for K channels in excitable tissues, especially data from an inward rectifier (Hille, 1984). Due to the lack of knowledge of spccitic gating rates as well as of specific apical driving forces, single-channel parameters have not been calculated for permeant ions other than for K . Frog skin apical K + channels belong, according to available data, to “mini,” +
5. APICAL POTASSIUM ION CHANNEL NOISE
181
rather than to "maxi," K + channels (Latorre and Miller, 1983). The latter exhibit single-channel conductances of up to several hundred picosiemens, thus almost reaching the theoretical limit discussed by Hille (1984). Maxi-K+ channels also seem to be strongly dependent on intracellular Ca?+ as an agonist (Latorre and Miller, 1983), whereas the K + channels from frog skin seem to be blocked by intracellular Ca? (Van Driessche, 1984). +
VIII. POTASSIUM ION CHANNEL CHEMISTRY Cation channels are charge specific when they present negative groups, in order to replace the cation's hydration shell for the permeation process. Physiological candidates for these negative groups are phosphate or carboxylate. The search for carboxylic groups as key functions of apical K + channels has been successful. They were detected (I)via their rather strong acidity when K + current across frog skin (Zeiske and Van Driessche, 1979), or its Lorentzian noise (Zeiske and Van Driessche, 1981) was titrated with mucosal H + and ( 2 ) by irreversible channel closure after chemical modification with the COO- reagent, N-ethoxycarbonyl-2-ethoxy- 1,2-dihydroquinoline(EEDQ) (Onken, 1985; Onken and Zeiske, 1986). The latter authors also repeated the pH titration experiments and revealed a two-step titration profile. It could be shown that the acidic group titrated first (pK, - 6) was blocked by H' in a fashion consistent with an uncompetitive mechanism, whereas the second, more acidic group, which had been the only one described before (pK, 3), was blocked competitively by H'. EEDQ depresses both titration steps, so that carboxylic groups seem to be involved. Furthermore, the pK, titration step can almost selectively be depressed by the thiol (SH) reagent, p-chloromercuribenzoate (PCMB). Onken and Zeiske ( I 986) interpret their results as an indication for two K' channel populations, the origin of which, however, remains in the dark. These findings partly support results obtained with noise analysis, in which a similar SH reagent, p-chloromercuriphenylsulfonate (PCMPS), reduced the spontaneous K + channel noise in an irreversible way (Van Driessche and Zeiske, 1982). Interestingly, this effect interfered with the Ba?' -like blocking action of Cd2+, which is also known to have a high affinity for thiol groups. When Cd?+ was given before PCMPS, the latter substance induced no further current or noise decrease, but the Cd2+-induced (Ba2+-like)blocker noise slowly disappeared. This was interpreted to be a sign of two sites for Cd2+action: one in reversible competition with Ba'+ and K +,the other in competition with PCMPS, thus leading to a permanent channel closure. Figure 14 presents a sketch of what we feel has been firmly established. When this scheme is compared to the minimal idea of the previously mentioned (cf. Section IV) two-site, three-barrier K + channel model (Zeiske and Van Driessche, 1983), the two descriptions cannot easily be interwoven, but neither do they exclude each other. Especially the ad hoc descriptions of molecular en-
-
182
WOLFGANG ZEISKE
muc 1
I cell
FIG. 14. Chemical iiiodel of frog skin apical K ’ channel. Basic features are wide openings toward extracellular and cytoplasinic apace, a selectivity liltcr (SF), and the spontaneously Iluctuating gate ( G ) . K ’ (or any other pcrnicant ion) is thought t o interact in a saturablc nianner with a carhoxylic group (B)with pK,, 3. Hcre, competitive blockers are Ba’+, C S ~Cd”, , Hi, and possibly TI ’ or Rb ’ , which also pernicate at high concentrations. Poorly reversible interaction occurs with the carhoxy reagent EEDQ at site B, and with thc thiol reagents Cd?+and PCMB at a more extcrnally located SH function. A second population of K ’ channels is thought to include a second 6 , showing irrcversihle block by EEDQ and carboxylic group (A; dashed-line box) with p K , reversible, uncompetitive inhibition by H ’ . I n an earlier two-siteithree-harrier iiiodcl (cf. Zciske and Van Driessche, 1983; Van Ilriesschc and Zeiske, 198Sa). an additional site along the access path to location B was proposed. It should enablc niultisite interactions hetween pcrnteant and/or blocking ions as well as with the gate, G . Thc chemical cqiiivalcnt for this site has still to be identilied, but another carboxylic group, inaybe with siniilzir properties, such as group B. is a reasonable candidate.
-
-
tities, such as “selectivity filter” or “gate,” still have no detailed structural correspondence. The only thing that can be said at present is that both structures should have some dipole/charged character. Schwarz et d.( 1988) found that in Drusophila neurons a whole family of K + channel proteins with known amino acid sequences is produced by alternative gene splicing, and K channel mRNA could even be expressed as functional K channels in Xmopus oocytes (Timpe Pt ul., 1988). These investigators describe K ’ channels as homo- or heterotctramers of a certain protein subunit. Indeed, the recently described K + channel in early distal tubules seems to be a “fourbarreled” structure (Hunter and Giebisch, 1987). +
+
IX. INFLUENCING APICAL POTASSIUM ION PERMEABILITY A. Hormone Action Increases Channel Density The activation of K channels by oxytocin in frog skin (Erlij et a / ., 1986) resulted in a last and large rise in Ba2+-blockableK ’ current (Fig. 1Sa);this rise was accompanied by a corresponding rise in spontaneous noise level (Fig. ISb), without changes in the corner frequency. +
183
5. APICAL POTASSIUM ION CHANNEL NOISE
-
AV-3mV
I2 m n
b
1
10 Frequency
100
loo0 HZ
FIG. 15. Hormonal stimulation of apical K ’ permeability in frog skin. (a) Oxytocin treatmcnt (0.1 U/ml, serosally) augments Ba’ ‘-hlockahle K current. Mucosa, KCI-Ringer’s; serosa, NaCIRinger’s. Vertical bars reflect current deflection during voltage pulses (from 0 to - 3 mV, outside negative, and back). (b) Exarnplc of oxytocin (OXYT) effect on spontaneous K Lorentzian. Control (CTR), before hormonc treatment. Oxytocin doubles plateau value whereas corner frequency is hardly aftectcd (100 vs. 97 Hz).[From Erlij ei a / . (1986) and Dc Wolf (1989).] +
+
Ba2+-inducedconduction noise had been analyzed for this case, and it turned out that practically all K t current rise induced by the hormone could be attributed to a rise in apical K channel density; single-channel current was somewhat reduced, which is predicted for the hormone-induced decrease in cellular negativity (Nagel and Hirschmann, 1980; Erlij e f a f . ,1986). In addition, it was noted +
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WOLFGANG ZEISKE
that Ba2+blockade kinetics are the same before and after hormone action. Because membrane-permeant derivatives of cyclic AMP and forskolin give similar results (De Wolf, 1989), CAMP is an internal messenger of the hormone; possibly, phosphorylation of silent K channels, could be instrumental for the observed effect. +
B. Long-Term Potassium Ion Exposure Also Increases Channel Number In vivo adaptation of frogs to KCI-containing water will also result in a considerable (up to sevenfold) increase in specific K + current after 7 days (Van Driessche, 1984; Onken, 1985), which is paralleled by a similar rise of the spontaneous K' channel relaxation noise (Van Driessche, 1984). Although it has not been investigated, hormones reacting to the K' load are probably involved in the response of the epithelium. Interestingly, but not yet understood, is the fact that several methods of obtaining long-term in vitro exposure of frog skin to high [K+] also lead to a substantial increase in specific K + current. For example, bathing a piece of skin in KCI-Ringer's for about 60 min increases K + current threefold when compared to a control group without such treatment (Van Driessche, 1984). A similar effect was observed when the skin, which was mounted in an Ussing chamber, had been bathed with KCI on both sides with ouabain in the serosal solution (Van Driessche, 1984). Here, current and K+-specific channel noise developed simultaneously. Simply exposing the apical side of in vitro frog skin to KCI-Ringer's for about 1 hr gave rise to a doubling of K+-specific current, although a Ba2+insensitive "shunt" pathway also seemed to become more permeable for K + (Zeiske and Van Driessche, 1979; Van Driessche and Zeiske, 1985b).
C. Channel Gating as Potassium Ion Current Determinant Specific K + currents that can be recorded macroscopically depend not only on the product of channel density M times single-channel current i (the latter parameter is again a product of conductance and net driving force), but also on the channel's open probability, P,, (cf. Section 11). Even if M and i are comparable for different tissues, Po might nevertheless be quite different and so give rise to large (Po + 1 ) or hardly detectable (Pcj+ 0) macroscopic currents. As was discussed above, P,, in the case of spontaneous channel fluctuations can be affected by several procedures. In a more descriptive manner, this means that the spontaneous K + channel fluctuations are governed by transition rates that are subject to voltage-dependent or ion concentration-dependent terms. The results obtained with the Ca2+-or pH-modified K' channel gating in frog skin (Zeiske and Van Driessche, 1981) also suggest two rather than one gating structure, whereby only the one responsible for channel opening is voltage dependent.
185
5. APICAL POTASSIUM ION CHANNEL NOISE
X. FAMILY OF "POTASSIUM ION-SPECIFIC" AND OTHER CATION CHANNELS A. Basolateral versus Apical Potassium Ion Channels Hille (1984) compiled data from throughout the animal kingdom and concluded that, in terms of phylogenic evolution, specific K + channels as well as less specific (also Ca2+-permeable)cation channels should have evolved first, because they have important tasks to fulfill. If it is accepted that cytosolic reactions can only take place when cell [ K + ] is high and "a+] is low, the formation of membrane K + channels will create a membrane voltage (assuming the extracellular [ K + ] is low). The so-generated electrical driving force may be of use for a multitude of processes, e.g., for Na+-coupled nutrient uptake or, in the case of absorbing epithelia, transport of NaCI. Naturally, serosal K + channels would also enable the K + recycling in parallel to the Na+,K+-ATPasein order to maintain a low intracellular "a']. So far, the characteristics of K+ channels in membranes bordering the blood side are almost indistinguishable from K + channels of apical origin. Great differences exist, however, for the blocking abilities of substances other than Ba2+,for example, TEA or Cs+. Furthermore, when investigated, apical epithelial K + channels do not respond to 4-aminopyridine but some may be blocked by quinineiquinidine.
B. Some Evidence for Kinship of Amphibian Apical Cation Channels Chemical relatives of 4-aminopyridine are the dihydropyridines, some of which do block whereas others open Calf channels (Hess et al., 1984). Among inorganic ions, Cd2+ is an established blocker of Ca2+channels in a variety of tissues (Hille, 1984; Lansman et al., 1986), which is probably due to an ionic charge and radius almost identical to the charge and radius of Ca2+.Up until now, there has been no study of Cd2+for a blockade of K + channels except for our search for the apical K + channel in frog skin (Zeiske and Van Driessche, 1980). At least for frog skin it is clear that Cd2+can interact, just like Ba2+,with the channel, resulting in typical blocker noise. A question that arises is whether the K + channel has a Ca'+ channel link. Cd2+ has been shown to interfere (Hillyard and Gonick, 1976; Scholtz and Zeiske, 1988) with the ability of the frog skin apical Na channel to demonstrate so-called Na+ self-inhibition (Fuchs et uL., 1977; Zeiske, 1978). Indeed, it has been established using the skin of the clawed frog that both Cd2+and Ca2+have this effect (Scholtz and Zeiske, 1988). Thus, specific binding locations at either a Na+ or K + channel for Ca2+or Cd2+ions could be assumed, and may suggest a certain structural relationship between the two channel types. Nonselective +
186
WOLFGANG ZEISKE
monovalent cation channels in the apical membranes of larval (Hillyard el ul., 1982) and adult frog skins (Van Driessche and Zeiske, 198%) do not discriminate between K+-like and Na+-like ions, and they can even pass Cs+ ions. In tadpole skin, these channels react with blockers of typical K' channels, such as BaZ+ or TEA, as well as with Nab channel agonists such as benzimidazole-2guanidine or antagonists such as amiloride (Hillyard et al., 1982): It appears that the agents with K + channel affinity lead to a blockage of the unspecific tadpole channel, but the Na ' -specific agents seem rather to stimulate current and noise originating from these channels. This sign of cross-reactivity might well point to channel congeniality. In contrast to nonselective apical cation channels in tadpole skin, those in the skin of adult frogs (Van Driessche and Zeiske, 1985~)and in toad urinary bladder (Van Driessche et d., 1987) are already blocked by submillimolar concentrations of polyvalent cations, i.e., CaL+or Cd?'. Moreover, mucosal exposure to nanomolar concentrations of Ag+ ions switches the selectivity of those channels from a N a + - and K+-permeable state (blocked by Ca2t or Cd") to a state that is Ca'l-, Mg?+-, or Ba2'-permeable. The block by Cd" and other Ca2+ channel blockers, such as La3+,the dihydropyridine nicardipine, or veraparnil, remains (Van Driessche, 1987). Finally, oxytocin and its messenger, CAMP, are regulators for all these different cation channels (cf. Van Driessche et ul., 1987). Thus, based on the these data, the assumption of close structural relationships between different types of epithelial cation channels seems justified. Diverse voltage- or ligand-gated cation channels from excitable membranes have been recognized to share several common structural features. This was revealed by molecular biology methods (cf. Stevens, 1987; Agnew, 1988; Schwarz et ul., 1988; Tinipe rt al.. 1988). It was far-sighted of Hille (1984) to propose a parental cation channel molecule that might have diversified, in the course of evolution, to become channels specific for K + , Ca?', or Na' .
Xi. SUMMARY 1 . Apical K + channels in epithelia are much like basolateral channels or channels related to the latter, i.e., the membrane potential-generating K t channels in symmetrical cells. 2. Some divergent peculiarities exist, including substrate-induced gating; block by Cd"; strong indication of homologies between K', N a + , and Ca2+ channels, such as the emergence of nonselective cation channels showing crossreactions with presumably specific inhibitors; or even interchangeability, such as that induced by silver ions. Oxytocin and its messenger, CAMP, are common activators. Finally, voltages play only a minor role in the gating process. 3. Permeation through K + channels displays rnultisite, single-file behavior as
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expressed by a broad spectrum of reactions toward permeant ions, blocking molecules, and even the gating mechanism. We have to stress that apical epithelial K + channels do not much differ in their basic aspects from K + channels in blood-sided membranes. Their physiology may differ among epithelia and may be of specific relevance there. It is their particular biological role that should be elucidated in the future. Of special interest will then be the question how epithelial cells organize the sorting of K + channels into apical or basolateral species, i.e., following their tracks from genetics to membrane location.
ACKNOWLEDGMENTS My thanks for preparing the manuscript are due to Ms. A. Pliimecke and Ms. A. Johannsen. I also gratefully acknowledge many helpful comments on the manuscript by Dr. I. De Wolf and Dr. H. Onken. Furthermore, I am indebted to Dr. Karin von Rosen for revising the English.
REFERENCES Agnew, W. S. (1988). A Rosetta stone for K+ channels. Nuture (London)331, 114- 115. Armda. J. A. L., and Sabatini, S. (1980). Effect of quinidine on N a + , Hi and water transport by the turtle and toad bladders. J . Memhr. Biol. 55, 141- 147. Chase, H. S . , Jr. (1984). Does calcium couple the apical and basolateral membrane permeabilities in epithelia'? Am. J . Ph-ysiol. 247, F869- F876. Clausen, C . , Machen, T. E . , and Diamond, J. M. (1983). Use of AC impedance analysib to study membrane changes related to acid secretion in amphibian gastric mucosa. Biophys. J . 41, 167- 178. De Wolf. I . (1989). "Gating, Inhibition and Stimulation of K'-Channels in thc Skin of Ranu temporuriu: A Biophysical Study," Ph.D. thcsis. Katolieke Universiteit Leuven, Leuven, Belgium. De Wolf, I . , and Van Driessche, W. (1986). Voltage-dependent Bale block of K'-channels in apical membrane of frog skin. Am. J . Phvsiol. 251, C696-C706. De Wolf, I . , and Van Driessche, W. (1988). Current-voltage relations of Cs+-inhibited K ' currents s 413, I I 1 - 1 17. through the apical membrane of frog skin. ~ / l u e g r rArch. Diamond, J. M. (1974). Tight and leaky junctions of epithelia: A perspective of kisses in the dark. Fed. Proc., Fed. Am. Soc. Exp. Biol. 33, 2220-2224. Dow, J . A. T., Gupta, B. L . , Hall, T. A., and Harvey, W. R. (1984). X-ray microanalysis of elements in frozen-hydrated sections of an electrogenic K+-transport system: The posterior midgut of tobacco hornworm (Munduca sexta) in vivo and in vitro. J . Membr. Biol. 77, 223-241. Erlij, D., Van Driessche, W., and De Wolf, 1. (1986). Oxytocin stimulates the apical K +conductance in frog skin. P/tuegers Arch. 407, 602-606. Frizell, R., and Schultz, S . G. (1978). Effect of aldosterone on ion transport by rabbit colon, in v i m . J . Memhr. Biol. 39, 1-26. Fromm, M . , and Schultz. S . G . (1981). Potassium transport across rabbit descending colon in virru: Evidence for single-tile diffusion through a paracellular pathway. J . Membr. B i d . 63, 93-98. Fuchs, W., Hviid Larsen, E., and Lindemann, B. (1977). Current-voltage curve of sodium channels and concentration dependence of Na +-permeability in frog skin. J . Physiol. (London) 267, 137- 166.
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Gogelein, H., and Greger, R. (1984). Single channel recordings from hasolateral and apical membranes of renal proximal tubules. F‘fiuegrrs Arch. 401, 424-426. Giigelein, H., and Van Driessche, W. (1981). The effect ofelectrical gradients on current fluctuations and impedance rccorded from Neciurits gallbladder. J. Membr. B i d . 60, 199 -209. Goldnian, L). E. ( 1944). Potential, impedance, and rectification in membranes. J . Grn. Physiul. 27, 37-60. Gregcr, R . ( 1985). Ion transport mechanisms in thick ascending limb or Henlc’s loop of mammalian nephron. Phvsiol. Rrv. 65, 760-797. Harvcy. W. R., Ciofti, M . , and Wolfersbergcr, M. G . (1983). Chemiosmotic potassium ion pump of insect epithelia. Am. J . Phvsiul. 244, R163-RI75. Hess, P., Lansnian, 1. B., and Tsien, R. W. (19x4). Different modes of Ca channel gating behaviour favourcd by dihydropyridine Ca agonists and antagonists. Narure (London) 31 I , 538-544. Hille, B. (1084). “Ionic Channels of Excitable Membranes.” Sinaucr, Sunderland, Massachusetts. Hillc, B . , and SchwarZ, W. (1978). Potaasium channcls as multi-ion single-file pores. J. G‘en. Physiol. 72, 409-442. Hillyard. S. D, and Gonick, H. C . (1976). Effects of Cd” on short-circuit current across epithelial membranes. I . Interactions with Call and vasoprcssin on frog skin. J . Membr. BicJ/. 26, 109- 119. klillyard, S . D., 7,eiske. W., and Van Driessche, W. (19x2). Poorly selective cation channels in the skin of the larval frog (stage 5 XIX). ?/luegrrs Arch. 394, 287-293. Hunter, M., and Giebisch, G. ( 1987). Multi-barreled K+-channels in rcnal tubules. Nature (Lundun) 327, 522-524 Koefocd-Johnsen, V., and Ussing, H. H. (IY58). Thc nature of the frog skin potential. Actu Phvsiol. Stwid. 42, 298 .~308. Koeppen. B. M., Biagi, €3. A,, and Gicbisch, G . H. (1983). lntracellular microelectrode characterization of the rabbit cortical collecting duct. Am. J . Physiol. 244, F35-F47. Lansman, J. B., Hess. P., and Tsien, R . W. (1986). Blockade of current through single calcium channels by Cd?’ , Mg!’ and Ca? ’ , Voltagc and concentration dependence of calcium-entry into the porc. J. Gen. Physiol. 88, 321-347. Latorre. R., and Miller. C. ( 1983). Conduction and selectivity in potassium channels. J . Memhr. B i d . 71, 11-30, Ihdemann, B., and Van Driesschc, W. (1978). The mechanism of Na-uptake through Na-selective channels in the cpitheliuiii of frog skin. In “Membrane Transport Processes” (J. f;. Hoffniann, cd.), Vol. I , pp. 1.5- 178. Raven, New York. Machen, T. E., and Zcuthen, T. (1982). CI- transport by gastric mucosa: Cellular CI- activity and membrane permeability. Pro(-. R . Soc. London B 299, 559-573. Macknight, A. D. C. (1977). Epithelial transport of potassium. Kidnrv Int. I I , 391 -414. Mandel, Id. J., and Curran, P. F. (1972). Response of the frog skin to steady-state voltage clamping. I . ‘lhc shunt pathway. J . Gen. Physiol. 59. 503-518. Maruyama, Y., Matsunaga, H., and Hoshi, T. (1986). Call- and voltage activated K+-channel in apical cell membrane of gallbladder epithelium from Trirurus. fflurgers Arch. 406, 563-567. Nagel, W., and Hirschmann, W. (1980). K + permeability of the outer border of the frog skin ( R . remporuriu). J . Memhr. R i d . 52, 107- 1 13. Niclben, K. (1971). Eftect of arnphotericin R on the frog skin i n v i m . Evidence for outward active potasbium transport across the epithelium. Acta Physiol. Scand. 83, 106- 114. O’Ncil, R . G . . and Boulpaep, E. L. (1982). Ionic conductive properties and clectrophysiology of the rabbit cortical collecting tubule. Am. J . Phvsiol. 243, F81-F95. Onken. H. (1985). “Studie zur Chernie und pH-ahhiingigen Lcitfiihigkeit van K+-Kanllen in der apikalen Menibran des Froschhautepithels (Runu temporaria),” M.S.thesis. Free University of Berlin. Berlin.
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Onken, H., and Zeiske, W. (1986). The pH titration curve of the K’ channel in the apical membrane of the frog skin (Rann trmporurio). Renal Pl7ysiol. 9, 93. Reuss, L.. and Finn, A. L. (1975). Electrical properties of the cellular transepithelial pathway in Necrurus gallbladder. 11. Ionic permeability of the apical cell membrane. J . Membr. B i d . 25, 141-161. RKUSS,L., Cheung, L. Y., and Grady, T. P. (1981). Mechanism of cation permeation across apical cell membrane of Nrcrurus gallbladder: Effects of luminal pH and divalent cations on K + and Na’ permeability. J . Memhr. B i d . 59, 21 1-224. Sachs, G . , Faller, L. D., and Rabon, E. (1982). Protodhydroxyl transport in gastric and intestinal epithelia. J . Mrmbr. B i d . 64, 123-135. Scholtz, E . , and Zeiske, W. (1988). A novel synergistic stimulation of Na+-transport across frog skin (Xenopus kieuis) by external Cd”- and Ca?+-ions.Pflueprs Arch. 413, 174- 180. Schwarz, T. L., Tempel, B. L., Papazian. D. M . , Jan, Y. N., and Jan, L. Y. (1988). Multiple potassium-channel components are produced by alternative splicing at the shaker locus in Drosophilu. Nurure (London)331, 137- 142. Stevens, C. F. (1987). Channel families in the brain. Narure (London)328, 198- 199. Timpe, L. C., Schwarz, T. L., Tempel, B. L., Papazian, D. M . , Jan, Y. N., and Jan, L. Y. (1988). Expression of functional potassium channels from Shaker cDNA in Xrnopus oocytes. Nufure (London) 331, 143- 145. Van Driessche, W. (1984). Physiological role of apical potassium ion channels in frog skin. J . Phvsiol. (London) 356, 79-95. Van Driessche, W. (1987). Ca” channels in the apical membrane of the toad urinary bladder. Pjuegers Arch. 410, 243-249. Van Driessche. W., and GBgclein, H. (1978).Potassium channels in the apical membrane of the toad gallbladder. Narurr (London)275,665-667. Van Driessche, W., and Giigelein, H. (1980).Attenuation of current and voltage noise signals recorded from epithelia. J . Theor. Biol. 86, 629-648. Van Driessche. W., and Zeiske, W. (1980a). Spontaneous fluctuations of potassium channels in the apical membrane of frog skin. J . Physiol. (London)299, 101- 116. Van Driessche, W., and Zeiske, W. (1980b). Ba”-induced conductance fluctuations of spontaneously fluctuating K+-channels in the apical membrane of frog skin ( R a m temporaria). J . Mrmhr. Biol. 56, 31-42. Van Driessche, W., and Zeiske, W. (1982). Effet of PCMPS on epithelial K+-channels. Arch. Inr. Phvsiol. Biochim. 90, PS7-P58. Van Driessche, W., and Zeiske, W. (198%). Ionic channels in epithelial cell membranes. Physiol. Rev. 65, 833-903. Van Driessche, W., and Zciske, W. (1985b). Apical K+-channels in frog skin: A pathway for K+ excretion. I n “Transport Processes, lono- and Osmoregulation” (R. Gilles and M. GillcBaillien, eds.), pp. 40-55. Springer-Verlag. Berlin. Van Driessche, W., and Zeiske. W. (1985~).Ca-sensitive, spontaneously fluctuating, cation channels in the apical mcmbrane of the adult frog akin epithelium. Pfluegers Arch. 407, 145- 152. Van Dricsschc, W., Aelvoet, I . , and Erlij, D. (1987). Oxytocin and CAMP stimulate monovalent cation movements through a Ca?+-sensitive, amiloride-insensitive channel in the apical membrane of toad urinary bladdcr. Proc. Null. At&. Sci. U.S.A. 84, 313-317. Wills, N. K . , and Biagi, B. (1982). Active potassium transport by rabhit descending colon epithelium. J . Memhr. B i d . 64, 195-203. Wills, N. K . , Zeiskc, W., and Van Driessche, W. (1982). Noise analysis reveals K+-channel conductance fluctuations in the apical membrane of rabbit colon. J . Membr. B i d . 69, 187- 197. Wolosin, I. M., and Forte, J . G. (1985). K ’ and CI- conductances in the apical membrane from
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secreting oxyntic cells arc concurrently inhibited by divalent cations. J . Mrmbr. B i d . 83, 261 -272. Wright, F. S. (1981). Potassium transport by successive segments of the mammalian nephron. Fed. Pro(... Fed. Am. Soc. E.rp. B i d . 40, 2398-2402. Yorio, T., and Bentley, P. S. (1977). The permeability of the rabbit colon, in v i m . Am. J . Phvsiol. 232, FS - F9. Zeiske, W. (1978).The stimulation of Na' -current through the outer surface of frog skin epithelium. Biochim. Bioplivs. Acta 352, 323-326. Zciskc, W., and Srhriidcr. H. (1988).Apical and serosal K'-channels in larval insect midgut. Comp. Biochem. Phvsiol. A YOA, 836. Zeiskc, W., and Van Dricsschc, W. (1979). Saturable K + pathway across the outer border of frog skin (Runu rempururiu): Kinetics and inhibition by Cs' and other cations. J . Memhr. B i d . 47, 71-96. Zciskc, W., and Van Dricsschc, W. (1980). Intcrdction of C d ? ' with K'-channels in the apical membrane of frog skin (Ratta tcmpnraria). Arch. Inr. Phvsiol. Biochim. 88, P23-P24. Zciskc, W., and Van Driesschc, W. (1981). Apical K+-channcls in frog skin (Runu remporuriu): Cation adsorption and voltage influence gating kinetics. Pfluegcrs Arch. 390, 22-29. Zeiskc, W., and Van Driessche, W. (1983) The interaction of "K+-like" cations with the apical K + channel in frog skin. J. M m h r . B i d . 76, S 7 ~72. Zeiske, W., Machen. T. E.. and Van Driessche. W. (1983). CI-- and K+-related fluctuations of ionic current through oxyntic cells in frog gabtric mucosa. Am. J. Phvsiol. 245, G797-G807. Zeiske, W., Van Driessche, W., and Ziegler, R . (1986). Current-noise analysis of the hasolateral route for K' ions across a K'-secreting insect niidgut epithelium (Munducu srxtu). Pfluegers Arch. 407,657-663. Zeuthen. T.. Christensen, 0..and Cherkscy, €3. (1987). Elcctrodiftusion of CI- and K + in epithelial rncinbrancs reconstituted into planar lipid bilayers. Pfirtrgers Arch. 408, 275-28 I .
C U R R E N T TOPICS IN MEMRRANES A N D IRANSPORT. VOLUME 37
Chapter 6 Basolateral Potassium Channel Noise: Signals from the Dark Side DAVID C . DAWSON, DANIEL J . WILKINSON, AND NEIL W. RICHARDS Deparfmenfof Physiology The University of Michigan Medical School Ann Arbor, Michigan 48109
I . Basolateral Memhrane: Dark Side of the Epithelial Cell A. Role of Basolateral Membrane in Transepithelial Salt Transport €3. Fluctuation Analysis: A Sensitive Listcning Device 11. Pcrmeabilized Cell Layers: Techniques and Limitations A . Pore-Forming Antibiotics: Selcctive Perrneabilization B . Detergents: I n Situ Reconstitution 111. Basolateral Membrane Noise A. Channels and Blockers B . Fluctuation versus Single-Channel Estimates of Blocker Kinetics C. Channel Subpopulations: Who Is Doing What? IV. Is the Noise Worth Listening to? References
1. BASOLATERAL MEMBRANE: DARK SIDE OF THE EPITHELIAL CELL The purpose of this chapter is to review the applications and contributions of current fluctuation analysis to the study of K channels in the basolateral membranes of epithelial cells. The paucity of information in this area will make this chapter mercifully brief, but we hope at least to alert the reader to some of the issues involved in the analysis of basolateral membrane ion transport and to point out areas in which the analysis of channel noise may make a contribution. We will discuss experimental difficulties that arise from two sources: the relative inaccessibility of the basolateral side of epithelial cells and the existence of mul-
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tiple K channel types. These difficulties make noise analysis a particularly appropriate device for “listening” to basokiterdl K channels. In addition, we will consider some of the experimental strategies that have been employed to gain access to the basolateral membrane, and we will compare estimates of K channel blocker kinetics gained from noise analysis with those obtained from singlechannel recordings.
A. Role of Basolateral Membrane in Transepithelial Salt Transport It has become increasingly apparent that the basolateral membrane plays a pivotal role in the regulation of transepithelial ion transport. For both absorptive (Schoen and Erlij, 1985; Schultz, 1981; Van Driessche and Erlij, 1988; Venglarik and Dawson, 1986) and secretory (Richards et al., 1989; Smith and Frizzell, 1984; Welsh, 1987) epithelia, there is now abundant evidence that alterations in salt transport can be accompanied by changes in the conductance of the basolateral membrane to potassium. A somewhat teleological rationale for this behavior can be obtained by considering the cell models shown in Fig. 1, which are meant to represent “generic” cellular mechanisms for salt secretion and absorption. Neither of the model cells effects net transcellular K transport, rather, K movements are confined to a pump-leak cycle at the basolateral membrane, which results in the continual “recycling” of K in the steady state. Conductive K flows at the basolateral membrane are, however, critical to the maintenance of charge balance within the cell. In the absorptive model, based on that proposed by Koefoed-Johnsen and Ussing ( 1958), transepithelial NaCl absorption is driven by transcellular Na flow from lumen to blood. Active Na absorption occurs in two steps: Na enters the cell through conductive channels in the apical membrane and exists via the basolateral Na-K pump, assumed here to have a stoichiometry of 3Na:2K. Because the pump extrudes only one net charge for every three Na transported, two-thirds of the inward current due to Na entry across the apical membrane must be balanced by an additional outwurd current due to K exit across the basolateral membrane. In the secretory model, NaCl transport from blood to lumen is driven by active CI secretion. The exit of CI at the apical membrane also comprises an inward current, the majority of which must be balanced by an outward K current across the basolateral membrane. In both secretory and absorptive epithelia it appears that the equality between inward apical and outward basolateral current is maintained during stimulation or inhibition of transport by a parallel regulation of apical and basolateral membrane conductances. For example, the activation of tracheal CI secretion by adrenergic agonists is accompanied by increases in both apical CI conductance and basolateral K conductance (Smith and Frizzell, 1984; Welsh, 1987). Similarly, the antidiuretic hormone (ADH)-induced activation of Na absorption in toad urinary bladder is associated with increases in apical Na conductance and
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6. BASOLATERAL POTASSIUM CHANNEL NOISE SALT SECRETION
SALT ABSORPTION
FIG. 1. Generic models for salt absorption and salt secretion. The absorptive model is esaentially that originally proposed by Koefoed-Johnsen and Ussing (1958). Na enters the absorptive cell via amiloride-sensitive channels in the apical membrane and exits via the basolateral Na ‘ ,K + -ATPase. The serosa-positive potential generated by active Na absorption drives passive CI absorption. The secretory model incorporates a basolateral Na-K-2Cl cotransport mechanism that mediates CI entry into the cell across the basolateral membrane. CI exits the cell via apical CI channels and the resulting mucosa-negative potential can drive passive Na secretion. Both of these models require a basolateral K conductance to allow for the “recycling” of cellular K and the exit of outward current.
basolateral K conductance (Van Driessche and Erlij, 1988). Conversely, inhibition of Na absorption appears to involve parallel decreases in apical and basolateral conductance. For instance, muscarinic inhibition of Na transport by turtle colon is due to inactivation of apical Na and basolateral K channels (Venglarik and Dawson, 1986; Wilkinson and Dawson, 1989).
6. Fluctuation Analysis: A Sensitive Listening Device The apical and basolateral membranes of epithelial cells differ not only in their properties but also in their relative accessibility to experimental investigation. The apical membranes received most of the attention in early studies of ion transport across epithelial cell layers. The “external” location of apical membranes makes them readily accessible for studies with ion substitution, channel blockers, microelectrodes, and, more recently, patch electrodes. In contrast, basolateral membranes are typically “buried” beneath layers of connective tissue or smooth muscle. Although some improvements in access can be obtained by dissection (Demarest and Finn, 1987; Wehner et al., 1990) or separation of cell layers (Erlij and Smith, 1973), the access still cannot approach that from the apical side. This means that even the simplest experiments involving an ion substitution or the addition of a blocker are potentially compromised by unstirred layers. Similarly, the basolateral surface is often inaccessible to the tips of patch pipets. Compounding the accessibility problem is the probable presence in the
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basolateral membrane of multiple channel types (Dawson, 1987; Germann et ul., 1986a,b). The results of single-channel investigations suggest that it is the rule, rather than the exception, that epithelial cells are endowed with more than one type of K channel (Dawson, 1987). Hence an important objective in the study of cellular transport regulation is that of identifying specific functional roles for particular subpopulations of K channels. It seems reasonable to suspect that the activation or inactivation of specific subpopulations of K channels permits a cell to respond appropriately to different environmental perturbations. The use of blocker-induced fluctuations as an assay for changes in basolateral membrane function has several advantages. First, the requirements for access are not as stringent as for other techniques, such as patch clamping. Analysis of channel noise requires only that the current through the basolateral membrane be directly measured. This can be accomplished by measuring transepithelial current using a cell layer in which the apical membrane has been artificially permeabilized or one in which the basolateral membrane dominates the transcellular resistance (Dawson et ul., 1988; Hanrahan et ul., 1986). A second and related advantage is that channel behavior can he assayed in an integrated macroscopic setting. This is particularly important for epithelia, in which cell isolation for single-channel recording can lead to loss of the very cell polarity that constitutes the basis for the function of the intact cell layer. Compounding this problem is the fact that the process of isolating epithelial cells is likely to seriously compromise specific cellular functions. A third advantage of fluctuation analysis is that channel noise can be produced in one specific subpopulation of channels if a suitable blocker is available. A point that is often not appreciated is the sensitivity of noise analysis. The technique analyzes fluctuations of current around the mean, so it is essential only that the jfuctuutions are discernible; the time-average current can be relatively small. For example, in the toad gallbladder and turtle colon current fluctuations due to apical K channels were detectable, although the macroscopic K current was not readily discernible (Van Driessche and Gogelein, 1978; Wilkinson and Dawson, 1990b).
II. PERMEABILIZED CELL LAYERS: TECHNIQUES AND LIMITATIONS In most isolated epithelial sheets, the shortest route to the basolateral membrane is through the apical membrane. Thus one approach to the study of basolateral membrane transport has been to chemically modify the apical membrane and rcnder it so leaky to ions that it no longer constitutes a significant barrier to transepithelial ion flows. In any such experiment there are two, sometimes competing, objectives. One is to eliminate the apical membrane as a barrier to the
6. BASOLATERAL POTASSIUM CHANNEL NOISE
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ion or molecule of interest. The second is to restrict or control changes in cell composition, which occur as a result of compromising the normal “barrier” function of thc apical membrane, in order to approach as nearly as possible the normal function of the cell. Ideally it would be possible, by means of different chemical modifications, to achieve different degrees of apical permeabilization and, consequently, different amounts of change in intracellular composition. We will discuss two methods that constitute, perhaps, the two limiting cases in the spectrum of permeabilization possibilities: antibiotics, which form pores selective for monovalent ions, and detergents, which disrupt membrane integrity to the point where cytosolic proteins are released from the cell.
A. Pore-Forming Antibiotics: Selective Permeabilization The polyene antibiotics, amphotericin B (Dawson, 1987; Dawson et al., 1988; Germann et al., 1986a,b; Halm and Dawson, 1983; Kirk and Dawson, 1983; Kirk et al., 1980; Lichtenstein and Leaf, 1965), nystatin (Garty, 1984; Lewis et al., 1977, 1978; Van Driessche et al., 1982; Van Driessche and Hillyard, 1985; Wills et ul.. 1979), and filipin (Nielsen, 1979), and the polypeptide antibiotic, gramicidin (Andersen, 1984; Finkelstein and Andersen, 198I ; Lewis and Wills, 1982; Wills, 1981), have been employed to permeabilize the apical membranes of epithelial cell layers to monovalent cations. Our review will focus on amphotericin B and nystatin because they are in widespread use. The polyene antibiotics are a class of antifungal compounds that markedly increase the ion conductance of sterol-containing membranes, biological or artificial (Cass and Dalmark, 1973; Cass er al., 1970; Kinsky, 1970; Kleinberg and Finkelstein, 1984). When added to the solutions bathing both sides of a planar lipid bilayer, nystatin and amphotericin B cause a permeability increase that is consistent with the introduction of an aqueous pore into the membrane. The permeability to water and small hydrophilic solutes (urea and glycerol) is increased, and the membrane develops an anion-selective conductance. In contrast, when polyenes are applied to only one side of a planar bilayer (the situation relevant to biological studies), membrane permeability to urea and glycerol is increased, but a cation-selective conductance develops. Holz and Finkelstein (1970), Kleinberg and Finkelstein (1984), and Marty and Finkelstein (1975) envision the polyene pores as oligomeric cylindrical structures formed from 8 to 10 monomers, which insert into the membrane like barrel staves. The polyene molecules, which are about 30 A long, have a polar head that anchors the molecule at the aqueous-membrane interface and a relatively nonpolar lactone ring that can insert into the hydrocarbon core of the bilayer. It is thought that polyene molecules added to one side of a planar bilayer form a single, barrel-like structure that is responsible for cation-selective conductance. When polyene is added to both sides of the membrane, however, it is thought that a dimer forms due to the end-to-end juxtaposition of two “single barrels” and that this structure is
DAVID C. DAWSON ET AL.
iTa B
Oi M A l
TIME
Rc;. 2. General experimental paradigm employed to measure basolateral K currcnts in cpithclial cell layers pernicabilizcd with amphotcr~cinB (AMPHO). Tissues are bathed on the mucosal side with a high K-Ringer's solution and on the seroaal side with a NaC1-Kingcr's solution. Addition of amphotcricin B to the niucosal bath lead.\ to the formation of polycne porcs in the apical membrane and the development of a transccllular K current, which can be eliminated by blocking basolateral K channels.
union selective. With regard to biological applications, it is noteworthy that in bilayers the one-sided, cution-selective effect generally does not gradually evolve into a two-sided, anion-selective effect, presumably because the polar head group of the polyene molecules effectively anchors them at the aqueousmembrane interface. Although the entry of polyene molecules into cells is not precluded, in epithelial cell layers the addition of arnphotericin B to the mucosal bath leads to permeabilization of the apical membrane but does not result in the formation of polyene pores in the basolateral membrane. Figure 2 shows an example of the experimental paradigm employed in our laboratory to study basolateral potassium currents in polyene-permeabilized eoIonic cell layers. Sheets of epithelium are mounted in conventional Ussing chambers or perfusion chambers (Dawson et ul., 1988; Kirk et a[., 1980), and a transmural K gradient is imposed by bathing the mucosal side of the tissue with a Na-free, K-Ringer's solution. This maneuver also eliminates active Na absorption, so the short-circuit current (I,,) in this condition is very low. The addition of amphotericin B to the mucosal bath (10 pM) leads to the development of a mucosal-to-serosal potassium current. This current can be blocked by the addition of K channel blockers (e.g., barium) to the serosal bath, and flux measurements confirm that the current is due to net K flow (Germann et al., 1986a,b; Kirk and Dawson, 1983). The ability to completely block the polyene-induced currents and conductance (Germann et ul., 1986a,b; Venglarik and Dawson,
6. BASOLATERAL POTASSIUM CHANNEL NOISE
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1986) using K channel blockers or a muscarinic agonist argues strongly that polyene added to the mucosal bath does not lead to the appearance of poiyene pores in the basolateral membrane. The advantage of amphotericin B as a permeabilizing tool lies in its ability to effect a rather large change in membrane conductance while causing changes in cellular composition that are limited to univalent ions. Isotope uptake assays (Germann er nl., 1986a,b) and measurements with microelectrodes (Lewis er al., 1977) show that it is possible to achieve a ratio of apical to basolateral conductance of 10: 1 in the presence of amphotericin or nystatin. In this condition, the cellular resistance will be dominated by that of the basolateral membrane. In our experience, however, there can be considerable variability in the efficacy of amphotericin. This has been attributed to variability in the amount of mucus on the apical surface of the colon, but we have also noticed that it is necessary to be very careful that the polyene is uniformly distributed in the mucosal solution. Sometimes a bolus dose added in dimethyl sulfoxide (DMSO) will appear to simply “precipitate out” and fall to the bottom of the chamber. One recent paper suggested sonicating solutions of nystatin to enhance the dispersion of the substance in salt solutions (Horn and Marty, 1988). When making up a large volume for perfusion, it is also helpful to add the polyene-DMSO stock solution slowly while stirring vigorously. The stability of the permeabilizing effect is also a potential problem. In Ussing chamber studies the effect of a bolus dose will sometimes wane but can be restored by adding additional polyene. Continuous perfusion is one way to combat this difficulty. The ratio of the apical and basolateral membrane resistances in the presence of mucosal polyene is an important consideration for two reasons. One is the possible attenuation of fluctuations at the basolateral membrane due to the series resistance of the apical membrane. This effect is discussed by Van Driessche and Gullentops (1982). (See also Wilkinson and Dawson, 1990a, and Van Driessche and Van Deynse, this volume.) A related problem is the inability to accurately determine the basolateral membrane potential. If apical membrane resistance ( R , ) is not much less than the basolateral resistance ( R J prior to the addition of blocker, then the progressive addition of blocker to the basolateral membrane will change the fractional resistance ( R , / R , ) and hence the basolateral membrane potential. This is a particularly acute problem if the blocking rate is expected to be voltage dependent (Hanrahan et al., 1986). Due to the potential variability in the permeabilizing efficacy of the polyene, it would be desirable to check this in each experiment. One possibility would be to routinely determine the apical-tobasolateral resistance ratio using impedance analysis (Van Driessche, 1986). The one-sided polyene pores that are presumably formed in biological and artificial membranes are cation selective, but not ideally so (Kleinberg and Finkelstein, 1984; Marty and Finkelstein, 1975). Thus the permeabilized apical membrane is ideal for the study of cation currents, but the finite permeability to univalent anions has some important consequences. The most important of these
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DAVID C. DAWSON ET AL.
is that the polyene pores can mcdiate salt entry into the cells, causing cell swelling (Cass and Dalmark, 1973; Dawson et af., 1988; Germann et al., 1986b). Thus it is necessary to “osmotically buffer” the external solution with an impermeant osinoticant (Cass and Dalmark, 1973; Germann et a/., I986b) or to use relatively impermeant anions such as gluconate or sulfate (see below, however) in the bathing solution. Germann et ul. (1986a,b) and Dawson et a / . (1988) exploited the finite salt permeability of the amphotericin pores in order to induce cell swelling and activate a specific basolateral K conductance. The permeability increase caused by amphotericin or nystatin appears to be limited to univalent ions (Na’, K ’ , H’, and CI-), water, and small nonelectrolytes such as urea. Divalent ions and organic molecules of the size of six-carbon sugars are excluded. This suggests that the introduction of polyenes can effect dramatic changes in cellular levels of Na, K , and C1 as well as cell pH. Even if the mucosal solution is designed to be cell-like (high K, low Na, etc.), it is likely that membrane potentials and cellular ion composition will be altered. It is noteworthy in this regard, however, that the cffects of the polyenes on transcellular transport are generally completely reversible. Figure 3 shows an example of an experiment with isolated turtle colon. Two tissues were perrneabilized, and a
200
1
WASH
i(
TIME (min) FIG. 3 . Reversibility of amphotcricin (AMPHO) pcrmeabilizdtion. I, versus lime is shown for two portions of turtle colon that were initially bdthcd by NaCl-Ringer’s on both sides. Subsequently, thc mucosal baths were changed to either K gluconatc-Ringer’s (dashed line) or KCI-Ringer’s (solid line). Arnphotericin (9 p M ) was added to the nlucosal bath of each and after a steady K current was achieved, the mucosal bath of each was washed and replaced with NaCI-Ringer’s. After I,, returned to it5 initial value, amiloride (50 p M ) was added to the mucosal bath. [Reprinted from G e r m d m Y I a / . (1986h) with pcrmission of the Rockefeller University Press.1
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6. BASOLATERAL POTASSIUM CHANNEL NOISE
large K current was induced in one by exposing it to mucosal KCI, which causes cell swelling. Despite these maneuvers the tissues were able to regain a normal amiloride-sensitive I,, after washing and incubation for about I hr. Thus although polyene-induced alterations in cell composition can be dramatic, they apparently do not irreversibly compromise cell regulatory mechanisms. Although the cation selectivity of the polyenes makes them most useful for manipulating and studying cation flows, their finite anion permeability (Kirk and Dawson, 1983; Marty and Finkelstein, 1975) has been exploited to effect changes in intracellular CI concentration in squid axon (Russell et a l . , 1977) and to study CI currents in the flounder urinary bladder (Dawson and Frizzell, 1989; Keller et al., 1984). In the latter tissue the basolateral membrane appears to be almost perfectly anion selective, but the apical membrane contains only a K conductance. Hence, apical permeabilization with amphotericin B creates an apical CI conductance, which allowed Dawson (1983) and Keller et al. (1984) to study basolateral CI conductance using transcellular CI currents. A potential concern with the use of channel-forming molecules to permeabilize apical membranes is the possibility that fluctuations in transepithelial current due to the gating of these molecules could introduce a Lorentzian component into the power density spectrum, possibly obscuring the desired basolateral membrane noise. Single-channel records published by Kleinberg and Finkelstein (1984) suggest that in nystatin-treated planar bilayers, dwell times in the conducting (f,,) and nonconducting ( t , ) states are of the order of 0.6-2 sec, t ; ' ) from about 0.2 to 0.6 corresponding to corner frequencies (27rf, = t;' Hz. These values lie within a range that is potentially discernible in transepithelial current noise, although they might be obscured by I/fnoise. It is possible, of course, that the gating of polyene channels could differ substantially in biological membranes so that the question of possible interference becomes largely an empirical one. Dawson et al. (1988) were not able to discern a spontaneous Lorentzian component in the fluctuations of transepithelial K current measured across amphotericin-treated turtle colon, although a Lorentzian component could be induced by lidocaine. On the other hand, Van Driessche (1984) reported that in nystatin-treated toad urinary bladder, fluctuations in transepithelial current showed evidence of a spontaneous Lorentzian component with a corner frequency around 1 Hz in the absence of blocker. The possibility that this component of the power density spectrum was due to nystatin could not be eliminated.
+
B. Detergents: In Situ Reconstitution Detergents such as digitonin produce a dramatic increase in the permeability of plasma membranes. In contrast to the relatively selective effect of the polyenes on univalent ions, digitonin causes the release of cytosolic proteins (Chang and Dawson, 1988). The basis for the permeability effect is the interaction of the
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DAVID C. DAWSON ET AL.
steroid glycoside,with cholesterol to form “porelike” structures (Fiskum, 1985). The requirement for cholesterol apparently confers a degree of selectivity on the permeabilization process such that intracellular membranes are less likely to be affected. Chang and Dawson (1988) used digitonin to remove the apical membrane of turtle colon epithelial cells. Mucosal concentrations of 20-40 pM led to the release of the cytosolic enzyme, lactate dehydrogenase, from the cells and rendered the apical membrane freely permeable to monovalent and divalent ions as well as organic buffers for calcium and pH. A somewhat surprising finding in this study was that the permeabilization of the apical membrane could proceed while the basolateral membrane retained its functional integrity as a resistance barrier. The long, columnar shape of the colonic cells may have contributed to this result. The principal problem with digitonin permeabilization is analogous to that encountered in the use of the whole-cell patch-clamp technique; namely, the extreme nature of the permeabilization is expected to lead to the loss of a variety of cellular constituents. These constituents could, in principle, include factors that are required to maintain the activity of certain channel populations as well as the components of intracellular signaling mechanisms that couple changes in channel activity to surface receptor binding events. It is interesting in this regard that Horn and Marty (1988) reported recently that nystatin could be used inside a patch pipet to increase the conductance of the patch membrane (“perforated patch”) and thus achieve a whole-cell voltage clamp while preventing the loss of intracellular constituents. The use of a digitonin-permeabilized preparation necessarily implies some attcmpt to “reconstitute in situ” the intracellular factors that are required to activate a particular conductive process. For example, Chang and Dawson (1988) used a mucosal solution that was designed to very rigidly control cytosolic pH and calcium activity by including appropriate amounts of organic pH buffer and 1,2-di(2-aminoethoxy)ethane-N,N,N’.N’-tetraacetic acid (EGTA) (Chang ef ul., 1988). The general paradigm of this experiment is illustrated in Fig. 4. Here, the permeabilization takes place in the presence of a mucosal calcium activity of less than 1 nM. The addition of digitonin, despite removal of the apical membrane as a barrier, did not lead to the transcellular current, due to the relative impermeability of the basolateral membrane in the presence of relatively low cellular free calcium. The addition of calcium to the rnucosal bath, sufficient to raise cytosolic calcium activity to about 0. I pA4, led to the prompt development of a K current that could be blocked by quinidine or inactivated by chelating the cellular calcium. Recent studies on symmetric cells suggest that pore-forming bacterial toxins such as a-toxin from Stuphyfococusuureus and streptolysin-0 from P-hemolytic streptococci may be useful for producing degrees of permeabilization that lie between those produced by amphotericin B or digitonin (Ahnert-Hilger and
6. BASOLATERAL POTASSIUM CHANNEL NOISE
I,
201
MUCOSAL
DIGITONIN
TIME FIG.4. Paradigm employed by Chang and Dawson (1988) to study calcium-activated K currents in digitonin-treated colonic cell layers. Tissues are bathed by K aspartate-Ringer’s on the mucosal side and Na aspartate-Ringer’s on the serosal side. The free calcium concentration in the mucosal bath is reduced to less than 10 nM by a Ca” -EGTA buffer. The addition of digitonin permeabilizes the apical membrane to the extent that the cytoplasmic enzyme, lactate dehydrogenase, can be detected in the mucosal bath, but in the absence of calcium then leads to only a small increase in I , . Raising the free calcium to 100- 1000 nM activates a K current that can be eliminated by blocking basolateral K channels.
Gratzl, 1988). The effects of these compounds have yet to be studied in epithelial cells, however.
111. BASOLATERAL MEMBRANE NOISE A. Channels and Blockers
After a suitable strategy for gaining access to the basolateral membrane is adopted, the application of fluctuation analysis hinges upon discovering the “right” channel blocker. As discussed elsewhere in this volume, it is blockerinduced fluctuations that provide the most useful results of noise analysis because the concentration dependence of the blocker-induced corner frequency leads directly to a determination of the kinetics of block and thereafter to estimation of the single-channel current and the number of channels. So-called spontaneous Lorentzians detected in the absence of a blocker may lead to some qualitative conclusions with regard to the existence of certain general classes of channels, but no calculation of single-channel parameters is possible because the opening
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and closing rates cannot be determined. The relationship between spontaneous and blocker-induced fluctuations can be appreciated in terms of a simple model that allows for three states of a channel: closed, open, and blocked,
where C and 0 represent, respectively, the closed and open states of the channel, B is the blocker, and OB is the blocked state of the channel, which is assumed here to result from the binding of the blocker to the open state. In the simplest physical model for open channel block, the blocking reaction is envisioned as the entry of the blocker into the open channel, where it obstructs the normal flow of permeant ions (Miller, 1987; Vergara and Latorre, 1983). Either one or both of the transitions in the three-state model (closed-open or open-blocked) can give rise to a Lorentzian component in the spectrum (Van Driessche and Zciske, 1980). The advantage of the blocker-induced noise lies in the fact that the frequency of the blocking reaction can be varied by varying the concentration of the blocker. For example, the corner frequency for a single blocker-induced Lorentzian is given by:
and the values for k,,,, and kb, can be determined from a plot of 25~f;versus [B]. A single-channel record for open channel block might appear as diagrammed in Fig. 5 . Single-channel recordings have provided tremendous advances in our understanding of the properties of ion channels, particularly with regard to the mode of action of blockers (Miller, 1987; Miller ot al., 1987; Neher and Steinbach, 1978; Vergara and Latorre, 1983). An important goal of future studies will be to determine if the estimates of channel blocking kinetics derived from fluctuation analysis can be confirmed by single-channel recordings, which allow a direct determination of the dwell times of the channel in various states. The current trace in the Fig. 5 depicts “slow” block. The dwell time in the blocked state is long relative to the dwell time in the normal closed state, so that a blocking event ( tblWked)can be easily distinguished from the shorter closing event ( rc,(,acd)as a long, nonconducting interval in the single-channel record. The inverse of the mean blocked time is an estimate of the off-rate for the blocker-channel reaction. The open channel block model predicts that the offrate should be independent of blocker concentration because konis simply a measure of the tendency of the blocker to dissociate from the channel. In contrast the on-rate, kAn[B], is expected to vary linearly with blocker concentration. If the blocking molecule is chbrged, then both the on-rate and the off-rate can be voltage dependent and the nature of the dependence can be used to determine if the blocker enters the channel from the cytoplasmic or the extracellular side.
203
6. BASOLATERAL POTASSIUM CHANNEL NOISE CLOSED
C-
OPEN
P
kbnbl
i-
a
s
" F A T"
koff
BLOCKED ~
08
"sLOW"
FIG. 5 . Idealized record of singlc-channel current for a channel that can exist in three states: closed, open, and blocked. The spontaneous gating (opening and closing) of the channel was arbitrarily chosen to bc "fast" with regard to the dwell time in the blocked state so that the blocking events can be clearly discerned as long. nonconducting intervals in the bingle-channel record. The coefficients a and /3 describe thc spontaneous closing and opening. rcspectively, and X.6, and A,,,, are, respectively, the rate coefficient for the pseudo-first-order blocking reaction and the dissociation vale for the blocker-channel complex.
In the following discussion we have attempted to examine the small amount of information available to determine if fluctuation and single-channel analyses provide a coherent picture of channel-blocker interactions in basolateral membranes.
B. Fluctuation versus Single-Channel Estimates of Blocker Kinetics Van Driessche and Hillyard ( 1985) analyzed fluctuations in basolateral K currents measured across nystatin-permeabilized skin from larval bullfrog, using quinidine as a blocker. Tadpole skin proved to be much easier to permeabilize with nystatin than did the skin of adult frog. In the presence of a transepithelial K gradient (mucosal to serosal), nystatin induced a transepithelial current that was blocked by quinidine. Concentrations of quinidine ranging from 25 to 300 pM produced Lorentzian components in the power density spectra (PDS) t with corner frequencies (,f;.) ranging from about 5 to 30 Hz. The linear dependence of ,f; on the concentration of quinidine allowed the kinetic parameters for a simple open-blocked reaction to be estimated as k,:" = 0.6 sec-I pM and k,,,, = 2 sec- where k,tn is the on rate coeficient for the first-order blocking reaction and k,,ffis the off-rate. It is of interest to compare these values with those obtained for block of single K channels by quinidine in turtle colon epithelial cells (Richards and Dawson, 1986, 1987b; N. W. Richards and D. C. Dawson, 3SION 13NNVH3 unpublished observations). In colonic cells, a 20-pSWnlSSVlOd K channel,lVtl3lVlOSVG which appears'9 EOZ
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DAVID C. DAWSON ET AL.
to be activated when cells are swollen, is characterized by slow quinidine block; i.e., quinidine induces long blocked periods (of the order of 10-I sec) in the current records, which are easily distinguished from the spontaneous gating. Analyses of the distribution of dwell times in the open and closed (or blocked) states in this case were consistent with a three-state, open channel block model in which a cationic form of quinidine blocked at the cytoplasmic face of the channel (Kichards and Dawson, 1987b). For example, the on-rate (k:,,[B]) increased with quinidine concentration, whereas the off-rate (k,,,,)was independent of quinidine concentration. At a membrane potential of 0 mV, the value of k,:,, was estimated to be 1 .S sec- p M -I and that for k,,,,was estimated to be 6.6 sec-I. Furthermore, both the on-rate and the off-rate were dependent upon membrane voltage in the manner expected for a cationic blocker that enters the channel from the cytoplasmic side: that is, thc on-rate increased with membrane depolarization (changing e-fold per 133 mV), while the off-rate decreased with depolarization (changing r-fold per 74 mV). The characteristics of the quinidinechannel interaction derived from analysis of single-channel records in turtle colon cells are remarkably similar to those estimated by applying fluctuation analysis to basolateral K currents in nystatin-permeabilized tadpole skin. Slow block of K channels by quinidine (reflected in the low off-rates) in these two rather different epithelial cells contrasts with the relatively fast block by quinidine (or its stereoisomer quinine) of large conductance, calcium-activated “maxi” K channels (GlavinoviC and Trifar6, 19XX). Slow quinidine block may therefore be an important “signature” for a particular class of epithelial K channel. Van Driessche (1986) employed nystatin to study basolateral K channels in toad urinary bladder and used transepithelial impedance analysis to estimate the changes in the apical and basolateral membrane resistance that occurred following exposure of the apical membrane to the antibiotic. The values obtained with Nyquist plots suggest the ratio of basolateral-to-apical membrane resistance was about 100: I . Lidocaine in concentrations ranging from 10 to 200 pM reversibly blocked transepithelial K currents and induced Lorentzian components in the PDS having corner frequencies from about 20 to about 90 Hz. Kinetic parameters estimated using a first-order model for the blocking reaction were k,,,, = 2.4 sec ’ pM I and k,,, = 100 scc-I. The off-rate for lidocainc in this case was about SO times faster than that determined for quinidine in tadpole skin, suggesting a block by lidocaine which is “faster” and more “flickery” than the slow quinidine block discussed above. Again, we can compare the characteristics of lidocaine block of toad bladder K channels with the kinetics derived from singlechannel records using turtle colon epithelial cells. Lidocaine block of the swelling-induced, 20-pS channel in these cells is “fast” relative to quinidine [i.e., the durations of blocking events are relatively brief, resulting in a flickery appearance of the current record (Richards and Dawson, 19X6)1. Lidocaine block is also voltage dependent in 21 manner similar to that of quinidine; that is, block
6. BASOLATERAL POTASSIUM CHANNEL NOISE
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was greatest at depolarized membrane potentials (Richards and Dawson, unpublished observations). Lidocaine-induced blocked intervals were clearly distinguishable in single-channel records at potentials of up to + 50 mV (cell positive), but could not be distinguished from normal closing events at -50 mV (cell interior negative). As for quinidine, the voltage dependence of block was consistent with a three-state model in which a positively charged form of lidocaine blocked the channel from the cytoplasmic side. From dwell time distributions k,,,, was estimated to be 104 sec-’ at V,,, = 0 mV (N. W. Richards and D. C. Dawson, unpublished observations), a value nearly identical to that obtained by analyzing lidocaine-induced fluctuations in toad bladder. A comparison of the on-rates is complicated somewhat by the fact that lidocaine is a weak base with a pK, of about 7.9, so that the relative amounts of the charged (protonated) form and the uncharged form will vary with pH. If we assume that it is the charged form that is active, then the measured on-rate can be corrected for the actual concentration of the charged form if the pH and the pK, are known (for a discussion, see Dawson ef ul., 1988). These calculations yield values for k,:” of 5.42 sec-] p M - ’ for toad bladder and I .84 sec-I p W i for the single-channel records from turtle colon cells. Comparable on and off rates for lidocaine were estimated from Huctuations in osmotically-induced basolateral K currents in turtle colon (Dawson et ul., 1988). The similarity between these values reinforces the notion that fast lidocaine block may also be a useful tag for this specific subpopulation of basolateral K channels, which, in some cells, may be activated by cell swelling (see also 111,B). Wills ( 1984) investigated basolateral membrane noise in the rabbit colon using nystatin to permeabilize the apical membranes, but variability in the results precluded a definitive interpretation. In this preparation a spontaneous Lorentzian was observed in only half of the tissues examined. This Lorentzian was abolished by the addition of serosal barium, suggesting that the signal originated in the K channel fluctuations. A barium-induced Lorentzian component was not evident in the spectra, however. Hanrahan et a / . (1986) used barium-induced fluctuations to study the basolateral K conductance in the locust rectum. Isolated cell layers were exposed to 1 mM CAMP in order to open apical K channels and “physiologically permeab i k e ” the apical membrane so that the basolateral K conductance was the primary determinant of transcellular K How. Transcellular K currents were induced by imposing a serosal-to-mucosal K gradient across cell layers. In the absence of barium, current fluctuations were dominated by 1 (f noise. Serosal barium at a concentration of 20 mM virtually abolished the transcellular current and concentrations from 0.5 to 10 mM induced Lorentzian components in the PDS that exhibited corner frequencies ranging from about 0.2 to 4 Hz. Analysis of the corner frequency plots by Hanrahan et al. (1986) suggested that the off-rate for barium was on the order of 0.7 sec-I, which corresponds to a dwell time in the blocked state of about 1.5 sec. This value is comparable to those observed in
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single-channel studies in which slow barium block has been reported (Vergara and Latorre, 1983). Similar corner frequencies were obtained by Van Driessche et al. (1985; W. Van Driessche, D. Chang, and D. C. Dawson, unpublished observations) for apiccrl K channels in flounder urinary bladder. For niucosal barium concentrations ranging from 0.5 to 5 mM. the corner frequencies ranged from 2 to 11 Hz. Interestingly, much faster barium block seems to characterize the apical K channel in frog skin (De Wolf and Van Driessche, 1986; Van Driessche and Zeiske, 1980). A quantitative comparison of the kinetics of barium blockade derived from noise analysis with those derived from single-channel recording is potentially compromised by the expected voltage dependence of barium block (Vergara and Latorre, 1983). The rate coefficients for the blocking reaction can vary enormously depending on the magnitude and orientation of the transmembrane potential, but this value is not always known in a permeabilized epithelial cell layer. Hanrahan ri ul. ( 1 986) noted an upward curvature in the relation between 2rh and [B] for basolateral K channel noise in locust rectum and presented evidence consistent with the notion that this behavior was due to a hyperpolarization of basolateral membrane potential with increasing serosal barium concentrations.
C. Channel Subpopulations: Who Is Doing What? Recent studies of epithelial cells suggest that there exists in the basolateral membrane a diversity of membrane channels that was not expected from earlier macroscopic experiments (Dawson, 1987). In the turtle colon, for example, it seems clear that the basolateral membrane is endowed with as many as four distinct types of K-conducting channels (Chang and Dawson, 1988; Dawson, 1987; Richards and Dawson, 1987a, 1989), and there is reason to bclieve that at least some of these have a specific functional significance. The experiments by Dawson ut ul. (1988) provide an example of the use of fluctuation analysis to provide an assay for the activation of specific channel subpopulutions. They also represent one of the few examples of specific cpithelial ion channels that have been studied using both single-channel recording and current noisc techniques. Germann et NI. (198ha,b) identified two major basolateral K conductances in aniphotericin-permeabilized turtle colon. One, tentatively dubbed the “resting” conductance of the cell, was the major conductance under normal or hyperosmotic conditions. This conductance was blocked by barium but was insensitive to quinidine or lidocaine. Swelling the epithelial cells, however, activated a second conductance that was not generally detectable under nonswclling conditions. This conductance was most easily identified by specific blockade with quinidinc or lidocaine. Richards and Dawson (1986) idcntified a single channel in isolated colonic cells that was specifically blocked by quinidine
6. BASOLATERAL POTASSIUM CHANNEL NOISE
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or lidocaine. This observation was important for several reasons. First, it was possible to associate the effect of blockers of a specific component of the membrane current with an effect on a particular channel. Second, the single-channel recording results were consistent with a direct action of the blocking molecule on the channel rather than an indirect effect of quinidine or lidocaine, on cell calcium, for example. Finally, the nature of the blocking by lidocaine (flickery block) suggested that this compound would be useful for the production of blocker-induced fluctuations with a corner frequency well separated from lowfrequency noise. Dawson et ul. (1988) measured fluctuations in basolateral K currents using portions of turtle colon that had been apically permeabilized by application of amphotericin B. They measured current noise under swelling and nonswelling conditions to determine if lidocaine-induced fluctuations could be used to monitor the appearance of the swelling-activated channels in the basolateral membrane. Under conditions of cell swelling a spontaneous Lorentzian component was not detectable in the power density spectrum, but the application of lidocaine (10-300 p M ) led to the appearance of a Lorentzian component. The corner frequencies increased linearly with increasing lidocaine concentration, as expected for a simple two-state, open-to-blocked reaction. The kinetic parameters were estimated as kc:“ = 1.3 sec-’ p M - ’ and k,,B = 247.2 sec-I, values that are comparable to those obtained from single-channel records obtained with turtle colon cells (see above). The on-rate coefficient, corrected for a pH of 8.3 assuming that only the charged form is a blocker, is 4.57 sec-’ p M - ’ . The single-channel conductance obtained from noise measurement, about 20 pS, was virtually identical to that obtained from single-channel recording (Richards and Dawson, 1986). The correspondence of the kinetic and conduction properties of the channels in these two experimental paradigms reinforces the conclusion that the measurements of lidocaine-induced noise reflect the properties of a specific subpopulation of basolateral K channels. The presence or absence of a lidocaine-induced Lorentzian component in the PDS also provided an assay for the activation of the lidocaine-sensitive channels by cell swelling. Under nonswelling conditions neither spontaneous nor lidocaine-induced Lorentzians were detected, but swelling the cells led to the appearance of the lidocaine-induced Lorentzian. Figure 6 shows a record of the macroscopic K current under nonswelling conditions (K gluconate-Ringer’s in mucosal bath). Successive addition of lidocaine (50 and 150 p M ) did not alter the current. Subsequently, the mucosal solution was switched to KCI-Ringer’s in the presence of lidocaine in order to swell the cells. Figure 7 shows the power density spectra recorded before and after cell swelling in the presence of lidocaine. Under nonswelling conditions lidocaine did not induce a Lorentzian component in the spectrum. Swelling the cells led to only a transient change in the macroscopic I , due to the presence of blocker, but now a lidocaine-induced Lorentzian was clearly discernible in the power density spectrum. This result is
DAVID C. DAWSON ET AL.
208
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TIME ( m i d FIG. 6. Basolateral K current measured using amphotericin-permcahilized turtle colon in the presence of mucosal K gluconate. Addition of 50 pLM (first L) and then I50 pM (second L) lidocaine did not alter I , appreciably. Mucosal bathing solution was changed to KCI-Ringerb (in the presence of lidocainc) to induce cell swelling. Small arrows indicatc times at which power density spectra were recordcd. [Reprinted from Dawson el a / . (1988)with permission of the American Physiological Society. I
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Frequency, Hz FIG. 7. Power density spectra recorded during experiment shown in Fig. 6 (arrows in Fig. 6) in the presence of 150 pM lidocaine under nonswelling conditions (mucosal K gluconate) and swelling conditions (mucosal KCI). Cell swelling was associated with the appearance in the power density spectrum of a Lorentzian component with a corner frequency of 49.2 H7.. [Reprinted from Dawson et u/. (1988) with permission of the American Physiological Society.]
6. BASOLATERAL POTASSIUM CHANNEL NOISE
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consistent with the notion that cell swelling was associated with the activation of a specific subpopulation of K channels in the basolateral membrane, a subpopulation that was not active under resting or isosmotic conditions.
IV. IS THE NOISE WORTH LISTENING TO? For an electrophysiologist there is a wonderful sort of irony about experiments in which the signal is the noise. Beyond this, however, it is pertinent to ask whether the technique of noise analysis has any role in future studies of the properties of the basolateral membrane. Although current fluctuations provide a unique way of “tuning in” to specific populations of channels that reside in the somewhat less accessible basolateral membrane, the technique also has limitations. The most significant of these is probably the need to adopt a specific model for channel kinetics in order to interpret corner frequencies in terms of channel gating. Here, the juxtaposition of single-channel data and fluctuations can provide important confirmation of the assumed channel kinetics. This sort of confirmation greatly increases one’s confidence in the values of single-channel current and channel number, which are ultimately derived from the blocker-induced noise. Noise analysis has the advantage of providing an assay for the behavior of a channel population in the functioning epithelium, but it is clear that the measurement of basolateral currents requires that the integrity of the cell be compromised to some extent in order to provide access to the “dark side” of the cell. This compromise between increased resolution and altered cell function, however, is similar to that which must be made in almost any experiment involving isolated cells, cell membranes, or membrane proteins. The use of specific blockers provides a partial resolution of this problem, because compounds that induce noise in specific channel populations can then be used to probe transport in intact tissues. Fluctuation analysis, particularly in conjunction with single-channel recording and intact tissue studies, can potentially provide important insights into the role of specific populations of basolateral membrane channels in salt transport, and may be an especially important tool for determining the functional role of specific populations of basolateral K channels. REFERENCES Ahnert-Hilger, G . , and Gralzl, M . (1988). Controlled manipulation of the cell interior by poreforming proteins. Trends Pharrnacol. Sci. 9, 195- 197. Andersen, 0. S . (1984). Gramicidin channels. Annu. Rev. Physiol. 46, 531-548. Cass, A , , and Dalmark, M . (1973). Equilibrium dialysis of ions in nystatin-treated red cells. Nature (London),New B i d . 244,47-49. Cass, A , , Finkelstein, A., and Krcspi, V. (1970). The ion permeability induced in thin lipid membranes by the polyene antibiotics nystatin and amphotericin B. J . Gen. Physiol. 56, 100- 124.
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Chang, D., and Dawson, D. C. ( 1988). 1)igitonin-permeabilized colonic cell layers: Ilcmonstration of calcium-activated hasolateral K ' and C 1 conductances. ~ J . Gen. P/ZV.S~J/. 92, 28 1-306. Chang, D., Hsich, P. S., and Dawson, D. C. (1988). Calcium: A program i n BASIC for calculating the composition of solutions with spccitied free concentrations of calcium, magnesium and other divalent cations. Cotttpu/. B i d . Med. 18, 35 1-366. Dawson, D. C. (1983). Baaolatcral chloride conductance in Rounder urinary bladder. Bull. M t . Dusrrt Island R i d . Lab. 23, 26-27. Dawson, D. C. (1987). Properties of epithelial K channels. Citrr. Top. Mumhr. Tronsp. 28, 41-71. Ilawson, D. C., and Frizzell, R. A. (1989). Mechanism of active K ' secretion by llounder urinary bladder. PJrrqrrs Arch. 414, 393-400. Ilawson, D.C.. Van Dricsachc. W., and Helnian. S . I. (1988). Osmotically induced basnlateral K + conductance i n turtle colon: Lidocaine-induccd K' channel noise. Am. J . Phvsiol. 254, C 165-C 174. Dcinarcst, I. R., and Finn, A. L. (1987). Characterization of the basolateral membrane conductance of Nec,/urit.s urinary bladder. J . Gen. Phvsiol. 89, 541 -562. I)e Wolf, I., and Van Driesachc, W. (1986). Voltage-dependent Ba" block of'K ' channel5 in apical membrane of frog akin. Am. J . Phvsiol. 251, C696-C706. Erlij, D., and Smith, M. W. (1973). Sodium uptake by frog skin and its modification by inhibitors of transcpithelial sodium transport. J . Phvsiul. (L(n7c/on)228, 22 1-239. Finkelstein, A,, and Andcrsen, 0. S . (IOXI). The gramicidin A channel: A rcview of its permeability characteristics with special reference t o the single-file aspect of transport. J . Mrmbr. B i d . 59, 155-171. Fiskum, C . (1985). lntracellular levels and distribution of Cali in digitonin-permeabilized cells. Cell Calcium 6, 25 - 37, Garty. H. (1984). Current-voltage relations of the hasolateral mcnlbranc in tight amphibian epithelia: Use of nystatin to depolarizc thc apical membrane. J . Memhr. B i d . 77, 213-222. Gcrmann. W. J . . lmwy, M. E . , Ernst, S. A , , and Dawson, D. C.(1986a). Differentiation of two distinct K conductances in thc basolateral membrane of turtle colon. J . Con. Phvsiol. 88, 237-251 Cermann, W. J . . Ernat, S.A., and Dawson, D. C. (1986b). Resting and osmotically induced basolateral K conductances in turtle colon. J . Gun. Phvsiol. 88, 253-274. Clavinovit, M. I., and 'Trifarci, I. M. ( 1988). Quinine blockade of currents through Ca'+-activdted K ' channel$ in bovine chroniaflin cclls. J . Plt.vsiol. (London)399, 139- 152. Halm, D. R . . and Dawson. D. C. (1983). Cation activation of the basolateral sodium-potassium pump in turtle colon. J . Gen. Phvsiol. 82, 315-329. Hanrahan. J . W., Wills. N. K., Phillips, J. E.. and Lewis. S. A. (19x6). Basolateral K channels in an inscct cpitheliuni. Channel density. conductance and block by barium. J . Gun. Physiol. 87, 443-466. Hulz. K..and Finkclstein. A . (1970). The water and nunclcetrulyte perineahility induced in thin lipid membrancs by the polyenc antibiotics nystatin and amphotericin B. J . Gen. Phvsiol. 56, 125- 145. Horn, R.. and Marty. A. (1988). Muscarinie activation (if ion currcnts incasttreed hy a new wholecell recording method. J . Gen. Phvsiol. 92, 145- 159. Kellcr. J . I d . . Ernst. S . A , , and Dawson. D. C. (1984). Anion currents across basolateral membranes of' urinary bladder of wintcr flounder (Psectdopleuron1,t.tc.sumericctnu3). Bull. Mt. Desert lsiatid B i d . Lab. 24, 94-95. Kinsky, S . C. (1970). Antibiotic interactions with model membranes. Annu. Rev. Pharmacol. 10, 119- 142. Kirk. K . I-., and Dawson, D. C. (1983). Basolatcrsl potassium channel in turtle colon: Evidence for single-lile ion Ilow. J . G m . Phvsiol. 82, 297-313.
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Kirk, K. L . , Halm, D. R., and Dawson. D. C. (1980). Active sodium transport by turtle colon via an electrogenic Na-K exchange pump. Nurure (London) 287, 237-239. Kleinberg, M. E., and Finkelstein, A. (1984). Single-length and double-length channels formed by nystatin in lipid bilayer membranes. J. Membr. B i d . 80, 257-269. Koefoed-Johnsen, V., and Ussing, H . H . (1958). The nature of the frog skin potential. Act0 Physiol. Scand. 42, 298-308. Lewis, S. A , , and Wills, N. K. (1982). Electrical properties of the rabbit urinary bladder assessed using gramicidin D. J. Membr. B i d . 67, 45-53. Lewis, S. A,, Eaton, D. C . , Clausen. C . , and Diamond, J. M. (1933). Nysvdtin as a probe for investigating the electrical properties of a tight epithelium. J . Gen. Phvsiol. 70, 427-440. Lewis, S. A., Wills. N. K . , and Eaton. D. C. (1978). Basolateral membrane potential of a tight epithelium: Ionic diffusion and clectrogenic pumps. J . Memhr. B i d . 41, 117- 148. Lichtenstein, N. S., and Leaf, A. (1965). Effect of amphotericin B on the permeability of the toad bladder. J . C h i . Inves/. 44, 1328- 1342. Marty, A , , and Finkelstein, A. (1975). Pores formed in lipid bilayer membranes by nystatin. Differences in its one-sided and two-sided action. J . Gen. Phvsiol. 65, 515-526. Miller, C. (1987). Trapping single ions inside single ion channels. f3iophy.s. J. 52, 123- 126. Miller, C . , Latorre, R., and Reisin, 1. (1987). Coupling of voltage-dependent gating and Ba+ block in the high-conductance of Cat +-activated K + channel. J. Gen. Physiol. 90,427-449. Neher. E . , and Steinbach, J. H . (1978). Local anaesthetics transiently block currents through single acetylcholine-receptor channels. J . Phvsiol. (London) 277, 153- 176. Nielsen. R . (1979). Coupled transepithelial sodium and potassium transport across isolated frog skin: Effect ofouabain, amiloride and the polyenc antibiotic filipin. J. Membr. B i d . 51, 161- 184. Richards. N. W., and Dawson. D. C. (1986). Single potassium channels blocked by lidocainc and quinidine in isolated turtle colon epithelial cells. Am. J . P!t.vsiol. 251, C85-C89. Richards, N. W., and Dawson, D. C. (1987a). Two types of Ca-activated channels in isolated turtle colon epithelial cells. Biophvs. J. 51, 344a. Richards, N. W., and Dawson, D. C. (1987b). Voltage-dependent quinidine block of single K ’ channels in turtle colon epithelial cells. Fed. Pro(,..Fed. Am. Soc. Exp. Biol. 46,496. Richards, N. W., and Dawson, D. C. (19x9). N-phenylanthranilic acid blocks specific classes of K-conducting channels in colonic epithelial cells. FASEB J . 3, A1 149. Richards, N. W., Lowy, R. J., Ernst, S . A,, and Dawson, D. C. (1989). Two K + channel types, muscarinic agonist-activated and inwardly rectifying, in a CI secretory epithelium: The avian salt gland. J . G m . Phj~siol.93, I171 1194. Russell, J. M . , Eaton, D. C . , and Brodwick, M. S . (1977). Effects of nyatatin on membrane conductance and internal ion activities in Aplvsiu neurons. J. Membr. B i d . 37, 137- 156. Schoen. H . F.. and Erli.1, D. (1985). Basolateral membrane responses to transport modifiers in the frog skin epithelium. P@egers Ardi. 405 (Suppl. I ) , S33LS38. Schultz, S. G . ( 19X I ) . Homocellular regulatory mechanisms in sodium-transporting epithelia: Avoidance of extinction by “flush-through.” Am. J . Phvsiol. 241, F579-FS90. Smith, P. L., and Frizzell, R . A. (1984). Chloride secretion by canine tracheal epithelium: IV. Basolateral membrane K permeability parallels secretion rate. J . Membr. B i d . 77, 187- 199. Van Driessche, W. ( 1986). Lidocainc blockage of basolateral potassium channels in the amphibian urinary bladder. J. Physiol. (London) 381, 575-593. Van Driessche, W.. and Erlij. D. (1988). Activation of K’ conductance in basolateral membrane of toad urinary bladder by oxytocin and CAMP. Am. J . Phvsiol. 254, CX16-C821. Van Driessche, W.. and Gegelein, H . (197X). Potassiuni channels in the apical membrane of the toad gallbladder. Norure (London) 275, 665-667. Van Driessche, W., and Gullentops. K. ( 1982). Conductance fluctuation analysis in epithelia. I n “Techniques in Cellular Physiology,” pp. 1- 13. ElsevierlNorth-Holland. Amsterdam. +
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Van Driessche, W., and Hillyard, S. D. (l98S). Quinidinc blockage of K ’ channels in hasolateral mcmhrane of larval hullfrog skin. Pjhregers Arch. 405 (Suppl. 1). S77-S82. Van Dricsschc, W., and Zciskc, W f 19x0). Ba’+-induced conductance fluctuations of spontaneously J . Mernhr. fluctuating K ’ channels in thc apical rnenihranc of frog skin (Xunci tern/~~ir~iricr). Biol. 56, 3 1-42, Van Driessche, W., Wills, N. K., Hillyard, S. D.. and Zeiske. W. (1982). K + channels in an epithelial “single membrane” preparation. Arch. Int. Pkvsiol. Biochirn. W, P12-PI4. Van Driessche, W., Chang, D . , and Ilawson, I). C:. (1985). Fluctuation analysis of apical K currents in the urinary bladder of the winter Hounder (Psercclo~leuronrcfescrmericunirs). Bull. Mt. Ucscrr lsland Biol. Lub. 25, 1-3. Venglarik, C. J . , and Dawson, D. C. (1986). Cholinergic regulation of Na ahsorption by turtle colon: Role of hasolateral K conductance. Am. J. Phvsiol. 251, C563LC570. Vergara, C., and Latorrc, K. (1983). Kinetics of fa’+-activated K’ channels from rabbit muscle incorporated into planar hilayers. Evidence for a Ca” and Ba” blockade. J . Cen. Physiol. 82, 543-568. Wehner, F., Carretson, I.., Dawson, K., Segal, Y . , and Kcuss, L. (1990). A nonensymatic prcparation of epithelial hasolateral memhrane for patch clamp. Am. J. Phvsiol. 258, C1159-CI 164. Welsh, M. J. (1987). Electrolyte transport by airway epithelia. P h y i o l . Rev. 67, 1143- I 184. Wilkinson, D. J . , and Dawson, D. C. (1989). Cholinergic modulation of apical Na channels in turtle colon: Current fluctuation analysis. FASEB J. 3 , A9X3. Wilkinson, 1). J . . and Dawson. D. C. ( 1990a). Cholinergic modulation of apical Na channels in turtle colon: Analysis of CDPC-induccd fluctuations. Rrn. J . Physiol. 259, in press. Wilkinson, D. J., and Dawson, D.C. (1990h). Apical K channcls in turtle colon: Current fluctuation analysis. FASEB J. 4, A447. Wills, N. K . (1981). Antibioti tools for studying the electrical propcrtics of tight cpithelia. Fed. Pro(.., Fed. Am. Sor.. Exp. R i d . 40, 2202-2205. Wills, N. K . (1984). Mechanisms of ion transport by the mammalian colon revealed hy frequency domain analysis techniques. Curr. Top. Mcmhr. Trump. 20, 61 XS. Wills, N. K . , Eaton, D . C., Lewis, S. A . , and Ifshin, M . S. (1979). Current-voltage relationship of the hasolateral mcmhranc of a tight epithelium. Biochim. B i o p h ~ .Actu ~ . 555, 5 19-523. -
Part III
Single-Channel Events in Epithelial Tissues
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CURRhNI TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 37
Chapter 7
Patch Clamp of Cation Channels SIMON A. LEWlS AND PAUL J. DONALDSON Department of Physiology utid Biophysics The Univrrsir?,($Texas Medicr~lBranch at Galveslon Galwslon. Texas 775.50
I. Introduction 11. Technical Aspects of Patch Clamping A . Patch Pipet Fahrication
B . Preparation of Epithelial Cells C. Patch Contigurations D. Idcntification and Analysis of Cation Channels 111. Scarch for Cation Channels in Epithelia A. Expected B . Unexpected I v. Use and Physiological Relevance of Single-Cation Channel Data V. Conclusions References
1. INTRODUCTION Since its introduction by Neher and colleagues (Neher et (11.. 1978; Hamill e l ul., 1981), the patch-clamp technique has been used to study ion channels at both the single-channel and the whole-cell level in animal, plant, and bacterial cells. The successful application of the technique to the study of ion channels in a particular tissue is critically dependent on the ability to form a high-resistance (10- 100 GSZ) seal between the patch pipet and the cell membrane of the tissue under study. Formation of a gigaseal effectively isolates the membrane patch both electrically and chemically. Electrical isolation of the patch allows the current flowing through a single channel to be resolved and the patch to be voltage clamped by simply applying a voltage to the pipet. Chemical isolation of a patch of membrane from a cell allows the normal ionic environment of the patch to be manipulated. The gigaseal is also mechanically very stable and enables the patch to 21 5 Copyright 0 1YW by Academic h e w Inc All rights or rcpruductam in any lomi rcaervcd.
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be either excised from the cell or ruptured, thus creating a number of different and useful recording configurations. Recently the patch-clamp technique has been used to study ion transport in epithelial tissues. The ability of an epithelial tissue to perform net ion transport is a consequence of the asymmetric distribution of ion channels and transporters in the apical and basolateral membranes of the epithelial cells. Application of patch-clamp techniques to the study of ion channels in epithelia is complicated by this assymmetric distribution of channel populations. It is the purpose of this chapter to discuss recent advances in the application of the patch-clamp technique to the study of cation channels in epithelia tissue. We define a cation channel as a proteinaccous pore that spans a lipid bilayer, and allows cations to How through this membrane-spanning protein as opposed to the lipid bilayer. Indeed, this would be an adequate definition if cations could not permeate anionselective channels (e.g., Hanrahan rt a!. , 1985). Thus we will rcstrict ourselves to channels that are more permeant to cations than anions, and for simplicity restrict ourselves to channels that are permeable to Na and/or K'. This chapter will consist of an initial review of the technical approaches used by investigators in the study of cation channels in epithelia. Next we outline the experimental approaches used to study cation channels in epithelia. Finally, the physiological relevance of the patch-clamp data obtained from three different epithelial tissues will be discussed. This chapter is not meant to act as a reference 5ource of all the new and wonderful epithelial cation channels that have been rcported since the last published extensive review. However, a summary (Table 11) of the epithelial cation channels studied to date has been included as a reference source for the interested reader. +
II. TECHNICAL ASPECTS OF PATCH CLAMPING EPITHELIAL CELLS To optimize the formation of a gigaseal, particular care has to be taken in the fabrication of patch pipets and in tissue preparation. Once a gigaseal has been formed the investigator has the choice of using several different patch configurations to record channel activity. It is the purpose of the following section to briefly consider the principles involved in patch pipet fabrication, epithelial tissue preparation, selection of an appropriate patch configuration, and analysis of the recorded channel activity. A. Patch Pipet Fabrication The instrumentation, techniques (fire polishing and coating), and geometries involved in constructing a patch-clamp electrode have been described in exten-
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sive detail by Hamill et al. (1981), Corey and Stevens (l983), and Sakmann and Neher (1983). Unfortunately, there are no hard and fast rules for producing a pipet that will form gigaseals on all cells, and as a consequence one is forced to proceed using a regimented, logical, and empirical approach for individual cell types. The first place to start is in the selection of the appropriate species of glass. Corey and Stevens (1983) suggest a series of rules to follow when determining the species of glass to use and in some cases the subspecies. The ideal pipet should have the following properties.
I . Low series resistunce. Series resistance is anatomically located from the tip of the pipet and extends toward the shaft. Although series resistance (1 - 10 MR) is negligible for excised or intact patch recording, in the whole-cell configuration (see below) membraneiseal resistance can be much lower and significant voltage drops can occur along the pipet, leading to an incorrect estimate of the voltage drop at the membrane. Thus rapidly tapering pipets (bullet shaped) made from soft glass will have a lower series resistance than more gently tapering pipets (wedge shaped) made from hard glass. 2 . Noise characteristics. As a rule of thumb, hard glasses such as borosilicate glass (e.g., Boralex, Kimax, and Corning 7040 and 7052) and aluminosilicate glass (e.g., Corning 1723) have low intrinsic noise characteristics, while soft glasses have higher intrinsic noise characteristics (e.g., soda glass or flint glass, available as blue-tip hematocrit tubing, Fisher and Kimble #R-6). An extensive survey of glass capillaries (over 20 types tried) by Rae (1985) uncovered a glass with a melting point less than soft glass and a noise level lower than the hard glasses. The authors describe Corning 8161 (potash lead glass) as the best general-purpose glass available for patch pipet fabrication (however, see below for possible problems). Noise levels of the pipet can be reduced by applying a layer of Sylgard # 184 to within 100 pm of the tip. 3 . Chemical inertness. Cota and Armstrong (1987) have shown that K currents recorded in the whole-cell mode from a primary cell culture of rat pituitary gland exhibit fast inactivation when either soda (VWR hematocrit tubing, blue brand) or Corning 8 161 (potash lead) glasses are used in the fabrication of patch pipets. No inactivation occurred when hard borosilicate glass (Kimax-5 I ) was used, or when high concentrations of 1,2-di(2-aminoethoxy)ethane-N,N,N',N'tetraacetic acid (EGTA) were used. The authors concluded that the current inactivation observed was due to a block of K + channels by di- or multivalent cations released from the soft glasses. 4. High yield of seals. A quick survey of the literature shows that borosilicate glass is used more frequently than soda glass, and in one case (Richards and Dawson, 1986) Corning 7052 produced seals in 80% of the attempts, whereas Kimax (Boralex) yields seals in less than 10% of the attempts on freshly isolated turtle colon enterocytes. 5. Pipette reusability. Dogma suggests that pipets, once used, cannot be re-
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SIMON A. LEWIS AND PAUL J. DONALDSON
used. The exception to this rule was reported by Rae and Levis (1984) in which multiple seals (up to seven times) were achieved using Corning 7052.
6. Preparation of Epithelial Cells
To obtain gigaseals, the membrane to be patched has to be relatively free from any substance that would restrict the pipet from making close contact with the membrane, thereby impeding the formation of a high-resistance seal (e.g., glycocalyx, basement membrane, or mucous). In a number of studies on epithelia (see Table I ) the enzymes hyaluronidase and collagenase have been used to clean the membrane surface and thus improve the success rate of acquiring highresistance seals. Caution should be exercised when using an enzyme treatment because the enzyme might modify channel kinetics or even channel conductance and selectivity. Although channels might be stable to enzymes such as collagenase and hyaluronidase, other enzymes (e.g., trypsin) have been shown to hydrolyze epithelial Na’ channels. If such hydrolysis only eliminated the channel, it would simply reduce the success rate of recording channel activity. However, it has been demonstrated that similar scrine proteases can alter channel selectivity, drug binding kinetics, and also spontaneous channel opening and closing kinetics (Lewis and Alles, 1986). Given this observation for some serine proteases, one should use enzymes with a degree of caution. If seals cannot be obtained without “cleaning” the membrane surface, the investigator should make every attempt to study the effect of the enzyme on the in vitro preparation to see if the enzyme of choice alters the basic epithelial transport properties. Without such assurances, one might want to interpret results with caution. In addition to cleaning the membrane surface, preparation of epithelial tissue for patch-clamp studies is governed by two other considerations. The first is the type of epithelium to be studied and the second is which membrane (apical or basolateral) is to be investigated. Three types of epithelial preparations have been studied: tubular preparations (for example, renal tubules, lacrimal and pancreatic acinar cells, and shark rectal gland), flat sheet epithelia (for example, lens, urinary bladder, small intestine, peritoneal epithelium, and colon), and a variety of cultured cells from renal tissues. Table I lists the protocols used for isolation and treatment of the cell surfaces prior to the patch clamping for all three types of tissues. We will briefly review the methods used to prepare the apical and then the basolateral membranes for patch-clamp studies in all three tissue types. For additional details concerning methodology, refer to the refcrences shown in Table 1. The morphological feature that allows epithelia to perform vectorial (directed) transport is two series membranes with widely differing transport properties. It has been demonstrated that, upon dissociation of an epithelium into individual
7. PATCH CLAMP OF CATION CHANNEL
219
cells, integral membrane proteins are redistributed over the entire membrane surface (Ziomek et d . , 1980; Dragsten et d . , 1981). Thus for studies of ion channels in the apical membrane, one must leave the epithelium intact and select cells away from the epithelial edges. For flat sheet epithelia and cultured epithelial cells this is a simple process, because there is easy access to the apical surface and, if necessary, the apical surface needs only to be clean prior to attempting to patch the membrane. However, for cultured epithelia, one must pick areas where the cells are confluent (have already formed tight junctions). Unfortunately, in renal tubules the apical surface is enclosed within the tubule lumen. Two approaches have been used to gain access. In one, the apical membrane is exposed by ripping the tubule lengthwise with a sharp needle. The tubule is then secured to a coverslip and transferred to the experimental setup (Hunter et ul., 1984). In the other approach, the tissue is perfused from one end and the apical membrane is approached from the opposing end (Gogelein and Greger, 1984). Interestingly, using either method, seals can be formed without the need for enzymatic cleaning of the exposed apical surface. Access to the basolateral membrane is more difficult because the basement membrane and basolateral membrane are intimately linked. Because the basement membrane acts as a mechanical support for the epithelial cells, removal of this structure normally leads to a dissociation of the cells. A novel approach has been used to circumvent the problem of' channel redistribution. Briefly, the lateral membrane at the open end of a single-ended perfused tubule can be directly patch clamped (Giigelein and Greger, 1984). Even under these conditions there is a possibility of channel mixing between the two membranes because of the disrupted tight junctional complex. Only time will tell whether other methods will become available and will allow one to measure only the channels in the basolateral membrane. The last point to be made in this section is that in some instances the bathing solution one uses in the patch-clamp chamber can be critical. Almost all studies to date have been performed at room temperature (which is appropriate for amphibian cells) with a NaCl bathing solution, usually buffered to pH 7.2-7.4 with 10 mM HEPES. For two studies (to date), one on the proximal tubule from Necturus (Lopes and Guggino, 1987) and the other in rabbit urinary bladder (Donaldson and Lewis, 1988), removal of bathing solution HCO,/CO, causes a 15- and 10-mV depolarization, respectively. In the rabbit urinary bladder this depolarization is a result of a decrease in basolateral membrane K + permeability (decrease in number of K t channels). In the epithelial kidney cell line A6, fluctuation analysis has shown that the number of Na+ channels in the apical membrane is dependent on the type of buffer used, with replacement of HCOy/CO, with either HEPES or TRlS producing a decrease in channel number and cellular current (Wills et uI., 1989). In patch-clamp studies, HEPES and related buffers
PROCEDURES N N 0
Species
Tubular epithelia Rabbit Rabbit Rat Rabbit Rat Mouse, rat, and pig Shark
Tissue
TABLE I USED TO PATCH CLAMP EPITHELIAL CELLS”
Isolation
Treatment
Membrane
Reference
Hunter et al. (1984) Koeppen et a/. (1984) Palmer and Frindt (1986) Gogelein and Creger (1984) Trautmann and Marty ( 1984)
CCT CCT CCT Prox i ma1
D
D D D
Slit open Slit open Slit open Open-end perfusion, collagenase
Lacrimal gland acinar cells Pancreatic acinar cells Rectal gland
D
Collagenase. trypsin, primary culture
Apical Apical Apical Apical. lateral BI?
D
Collagenase
BI?
Maruyama and Petersen
D
Open-end perfused
Apical
Creger et al. (1985)
D
Fresh-scraped cell clumps. collapenase, primary culture
BI
Hanrahan eta!. (1985)
Flat sheet epithelia Rabbit Urinary bladder
(1982)
D
Rat. frogs Toad Rat
Lens epithelium Colon Small intestine
Frog
Peri toneum
D 0 Ca' dissociation D
Human
Trachea
Autopsy
None Fresh-scraped hyaluronidase Hyaluronidase
Apical BI?
None
Apical
Collagenase, primary culture
Apical
Grygorczyk and Simon (1986) Welsh and Liedtke ( 1986)
Collagen-coated coverslips Coverslips
Apical Apical
Guggino et a / . (1985) Kolb et a / . (1986)
Coverslips, trypsin Coverslips
Apical Apical
Hamilton and Eaton (1985) Bolivar and Cereijido (1987)
BI
Rae (1985) Richards and Dawson (1986) Morris a / . (1986)
Cultured epithelia Rabbit Monkey Frog Dog
MTAL Renal JTC- 12. P.3 Renal A6 Distal renal MDCK
"CCT, Cortical collecting tubule; MTAL, medullary thick ascending limb: D. dissection procedure used to isolate and study the epithelium in vitro. Autopsy material was human trachea obtained within 18 hr of death from cystic fibrosis patients. dissection followed methods used for dog trachea; slit open, after single-tubule dissection. Tubule laid on coverslip. pinned using aluminum foil clips, and then opened up using a sharp needle. BI?. single isolated cells-because of possible channel mixing, cation channels observed might be of apical origin.
222
SIMON A. LEWIS AND PAUL J. DONALDSON
were found to reduce open channel conductance of outwardly rectifying anion channels in excised patches obtained from a human pancreatic duct epithelial cell line (Tabcharani and Hanrahan, 1989). The effect of buffers on cation channels in this tissue was not reported. Similar reductions might also occur if the epithelium or epithelial cells are not maintained at 37°C. Thus when studying epithelial ion channels (and in particular cation channels), it is best to incubate the cells in the same solutions and at the same temperature in which the in v i m epithelium was first studied.
C. Patch Configurations The different patch configurations that can be obtaincd are displayed diagrammatically in Fig. I . Formation of each configuration starts in the cell-attached mode with the formation of a gigaseal. From this position the combination of either rupturing the membrane patch with suction and/or withdrawal of the pipet will produce one of three well-defined configurations. For a detailed review of configuration formation, the reader is once again referred to the seminal paper by Hamill et d.(1981) and to the article by Sakniann and Neher (1984). In the cell-attached mode the measurement of current is limited to a small area of the cell membrane that is chemically and physically isolated from the bathing medium, so the addition of any substance to the bathing medium that produces changes in the activity of channels within the patch must be acting via intracelMar signals. Thus the cell-attached mode has been extensively used to study the mechanisms underlying hormone and transmitter activation of channel activity. This configuration bas a number of disadvantages. First, to accurately determine channel selectivity requires independent estimates of membrane potential and cell ion activities. Second, alteration of the pipet solution requires a sophisticated solution changing system. Last, the time course of pharmacological agents cannot be quantitated. After the formation of a cell-attached patch, the pipet may be drawn away from the cell surface to form an inside-out patch. The inside-out configuration is the one of choice for the construction of i-V relationships, because the voltage across the patch can be quickly and accurately clamped. In this configuration the cytoplasmic face of the membrane is exposed to the bath solution. This allows the composition of the solutions on either side of the membrane to be controlled. Typically, the solution in contact with the intracellular surface of the membrane is changed repeatedly (for methods, see Yellen, 1982; Fenwick et ul., 1982; Kakei and Ashcroft, 1987) in order to study its influence on membrane currents and channel selectivity. Considering the large number of channels that are regulated by diffusible intracellular components (Greengard, 1978), the possibility of alteration in channel activity by depletion of these components following formation of an excised
223
7. PATCH CLAMP OF CATION CHANNEL CELL AlTACHED MEMBRANE PERMEABILIZED
SLOW WHOLE CELL
FAST WHOLE CELL
INSIDE-OUT
FIG. 1 .
OUTSIDE-OUT
Schematic diagram of the different patch configurations available.
patch must be considered. A number of studies have addressed this problem by comparing ion channel properties in cell-attached and excised patches and indeed did find differences between the two (Trautmann and Siegelbaum, 1983; Trube and Hescheler, 1984; Cachelin et a!., 1983; Kunze ef al., 1985; Fernandez et al., 1984; Horn and Vanderberg, 1986), although one study has found no differences between the two modes (Pallotta et al., 1987). Obviously this is a potential problem with the technique, but it can also prove advantageous, as for example in the study of ATP-sensitive K' channels. These channels are found in cardiac and skeletal muscle and insulin-secreting pancreatic islet cells and exhibit channel run-down after patch excision (Findlay et al., 1985; Trube and Hescheler, 1983), making the advantages of using the insideout configuration to study these channels unavailable to these investigators. However, studies into the cause of channel run-down have determined that it is due in part to the loss of GTP, GDP, and other nucleotides, and that addition of GTP or GDP to the bath will act to restore channel activity (Dunne and Petersen, 1986). To obtain the whole-cell recording mode, the cell-attached patch is broken by either high voltage or suction. In the whole-cell configuration the cytoplasm of the cell is in direct contact with the pipet solution and the composition of the
224
SIMON A. LEWIS AND PAUL J. DONALDSON
pipet solution can be used to dialyze the intracellular environment of the cell, enabling intracellular contents to be changed as the pipet solution is changed. The ability to alter the intracellular environment has obvious advantages, but in some cases dialyzing the cell may result in the loss of essential cytoplasmic components. To prevent the loss of such components a new recording contiguration has been developed. In this configuration a gigaseal is first formed, but, instead of gaining access to the interior of the cell by disrupting the membrane patch by suction, the membrane patch is permeabilized by the addition of ATP4to the patch pipet (Lindau and Fernandez, 1986). The permeabilized patch provides electrical access to the cell and allows for the exchange of small ions, but prevents the rapid diffusion of large molecules from the cell. Electrically the only difference between this new approach and the standard whole-cell configuration is that the time constant of the capacitive transient is longer for the permeabilized patch. Hence, Lindau and Fernandez (1986) have called this new configuration the “slow whole-cell” configuration so as to distinguish it from the standard whole-cell or “fast whole-cell” configuration obtained when the membrane patch is disrupted by suction. A modification of this method was recently reported by Horn and Marty (1988) for the lacrimal gland epithelium. These authors reported that in the whole-cell recording mode. K + channel activity disappears 5 min after the patch is disrupted, suggesting the loss of an intracellular channel regulator. To eliminate the loss of this or other cytoplasmic Components, these authors permeabilized the membrane patch using the pore-forming antibiotic nystatin. This procedure eliminated the loss of channel run-down and allowed recordings of channel activity for an hour or more. Whole-cell recording can be done under voltage or current clamp conditions. In the current clamp mode, and in the absence of current injection, the potential recorded will be the cell membrane potential. This approach has been used to measure membrane potential in small cells such as red blood cells, for which conventional microelectrode techniques have failed to give reliable measurements of membrane potential (Hamill, 1983). Voltage clamping the membrane potential of a cell in the whole-cell configuration is simply a matter of applying a potential to the pipet electrode. This allows conventional voltage-clamp experiments to be carried out on cells not normally amenable to the approach using the more traditional microelectrode technique. An interesting application of patch-clamp recording in the whole-cell configuration is the ability to record discrete changes in membrane capacitance (Neher and Marty, 1982). These changes were identified as exocytotic and endocytotic events that add or delete membrane area, respectively. This method has been used on isolated exocrine pancreatic epithelium to study the regulation of zymogen granule release (Maruyama, 1989). Because regulation of cation channels might occur by the insertion of channels into the plasma membrane, the
7. PATCH CLAMP OF CATION CHANNEL
225
whole-cell recording method in conjunction with capacitance measurements might yield crucial information about such a mechanism. The outside-out mode is obtained from the whole-cell mode by withdrawing the pipet from the cell surface, causing a patch of membrane to reform across the pipet tip, but now with the extracellular membrane facing the bathing solution. The outside-out patch is the one of choice for examining ionic channels controlled by externally located receptors. The extracellular solution can be easily exchanged, allowing the effects of different agonists and permeating ions to be tested. However, results should be interrupted with care, because the formation of the outside-out patch may cause major structural rearrangement of the membrane and cytoskeletal elements and, as a consequence, alter channel properties (Trautmann and Siegelbaum, 1983).
D. Identification and Analysis of Cation Channels Once an adequate seal has been formed and channel activity has been observed and recorded, the next challenge is to identify the channel. However, before proceeding with the analysis, we must ask ourselves a very straightforward question. To what detail or depth do we want to analyze this channel? This question is dependent upon the quality and quantity of the collected data, the assurance that there is only one channel in the patch of membrane being studied, and the level of the computer software analysis programs either commercially available or currently under development. Whatever the quality of the data, some basic information concerning channel identity can be obtained by generating amplitude histograms from the channel data. Information concerning the kinetics of channel opening and closing is obtained from duration histograms but is more difficult to obtain, requiring highquality single-channel recordings that exhibit long periods of channel activity. 1. AMPLITUDE HISTOGRAMS
Channels isolated by the patch pipet are integral membrane proteins that catalyze the diffusion of ions across cell membranes. Conformational changes in these channels will be resolved electrically as pulses of current with a fixed amplitude, which are typically rectangular in shape and rise and return to a set baseline (Fig. 2A). This baseline value is a function of the seal resistance, voltage-clamp potential, and solution composition. By measuring the distribution of these single-channel amplitudes, amplitude histograms can be constructed. Amplitude histograms (Fig. 2B) generated at different clamp voltages and under varying ionic conditions are used to obtain initial estimates of single-channel conductance, channel selectivity, and the open time probability of the channel and its possible modulation by agonists and antagonists. The shape of a current-
A
Counts
0
Nernrt Potentiol I
FIG.2. (A) Single-channel current records from an excised patch of menihrane obtained from a primary culturc of rabbit bladder cells grown on plastic supports. The cells werc removed from the plastic substrate hy a 15-rnin exposure to 0.?S% trypsin and resuspended in a bathing media (NaCI, 1 1 I.2 mM; KCI, 5.8 mM; NaHCO,, 25 m M : KH,PO,, 1.2 n M ; MgSO,, I .2 mM: CaCI,, 2 niM: glucose, 1 I mM) that was kept at 37°C and huhhled with 95% 0,-5% CO? to maintain pH at 7.4. The pipet solution contained 150 mM KCI. 10 mM HEPES, and 80 p M ECTA. ( B ) Amplitude histogram of channcl activity recorded from the above channcl at a holding potential of 8 rnV. (C) The i-V rclationship of thc channel shown in A . Tho conductance of the channel in asymmetric solutions was I 1 3 pS at a holding potential of 48 mV. The i- V relationship is curvilinear and the last three points have becn extrapolated (dashed line) to give an estiniate of the reversal potential of 65.2 mV, which gives a P K I P N of23. a In addition, the i-V has been fitted with the constant field equation (solid line) to yield a reversal potential that is not significantly different from the Nernst potential for K ' , indicating that the channel is highly selcctive for K' and irnpcrrneant to Na ' .
7. PATCH CLAMP OF CATION CHANNEL
227
voltage (i-V) plot (Fig. 2C) contains information on the type of conduction process. A linear plot is obtained if the channel is acting as an ohmic conductor, and rectification is indicated by a curvilinear plot. To date, in epithelia (in symmetric solutions), an inwardly rectifying K + channel (Kolb et u l . , 1987; Hunter et ul., 1986), an outwardly rectifying K + channel (Kolb et al., 1987), and a nonrectifying K + channel (Lewis and Hanrahan, 1985) have been reported. The slope of the i-V relationship gives the conductance, which may depend on the voltage and the ionic conditions. When symmetrical solutions are used to bathe the membrane patch, the i-V curve passes through the origin. Imposition of an ionic gradient across the patch should simply displace the plot to either the left or the right, depending on the direction of the imposed ion gradient and channel selectivity. The point where the plot intersects the voltage axis is the reversal potential (i.e., the voltage at which there is no net flow of ions through the channel). Thus, if the ion activities on either side of the patch are known, the reversal potential can be used to determine channel selectivity. For a channel that is perfectly selective for one ion species, the reversal potential will equal the Nernst potential for that ion. Commonly, however, there is a deviation from the ideal Nernst potential, which reflects the channels' selectivity to other ions. For example, to determine the relative permeabilities of CI- and Na+ in a K' channel, i-V curves are constructed in the presence of a number of different ion gradients. The first is a pure KCI gradient. This yields a value for the K+-to-C1 selectivity of the channel. Next is to use a mixed solution of NaCI and KCI to determine the K +-to-Na+ selectivity. The observed reversal potentials are then inserted in the Goldmann equation and are used to determine the relative permeabilities. A potential problem with this approach, which can lead to an underestimation of channel selectivity, is illustrated in Fig. 2C. Here the i-V data obtained from an excised patch of membrane from rabbit urinary bladder cells grown in primary culture are displayed. Although the single-channel current did not reverse, an estimate of the reversal potential can be obtained by extrapolation (dashed line) of the last three data points. Such a procedure gives a reversal potential of some 65 mV and a permeability ratio (PK/PNL,) of 23. Alternatively the reversal potential can be calculated by fitting the data to the constant field equation (solid line). The best fit gives a reversal potential that is not significantly different from the Nernst potential for K +,indicating the channel is impermeant to Na'. This example illustrates the dangers of extrapolating nonlinear i-V curves to obtain an estimate of the reversal potential and channel selectivity. The area under an amplitude histogram can be used to determine the fraction of time that the channel spends in the open state, i.e., the probability that the channel is open ( P J . The amplitude histogram is ideally composed of two current peaks, one that represents the closed state of the channel and another that represents the open state of the channel. The amount of time the channel spends in the open state is equal to the total number of times the current is above a
228
SIMON A. LEWIS AND PAUL J. DONALDSON
specified value, multiplied by the rate at which the data were sampled by the computer (time per point). Similarly, the amount of time the channel spends in the closed state is equal to the total number of times the current is below a specified value, multiplied by the rate at which the data were sampled by the computer. The open probability is then the ratio of the amount of time spent in the open state divided by the sum of time in both the open and closed states. Measurement of P,, can be used to quantify the effect channel blockers have on channel activity. This approach has the added advantage that blockers can be used to quantify a channel's contribution to the macroscopic current, enabling comparisons between microscopic and macroscopic currents, which will give an insight into whether channel function is altered by the mechanics of membrane patch isolation. A number of epithelial cation channels have been studied by this approach (see Table I I). In addition to comparisons between macroscopic and microscopic currents, interesting information can be obtained by the comparison of P,, values obtained in different recording modes. Serotonin increases intracellular Ca?+ activity in Madin-Darby canine kidney (MDCK) cells. Friedrich et uI. (1988) used the excised inside-out patch configuration to establish the presence of an inwardly rectifying K+-selective channel in MDCK cells and characterized the channel's sensitivity to Ca' +.The authors then used the cell-attached configuration to establish that this channel could be activated by application of serotonin to the bath. The P,, values obtained using these two recording modes were then compared. It was found that after the application of the serotonin, the P,, values in cell-attached patches approach values for P,, obtained in excised patches exposed to lo-" M Ca?'. This type of experiment can then be used as a bioassay for cell Ca' ' activity.
2.
DURATION HISTOGRAMS
The current flowing through a single channel can be thought of as a series of rectangular current pulses having a relatively fixed amplitude but undergoing time-independent transitions between any number of different conformational states. The time a channel resides (dwell time) in any given conformational state is an exponentially distributed random variable. For construction of open-time histograms, the time taken for the opening and subsequent closing of a channel (termed an event) is first recorded and then placed in an appropriate bin, the width of which is a prespecitied time interval. The number of events that occupy a specific bin is then plotted against the time interval represented by the bin. The process is similar for construction of a closed histogram, but the event measured is the time between closing of and subsequent opening of a channel. Technically, the challenge is to be able to resolve as many of the actual channel transitions as possible, including the briefest openings and closings, and to perform this task as quickly and as accurately as possible.
TABLE 11 PROPERTIES OF EPITHELIAL CATION SELECTIVE CHANNELS" Animal
K
channels Necrurlrs
Tissue
Membrane
Regulation
y (pS)
Kinetics
Other
Reference
+
Proximal tubule
Basolateral
36
Basolateral Basolateral
47
Proximal tubule
50
Stretch activated, Po 1 , V depolarizes
Linear i-V
Sackin ( 1 987) Sackin and Palmer (1987)
-
Ba2+ block
P,, t . V depolarizes Ca" activated, PH 1' P" T V depolarizes Ca2+ activated, P, t , V depolarizes P,, T , V hyperpolarizes
-
-
2 open, 2 closed
-
Kawahara cf a/. (1987) Kawahara ef 01. ( 1987) Christensen and Zeuthen (1987)
Aggregates of subunits with cooperative gating
TEA block
Grygorczyk and Simon (1986)
Disappears upon excising patch Ca2+ activated, P, T , V depolarizes Po , V depolarizes, Ca'+ activated
-
Lidocaine, quinidine block B a + block, TEA block
Richards and Dawson (1986) Frindt and Palmer (1987)
Short open
Long open
P,,
-1 , V depolarizes
Proximal tubule
Apical
60
Choroid plexus
Apical
200
3
Triturus
Gallbladder
Apical
140
Frog
Peritoneum
Apical
22
Turtle
Colon
Basolateral
17
Rat
CCT
Apical
135
Lacrimal gland
?
Large
9
Maruyama cf a/ (1986)
Trautmann and Marty ( I 984) (continuFd)
TABLE II (continued) Animal
Tissue
Enterocytes
Membrane
y (pS)
Basolateral
250
Regulation
P,, f , V depolarizes. Ca? activated Ca?' activated. P,, t V depolarizes C a ? * activated. P,, f , V depolarizes
Kinetics
-
Other
Reference
Morris et ul ( 1986)
Ba2+ block
+
Pip
Pancreatic acinar cell
?
Rabbit
CCT
Apical
Proximal urinary bladder
Basolateral
30-40
-
Basolateral
220
Ba
Continuous culture Dog Renal MDCK
Monkey
200
42
Maruyama et ul. (1983)
.
+
inhibited
Apical
70
Epinephrine and Ca?' activated
Apical
79
MDCK
Apical
220
Renal JTE-12.P.3
Apical
220
Ca?' activated. P,, 1 V depolarizes Ca?' activated. P,, f V depolarizes Ca?' activated. P,, bell shaped, Ca?' sensitive
2 open. 2 closed
Complex (2 open, 2 closed)
Linear i-V. P,, f V depolarizes. Ca2+ insensitive, Ba?' block
Hunter et a1 ( 1986)
.
Gopelein and Greger (1986) Lewis and Hanrahan (1985)
Outward rectification, clustered 3-9 Inward rectification
Kolb et a / . (1987)
-
TEA blocked. quinidine block
Bolivar and Cereijido (1987)
2 closed, I open
-
Kolb el ul. (1986)
.
.
-
Kolb et a / . (1987)
Primary culture
T , V depolarizes, CaZ' activated, ADH activated P f , V depolarizes, Ca2' activated
Chick
Renal
Apical
107
Rabbit
CCT
Apical
I80
Na channels Rat CCT
Apical
6
Apical
12
Amiloride inhibits, P, as PH T Amiloride
Tissue culture Frog A6
Apical
84
Cation selective Necturus Choroid plexus
Apical
?
Ba2+ inhibited
Guggino et a / . (1985)
2 open, 2 closed
Ba" inhibited, Nai inhibited
Gitter et a / . ( 1987)
1 open, I closed
Ca2+ insensitive
Palmer and Frindt (1986, 1987)
-
Linear i-V voltage ins.
Gogelein and Greger ( 1986)
Amiloride
1 open. 1 closed
Nonlinear i-V. 4: I Na:K+ selectivity
Hamilton and Eaton (1985)
40
Stretch activated
1 open, 3 closed
Not Ca2+ activated, Na' = K + = Ca2+
Christensen (1987)
35
CCK activated, ACh activated
-
P,
+
Rabbit
R,
2
Mouse
Proximal
Pancreatic acinar
t
Maruyama and Petersen (1982)
'y , Single-channel conductance; CCT,cortical collecting tubule; MDCK, Madin-Darby canine kidney cell line; A6, continuous cell line from toad kidney; ?, single isolated cells-because of the possibility of channel mixing the actual membrane that was patched is not specified; Po,the open probability of the channel; ADH, antidiuretic hormone; ACh, acetylcholine; TEA, tetraethylammonium; CCK, cholecystokinin; T increase in Po; S. , decrease in Po;V depolarizes, membrane potential depolarizes.
.
232
SIMON A. LEWIS AND PAUL J. DONALDSON
Obviously, the use of computer-aided analysis is necessary and for a full discussion of the theory behind the process of kinetic analysis the reader is referred to work by Colquhoun and Sigworth ( 1983). The histograms are then fitted with an exponential distribution, the fitted distribution being usually tested to determine the goodness of the fit. This fitted exponential distribution can be used to formulate a model to account for the observed number of conformational states. The time spent in each state is described by a sum of exponentials, the number of exponentials indicating thc number of open or closed states. Thus, deriving kinetic parameters from multistate channels requires the investigator to choose a kinetic model. For the case of the channel that has one open state and two closed states, the number of possible kinetic models one can choose from is three (see Dionne, 1981). Thus the derived kinetic parameters are not unique, that is, they are model dependent. Table I1 summarizes the types of different kinetic schemes reported to date for epithelial cation channels. Kinetic analysis is complicated by a number of factors. For an accurate analysis of channel kinetics, long recordings containing many channel openings and closings are required because the standard deviation of an exponential distribution is equal to its mean. It is also necessary that the statistical probabilities governing the channel fluctuations, recorded over these long time periods, do not change. This does not always hold true and alterations in channel environment (such as channel run-down; see above) or drug desensitization can alter open and close times. An example uf cation channel run-down in epithelial cells was reported by Richards and Dawson (1986) i n the turtle urinary bladder. Formation of an inside-out patch demonstrated channel activity that rapidly became quiescent. Even if the above conditions are met, kinetic analysis can be greatly complicated if there is more than one channel in the patch. It is becoming increasingly evident that epithelial ion channels appear in clusters, i.e., more than one channel in the membrane patch. This occurrence of more than one channel in a patch is serious because it will yield overestimates for the open probability of a single channel and incorrect estimates for calculated kinetic parameters. The probability of finding r identical and independent channels open ( P , ) in a patch of membrane containing N channels is given by P,
N! =
r!(N - r ) !
Pi,( I - P
J - r
where P,, is the probability of a single channel opening (see Labarca et d., 1980; Colquhoun and Hawkes, 1983). When r = N , then P , = Y;,.This type of equation has been used on epithelial Na' channels to determine the number of channels in an obviously multiple-channel patch (Palmer and Frindt, 1986). An alternative approach to determine the P,, for suspect, multiple-channel
7. PATCH CLAMP OF CATION CHANNEL
233
patches (i.e., patches that demonstrate a low value for P J is to simply state that the reported open time probability for the channels in a patch is equal to N (number of channels in the patch) times P,, (open probability of a single channel) (see Sackin and Palmer, 1987). Such a relationship, although qualitative in terms of the absolute P,,, nevertheless yields important information if, for instance, the channel is voltage or agonist gated. Determination of kinetic parameters in obviously multichannel patches is, if not impossible, extremely difficult and requires not only picking what one hopes is an adequate model but assuming that the channels are identical and functionally independent. This can be further complicated by the existence of multibarrelled channels such as the K + channel recently described by Hunter and Giebisch (1987) in the apical membrane of the early distal tubule of Amphiuma kidney. Initial examination of channel activity is indicative of a multichannel patch recording; however, closer inspection reveals frequent closures to the zerocurrent level, indicating the presence of a common gating mechanism. Hunter and Giebisch were able to model the activity for this K + channel as four parallel subunits, each with its own gate controlled by a main gate that is capable of simultaneously closing all channel subunits.
111. SEARCH FOR CATION CHANNELS IN EPITHELIA In this section the principles previously discussed are applied to the study of ion channels in epithelial tissues. However, before proceeding, we would like to stress the need to have a good macroscopic “fingerprint” of the channel that one is looking for, before initiating patch-clamp studies. Indeed, there was a burst of patch-clamp activity, in many cases searching for a channel that might correspond to a known membrane conductance of known pharmacology. Many of these preliminary reports tended to be qualitative in nature, lacking extensive selectivity data, voltage dependence, simple kinetics, etc. Other reports demonstrated the existence of a conductance that could not be localized to a particular membrane or was not found in the intact epithelium. This fingerprint can consist of any one or all of the following points.
1. 2. 3. 4. 5.
Specific pharmacological blockers. Ion selectivity data. Current-voltage relationship. Kinetic data from fluctuation analysis. Channel density from which rough estimate of single-channel conductance can be estimated (ligand binding studies). 6. Single-channel currents (or conductance) from fluctuation analysis. 7. Known regulators of channel activity (e.g., Caz+,cyclic nucleotides, protein kinases, or phosphatases).
234
SIMON A. LEWIS AND PAUL J. DONALDSON
By comparing this macroscopic fingerprint to the single-channel fingerprint, one can feel confident that the channel measured is at least partially involved in determining the macroscopic conductive properties of that membrane. Indeed, a great number of investigations have found the expected channel or channels that correspond to the macroscopic conductance properties of the intact tissue. However, there are also many reports of channels, the existence of which were not predicted on the basis of macroscopic data. These expected and unexpected cation channels will be discussed in turn.
A. Expected The asymmetric distribution of cation channels in Na+-transporting epithelia predicts that the apical membrane will contain an amiloride-sensitive highly selective Na' channel and that the basolateral membrane will contain a K + selective channel. In K +-secreting epithelia such as the cortical collecting tubule (CCT), an apical K' channel would also be predicted.
I . SODIUM ION CHANNELS In line with these predictions, highly selective amiloride-sensitive Na+ channels have been found in the apical membrane of rat CCT, rabbit proximal tubule, and the A6 kidney cell line [see Garty and Benos (1988) for a review of amiloride-sensitive Na channel and Table II for specific epithelial Na' channel characteristics and referenccs]. 2 . POIASSIUM IONCHANNELS Apical membrane K + channels have been found in a variety of epithelial tissues (see Table 11). Although the single-channel conductance, voltage dependency, and pharmacology of these different channels varies among tissues, in the majority of instances an increase in the concentration of intracellular Ca'+ produces an increase in K channel p<,.This property of the apical K + channel is consistent with a modulation of transepithelial K + secretion that is regulated by agents that act to increase intracellular Ca?+concentration (Guggino et a / . , 1985; Kolb Ct al., 1987). Such a finding is, however, not necessarily applicable to all tissues, as was borne out in the rat CCT. The search for the apical membrane K' channel responsible for K secretion in the rat CCT is also an excellent example of how a comparison between the macroscopic fingerprint and the single-channel fingerprint can be used to identify the involvement of a particular channel in a conductive pathway. Frindt and Palmer (1987) patch clamped the apical membrane of the rat CCT, an epithelium that is known to secrete K + via a K + conductive +
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pathway in the apical membrane. The K + conductance of the apical membrane is increased when plasma aldosterone levels are increased using dietary manipulations. At the single-channel level one would predict that this increase is due either to an increase in channel density, single-channel conductance, or singlechannel kinetics. This apical membrane K + conductance is blocked by Ba2+but not by TEA, and is only slightly more selective for K + than Rb+ . Patch-clamp studies of the CCT apical membrane showed the existence of a Ca? +-activated voltage-dependent K channel. A comparison of the microscopic properties to macroscopic conductance revealed some striking differences in the conductance and the channel. First, the Ca”-activated K + channel is blocked by both Ba’+ and TEA, but TEA is without effect on the apical membrane K + conductance. Next, even though the apical membrane K + conductance is only slightly more conductive to K + than to Rb+, the Ca’+-activated K + channel is highly K + selective compared to R b + . Last, dietary manipulations did not alter channel kinetics, the probability of finding a Ca’+-activated K + channel in the patch, nor the density of channels in a patch. The conclusion that Frindt and Palmer (1987) reached is that the observed Ca’+-activated K + channel is not the major mechanism or pathway of K + secretion in the CCT, at least under their experimental conditions. In support of this conclusion, Frindt and Palmer (1989) have recently reported the existence of a second K + channel in the apical membrane of the CCT that does appear to correspond to the macroscopic fingerprint for apical K conductance. This K + channel has a lower conductance and a higher open probability than the Ca*+-activated K channel found in earlier studies. The low-conductance K + channel is blocked by Ba2+ and is also conductive to Rb+. The correlation between the microscopic conductance data and the macroscopic conductance data strongly suggests that the low-conductance K + channel is responsible for apical K + conductance and transepithelial K + secretion in the rat CCT. The identification of K + channels in the basolateral membrane of epithelial has proved more difficult due to the technical limitation of gaining access to this membrane. However, using the methodological approaches described in Section II,B, K + channels have been found in the basolateral membranes of Necturus proximal tubule, turtle colon, rat enterocytes, and rabbit proximal tubule and urinary bladder (see Table I1 for channel characteristics and references). +
+
B. Unexpected 1. SODIUM ION CHANNELS As seen above, the selectivity of the amiloride-sensitive apical membrane Na’ channel has been extensively studied using macroscopic and patch-clamp techniques and the high selectivity of apical Na+ channel for Na’ over K + has
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essentially been confirmed for the majority of tight epithelia (Palmer, 1987). However, in some tissues the patch-clamp technique has isolated Na' channels with unexpectedly low values for channel selectivity. In the A6 kidney cell line (Hamilton and Eaton, 1986) and the toad urinary bladder (Frings et al., 1988), a variety of amiloride-sensitive Na+ channels that exhibit different selectivities have been found. In the A 6 cell line, two distinct types of channels were found in the apical membrane. The first type of channel is characterized by a conductance of 7-10 pS, a low selectivity for Na+-K' of 3-4: I , and was seen predominantly in cells grown on plastic supports. The second type had a lower conductance (3-5 pS), slower spontaneous kinetics, a high selectivity for Na+-K' of >20: I , and was seen predominantly in cells grown on permeable supports. One possible explanation for the existence of these two types of channels is that the low-selectivity channel is the precursor of a highly selective mature channel. This appears to be the case in the larval bullfrog. Here fluctuation analysis has shown that during the molting cycle, the amiloride-sensitive Na' channel loses its selectivity in a way consistent with the age-related changes in channel selectivity (Hillyard et al., 1982).
2. POTASSIUM ION CHANNELS Patch-clamp studies o n both the apical and basolateral membranes of epithelial cells have uncovered, wherc appropriate, the expected type of K ' channel. Here the unexpected finding is the diverse nature of the channel characteristics not only between species and across epithelia, but within an epithelium. For example, Rae and his colleagues, having characterized the macroscopic conductance propcrties of the lens with conventional techniques, turned to the patch-clamp technique in the hope of localizing the macroscopic conductance properties of the lens to specific membranes and stereotypic channel populations. Instead, they found an ever-increasing number of diverse channel types with at least 11 different types of K + channels appearing responsible for the K + conductance of the lens. The physiological relevance of this collection of channels to the normal function of the lcns is at present unknown. A physiological function has, however, been assigned to the two types of K' channels located in the basolateral membrane of Nucturus proximal tubule (Sackin and Palmer, 1987). The two K + channels were classified by the authors as either long opcn-time or short open-time channels (for further differences, see Table 11). The most striking property of the short opcn-time channel was that in either cell-attached or cell-excised inside-out patches, application of a negative pressure to the pipct increased P,, of this K + channel (Sackin, 1987). In a more recent study, Sackin ( 1989) has shown that the short open-time channel in cellattacheu patchcs can be activated by decreasing the osmolarity of the bathing
7. PATCH CLAMP OF CATION CHANNEL
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solution and causing the cell to swell. This finding is consistent with the short open-time stretch-activated K + channel having a role in cell volume regulation in Necturus proximal tubule. It remains to be determined that the multiple types of K + channels found to coexist in the same membrane of other epithelial cells also have distinct physiological functions.
3. NONSELECTIVE CATIONCHANNELS Recent patch-clamp studies in a variety of epithelia have reported the existence of nonselective cation channels that were not initially predicted from macroscopic measurements. These channels do not discriminate between Na' and K + but are impermeable to CI- ions. In addition, a number of these channels are strongly dependent on intracellular Ca2+ concentration or membrane stretch, or can be activated by endogenously applied agonists (see Table 11). The assignment of a physiological function to these newly found channels has been hampered by the lack of specific channel blockers that can be used to quantify a channel's contribution to the macroscopic conductance. However, this may change following two recent reports that 3',5-dichlorodiphenylamine-2-carboxylicacid (DCDPC) inhibits nonselective cation channels in the basolateral membrane of rat pancreas (Gogelein and Pfannmuller, 1989) and that gadolinium inhibits nonselective stretch-activated cation channels in Xenopus oocytes (Yang and Sachs, 1989). Although use of such pharmacological agents should speed the study of nonselective cation channels, the physiological relevance of these channels in most cases is still in doubt. For example, three distinct types of nonselective cation channels have been found in the lens (Rae et ul., 1988). However, it could be that the ma.jority of these channels are normally inactive in the lens membrane. In particular, a nonselective stretch-activated cation channel is seen frequently in membrane patches but is known not to normally contribute to the resting membrane conductance, as witnessed by a comparison of its reversal potential to the membrane resting potential. It is possible then that this cation channel becomes activated by the mechanical stress produced by formation of a gigaseal.
IV. USE AND PHYSIOLOGICAL RELEVANCE OF SINGLE-CATION CHANNEL DATA In this last section we will illustrate how the patch-clamp technique has yielded unique information about the role and regulation of cation channels in epithelia. Three examples will be described. First, additional information can be gleaned from patch-clamp data of cation channels other than the existence of a particular channel in a given membrane. Second, the patch clamp has been used to study the role of cation channels in cell volume regulation. Last, the role of
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second messengers and cytoplasmic factors in epithelial cation channel regulation is of interest. In addition to identifying a particular channel's contribution to a known membrane conductance, other useful information that can be obtained from interpretation of patch-clamp data includes the following considerations. 1 . Determination of the channel density in the membrane. 2. For a basolateral membrane K + channel of known properties, one can estimate the number of Nai ,K+-ATPases required to counter the calculated K + flux through the channel. 3. A clustering of channels might suggest a cellular mechanism of channel turnover or regulation by insertion and withdrawal. 4. The appearance or disappearance of a channel in a patch might suggest the loss of a regulatory component. 5 . Microscopic selectivity of a channel, e.g., a finite Na permeability, might account for a paradoxical Nai conductance of the membrane. +
This is only a partial list, which will grow as more epitheliologists employ the patch clamp to study epithelial transport. This type of approach has been used in the study of basolateral membrane permeability in the rabbit urinary bladder. This tissue is a sodium-transporting tight epithelium that has been extensively studied using conventional techniques (Lewis, 1986); in particular, the macroscopic fingerprint of the basolateral membrane is well established. Briefly, the basolateral membrane contains the Na ' ,K -ATPase, which removes 3Na' ions from the cell in exchange for 2K ions (Lewis and Wills, 1983). In addition, the basolateral membrane is highly conductive to both K + and C1-- ( P J P , = 1.2) and shows a small but finite conductivity to Na' (PNi,IPK= 0.04). These conductivity properties coupled with a K activity gradient (14: 1) combine to produce a resting membrane potential of around - 5 2 mV (Lewis et ul., 1978). Application of Ba2+,a known K' channel blocker, to the basolateral bathing media produces a sustained depolarization of this potential but micromolar concentrations of the sodium channel blocker amiloride have no effect. The first step and the first challenge in patch clamping the basolateral membrane of any epithelium is gaining access to the basolateral surface. In flat epithelial tissues this can be achieved by simply scraping the cells from their luminal surface with a glass microscopc slide. However, in many epithelial tissues this approach is limited because cell polarity is lost. Fortunately, in the rabbit bladder the epithelium is transitional in nature, consisting of three distinct cell layers that can be distinguished from each other on the basis of size and shape. Here, scraping the cells and then briefly exposing them to collagenase leaves clumps of cells; a few intermediate cells are left attached to those from the surface layer. The intermediate cells act as a marker for the basolateral membrane of the surface +
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cells, and patches of basolateral membrane can be obtained by simply placing the pipet against a surface cell membrane that was next to an attached intermediate cell (Hanrahan et ul., 1985). The above considerations would point to K’ or C1- channel activity as being the most likely to be observed upon isolation of a patch of basolateral membrane. To optimize this possibility, the pipet should be filled with a KCl solution. In the data to be summarized, 150 mM KCI, 10 mM HEPES (pH 7.2), and 80 pM EGTA were used to fill the pipet. The initial extracellular solution was 140 mM NaCI, 6 mM KCl, 2 mM MgCI,, 2 mM CaCI,, and 20 mM HEPES (pH 7.2), giving a 20 mOsmol difference in osmolarity across the membrane to help promote seal formation. All experiments were carried out at room temperature. In this system, three types of channels were observed in the basolateral membrane. The first is a maxi K + channel, which has a single-channel conductance of 220 pS in symmetric 150 mM KCI solutions, a linear i-V relationship, and a voltage-dependent P,, (such that depolarizing the membrane increases P,,); it is insensitive to Ca’+ over the range 10-x-10-3M , is Ba2+blockable, appears in clusters of two to four, and in some instances was not active until the cellattached patch was excised (see Lewis and Hanrahan, 1985). The second type of channel was cation selective, had a conductance of 26 pS in symmetric 150 mM KCI solution, and had a linear i-V relationship (J. W. Hanrahan, personal communication). The last type of channel we mention not only for completeness but also because it has a finite cation permeability. The details of the channel have been published (Hanrahan ct al., 1985). in brief, it is an anion-selective channel (C1- = Br- = NO, = I - = SCN- > F- > acetate- > gluconate- > Na+ = K’; HCO, is permeable but has not been quantified), is an inward rectifier with a slope conductance of 64 pS at - 50 mV, inactivates at membrane voltages more negative than - 80 mV, has a P,, of 0.92 between membrane potentials of - 100 and - 20 mV and a P,, of 0.25 at + 40 mV, is insensitive to cytoplasmic Ca2+,and irreversibly blocked by 0. I mM 4,4’-diisothiocyanatostilbene-2,2’disulfonic acid (DlDS) in the outside solution. This channel is kinetically composed of one open and two closed states, and as in the case of the K + channel, in some instances was not active until the cell-attached patch was excised (Hanrahan et d . , 1985). The existence and quantification of these three types of conductances in the basolateral membrane were determined by Lewis et al. (1978) using microelectrode measurements and ion substitutions. Thus a Ba2+-blockable, Ca2+insensitive basolateral conductance can be accounted for by the K + channel outlined above. The large C1- permeability is consistent with the measured anion channel. Last, the finite Na+ permeability can be explained by both a cation channel as well as the finite permeability of the anion channel to Na’. From the calculated values of basolateral membrane K + and CI- permeabilities and the
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SIMON A. LEWIS AND PAUL J. DONALDSON
single-channel permeabilities (taking into account their rectification properties and open probabilities), Lewis and Hanrahan ( 1985) calculated that the density of both K + and CI- channels is about 1/15 p m 2 . However, because both K + and CI- channels do appear in clusters, the above-calculated density cannot be used to determine the probability of finding a channel in a patch of membrane. This observed clustering of channels suggests that channels might be inserted into membranes via lipid vesicles and also that the channels might be maintained in these clusters either by channel-channel interactions, cytoplasmic filament attachments, or perhaps lipid-specific domains. What is not known is whether clusters are evenly distributed over the entire basolateral surface or whether they are restricted to either the lateral or basal aspects. Given that the K' channel is (sometimes) active in cell-attached patches, one can calculate the number ofNa+,K+-ATPasesrequired to offset the net K + flux through the K channel. The details of this calculation are given by Lewis and Hanrahan ( 1985) and yield the startling number of 36,000 pumps per channel, i.e., 2200 pumps/pm2 of basolateral membrane. One of the interesting features of the K + (and also anion) channel was that in some instances channel activity was only observed after excising the patch. It is only in retrospect that this observation becomes interesting and perhaps reflects a regulatory role of K + and anion channel activity, In a recent series of experiments on intact bladder epithelium, Donaldson et ul. (1989) and Donaldson and Lewis ( 1988) determined that the basolateral membrane K + and C1- permeabilities were dependent upon cell volume. This volume can be manipulated by modification of the bathing solution anion composition. Thus removal of HCO, and CO, resulted in a decrease in the basolateral membrane K ' and CI- conductance (i.e., permeability). This decrease is perhaps mediated by a decrease in cell volume. Because the experiments by Hanrahan et al. (1985) and by Lewis and Hanrahan ( I 985) were performed in HCO, /CO,-free solutions, an experimental condition that leads to a decrease in membrane permeability (i.e., number of active channels), the appearance of channel activity upon excising the patch suggests that the external bathing solution lacks a regulatory component present in the cell cytoplasm. In keeping with our own dogma, experiments are now being performed in HCO, /CO, buffered solutions. Finally, we can use patch-clamp data to test assumptions made in determining macroscopic membrane ionic permeability. The initial calculation of basolateral membrane selectivity by Lewis et al. (1978) assumed that this membrane was only permeable to Na +,K t , and CI -. The patch-clamp data have shown that this is not correct, because the anion channel has a finite permeability to HCO, . From the measured cell pH of 7.1 (Eaton rr u l . , 1984) and the extracellular pH of 7.4, one calculates that cell HCO, is 12.8 mM and extracellular HCO, is 25 mM. This small HCO; gradient will tend to depolarize the basolaterdl membrane, suggesting that the actual Na+ permeability will be less than originally
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reported. Whether the HCO; or the anion channel plays a significant role in cell pH regulation has yet to be determined. It is in light of these results obtained using the patch-clamp technique that the above questions can be studied in detail using the intact epithelium. Our second example involves patch-clamp studies on the choroid plexus, a secretory epithelium responsible for the production and maintenance of the cerebral spinal fluid. Patch-clamp studies on this tissue have revealed a nonselective cation channel (Christensen, 1987) and Ca2+-sensitive, voltage-gated K + channel (Christensen and Zeuthen, 1987). Christensen (1987) has found that the cation channel is activated by membrane stretch and is permeable to calcium. In a number of cell-attached patches both channels were occasionally present. In such patches activation of the stretch-activated Ca2+channel by application of a negative pipet pressure also produced an increase in K + channel activity, which was dependent on the presence of Ca2+ in the pipet. Thus it appears that external Ca2+can enter the cell through the stretch-activated channel and activate neighboring K + channels. K i channel activity in cell-attached patches could also be increased by lowering the osmolarity of the external media and swelling the cell, but only in the presence of Ca?+. Therefore, it appears that cell swelling increases membrane tension, which increases the P,of the stretch-activated channel. This will lead to an increase in cell Ca2+concentration, which in turn will activate the Ca'+-sensitive, voltage-gated K channel in the same membrane. Activation of the K + channel will then allow the cells to lose K + and C1- (the latter through a C 1 conductance) ~ and water, the end result being cell shrinkage. Thus the stretch-activated channel appears to be the transducing element for volume regulation in the choroid plexus via a Ca'+-sensitive K + channel. This last example illustrates how the patch-clamp technique has been useful in elucidating the physiological and cellular mechanism(s) responsible for the regulation of cation channels from the inner medullary collecting duct (IMCD) of the kidney. The epithelial cells from this segment of the kidney are known to actively absorb Na+ in a manner similar to that described for the rabbit urinary bladder, i .e., conductive Na+ entry across the apical membrane and electrogenic extrusion via the Na+,K+-ATPase across the basolateral membrane. Atrial natriuretic peptide (ANP), a peptide released from cardiac atrial cells, elicits a diuresis of renal origin, with the IMCD being a primary site of action. Zeidel et al. (1987), using oxygen consumption studies, concluded that ANP inhibited Na+ absorption in this segment by a cGMP-mediated inhibition of Na+ entry. The mechanism of ANP and cCMP modulation of the Na+ entry step was studied by Light et al. (1989b) using the patch-clamp technique in the cell-attached and excised inside-out configurations. These authors found that primary cultures of lMCD cells contain in their apical membrane a cation-selective (Na+ = K + ) and amiloride-inhibited ion channel of 28 pS conductance. Of interest is that, as in the case of other epithelial channels, this cation channel appears in clusters, with +
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three to four channels being present in a membrane patch. Using cell-attached patches, the addition of 10 II M ANP to the bathing medium reduced the open probability of the cation channel cluster from 0.64 to 0.44. Similar decreases in open channel probability were observed in cell-attached patches when exogenous cGMP was added to the bath. Conversely, decreasing cell cGMP levels resulted in an increase in cation channel activity. This inhibition of channel activity by cGMP was also present in excised inside-out patches, suggesting that the effect of cGMP on the channel activity is direct. However, the authors add a warning that other cGMP-dependent mechanisms might also be involved in channel regulation because a submaximal concentration of cGMP is less effective for inhibiting the cation channels in excised patches compared to cell-attached patches. More recent work on this channel (Light et d.,1989a) has demonstrated the involvement of G proteins (in this case the activated aI subunit of the pertussis toxin-sensitive G protein) in the up-regulation of this cation channel. It seems then that this channel has a number of regulators. At this point we must comment that although an amiloride-sensitive conductance has been demonstrated in the isolated perfused IMCD, the selectivity of this channel for Na’ to K + has not been reported. It will be interesting to determine whether this tubule segment does indeed possess a nonselective cation channel that is blocked by amiloride or whether, as in the case of the A6 cells, the patch-clamp technique “selects” for poorly selective amiloride-sensitive channels (see above). Independent of the outcome of the channel selectivity, the studies on the IMCD are a good demonstration of the power of the patch-clamp technique in studying the mechanism(s) of epithelial channel regulation by second messengers.
V. CONCLUSIONS We have attempted to outline the most important aspects of patch-clamp technology and how this technology can be used to study epithelial cation channels. For a more detailed discussion, the interested researcher should consult the definitive publication by Sakmann and Neher (1983). ACKNOWLEDGMENTS One of the authors (S. A. Lewis) thanks Dr. J. W. Hdnrahan for past collaborations. This work was supported by NIH Grant DK 33243. We would also like to thank Dr. J. S. Stodddrd for reading this manuscript REFERENCES Bolivar, J. J . , and Cereijido, M. (19x7). Voltage and Ca’+-activated K’ channcl in cultured cpithelial cells (MDCK). J . Mernhr. R i d . 97, 43-51. Cachelin, A. B . , DePeyer, J. E . , Kokubun, S . , and Rcuter, H. (19x3). Sodium channels in cultured cardiac cells. J Physiol. (London) 340, 389-401.
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Christensen, 0. (1987). Mediation of cell volume regulation by Ca’+ influx through stretch-activated channels. Nurure (London) 330, 66-68. Christensen, O., and Zeuthen, T. (1987). Maxi K t channels in leaky epithelia are regulated by intracellular Caz+,pH and membrane potential. Pfluegrrs Arch. 408, 249-259. Colquhoun, D., and Hawkes, A. G. (1983). The principals of the stochastic interpretation of ionchannel mechanisms. In “Single-Channel Recording” (B. Sakmann and E. Neher, eds.), pp. 135-176. Plenum, New York. Colquhoun, D., and Sigworth, F. J . (1983). Fitting and statistical analysis of single channel records. I n “Single-Channel Recording” (B.Sakmann and E. Neher, eds.), pp. 191-263. Plenum, New York. Corey, D. P., and Stevens, C. F. (1983). Science and technology of patch recording electrodes. In “Single-Channel Recording” (B. Sakmann and E. Neher, eds.), pp. 53-68. Plenum, New York. Cota, G., and Armstrong, C. M. (1987). Potassium channel “inactivation” induced by soft glass patch pipettes. Biophys. J . 53, 107-109. Dionne, V. E. (1981). The kinetics of slow muscle acetylcholine-operated channels in the garter snake. J. Physiol. (London) 310, 159- 190. Donaldson, P. J., and Lewis, S. A. (1988). The effect of serosal hypertonic challenge on basolateral membrane potential in the rabbit urinary bladder. FASEB J. 2, A 1284. Donaldson, P. J., Chen, L. K., and Lewis, S. A. (1989). Effects of serosal anion composition on the permeability properties of rabbit urinary bladder. Am. J . Physiol. 256, F1125-FI 134. Dragsten, P. R . , Blumenthal, K., and Handler, J. S. (1981). Membrane asymmetry in epithelia: Is the tight junction a barrier to diffusion in the plasma membrane? Nature (London) 294, 718-722. Dunne, M. J., and Petersen, 0. H. (1986). GTP and GDP activation of K t channels that can be inhibited by ATP. Pfluegers Arch. 407, 564-565. Eaton, D. C., Hamilton, K . L., and Johnson, K. E. (1984). Intracellular acidosis blocks the basolateral Na-K pump in rabbit urinary bladder. Am. J. Phvsiol. 247, F946-F954. Fenwick, E. M., Marty, A , , and Neher, E. (1982). A patch-clamp study of bovine chromaffin cells and their sensitivity to acetylcholine. J . Physiol. (London) 331, 577-597. Fernandez, J. M., Fox, A. P., and Krasne, S. (1984). Membrane patches and whole-cell membranes: A comparison of electrical properties in rat clonal pituitary (GH,) cells. J. Physiol. (London) 356,565-585. Findlay, I . , Dunne, M. J., and Petersen, 0. H. (1985). ATP-sensitive inward rectifier and voltageand calcium-activated K + channels in cultured pancreatic islet cells. J. Membr. B i d . 88, 165-172. Friedrich, F., Paulmichl, M., Kolb, H. A,, and Lang, F. (1988). Inward rectifier K channels in renal epithelial cells (MDCK) activated by serotonin. J. Membr. B i d . 106, 149- 155. Frindt, G., and Palmer, L. G. (1987). Ca-activated K channels in apical membrane of mammalian CCT, and their role in K secretion. Am. 1. Physiol. 252, F458-F467. Frindt, F., and Palmer, L. G. (1989). Low-conductance K channels in apical membrane of rat cortical collecting tubule. Am. J. Physiol. 256, F143-FI51. Frings, S., Purves, R. D., and Macknight, A. D. C. (1988). Single-channel recordings from the apical membrane of the toad urinary bladder epithelial cell. J. Membr. B i d . 106, 157- 172. Garty, H., and Benos, D. I. (1988). Characteristics and regulatory mechanisms of the amilorideblockable Na+ channel. Physiol. Rev. 68,309-373. Gitter, A. H., Beyenbach, K. W., Christine, C. W., Gross, P., Minuth, W. W., and Fromter, E. (1987). High-conductance K’ channel in apical membranes of principal cells cultured from rabbit renal cortical collecting duct anlagen. Pflueger.7 Arch. 408, 282-290. Gogelein, H., and Greger, R . (1984). Single channel recordings from basolateral and apical membranes of renal proximal tubules. Pffuegers Arch. 401, 424-426.
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Gogelcin, H., and Greger. R. (1986). Na+ selective channels in the apical memhrane of rabbit late proximal tubules (pars recta). Pjuugers Arch. 406, 198-203. Gogclein, H., and Pfannmuller, R. (1989). The nonselective cation channel in the hasolatcrdl meiiiacid branc of the rat exocrine pancreas. Inhihition by 3’,5-dichlorodiphcnylamine-2-carboxylic (DCDPC) and activation by stilhene disulfonates. PflueKers Arch. 413, 287-298. Greengard. P. ( 1978). Phosphorylated proteins as physiological effectors. Science 199, 146- 152. Greger, R . , Schlatter, E., and GBgelein, H. (1985). CI--channels in the apical cell membrane of the rectal gland “induced” by CAMP. Pfluegers Arch. 403, 446-448. Grygorczyk, R . , and Simon, M. (1986). Single K ’ channels in the apical membrane of amphibian pcritoneurn. Biochim. Biuphys. Actu 861, 385-388. Guggino, S. E., Suarez-lsla, B. A,, Guggino, W. B., and Sacktor, R. (1985). Forskolin and antidiuretic hormone stimulate a Ca” -activated K ’ channel in cultured kidney cells. Am. J . Physiol. 249, F448-F455. Hamill. 0. P. (1983). Potassium and chloride channels in red blood cells. I n “Single-Channel Recording” (B. Sakmann and E. Neher. eds.). pp. 451-471. Plenum, New York. Hamill, 0 . P., Marty, A , . Neher, E., Sakmann, B., and Sigworth, F. J. (1981). Improved patch c h i p techniques for high resolution current recording from cclls and cell-frcc membrane patches. Pfluegers Arch. 391,85- 100. Hamilton. K. I>.*and Eaton, D. C. ( 1985). Single-channel recordings f r o ~ ramiloride-scnsitive ~ cpithelial sodium channel. Am. J . Phvsiol. 249, C200-C207. Hamilton, K . L., and Eaton. D. C. (1986). Regulation of single sodium channels in renal tubules. A rolc of sodium honicostasis. Fed. Proc.. Pod. Am. Sor. Exp. B i d . 45, 2713-2717. Hanrahan, J. W., Alles, W. P.. and Ixwis, S . A. (1985). Single anion-selectivc channcls in hasolatera1 nieinbranc o l a manimalian tight epithelium. Proc. N u t / . Arcid. Sci. U.S.A. 82, 7791 -7795. Hillyard, S . D.. Zeiske, W., and Van Driesschc, W. (1982). Poorly selective cation channels in the skin of the larval frog (stage S XIX). y/litegers Arch. 394, 2x7-293. Horn, R., and Marty, A . (1988). Muscarinic activation of ionic currents measured by a new whole cell recording method. J . Gm. Phvsiol. 92, 145- 159. Horn, R.. and Vandcrberg, C. (1986). lnaclivation of single sodium channels. I n “Ion Channels in Neural Membranes” (J. M. Ritchie, R . D. Keynes, and L. Rolis, eds.), pp. 71-83. Liss, New York. Hunter. M., and Giehisch. G. ( 1987). Multi-barrelled K channels in renal tubules. Nurirrr (Lortdow) 327, 522-524. Hunter, M., Lopes. A. G.. Boulpacp. E. I , . , andGicbisch. ti. 14. (1984). Single channel recordings of calcium-activated potassium channels in the apical membrane of rabbit cortical collecting tubules. Proc. Nurl. Accid. Sci. U S A 81, 4237-4239. Hunter, M., Lopes, A. G . , Boulpaep, E. L., and Giebisch, G. (1986). Regulation of single potassium ion channels from apical niembranc of rabbit collecting tubule. Am. J . Phvsiol. 251, F72S- F733. Kakei, M . . and Ashcroft, F. M. (1987). A microtlow superfusion system for use with excised nicmbrdne patches. ~flrrcyersArch. 409, 337-341. Kawahara. K.. Hunter, M , and Giebisch, G . (1987). Potassium channels in N~cturusproximal tubule. Am. J . Physiol. 253, 1;48X-F494. Kocppen, H. M.. Heycnbach, K. W., and Helman, S . I . (1984). Single channel rccording in renal tuhulcs. Am. J. P h y i o l . 247, F380-F384. Kolh. H. A . . Brown, C. D. A , , and Murer, H. (1986). Characterization of a Ca-depcndent maxi K channcl in thc apical membrane of a cultured renal epithelium (JTC-IZ.P3). 1.Memhr. B i d . 92,207 21.5. Kolh. H. A,, Pdulmichl, M., and Lang, F. (19x7). Epinephrinc activates outward rectifying K channel in Madin-Darby canine kidney cells. pfluugcvs Arch. 408, 584-591. Kunze, D. L., Ldcerda, A. E., Wilson, D. I,.. and Brown, A. M. (lY85). Cardiac Na currents
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and the inactivating, reopening and waiting properties of single cardiac Na channels. J . Gen. Phvsiol. 86, 691-719. Labarca, P., Coronado, R., and Miller, C. (1980). Thermodynamics and kinetic studies of the gating behavior of a K +-selectivechannel from the sarcoplasmic reticulum membranes. J . Gen. Physiol. 76, 397-424. Lewis, S . A. (1986). The mammalian urinary bladder: It's more than accommodating. News Phvsiol. Sci. I , 61-65. Lewis, S. A., and Alles, W. P. (1986). Urinary kallikrein: A physiological regulator of epithelial Na+ absorption. Proc. Natl. Acad. Sci. U.S.A. 83, 5345-5348. Lewis, S. A., and Hanrahan, 1. W. (1985). Apical and basolateral membrane ionic channels in rabbit urinary bladder epithelium. ffluegers Arch. 405, 583-588. Lewis, S . A,, and Wills, N. K. (1983). Apical membrane permeability and kinetic properties of the sodium pump in rabbit urinary bladder. J . Phv.siol. (London)341, 169- 184. Lewis, S. A , , Wills, N. K., and Eaton. D. C. (1978). Basolateral membrane potential of a tight epithelium: Ionic diffusion and electrogenic pumps. J . Memhr. B i d . 41, 117- 148. Light, D. B . , Ausiello, D. A , , and Stanton, B . A. (1989a). G, protein gates a cation channel in renal inncr medullary collecting duct cells. J . Gen. Phvsiol. 94, 3 la. Light, D. B., Schwiebert, E. M., Karlson, K. H., and Stanton, B. A. (1989b). Atrial natriuretic peptide inhibits a cation channel in renal inner medullary collecting duct cells. Science 243, 383- 385. Lindau, M . , and Fernandez, J. M. (1986). IgE-mediated degranulation of mast cells does not require opening of ion channels. Nuture (London) 319, 1.50- 15.7. Lopes, A. G., and Guggino, W. B . (1987). Volume regulation in the early proximal tubule of the Necrurus kidney. J . Membr. B i d . 97, 117-125. Maruyama, Y. (1989). Control of exocytosis in single cells. News Physiol. Sci. 4, 53-56. Maruyama. Y . , and Petersen, 0. H. (1982). Single-channel currents in isolated patches of plasma membrane from basal surface of pancreatic acini. Nuture (London) 299, 159- 161. Maruyama, Y . . Petersen, 0. H., Flanagan, P., and Pearson, G. T. (1983). Quantification of Ca'+activated K channels under hormonal control in pig pancreas acinar cells. Nurure (London) 305, 228-232. Maruyama. Y . . Matsunaga, H., and Hoshi, T. (1986). CaL+-and voltage activated K + channel in apical cell membrane of gallbladder epithelium from Trirurus. Pfluegers Arch. 406, 563-567. Morris, A. P., Gallacher, D. V., and Lee, J. A. C. (1986). A large conductance, voltage- and calcium-activated K ' channel in the basolateral membrane of rat enterocytes. FEES Lett. 206, 87-92. Neher, E.. and Marty, A. (1982). Discrctc changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc. Nurl. Acud. Sci. U.S.A. 79, 67 12-67 16. Neher, E., %dkmdnII, B . , and Steinbdch, J . H. (1978). The extracellular patch clamp: A method for resolving currents through individual open channels in biological membranes. ffluegers Arch. 375, 219-226. Pallotta, B . S . . Hepler, K. R.,Oglesby, S . A , , and Harden, T. K . (1987). A comparison of calciumactivated potassium channel currents in cell-attached and excised patches. J . Gen. Phvsiol. 89, 985 997 Palmer, 1.. G . (1987). Ion selectivity of epithelial Na+ channels. J . Memhr. B i d . 96, 97-106. Palmer. L. G., and Frindt. G . (1986). Amiloride-sensitive Na channels from the apical membrane of the rat cortical collecting tubule. Proc. Nut/. Arad. Sci. U.S.A. 83, 2767-2770. Palmer, L. G., and Frindt. G . (1987). Effects of cell Ca and pH on Na channels from rat cortical collecting tubule. Am. J . Phvsiol. 253, F333-F339. Rae, J. L. (1985). The application of patch clamp methods to ocular epithelia. Curr. Eve Res. 4, 409-420. +
-
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Rae, J. L., and Lcvis, R. A. (1984). Patch clamp recordings from the epithelium of the lens obtained using glasses selected for low noise and improved sealing properties. Biophvs. J. 45, 144- 146. Rae, J. L., Levis, R . A., and Eisenbcrg, R . S. (1988). Ionic channels in ocular epithelia. I n “Ion Channels” (T. Narahashi. ed.), Vol. I . Plenum. New York. Richards, N. W., and Dawson, D. C. (1986). Single potassiuni channels blocked by lidocaine and quinidine in isolated turtle colon epithelial cells. Am. J. Physiol. 251, CXSSC89. Sackin, H. ( 1987). Stretch-activated potassium channels in renal proximal tubule. Am. J. Physiol. 253, FI 253- F 1262. Sackin. H. (1989). A stretch-activated K + channel sensitive to cell volume. P ror. Natl. Acad. Sci. U.S.A. 86, 1731- 1735. Sackin, H., and Palmer, L. G . (1987). Basolateral potassium channels in renal proximal tubules. Am. J . Ph.vsinl. 253, F476-F487. Sakmann, B., and Neher, E. (1983). Geometric parameters of pipettes and membrane patches. I n “Single-Channel Recording” (B. Sakmann and E. Neher, eds.), pp. 37-52. Plenum, New York. Sakmann, B., and Neher, E. (1984). Patch clamp techniques for studying ionic channels in excitablc mernbrancs. Annu. Rev. Physiol. 46,455-412. Tabcharani, J. A,, and Hanrahan, J. W. (1989). Inhibition of epithelial anion channels by HEPES and related buffers. J . Gen. Physiol. 94, 34a. Trautmann, A , , and Marly, A. (1984). Activation of Ca-dependent K channels by carbdmoykholine in rat lacrimal glands. Proc. Nad. Arad. Sci. U.S.A. 81, 61 1-615. Trautmann, A , , and Siegelbaum, S. A. (1983). The influence of membrane patch isolation on single acetylcholine-channel current in rat myotubules. I n ”Single-Channel Recording” (B. Sakmann and E. Neher, eds.), pp. 473-480. Plenum, New York. Trube, G . , and Hescheler, I. (1984). inward-rectifying channels in isolated patches of heart cell membrane: ATP dependence and comparison with cell-attached patches. PJiuegers Arch. 401, 178-184. Welsh, M. J., and Licdtke, C. M. (1986). Chloride and potassium channels in cystic fibrosis airway epithelia. Nurure (London) 322, 467-470. Wills, N. K . , Donaldson, P. J., Purcell, K . , and Millinoff, L. (1989). Bicarbonate dependence of Na+ channel activity in A6 cultured renal epithelia. FASEB J . 3, A860. Yiang, X.-C., and Sachs, F. (1989). Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science 243, 1068- 107 I . Yellen, G . (1982). Single C a z t activated non-selective cation channels in neuroblastoma. Nature (London) 296, 357-359. Zeidel, M. L., Silva, P., Brenner, B. M., and Siefter, J. L. (1987). cGMP mediates effects of atrial peptides on medullary collecting duct cells. Am. J. Physiol. 252, F5.57- F559. Ziomek, C. A , , Schulman, S., and Edidin. M. (1980). Redistribution of membrane proteins in isolatcd mouse intestinal epithelial cells. J . Cell B i d . 86, 849-857.
CIIRRCNT TOPICS IN MEMBRANESAND TRANSPORT, VOLUME 37
Chapter 8
Chloride Channels in Epithelial Cells RAYMOND A . FRIZZELL AND DAN R . HALM Department of Physiology and Biophysics The University of Alabama ar Birmingham Birmingham, Alabama 35294
I. Chloride Channels in Absorptive Epithelia A. Tight Epithelia B. Leaky Epithelia C. Regulation of Chloridc Channels in Absorptive Cells II. Chloride Channels in Secrctory Epithelial Cells A. Properties of Secretory Chloride Channels B . Regulation of Sccretory Chloride Channels 111. Volume-Sensitive Chloride Channels 1V. Comparisons among Anion Channels A. Regulatory Mechanisms B . Anion Permeation References Note Added in Proof
Chloride (CI) channels play a variety of functions in the physiology of cells. In excitable cells, C1 channels act to determine levels of electrical excitability, and they function at inhibitory synapses of the central nervous system (Hille, 1984). Chloride channels contribute to the capacity of many cell types to regulate their intracellular composition and volume (Hoffmann, 1987). In the response to cell swelling, C1 channel activation appears to be the rate-determining step in the ability of cells to reduce their volume toward normal values. In epithelial cells, CI channels function in salt absorption and secretion. Chloride movements are secondary to the active transport of Na across salt-absorbing epithelia, in which the mechanism that links CI to Na transport varies from passive electrical coupling to direct coupling via shared transport mechanisms. In salt-secreting epithelia, C1 is the “driven ion,” and agonist-regulated CI channels play a primary role in determining the magnitude of salt and water secretion. In this chapter, we will examine the properties and regulation of the CI chan247
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nels that contributc to the physiology of absorptive and secretory epithelial cells. It will be apparent that our knowledge of CI channels in secretory cells is more extensive than that for absorptive epithelia. Where possible, we will categorize the CI channels of different cell types on the basis of their membrane location, biophysical propertics, and mode of regulation. Our goal is not to review the transport physiology of these systems, but we will touch on the relation of CI channel properties to the physiology of absorptive and secretory cells. In the final section, we will compare epithelial cell CI channels with those observed in a variety of other cell types. The reader is also referred to a review of this subject by Gogelein (1988).
1. CHLORIDE CHANNELS IN ABSORPTIVE EPITHELIA
A. Tight Epithelia These tissues form barriers between solutions of markedly different conipositions and are capable of absorbing salt from dilute outer or luminal solutions. Examples are amphibian skin, urinary bladder, and distal nephron. The cellular model that applies to these tissues is that formulated by Koefoed-Johnson and Ussing (1958), and is illustrated in Fig. I A . According to this model, Na enters across the apical membrane through amiloride-sensitive Na channels and is extruded from the cell by the basolateral Na-K pump. Transepithelial CI flow is diffusional and parallels that of Na. The absorptive flow of C1 is thought to traverse both cellular and paracellular pathways, as shown in the figure. The ability of CI to cross the cells by diffusion requires the presence of a conductive pathway for CI rind a favorable driving force for C1 to enter absorptive cells across their apical membranes. Conductive Na entry can assist in establishing the electrical driving force appropriate for CI entry by depolarizing the apical membrane voltage, V : , , below the equilibrium potential for CI, ES', so that V , > EC'. In toad skin, the magnitude of the cellular chloride conductance and the driving force for CI absorption appear to be related. Positive transepithelial voltages activate a transepithelial CI conductance, as shown by the conductance-voltage relation of Fig. 2. As the inside of the skin becomes more positive due to increased Na transport, a proportional change in CI conductance occurs (Willumsen and Larsen, 1986). This voltage-dependent C1 conductance pathway is thought to reside in the apical membranes of the mitochondria-rich cells, which parallel (shunt) the cells that actively transport Na (Larsen rt a!., 1987; Voute and Meier, 1978; see Fig. IA). Its voltage sensitivity ensures that the CI conductance pathway is open only when an appropriate electrical driving force exists for diffusional CI absorption. Single-channel events possessing these interesting properties have not been recorded from toad skin, however, due to the
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aplcal membrane
B
rnucosal solution
apical membrane
t
I
absor tive
ceFl
I
ssrosal solution
7
FIG. 1 . Cellular models for CI transport across absorptive epithelia. (A) Tight epithelia, ( B ) leaky epithelia. See text for further discussion.
presence of the cornified cell layer that prevents access of patch pipets to the transporting cells. Among tight epithelia, apical membrane CI channels have been detected only in A6 cells (Nelson cr al., 1984). This toad kidney cell line is presumably of distal nephron origin, as suggested by its transport properties and hormone responsiveness (Perkins and Handler, 1981).Apical membrane CI channels in A6 cells have a large single-channel conductance (350 pS) and are active only when the transmembrane voltage is near 0 mV. The activity range varies, but is gen-
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RAYMOND A. FRIZZELL AND DAN R. HALM
Te 2
1
1
1
1
1
mS’cm2
1
1
1
1
1
1
1
1
-100 -50 50 100 FIG.2 . Voltage-dependent CI conductance of amphibian skin. Chloride-dependent transepithclial current was measured acrosb toad skin ~ v e ar range o f transepithelial clcctrical potential differences (Ldrs.cn rt ul.. 1987; Willumsen and Larsen, IYXh). Potentials are referenced to the cxterior (outer) solution such that positive voltages provide a driving force for anion absorption.
erally between +- 20 mV. As discussed above, Nelson er a/. ( 1984) reasoned that these channels would provide a pathway for CI entry across the apical membrane when apical Na conductance is high, which would depolarize the apical membrane voltage to values appropriate for opening of this maxi CI channel. An anion conductance with similar properties (Schein ef ( J I . , 1976) has been identified in the outer mitochondrial membrane. Its conductance-voltage relation is illustrated in Fig. 3. Maxi C1 channels with these characteristics have been found in a variety of cultured cells, including nonepithelial cells, at the singlechannel level (see Table 1). ‘The contribution of this channel to the physiology of the many cell types in which it i s present has not been defined, and its relation to the anion channel of the mitochondrial membrane requires clarification. A brief report (Ashford, 1986) of single C1 channel currents in cultured epididymal cells also may be relevant to this discussion of apical conductance properties in tight epithelia. An outwardly rectifying 35-pS CI channel was present in excised patches bathed by Ca-free media. It may play a role in NaCl absorption by this tissue. and interestingly, its properties resemble those of the apical mem-
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8. CHLORIDE CHANNELS IN EPITHELIAL CELLS
FIG. 3. Voltage-dependent anion conductance in mitochondria. The conductance G (relative to that at 0 mV, G<,)induced by reconstitution of a mitochondria1 protein (VDAC) into planar lipid bilayers is plotted as a function of trdnSInembrdne voltage (Schein e t a / . . 1976). The channel is open primarily near 0 mV and open probability falls markedly at hyperpolarizing or depolarizing voltages.
brane C1 channel that is present in salt-secreting epithelial cells (see Section II,A, Properties of Secretory Chloride Channels). Two types of CI channels have been detected in the basolateral membranes of freshly isolated mammalian urinary bladder cells (Hanrahan et a l . , 1985). A 360-pS maxi C1 channel was detected, similar to those discussed above, whose role in CI absorption was uncertain due to the restricted voltage range (around 0 mV) over which this channel is active. A medium-conductance (30 pS) anionselective channel also was present, and its properties are more appropriate to a physiological role in C1 absorption. As illustrated in Fig. 4A, this channel is inwardly rectified, that is, it passes inward current (outward C1 flow)' more readily than outward current (inward Cl flow) for an equivalent driving force. In addition, the probability of channel opening is high in the physiological voltage range but decreases toward zero with depolarization above 20 mV (Fig. 4B).
+
' By biological convention, current represents the flow of positive charge. Thus, currents carried by anions flow in the direction opposite to anion movement: outward CI current is carried by inward CI flow and inward CI current is due to outward CI How.
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RAYMOND A. FRIZZELL AND DAN R. HALM
A
c
# J
A
-.+ t
B
T
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100
Flci. 4. Properties of the basolateral membrane CI channel in mammalian urinary bladder (Hanrahan el d.,1985). ( A ) Single-channel current-voltage relation. Memhrane voltage is referenced to the cell exterior; inward currents rcprcscnt CI flow from inside to outside. (B) Channel open probability as a function of membrane voltage. The observed rclation indicates that thc channel opens at physiological voltages, favoring CI cxit across the basolateral membrane.
8. CHLORIDE CHANNELS IN EPITHELIAL CELLS
253
The inward rectification and voltage dependence of this channel would be appropriate to optimize basolateral membrane CI conductance when a driving force for CI diffusion from cell to serosal solution is present. The authors concluded that this channel provides a major pathway for CI absorption across the basolateral tnembranes of urinary bladder cells. 8. Leaky Epithelia Leaky epithelia absorb relatively large volumes of fluids isotonically. Because they cannot sustain significant transepithelial gradients of small solutes, the salt concentrations of the fluid compartments that bathe these tissues are near equilibrium. The epithelial sheet is relatively leaky because of the high-conductance paracellular pathway between adjacent cells, which serves to shunt (minimize) voltage differences between the apical and basolateral membranes. The cells display a relatively large cell-negative apical membrane potential that arises from two factors: ( I ) a high basolateral membrane K conductance and (2) a high ratio of paracellular-to-apical membrane conductance, which permits this basolateral K conductance to hyperpolarize V,. Accordingly, the diffusional driving force for CI at the apical membrane opposes CI absorption because V:, < Err. If CI channels were present, C1 exit from cell to lumen would occur. However, these epithelia generally do not display a significant apical C1 conductance; rather, salt absorption is mediated by coupled NaCl entry mechanisms. In different tissues, coupled entry involves Na-H plus CI-HCO1 exchangers, Na-K-2CI cotransport, or Na-CI cotransport. A generic cellular model that illustrates the transport properties of these cells is given in Fig. 1 B. During salt absorption across leaky epithelia, a favorable electrochemical gradient for C1 exit across the basolateral membrane is present. Microelectrode recordings support the concept of a basolateral CI conductance that would permit CI exit across this barrier by diffusion (Greger, 1985; Greger and Schlatter, 1983; Halm et ul., 1984); nevertheless, a single-channel correlate of this conductance has not been adequately defined (See Note Added in Proof 1, p. 276). In addition, estimates of the magnitude of the basolateral C1 conductance from microelectrode studies suggest that it is insufficient to permit cell-to-serosa CI transport at the rate that is observed during absorption (Duffey et al., 1978). Evidence that KCI cotransport contributes to CI exit across the basolateral membranes of leaky epithelia has been presented by Reuss (1989). The relative magnitudes of C1 exit via conductive and coupled processes probably vary among tissues. The only report of a channel conductive to CI in the basolateral membranes of leaky epithelia is that by Gogelein and Greger (1986), who detected a channel that was relatively nonselective for anions versus cations in late proximal tubule cells. Although this channel could play a role in CI exit across the basolateral membrane, its physiological importance is uncertain because it would lead also to dissipation of transmembrane cation gradients.
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C. Regulation of Chloride Channels in Absorptive Cells From available data, the Cl conductance of the cellular pathway in tight epithelia appears to be primarily voltage regulated. The serial arrangement of the apical and basolateral CI conductances can provide a pathway for anion diffusion that parallels the active Na transport pathway. However, if CI is to both enter and exit by diffusion, then a precise relation between the cell CI activity and the apical and basolateral membrane voltages must be maintained. For example, the apical CI channel must open only when the transmembrane driving force favors C1 entry, and this occurs when V , is depolarized by a high apical Na conductance. Under open-circuit conditions, this will be true even if Na and CI channels reside in different cell types, because the transepithelial voltage is proportional to Na transport rate. Thus, a relatively steep dependence of C1 channel open probability on voltage (presumably responsible for the relation shown in Fig. 2) would serve to switch the apical CI conductance off when the driving force for C1 movement no longer favors entry by diffusion. This would maintain the barrier function of these tissues by restricting free diffusion of CI under nontransporting conditions. The voltage dependence of the maxi C1 channel of A6 cells (Nelson er al., 1984)and the voltage-activated C1 conductance of toad skin (Willumsen and Larsen, 1986) appear to satisfy this constraint, although a match between P , and driving force (including both the electrical and chemical gradients) has not been quantified experimentally. At the basolateral membranes of tight epithelia, a driving force for net C1 exit will exist unless cell CI activity falls into equilibrium with the basolateral membrane voltage, V,. The basolateral C1 channel detected in rabbit urinary bladder by Hanrahan et ul. (1985) has a high open probability at membrane potentials of -30 to - 100 mV, so that physiological values of V,, are consistent with a high CI conductance (Fig. 4). Chloride absorption across leaky epithelia proceeds via coupled uptake of Na and C1 at the apical membrane. Chloride exit across the basolateral membrane occurs, at least in part, by diffusion through CI channels. Regulation of CI absorption by CAMP-dependent conductance pathways has been described in the thick ascending limb of Henle’s loop (Greger, 1985). Stimulation of salt absorption by antidiuretic hormone (ADH) increases both basolateral C1 conductance and apical K conductance. A preliminary report from patch-clamp studies (Gogelein, 1988) indicates that the single-channel events corresponding to this CAMP-activated C1 conductance have been detected, but details of their properties were not provided (See Note Added in Proof 1, p. 276). Regulated CI channels in the apical membranes of leaky epithelia have also been reported, although their activation would have antiabsorptive effects. Petersen and Reuss (1983) and Garcia-Diaz ~t ul. (1983) found a CAMP-activated CI conductance in the apical membranes of Necturus gallbladder, which allows CI to exit from cell to lumen; CAMP concomitantly inhibits NaCl entry into gallbladder cells (Reuss, 1989). A 10-pS CI channel has been detected in the apical
255
8. CHLORIDE CHANNELS IN EPITHELIAL CELLS
membranes of cells dissociated from Necturus gallbladder after pretreatment with CAMP-mediated agonists (Segal and Reuss, 1989). A significant CI conductance is present also in the apical membranes of Necturus enterocytes (Giraldez and Sepulveda, 1987; Giraldez et al., 1988). The relative CI conductance of this barrier increased about sixfold to eightfold during Na-coupled alanine absorption. This appears to be part of the volume regulatory response of intestinal cells to the cell swelling that occurs during Na-coupled nonelectrolyte transport. It is possible that the apical CI conductance of the intestine is activated when high luminal solute entry rates exceed basolateral exit, which would lead to an increase in cell solute and volume. Activation of the apical CI conductance would decrease solute entry (by recycling CI and/or depolarizing V , ) to adjust solute entry and exit rates and protect cell volume during high rates of transport.
II. CHLORIDE CHANNELS IN SECRETORY EPITHELIAL CELLS A variety of tissues have the capacity to secrete salt by virtue of a transcellular CI transport mechanism in which CI channels at the apical membranes play a vital role. Chloride secretion is regulated by a variety of hormones and neurotransmitters, and the actions of these secretory agonists have been used to identify physiologically relevant C1 channels in patch-clamp studies. The model shown in Fig. 5 illustrates the cellular transport events in CI secretion (Frizzell mucosai solution
apical membrane
I
secretory cell
I
serosal solution
7
Fic;. 5. Cellular model for transepithelial C1 secretion. Chloride enters secretory cells across the basolateral membrane coupled to the entry of Na and K via a loop diuretic-sensitive Na-K-2CI cotransport process. Sodium is returned to the serosal (plasma-facing) solution by the ouabainsensitive Na-K pump, which also brings K into the cell. Cell K is returned to the serosal solution via basolateral K channels. These three processes lead to cellular C1 accumulation to levels that exceed the electrochemical potential of extracellular CI. Thus, CI exits across the apical membrane by diffusion through CI-selective channels. Sodium accompanies C1 to the luminal solution by diffusing across the paracellular pathway. driven by the transepithelial voltage arising from electrogenic CI transport.
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RAYMOND A. FRIZZELL AND DAN R. HALM
et al., 1979). The roles of Na and CI are reversed from the situation in leaky absorptive cells; that is, NaCl cotransport resides at the basolateral membrane and CI diffuses iicross the apical membrane. Sodium secretion is driven by the transepithelial electrical potential difference that arises from electrogenic C1 transport, and this diffusional flow of Na traverses the paracellular pathways. These processes are present in a variety of secretory epithelia, including the intestines, the airways, and the exocrine glands. Slight modifications on this basic theme will alter the secretory product, e.g., addition of apical CI-HCO, exchange and basolateral Na-H exchange (replacing Na-K-2CI cotransport) results in HCO, secretion, as in skate alkaline gland (Smith, 1985), turtle urinary bladder (Stetson et NI., 1985), and rat pancreatic duct (Novak and Greger, 1988). Relatively few, if any, secretory tissues represent pure populations of saltsecreting epithelial cells. For this and other technical reasons, much of our detailed knowledge of the properties of secretory CI channels derives from studies of primary cell cultures and epithelial cell lines.
A. Properties of the Secretory Chloride Channels The apical membrane CI channel that is most frequently activated by secretory agonists has distinct biophysical features (Fig. 6 and Table 1). Single-channel records from trachea (Frizzell et u / . , 1986; Shoemaker et al., 1986; Welsh, 1986a,b), sweat gland (Krouse et d., 1987; Schoumacher et a / . . 1987b), colon (Reinhardt ct d.,1987), colonic tumor cells (Halm et c d . , 1988; Hayslctt et a / ., 1987), and pancreatic tumor cells (Schoumacher et a / ., 1988) provide evidence of a CI channel with relatively well-defined conductance properties, ion selectivity, and kinetics (Fig. 6A). Its widespread distribution in secretory tissues and its agonist-induced activation have led to its designation as the secretory CI channel (Frizzell, 1987). A channel with similar properties has been detected recently in human skin tibroblasts (Bear, 1988) and lymphocytes (Chen et al., 1989); however, its function in those systems has not been identified. Single-channel measurements have begun to provide ti better understanding of the anion permeation and kinetic properties of this channel. Other anion-selective channels are present in secretory cells, e.g., low-conductance CI channels in human tracheal (Frizzell et a/., 1986), pancreatic duct (Gray et d.,1988), and lacrimal gland (Evans and Marty, 1986a) cells (see below and Table I), but their properties are less well characterized because they have been observed less frequently. 1 . I O N SEI.EC.TIV1 I
Y
Once activated, the secretory CI channel must permit CI flow in preference to cations, particularly Na, because the electrochemical gradient favoring Na entry (- 120 mV) across the apical membrane is much greater than that favoring CI exit (20-30 mV). Single-channel cation-anion selectivities have been derived
8. CHLORIDE CHANNELS IN EPITHELIAL CELLS
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from reversal potential measurements with imposed NaCl concentration gradients. The selectivity for CI over Na found in the apical CI channel of several secretory cell types, calculated from the Goldman-Hodgkin-Katz equation, varies between 10 and 100 (see Table I).? The anion selectivity of the secretory C1 channel has been determined from biionic reversal potential measurements. The halide anion selectivity sequence is I( 1.7)/Br(1.3)/C1(1 .O)/F(0.4). The relative bicarbonate permeability (PHco3/Pc.) was 0.4 for the channel incorporated into planar lipid bilayers from rat colon (Reinhardt et a l . , 1987). This suggests that the secretory CI channel has a significant bicarbonate permeability, whose implications will be discussed below. In addition to reversal potential measurements, anion selectivities also can be evaluated from the individual anion conductances, measured as the currents carried by various anions. Inward currents (outward anion flow), measured during anion substitution at the inner membrane surface, indicate that I, Br, and C1 are equally effective current carriers (Halm et al., 1988; Hayslett et al., 1987). Similar studies show that the secretory CI channel in shark rectal gland is significantly more conductive to C1 than to Br (Greger et al., 1987). However, permeabilities calculated from reversal potentials and anion conductances do not provide equivalent measures of ion selectivity, because different properties of the conduction pathway are assessed by these measurements. In the case of secretory cells, the conductance measurement is probably more relevant because it assesses the rate at which the secretory channel transfers various anions across the apical membrane. Importantly, the conductive selectivity for HCO, has not been determined (See Note Added in Proof 2, p. 276). Transepithelial flux measurements provide a selectivity for anion secretion of CI > Br >> I (Widdicombe and Welsh, 1980), which differs from the selectivity sequence of the apical membrane C1 channel, as assessed by either method. Thus, the channel does not determine which anions are secreted (except perhaps in the shark rectal gland, where this sequence is observed), rather the selectivity of Na-K-2CI cotransport (Palfrey and Greengard, 1981), CI > Br >> I, which mediates anion uptake from the serosal solution, defines the capacity of the epithelium for secretion of various anions. Complete substitution of CI by another anion allows the channel's conductance for that anion to be measured over the entire voltage range. In contrast, permeabilities derived from reversal potentials are determined at zero current flow at a single voltage. Nevertheless, both of these selectivity measurements can provide information on the interaction of ions with structural features of the channel. Flow through the channel can be represented as the free-energy profile experienced by an ion. This profile will consist of a series of peaks and valleys that correspond to the transitions that occur as the ion leaves its association with the ' A significant Na conductance might account for the small amount of amiloride-insensitive Na absorption detected in some secretory tissues.
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RAYMOND A. FRIZZELL AND DAN R. HALM
aqueous phase and enters regions within the channel that provide more or less favorable interactions. The highest peak (energy barrier) within the channel acts as the selectivity filter, and an ion that cncounters the lowest peak energy will traverse this pathway most easily and therefore will have the highest permeability (liille, 1984). The halide permeabilities of the secretory CI channel suggest that anions with largcr ionic diameters experience lower peak energies. In the formalism of Eisenman (Eisenman and Horn, 1983), this sequence is consistent with a weak site of interaction at the selectivity filter. This designation of the site as weak is relative to the ion-water interaction energy. Because the larger anions have the lower hydration energies, dehydration of the anion is a major energetic determinant of anion permeability in the secretory CI channel. Conductance includes the intluences of valleys as well as peaks, because the rate of passage is determined by the residence time of the ion at each site. For this reason, selectivity based on conductances need not be the same as that derived from pcrmcabilities. The y-aminobutyric acid (GABA)- and glycine-
A 1 2 . 5 PA 20 m e e c
+100mV
+50mV
-1OOmV
FK;. 6 . Properties of the sccrctory cell CI channcl. ( A ) Single-channel currents derived lrom patch-clamp records of excised. insidc-out patches from the apical membrane of TX4 colonic tumor cells (Halm zt (I/.. 1988). The closed states are indicated by horizontal lines at each transmcmhranc voltage (referenced to the cell cxtcrior). ( B ) Current-voltage rclation obtained with symmetrical I60 rriM NuCl at both membrane surfaccs, illustrating outward rectification of the secretory CI channel. (C) Channel open prohahility (f,,) as a function of membrane voltage.
C
1
.o
PO
0.5
I
I
I
:
-100 FIG. 6 . ( B ) & (C)
I
: -50
I
:
;
:
l 1
1
1
1
1
1
I
I
I
I
l
I
I
I
I
I
I
50
mV 100
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RAYMOND A. FRIZZELL AND DAN R. HALM
activated CI channels show such a difference in anion selectivities from permcability and conductance measurements (Bormann el d., 1987). Permeability measurements provide a selectivity sequence of I > Br > CI >> F, similar to that seen for the secretory CI channel. Single-anion conductances yield a different selectivity, CI > Br 2 1 >> F. Clearly, the channel features that determine anion conductance are not identical to those that define its permeability, and these two measures of selectivity highlight different features of the free-energy profile. One possibility for the discrepancy in selectivitics is that the energy profiles are qualitatively similar for each ion but that larger anions have lower overall energy profiles. This would result in lower peaks for the larger anions (and thus higher permeabilities) but would also lower the valleys. Making the valleys lower would increase the dwell time of the ion in the channel, and decrease its conductance. In the case of fluoride, the high peaks encountered by this small mion would appear to be sufficient to also limit its conductance, because it lies at the end of the selectivity order for both conductance and permeability. These mcasures of ion selectivity provide an energetic topology of the permeation pathway and, together with other information, can provide evidence of the chcmical groups that determine selectivity.
2 . S I N G L E - C I I A N CONDUCTANCE NEL A distinguishing feature of thc CI channel found in most secretory cells is rectification of current flow, favoring outward currents ovcr inward currents by a factor of about 4 in the range + 100 to - 100 mV (Fig. 6A and B). During cellattached recording, a low intracellular CI concentration could account for the smaller inward currents (outward CI flow), but, in excised inside-out patches with equal C1 concentrations bathing both sides o f the channel, the rectification of current is similar. With symmetrical 160 mM CI, the single-channel chord conductance is 40 to 50 pS at zero voltage. Increasing voltage to bctween +60 and 80 mV nearly doubles this conductance. whereas reversing the voltage to between 60 and - 80 gives a conductance about half that at 0 mV. Thus, CI currents are sniallest in the physiological voltage range. Although the utility of this voltage dependence is not apparent, rectification of current flow must be due to some asymmetry in the structure of the channel. An exception to this pattern is observed in the C1 channel from shark rectal gland, whose current-voltage relation is linear (Gregcr et a / . . 1987). However, the kinetics and conductance of the rectal gland channel are similar to those of other secretory CI channels. The rectification of current flow has its basis in the free-energy profilc that CI experiences as it traverses the channel in different directions. The initial interaction between CI and the channel occurs at the mouth of the channel, where the effects o f the aqueous solution diminish. Electrostatic forces from fixed charges
+
~
8. CHLORIDE CHANNELS IN EPITHELIAL CELLS
261
or dipoles on the channel (or the lipid bilayer) could lead to an increase or decrease in local CI concentration relative to its concentration in bulk solution. A difference in CI concentrations at the mouths of the channel would rectify current flows. For example, outward rectification could result from excess fixed negative charge (perhaps due to phosphorylation) on the intracellular channel surface relative to that outside. This would result in a lower CI concentration adjacent to the inner mouth of the channel compared with that outside, so that fewer charge carriers would be available to carry inward current than outward current. Whether surface charge could account for rectification has not been examined directly, but the reconstitution experiments by Reinhardt et ul. (1987) are informative in this respect. When the secretory CI channel was reconstituted into a planar bilayer consisting of neutral lipids, currents were outwardly rectified, suggesting that any surface charges that influence current flows must reside on the channel rather than on the bilayer. Another source of rectification could be the interaction of CI with sites inside the channel. These sites could bear net charge (e.g., side chains of lysine, arginine, or histidine) or be composed of dipoles (e.g., peptide linkages or side chains of asparagine, glutamine, tyrosine, threonine, or serine) where anions reside momentarily on their way through the channel. As in the discussion of ion selectivity, ions jump over peaks (relatively unfavorable sites) in free energy to arrive at more favorable sites of interaction. The complexity of the profile depends on the number of peaks and valleys. Normally an attempt is made to define the least complex model that describes the observed currents. Eyring rate theory (Eyring, 1936; Hille, 1984) can be used to calculate ion flow, with each transition characterized by a barrier whose height represents the energy necessary to traverse it. In the case of secretory CI channels, a one-site, two-barrier model can qualitatively account for the observed rectification. Figure 7A shows two types of free-energy profiles that satisfy this requirement: ( 1 ) asymmetric peak energy heights and (2) asymmetric placement of the internal interaction site within the electric field experienced by the ion. In the first scheme (Fig. 7 top), an asymmetry in the heights of the peak energies experienced by CI will produce rectification of the current-voltage relation. A larger peak at the cytoplasmic face (inside) of a two-barrier channel creates a voltage dependence of CI conduction because equal but opposite voltages do not distort the free-energy profile equally. An inside-positive potential increases the free energy of extracellular CI relative to cytoplasmic CI, so that C1 flows in (outward current). With this model, positive potentials cause a large drop in the height of the larger inner peak so that it is no longer higher than the outcr peak. This creates an exponential rise in outward current because the smaller outer peak becomes the rate-determining barrier within the electric field. Outward C1 flow (inward current) occurs when the cytoplasmic side is made negative. With the electric field reversed, the larger inner peak remains the rate-
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RAYMOND A. FRIZZELL AND DAN R. HALM
B
A
in
out
-
current
C
a--current
Free-energy profiles across outwardly rectifying CI channels. The free energy of the CI ion is dcpictcd along the length of the channel at 0 mV (A), and at positive (B) and negative ( C ) voltages. Sites of relatively unhvorahlc interactions are shown as peaks. and valleys indicate relatively favorahlc sites of interaction. Application of a n electrical potential distorts thc frcc-energy profile. producing rcctilication. See text for further discussion. FI(; 7
determining barrier as the voltage is made more negative. Thus, rectification occurs because the rate-determining barrier shifts from one peak to the other as the electric field is reversed, and these peaks are different in sizc. Indeed, rectification can occur in any model in which the rate-determining barrier shifts within the electric field (Krasne, 1978). A second suitable model can be made by placing energy barriers of identical height asymmetrically within the electric field (Fig. 7 bottom). To achieve outward rectification, the outer barrier is placed deep in the electric field. In this case, inward CI flow (outward current) will be limited by the outer barrier, but it senses a large fraction of the electric field, so that its height drops rapidly as the voltage increases. For outward C1 flow (inward current), the rate-determining inner barrier falls more slowly with the voltage change because it senses a smaller fraction of the electric field. A more definitive model of rectification in the secretory CI channel cannot be obtained without experiments that will exclude some of the possibilities. Although the singlc-site models of Fig. 7 can account for rectification of current flow, they do not allow for interactions between ions within the channel. With multiple sites, two or more ions can occupy the channel simultaneously. Thcy
8. CHLORIDE CHANNELS IN EPITHELIAL CELLS
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would be repelled by electrostatic forces between them, which can be represented schematically as differences in the free-energy profile for single and double occupancy. Preliminary results suggest that there is interaction between anions within the secretory CI channel (Halm and Frizzell, 1988). The evidence for interaction comes from conductance measurements performed using mixtures of two permeant anions. Mixtures of intermediate concentrations (e.g., 130 mM NaCl plus 30 mM NaSCN) produce lower current flow than that expected from the algebraic sum of the independent anion currents. This behavior represents a deviation from independence in the flows of ions and also has been observed for the GABA-activated CI channel (Bormann et al., 1987). These results can be interpreted as indicating multiple sites for anion interactions within the channel. Most simply stated, the presence of an anion in the channel will interfere with the passage of a different anion, producing a slower rate of total anion flow (current). However, detecting ion interactions is not sufficient to establish a model wherein two (or more) sites reside within the permeation pathway, because a conformational change in the channel that alters conductance also could result from an anion binding site external to the conduction pathway. A better understanding of anion-anion interactions in the secretory CI channel could enhance our understanding of epithelial CI secretion. For example, interactions between C1 and bicarbonate or phosphate might be physiologically relevant in determining the rate of channel-mediated C1 exit during secretion. 3. OPENPROBABILITY The activity of secretory CI channels recorded from different cell types shows similar open-closed kinetics (Bridges et al., 1989; Frizzell et al., 1986; Greger et al., 1987; Halm et al., 1988; Hayslett et a l . , 1987; Reinhardt et al., 1987; Welsh, 1986a,b). Bursting behavior is apparent as frequent but brief ( 5 1 msec) closures during periods of primarily open channel activity (Fig. 6A). Another characteristic feature of the secretory C1 channel is the dependence of its open probability ( P , , )on membrane voltage (Greger et al., 1987; Halm et ul., 1988; Hayslett et al., 1987): P,, increases as the cytoplasmic side is made more positive (Fig. 6C). The median value of open probability occurs at about 0 mV in excised patches, with P,, increasing from about 0.15 at -80 mV to about 0.75 at 80 mV. The physiological consequence of this voltage dependence would be to sustain CI diffusion from cell to lumen as apical CI channels are opened by secretory agonists. That is, activation of CI channels causes V , to depolarize toward the CI equilibrium potential, ES', as apical membrane CI conductance rises (Welsh et al., 1983). The voltage dependence of P ,would tend to offset this decrease in driving force by increasing the probability that CI channels are open, and would tend to maintain diffusional CI exit, despite a reduced driving force.
+
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RAYMOND A. FRIZZELL AND DAN R. HALM
4. BLOCKERS Drugs that specifically block the flow of ions through channels have been useful for perturbing channel kinetics, so as to achieve a phenomenological description of channel gating properties. Compounds of three different chemical classcs have been shown to block CI channels; however, all of these agents influence other transport or cellular processes. At this time, high-affinity, selective blockers of C1 currents through the secretory channel have not been identified. The disulfonic stilbene derivatives, 4,4’-diisothiocyanatostilbene-2,2‘-disulfonic acid (DIDS) and 4-acetamido-4’-isothiocyanatostilbene-2,2’-disulfonic acid (SITS), which traditionally have been used as inhibitors of anion exchange, also block CI flow through some anion-selective channels (Nelson et al., 1984; White and Miller, 1979, 1981). The reversible stilbene analog, 4,4’-dinitrostilbene2,2’-disulfonic acid (DNDS), produces a flickery block of the secretory C1 channel from rat colon, reconstituted into planar lipid bilayers (Bridges rt al., 1989; see Chapter 9 by Bridges and Benos). DNDS was effective only when added to the solution bathing the external (extracellular) surface of the channel.’ The results suggest that DNDS is an open-state channel blocker than enhances the open-closed transition frequency and produces long periods of flickering current with reduced P,,, but with fewer long closed periods. A 50% reduction in P,, was obtained from 2-3 p V l DNDS. Despite their evident blocking effect on the single-channel current, stilbenes do not significantly affect transepithelial C1 secretion (e.g., short-circuit current or C1 lluxes). However, macroscopic CI transport events might not be affected by substances that function as open channel blockers if the kinetics are modified without markedly affecting the percentage of open time of the channel (e.g., an’ increase in transition frequency, but with fewer long closed periods). This can occur if the channel has a relatively low P,, at physiological voltages, as is true for the secretory CI channel (see above). Alternatively, the transmembrane voltage may influence blocker effectiveness. Most stilbenes are divalent anions, so that a high-affinity block may require an inside-positive membrane voltage, which does not exist physiologically. Derivatives of anthrdcene-9-carboxylic acid (DiStefano et al., 1985; Oberleithner et a/. , 1983; Wangemann rt ul., 1986) also block secretory C1 channels (Dreinhofer ot a / ., 1988; Greger et a / ., 1987; Hayslett et a / . ,1987). Flickery block of single-channcl currents has been observed when one of these derivatives, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), is added to the inside of inside-out membrane patches from HT29 cells (Dreinhofer et al., 1988). However, /‘, was reduced by only SO%, even at high NPPB concentrations, which may account for the inability of these agents to serve as high-affinity inhibitors of CI secretion. In addition, if flickering kinetics predominate under the condi’Thc orientation of the channel in the bilaycr was dcduced from the rectification of current flow in symmetrical salt concentrations.
8 . CHLORIDE CHANNELS IN EPITHELIAL CELLS
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tions of voltage and anion concentration that prevail in the intact epithelium, the problem of a relatively weak effect of an open channel blocker on mean open time will also influence their effectiveness, as discussed for the stilbenes. Another series of blockers has been derived from ethacrynic acid (Landry et a / . , 1987). The blocking actions of these compounds have been reported only for anion fluxes in renal membrane vesicles. A common structural feature among the stilbenes, anthracenes, and ethacrynate derivatives is a relatively planar hydrophobic backbone with a negatively charged group attached near the end of the molecule. This similarity in structure suggests a common mode of action for these compounds that may relate partly to hydrophobic interactions and partly to an association with a positively charged group on the channel. All C1 channel blockers have significant affinities for other anion transporters, e.g., the stilbenes inhibit anion exchange. An apical membrane anion exchanger in gallbladder is inhibited by the anthracene derivative diphenylamine-2-carboxylate (DPC) (Reuss et a / . ,1987), and ethacrynic acid and some anthracene derivatives inhibit NaCl cotransport processes (Al-Awqati et a / ., 1974; Wangemann et ul., 1986). The loop-diuretics furosemide and bumetanide are commonly used to inhibit Na-K-2CI cotransport, but they reduce CI currents in lacrimal gland cells as well (Evans et d . ,1986). This effect requires I mM bumetanide, however, a concentration that is about three orders of magnitude higher than that necessary to inhibit Na-K-2CI cotransport. Whereas the disulfonic stilbenes are relatively hydrophilic and do not cross biological membranes, the high lipid solubilities of the anthracene and ethacrynate derivatives could lead to other nonspecific effects at high concentrations. For example, Weymer ef al. (1985) found that 10 mM DPC or anthracene-9carboxylate abolished prostaglandin-induced increases in intracellular CAMP levels in colonic tumor cells. This would abolish C1 secretion without perturbing channel gating and implies that inhibitor studies alone are insufficient to implicate a role for C1 channels in transport events when only macroscopic currents or fluxes are assessed.
5 . CHEMICAL MODIFICATIONS
In principle, channel blockers can act by obstructing the passage of ions through the channel’s pore (as a plug) or by interfering with gating mechanisms. Selective chemical modification of these properties has been produced in studies of the mitochondria1 anion channel, using a voltage-dependent anion conductor (VDAC). Addition of aluminum chloride to planar bilayers containing VDAC eliminated the voltage sensitivity of channel conductance, so that the channels remained open over a large voltage range (Dill et a / . , 1987). This change in gating occurred without altering single-channel conductance or ion selectivity. These results suggest that aluminum [as AItOH), or Al(OH),] binds to the volt-
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RAYMOND A. FRIZZELL AND DAN R. HALM
age sensor and thus removes voltage-sensitive channel gating. In contrast, prolonged treatment of VDAC with succinic anhydride (Adelsberger-Mangan and Colombini, 1987) produced cation-selective channels with the same bell-shaped conductance-voltage relation observed under control conditions (Fig. 3). This is consistent with a modification of thc selectivity filter in which amino groups are changed to carboxyl groups. Probing of other C1 channels by chemical modilication might assist In identifying the chemical groups that determine their ion selectivity and gating properties. B. Regulation of Secretory Chloride Channels Thc regulatory modes of the secretory CI channel that have been described thus far are illustrated in Fig. 8. Regulation occurs at two Icvels. In the absence of stimuli, CI channels are in an inactive state. Oncc activated, the biophysical properties of the channel determine the magnitude of secretory CI flow. As discussed above, channel open probability increases with depolarization over the physiological voltage range (Halm et ul., 1988). However, CI channels must be activated by intracellular second messengers before more subtlc influences, such as thc cffect of voltage on channel kinetics, can occur. The primary event in stimulation of CI secretion is the activation of the apical mcmbranc CI conductance (Welsh et d . , 1983). At the single-channel level during cell-attached recording, this is detected as a transition from an inactive to an active state, rather than as a change in relative channel activity (kinetics). Channel activation can be evoked by several regulatory pathways, and a defect in one of thcsc channel regulatory pathways occurs in the secretory epithelial cells of cystic fibrosis (CF) patients. The primary pathway for activation of secretory CI channels in many epithelia involvcs CAMP-dependent phosphorylation. In tissues such as the intestines, airways, and pancreatic ducts, primary agonists such as vasoactive intestinal pep-
depolarization temperature
phosphatases FIG.8. Regulatory modes aflecting channel activity. The tirst reaction illustrates the activation of electrically quiet membrane patches during cell-attached recordings hy CAMP or calcium or the activation of CI channels in excised patches by PKA, PKC, depolarization, iernpera(ure, or high salt. The second rcaction illustrates the effect of membrane voltage on channel kinetics, which is observed in both cell-attached and excised patch recordings. See text for dctailcd discussion.
8 . CHLORIDE CHANNELS IN EPITHELIAL CELLS
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tide (VIP), norepinephrine, and secretin bind to basolateral membrane receptors to activate adenylate cyclase-dependent cAMP accumulation. In primary cultures of airway cells (Frizzell et al., 1986; Welsh, 1986a,b; Welsh and Liedtke, 1986), colonic tumor cell lines (Halm et al., 1988), shark rectal gland tubules (Greger et a / ., 198S), and isolated Necturus enterocytes (Giraldez et a/., 1988), CAMP-dependent agonists activate CI channels during cell-attached recording. As discussed above, this provides a physiological assay for the channels that underlie the high apical membrane CI conductance of secretory cells during stimulation. An outwardly rectifying 40- to SO-pS C1 channel is frequently activated by secretory agonists, but occasionally other channels are detected. These generally have lower conductances (4-20 pS) and linear current-voltage relations (Table 1). (See Note Added in Proof 3.) The infrequency with which these channels are observed in most systems has hampered a more detailed description of their properties, but low-conductance channels (4 pS) were found to predominate in the apical membranes of pancreatic duct cells during cAMP stimulation (Gray et ul., 1988). (See Note Added in Proof 4.) The medium-conductance, outwardly rectifying C1 channel has been detected in pancreatic cell lines (Schoumacher et al.. 1988). The CAMP-mediated activation pathway has been experimentally dissected using the purified catalytic subunit of the CAMP-dependent protein kinase (PKA) and excised membrane patches (Li et a/., 1988; Schoumacher et al., 1987a). Addition of the catalytic subunit of PKA to the cytoplasmic surface of channelcontaining patches activates the outwardly rectifying 40- to 50-pS CI channel. This requires both PKA and ATP, so that phosphorylation is responsible for eliciting channel activity. In cystic fibrosis airway cells, the 40- to 50-pS C1 channel is present, but it cannot be activated by CAMP-mediated stimuli during cell-attached recording. This defect in CAMP-induced channel activation is observed also in excised membrane patches challenged with the purified catalytic subunit of PKA. Cells from cystic fibrosis patients also have normal levels of PKA activity (Barthelson and Widdicombe, 1987). Thus, the block in the cAMP regulatory cascade in CF exists at the level of the isolated membrane patch, and lies beyond the step involving activation of PKA. These findings imply that CF channels are not a suitable substrate for phosphorylation by PKA, or that once phosphorylated, the conformational changes that are necessary to activate GI conduction do not occur. Calcium-dependent activation of secretory C1 channels has been detected in a variety of systems. In certain exocrine glands, including pancreas (Randriamampita et al., 1988), sweat gland secretory coil (Sato and Sato, 1981), and lacrimal gland (Evans and Marty, 1986a; Findlay and Petersen, 1985; Marty et al., 1984), intracellular Ca serves as the second messenger mediating agonistinduced secretion. During whole-cell recording, Evans and Marty (1986b) observed a stimulation of C1 currents by muscarinic agonists, and C1 conductance was further enhanced by intracellular dialysis with nonhydrolyzable GTP ana-
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RAYMOND A. FRiZZELL AND DAN R. HALM
logs or inositol-1.4.5-trisphosphate(lP3). The effects of GTP and IP3 were attributed to stimulation of Ca release from intracellular stores, perhaps from the endoplasmic reticulum, as well as a slow increase in Ca entry across the plasma membrane (Evans and Marty, 1986b; Llano c’t a/., 1987). It is not certain whether the activation of CI conductance in lacrimal gland is due to a direct action of Ca on C1 channels or is mediated by other Ca-dependent regulators. The CI channels corresponding to the muscarinic receptor-activated whole-cell conductance have not been detected at the single-channcl level, but this would facilitate a more direct assessment of the regulatory mechanism. Calcium-dependent regulation also has been demonstrated in airway epithelial cells, in which cell-attached activation of CI channels has been observed in response to Ca ionophore (Frizzell rr d., 1986). Interestingly, this pathway for channel activation remains intact in cystic fibrosis epithelia, including airway and sweat gland cells (Frizzell et ul., 1986; Schoumacher c’t ul., 1987b). The effect of Ca on the secretory CI channel is probably not a direct, ligand-type interaction. Once the channel is activated by Ca ionophore or other procedures in the cell-attached mode, its activity in excised patches cannot be altered by changing the free-Ca concentration bathing the cytoplasmic surface. However, recent studies (Cliff and Frizzell, 1989) show that the whole-cell CI conductance of colonic tumor cells i s activated by Ca ionophorcs or by buffering the cell Ca to lcvels of 300-400 nM. Thus, the factors that confer Ca sensitivity on this pathway are present during whole-cell recording, but do not remain intact in the excised membrane patch. The mediators of Ca-dependent channel activation require resolution, because their manipulation represents a potentially effective therapy in cystic fibrosis. Activation of protein kinase C (PKC), induced by exposure to phorbol esters, also has been shown to influence CI secretory processes in both intestinal and airway epithelia (Barthelson et a / . , 1987; Chang el nl., 1985; Fondacaro and Ilenderson, 1985; Welsh, 1987). Phorbol esters promote transient stimulation of sccretion, followed by prolonged inhibition. In experiments conceptually similar to those described above for PKA, PKC-induced activation of secretory CI channels in membrane patches excised from airway cells has been observed (Hwang et a / ., 1989; Li et al., 1989). Channel activation required the presence of PKC, ATP, and a n activating lipid such as a phorbol ester. Although channels in patches excised from normal airway cells were activated by PKC, those from CF cells were not. The similarity of PKA and PKC effects may reflect phosphorylation of a common substrate, perhaps at the same site. At higher concentrations or with longer exposure times, CI secretion across intact epithelia is inhibited by phorbol esters, and under these conditions secretory cells bccome refractory to CAMP-mediated agonists (Barthelson et ul., 1987; Welsh, 1987). The PKC pathway could be an important factor mediating the down-regulation of apical C1 channels and is therefore potentially important in the failure of CAMP-dependent processes to activate CI conductance in cystic
8. CHLORIDE CHANNELS IN EPITHELIAL CELLS
269
fibrosis epithelial cells. PKC-induced inhibition at the single-channel level requires further study, in particular, to determine the conditions under which the activation by PKA can be reversed by PKC. It is possible also that basolateral transport pathways involved in CI secretion are targets for the inhibitory effects of protein C kinase. Franklin et ul. (1988) showed that Na-K-2CI cotransport in colonic tumor cells is inhibited by phorbol esters. Finally, although calmodulin antagonists have been shown to inhibit intestinal CI secretion (Smith and Field, 1980), the possibility that calmodulin is involved in regulating the activity of single CI channels has not been examined directly. Apart from the second messenger pathways discussed above, several nonphysiological activation techniques have been identified experimentally; these seem to be effective only in the excised membrane patch. Exposure of excised patches to large depolarizing voltages for a time ranging from seconds to minutes activates the secretory C1 channel (Halm et al., 1988; Li et ul., 1988; Schoumacher et d.,1987a). This occurs in patches derived from both normal and cystic fibrosis epithelial cells and has provided a convenient assay for detecting the presence of CI channels in patches when negative results are obtained from exposure to regulatory proteins. As discussed above, channels remain inactive in excised membranes at room temperature (the condition for all of the kinase studies), but they activate when the temperature is raised to 37°C (R. Greger and M. J. Welsh, personal communications). Exposure of the internal membrane surface to high salt concentrations will also activate CI channels (J. W. Hanrahan, personal communication). The effects of voltage, temperature, and high salt could involve dissociation of a channel regulatory factor from the excised membrane, as suggested previously for voltage-induced activation (Schoumacher et ul., 1987a). Removal of a regulatory protein by these procedures could expose the channel’s conduction pathway, which is normal. However, Li et (11. (1989) recently reported that voltage- and PKA-induced activations could bc reversed by exposing the inside surface of excised patches to PKC so that physical dissociation of a channel regulatory factor by voltage seems unlikely. It should be stressed that voltage- and temperature-induced activation occurs only in the excised patch and is not observed during cell-attached (Schoumacher et a/., 1987a) or whole-cell recording (Cliff and Frizzell, 1989). This serves to reinforce the need to define the compositional differences that exist in the intact cell versus the excised membrane patch, as noted in our discussion of Ca-induced activation. (See Note Added in Proof 5 . )
111. VOLUME-SENSITIVE CHLORIDE CHANNELS Activation of CI and K conductance pathways has been implicated in the volume regulatory response to cell swelling in both epithelial and nonepithelial cells (Hoffmann, 1987). The resulting loss of cell solute (as KCI) and water constitutes a regulatory volume decrease (RVD) that returns the cells toward their nor-
270
RAYMOND A. FRIZZELL AND DAN R . HALM
T 4
nA
mV
-1
0
-2
1
Fici 9 Properties of the swelling-induced CI conductance in secretory epithelial cells. Wholecell CI currents ah a function of niemhrane voltage during swelling of T84 colonic tumor cells (Worrcll PI a / . , 1989). Both instantaneous (lillcd circles) and steady-state (open circles) currents are bhown. illustrating the outward rcctitication of instantaneous currents and the time-dependent inactivation of steady-state currcnts at dcpularizing voltagcs.
ma1 volume. The CI channels involved in this response in secretory epithelial cells have been detected recently (Worrell ef al., 1989). Their single-channel conductance and ion selectivity properties are similar to those of the secretory CI channel, but their voltage dependence differs markedly (Fig. 9). During depolarization to voltages in excess of +40 mV, the secretory CI channel shows continuous activity with a relatively high open probability (Halm et al., 1988), whereas the swelling-induced CI channel undergoes inactivation during a depolarizing voltage pulse (Worrell ct a / . , 19x9). The processes that activate the swelling-induced epithelial C1 conductance have not been identified. Ubl et ( I / . (1988) recently described a nonselective channel in an opossum kidney cell line that could be activated by mechanical stretch of the plasma tnembranc. However, the swelling-induced CI channel discussed above could not be activated by stretch, and it seems likely that metabolic pathways are involved in its activation, as observed in Erhlich ascites tumor cells (Lambert, 1987). It is important to determine whether the swelling-induced CI conductance and the secretory CI conductance reflect the presence of different CI channels or
8. CHLORIDE CHANNELS IN EPITHELIAL CELLS
271
whether the same conductance pathway has a different voltage sensitivity when activated by diRerent regulators. For example, phosphorylation could lead to an alteration in the voltage dependence of channel kinetics because negatively charged phosphate groups are added to a site that influences channel gating. The presence of two types of CI currents in secretory cells would complicate the analysis of whole-cell currents and may interfere with attempts to experimentally resolve the regulators of these CI conductance pathways.
IV. COMPARISONS AMONG ANION CHANNELS Many of the CI-selective channels that have been recorded in different cell types have not been linked with a cellular function. In this section, we compare some of the properties of CI channels obtained from epithelial and nonepithelial cells in the hope that resolving some of their distinguishing features may lead t o a preliminary classification of channel types. Ultimately, this information can provide a basis for relating channel properties to the primary structure of these proteins. Two general features that can be used to broadly classify channels are their anion permeation properties and regulatory mechanisms. Table 1 provides a listing of CI channels based on these properties.
A. Regulatory Mechanisms A number of the CI channels that have been recorded from different cell types are constitutively active. The experiments that would assess regulatory influences have, in general, not been performed. A distinction useful for grouping ion channels according to their regulatory modes separates those activated by extracellular ligands (e.g., the GABA- and glycine-gated C1 channels) from those that respond to intracellular second messenger systems. Ligand-gated CI channels have not been described in epithelial cells. The CAMP- and Ca-mediated regulation of epithelial CI channels has been discussed above. It is interesting that the CI channels detected in different secretory epithelial cells have similar properties. They are of intermediate conductance, show similar anion selectivities, and, in general, exhibit outward rectification of anion currents under symmetrical salt conditions. These similarities in their biophysical properties and the defect in their activation by PKA-mediated phosphorylation in cystic fibrosis suggests that the C F defect is linked closely to this channel and argues, together with the excised-patch activation studies summarized above, that the CF defect is linked closely to the secretory C1 channel. Nearly all CI channels show at least a weak dependence of channel open time on membrane voltage, and the voltage dependence of P,,should not be overlooked as a regulatory mechanism. In no case, however, is the voltage depen-
TABLE I CHLORIDE CHASNELS" Cell Type
Condition
Conductance
Po
P C l 'Pha
Anion selectivity
Referenceh
~~
Iu -l Iu
Epithelia Absorptive cells 0.15 M IP) Urinary bladder (rabbit) Skin (toad) 0.11 M ( E I Epididymal duct (rat) 0.14 M IP) Seuerory cells ((-AMP-dependem Cl c~hui~riels~ Trachea Human 0.15 M IP) Canine 0.15 M fP) Bovine 0.15 M ( B ) Colon T84 (human) 0.15 M (PI HT29 (human) 0.15 M (P) Rat 0.20 M ( B ) Sweat gland secretory 0.1s M (PI coil (human) Pancreatic duct Human 0.15 M IP) Rat 0.15 M (PI Rectal gland (shark) 0.29 M (PI Enterocytes (Necturus) 0.10 M (PI 0.10 M (PI Gallbladder (Necturus) Secretory cells (other CI chatinels) Trachea Human 0.15 M (P) Canine 0.15 M (PI 0. 15 M ( P ) 0. I5 M ( W ) Colon, T84 (human) Rectal gland (shark) 0.29 M (PI 0.15 M ( W ) Lacrimal gland (rat) Pancreatic acinus (rat) 0.15 M ( W )
[PI I = Br = CI > F [g] Br > CI > I -
4s ps 40 p s 70 pS
40 p s
(0) (0)
P
>I0 8 25
[PI I > Br > CI -
[gl CI > Br > F [PI I > Br > CI > F
100
[g] I = Br = CI
-
16
[PI I > Br > CI > F
10 II 12. 13
[PI I > Br > CI
I?, 15
-
[g] CI
> Br
10 10
N -
20
10
P P
30
- (0)
10 10
16 17
18
-
19
-
20
[PI CI > I = Br P P (0)
6. 7 8 9
50
(0)
ps 2-5 ps
5
P P
[PI I > Br > CI
20 p s 40 p s 20 p s 75 ps
I 2-4
-
[g] CI > Br > I [PI I > Br > CI > F
6 21 21 22 23 24. 25 26
Neural Amino ucid activated
0.15 M (P)
45 p s (s)
P
20 *
[PI I > Br > C1 >> F [g] C1 > Br 2 I >> F
Hippocampal neurons
0. I5 M (P) 0.15 M (P)
60 pS 30 pS
P
6
P
5
[PI Br > CI [PI I > Br > CI > F
0.60 M 0.14 M 0.14 M 0.10 M
(P) (P) (P) (W)
15 PS
0.10 M 0.10 M 0.10 M 0.10 M
(P) (B)
Invertebrate neurons Apl~sia Drosophilu
Squid
is)
10 p s 35 ps
- (0)
N
20 *
N
-
Z 0
-
[PI CI > F [PI CI > F
27-29 30 31 32 33 34 34
35
Muscle Skeletal Cardiac Electric organ
(P)
(B)
45 ps 60 pS 70 pS 20 ps (s)
P P
6* 6*
N
10*
N
60 *
[PI CI > Br >> F [PI C1 > F > Br [g] CI > F > Br > I
~
36. 37 36 38 39-42
h)
2
Bacteria Outer membrane
0.10 M (B)
100- 160 pS
0
I00
0.10 M (B) 0.15 M (P)
450 pS 105 pS
Z P
7*
0.15 M (P)
250-460 pS (s)
Z
4-70
0.15 M (P)
260-430 pS (s)
z. P
4- 100
43. 44
Mitochondria Outer membrane (VDAC) Inner membrane
45-47 48
5*
Maxi CI channels Epithelia Muscle Skeletal
[PI 1 2 Br = C1 > F -
1, 49-53
54-57 (cotztinued)
TABLE I (confinued) Cell Type Sarcoplasmic reticulum Smooth Neural Schwann cell Neuroblastoma Retinal rod Ganglionic neurones Blood Cells Macrophage Lymphocyte
Tumor cells Ehrlrch ascifes cells
Condition
Conductance
PCl ‘Ph,
Po
0.10 M (B)
200 p s
N
7%
0.15 M (P)
340-420 pS (s)
Z
10
0.15 M 0.10 M 0.11 M 0.05 M
450 pS 400 p s ( 5 ) 200 ps 200 p s Is)
Z N, Z
5
(P)
[P’I (P) (PI
0 I6 M (P)
220-400 p s 420 p s ( 5 )
0.15 M (P)
25 p s
0.15 M (PI
(0)
P P (5)
Z
Z
8* 100
> 10 5 10
10*
Anion selectivity (PI Br 1 CI [g] Br Z CI 2 1 -
[PI I > Br > CI
Reference’ 58 59. 60
-
61 62 63 64
-
55
-
[PI CI > F
[PI F 2 I > Br z C1
65
66
“Chloride channels are listed by the tissue type and species in which they are found. ”Condition” indicates C1 concentration and recording method: transepithelial (E), excised patches (P). bilayer incorporation (B), or whole-cell patch (W). Conductance is given for zero voltage in symmetric solutions: inward rectifying (i), outward rectifying ( 0 ) . or substates ( s ) .Voltage dependence of open probability ( P o )is shown as open primarily at negative voltages (N), open primarily at positive voltages (P), open primarily at zero voltage (Z). or no voltage dependence ( 0 ) .Dilution potentials provide a measure of relative anion-cation permeability, Pc,/PNa, with the asterisk (*‘I denoting that it is relative to PK.Anion selectivity sequences are given as [PI (from biionic reversal potentials) or [g] (from conductances). bReferences: (1) Hanrahan era/.. 1985; (2) Harck and Larsen, 1986; ( 3 ) Larsen et a/.. 1987: (4) Willumsen and Larsen, 1986: (5) Ashford, 1986; (6) Frizzell ef al.. 1986; (7) Welsh, 1986a: (8’1Welsh, 1986b: (9) Valdivia e f al.. 1988; (10) Halm e r a / . , 1988; (11) Hayslett etal.. 1987; (12) Bridges etal.. 1989; (13) Reinhardt ef a!.. 1987; (14) Krouse etal.. 1987: (15) Schoumacher etal.. 1987b; (161 Schoumacher e r a l . , 1988; (17)Gray etal.. 1988; (18) Greger etal.. 1987: (19)Giraldez et al.. 1988; (20) Segal and Reuss, 1989; (211 Shoemaker ef al., 1986; (22) Worrell er al.. 1989; (23) Gogelein eta!.. 1987; (24) Evans and Marty. 1986a: (25) Marty ef a / . , 1984; (26) Randriamampita etal. 1988: (27) Bormann er al., 1987; (28) Cull-Candy and Ogden. 1985; (29) Gray and Johnston, 1985; 130)Hamill ef a/..1983; (31) Franciolini and Petris. 1988: (32)Franciolini and Nonner, 1987; (33) Chesney-Marchais and Evans. 1986; (34) Yamamoto and Suzuki. 1987: (35) Inoue, 1985. (36) Blatz and Magleby, 1985; (37) Blatz and Magleby. 1986; (38) Coronado and Latorre, 1982; (39) Kanemasa et a l . . 1987: (40) Miller. 1982; (41) Miller and White, 1980;(42) White and Miller. 1979; (43) Armstrong etal. . 1986; (44)Benz and Hancock, 1987; (45) Colombini, 1979; (46) Forte ef a/., 1987: (47) Schein et a / ., 1976; (48) Sorgato e t a / ., 1987; (49) Kolb et al. , 1985: (501 Krouse et a/. , 1986: ( 5 I ) Nelson et ul., 1984: ( 5 2 )Schmid et a/.. 1988; (53) Schneider et a / . . 1985; (54) Blatz and Magleby. 1983; (551 Schwarze and Kolb. 1984; (561 Woll et a/..1987; (57) Woll and Neumcke, 1987: (58) Tanifuji et a l . , 1987; (59) Coleman and Parkinton, 1987; (60) Soejima and Kokubun, 1988; (61) Gray et a/. , 1984; (62) Bolotina et a / . , 1987; (63)Kolesnikov et al., 1984; (64)Geletyuk and Kazachenko, 1985: (65) Bosma. 1989; (66) Hudson and Schultz. 1988. ~
8. CHLORIDE CHANNELS IN EPITHELIAL CELLS
275
dence of C1 channel activity as steep as that observed for the Na and K channels of excirable membranes (Hille, 1984). Thus, voltage dependence may not be a primary mode of channel regulation, but is probably secondary to other modes of activation, as discussed above for secretory CI channels. Another example of voltage dependence secondary to other transport events is the C1 conductance of amphibian skin, which is activated when the transepithelial voltage is depolarized during stimulation of Na absorption. 8. Anion Permeation Classification of' Cl channels on the basis of their conductances yields three basic categories. Small-conductance CI channels (4-20 pS) are found in electroplax and a few other cell types, including some secretory epithelial cells. Studies of small-conductance CI channels is hindered by technical difficulties associated with detecting small current steps within the background noise and the activity of other channels. The medium-conductance CI channels, ranging from 30 to 70 pS, and the maxi CI channels, at 250-500 pS, appear to be widely distributed. The anion channels of secretory epithelial cells fall predominantly into the intermediate-conductance range. Conductance substates have been described in a number of channel types. Substates indicate that channels have open configurations with different singlechannel conductances, which appear as distinct current levels. For example, the electroplax Cl channel behaves as though it is composed of two equally conductive pores (Miller, 1982). These pores gate independently, but when the channel is reconstituted into lipid bilayers, they always appear together. Substate conductances also are observed in the maxi CI channel (Geletyuk and Kazachenko, 1985; Krouse et a l . , 1986). Many equal-sized current steps often are evident, and they frequently appear as small steps near the maximal current level. This suggests that the maxi channel may be composed of multiple (5 to 15) pores that gate together, so that a single large current step is commonly seen. Substates also may reflect changes in channel conformation that led to a change in conductance. The amino acid receptor channels show four open-state conductances (Bormann el u l . , 1987; Hamill et a / . , 1983), some of which may be multiples of lower levels. Interestingly, the ligand that gates channel activity determines the predominant open-state conductance. When glycine opens the channel, over 70% of the open time is spent in the largest conductance state (45 pS), whereas GABA opens the channel such that about 80% of the open time is spent in the third conductance state (30 pS). Anion selectivity sequences could provide an additional means of grouping C1 channels into different classes, but little discrimination can be made on the basis of the available data. Three selectivity sequences have been observed, as determined from reversal potentials under biionic conditions. By far, the most common pattern is Eisenman sequence I, I > Br > C1 > F (Wright and Diamond,
276
RAYMOND A. FRIZZELL AND DAN R. HALM
1977). Two other sequences have been observed: sequence V in the electroplax (CI > Br >> F) and sequence VI in the outer mcmbrane of bacteria (CI > F > Br). These findings indicate that the conduction pathway of most CI channels behaves similarly with regard to ion-channel interactions. In addition, pcrmeability ratios greater than 3 : 1 are uncommon. This situation contrasts with many cation-selective channels, which discriminate strongly among cations of identical charge. All anion-selectivc C1 channels observed thus far select for CI over HCO,, the next most common anion in biological systems. It will be interesting to see if channels primarily selective to anions other than C1 are discovered. We could learn more about the factors that determine anion selectivity from such comparisons. As more anion channels reveal their primary structures, it will be interesting also to assess their similarities to each other and to the cation channels; e.g., marked homology exists in the primary structures of the amino acid receptor CI channels and Na channels (Schofield et a / . , 1987). The factors that determine the channel’s architecture, as opposed to its selectivity and gating, may be revealed by such comparisons. REFERENCES Adelsherger-Mangan, D. M., and Colombini, M . ( 1987). Elimination and restoration of voltage dependence in the niitochondrial channel, VDAC. hy graded modification with succinic anhydride. J . Mo?ihr. B i d . 98, 157-168. AI-Awqati, Q., Field, M.. and Creenough, W. B . . 111 (1074). Reversal of cyclic AMP-mediated intestinal secretion hy cthacrynic acid. J. C’liri. f r t w s i . 53, 687-692. and Hancock, K. E. W. (1986). Bnrt/e/el/a Armstrong, S. K . . Parr, T. R . , Jr., Parkcr, C. I)., pur/u.ssis major outer membrane porin protein forms small. anion-selective channels in lipid bilayer mcntbranes. 1.Bacruriol. 166, 212-216. Ashford. M . L. J. (1986). Single chloride channel currents in cultured rat epididymal cells. J. Phvsiol. ( L ~ ) i d o r371, i ) 142P (abstr.). Barthelson, R. A , , and Widdiconibe, J. H. (1987). Cyclic adenosine tilonophosphate-dependent kinase in cystic librosis tracheal epithelium. J . Clin. /rivesf.80, 1799- 1802 Rarthelson, R . A,, Jacohy. 1). R . . and Widdicornbe. J. H. (1987). Regulation of chloridc secretion in dog tracheal epithelium hy protein kinase C. Am. J. Physiol. 253, C802-CX08. Bear. C. E. ( 19x8). I’hosphorylation-activated chloride channels in human skin fibroblasts. FEBS L w . 237, 145 140. Hcnz, R., and Ilancock, R . E. W. (19x7). Mechanism of ion trdnsport through the anion-selective channel of Pscwforrtowtts trtwginosti outer membrane. J , Gm. Phvsiol. 89, 275-295. Rlatz, A. L.. and Magleby, K . L. (1983). Single voltage-dependent chloride-selectivc channels of large conductance in cultured rat muscle. R i ~ p h J. ~ 43, . 237-241. Blatz, A. L., and Magleby, K . L. (19x5). Single chloride-selective channels active at resting membrane potentials in cultured rat skeletal muscle. Biophvs. J . 47, 119- 123. B l a t ~A , . L.. and Magleby, K . L (1986). Quantitativc description of three modes of activity of fast chloride channels from rat skeletal muscle. J . Phvsiol. (Loridori) 378, 141- 174. Bolotina, V.. Borecky, J.. Vlachova, V., Baudysova, M., and Vyskocil. F. (19x7). Voltagcdependent chloridc channels with sevcral suhstaies in excised patchcs from niouse neuroblastoma cells. N t w o x i . L ~ r r 77, . 298-302. Bormann, J.. Hatiinrill. 0. P.. and Sakinann. R . (1987). Mechanism of anion permeatton through ~
8. CHLORIDE CHANNELS IN EPITHELIAL CELLS
277
channels gated by glycine and y-aminobutyric acid in mouse cultured spinal neurones. J . Physiol. (London) 385, 243-286. Bosnia, M. (1989). Anion channels with multiple conductance levels in a mouse B lymphocyte cell line. J . Phvsiol. (Lodon) 410, 67-90. Bridges, R . J . , Worrell. R . T., Frizzell, R . A . . and Benos, D. I. (1989). Stilbene disulfonate blockade of colonic secretory CI channels in planar lipid bilayers. Am. J. Physiol. 256, C902-C912. Chang. E. B., Wang. N. S . , and Rao, M. C. (1985). Phorbol ester stimulation of active anion secretion in intestine. Am. J . P h w i o l . 249, C356-C36l. Chen, J . H., Schulman, H., and Gardner, P. ( 1989). A CAMP-regulated chloride channel in lymphocytes that is affected in cystic fibrosis. Sciertcr 243, 657-660. Chcsncy-Marchais. D., and Evans, M. G . (1986). Chloride channels activated by hyperpolarization in Aplwiu neurons. F‘flurgers Arch. 407, 694-696. ClitT, W. H., and Frizzcll, R. A. (1989). CAMP- and Ca-mediated regulation of CI conductances in the human colonic cell line T84. Ped. Pulmo,i. Suppl. 4, 123. Coleman, H. A , , and Parkinton. H. C. (1987). Single channel chloride and potassium currents from cells of uterus not treated with enzymes. eflur,qer.s Arch. 410, 560-562. Colombini. M. ( 1979). A candidate for the permeability pathway of the outer mitochondrial membrane. Nuturr (London) 279, 643-645. Coronado, R . , and Latorre, R . (1982). Detection of potassium and chloride channels from calf cardiac sarcolemma in planar lipid bilayer membranes. Nature (London) 298, 849-852. Cull-Candy, S . G . , and Ogden. D. (1985). Ion channels activated by 1.-glutamate and GABA in cultured cerebellar neurons of the rat. Pror. R . Soc. Loridon B 224. 367-373. Dill. E. T., Holden. M. J . . and Colombini. M. (1987). Voltage gating in VDAC is markedly inhibited by micromolar quantities of aluminum. J . Memhr. Biol. 99, 187- 196. DiStefano. A . Wittncr. M., Schlatter, E . , Lang, H. J., Englert. H., and Greger, R . ( 1985). Diphenylamine-2-carboxylate, a blocker of the chloride conductive pathway in chloride transporting epithelia. PJuegers Arch. 405 (Suppl. I), S95-Sl00. Drcinhofcr. J . , Giigelcin, H.. and Grcger. R . (1988). Blocking kinetics of chloride channels in colonic carcinoma cells (HT29) as revealed by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Biochim. Biophys. Actu 946, 135- 142. Duffcy, M. E., Turnheim, K . , Frizzell, R . A,. and Schultz, S . G . (1978). Intracellular chloride activities in rabbit gallbladder: Direct evidence for the role of the sodium gradient in energizing “uphill” chloride transport. J . M r m b r . B i d . 42, 229-239. Eisenman, C . , and Horn, R . (1983). Ionic sclcctivity revisited: The role of kinetic and equilibrium processes in ion permeation through channels. J . Memhr. B i d . 76, 197-225. Evans, M. G . , and Marty, A. (1986a). Calcium-dependent chloridc currcnts in isolated cells from rat lacrimal glands. J . P h w i o l . (London) 378, 437-460. Evans, M. G . . and Marty, A . (1986b). Potentiation of niuscarinic and a-adrenergic responses by an analogue of guanosinc S’hphosphate. Pro(.. N u t / . Acud. Sci. U . S . A . 83, 4099-4103. Evans. M. G.. Marty, A , , Tan, Y. P.. and Trautmann. A . (1986). Blockage of calcium-activated chloride conductance by furosemide in rat lacrimal glands. fflurgers Arch. 406, 6.5-68. Eyring, H. (1936). Viscosity, plasticity and diffusion as examples of absolute reaction rates. J. Chrm. Phys. 4, 283-291. Findlay, I . , and Petersen. 0. H. ( 1985). Acctylcholinc stimulates calcium-dependent chloride conductance in mouse lacrimal acinar cells. PJuegrrs Arch. 403, 328-330. Fondacaro, J . D., and Henderson. L. S . (1985). Evidence for protein kinase C as a regulator of intestinal electrolyte transport. Am. J . Phvsiol. 249, G422-(3426. Forte, M . , Adelsbcrger-Mangan. D., and Colombini, M. (1987). Purification and characterization of the voltage-dependent anion channel from the outer mitochondrial membrane of yeast. J . Memhr. B i d . 99, 65-72.
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Franciolini, F., and Nonner, W. (1987). Anion and cation permeability of a chloride channel in rat hippocanipal neurons. J . Gun. Plty.sio/. 90, 453-478. Franciolini. F., and Pctris, A. (19x8). Single chloride channels in cultured rat neurons. Arch. Riorhrm. Rinphys. 261, 97- 102. Franklin, C. C., Turner. J . T.. and Kin), H. D. (1988). Down-regulation of 'H-bunictanide binding sites accompanies inhibition of NaIKICI cotransport in response to PMA in HT29 cells. J . Gen. Physiol. 92, 27a (abstr ) Frizzell, R. A. (1987). Cystic fibrosis: A disease of ion channels? Trer7ds NeuroSci. ( P u s . E d . ) 10, 190- 193. Frizzell, R . A , , Field. M . , and Schultz, S . G. (1979). Sodium-coupled chloride transport by epithclial tissues. Am. J . Phv.siol. 236, FI -F8. Frizzell, R . A . . Rcchkemnrcr. G . , and Shncmakcr, R . 1.. (19x6). Altered regulation of airway epithelial cell chloride channels in cystic libroais. Scirnce 233, 558-560. Garcia-lhz, J. F., Corciu, A,, and Armstrong. W. ( 1983). lntracellular chloride activity and apical iiicmbranc chloridc conductance in N w f u r w Falibladder. 1. Membr. B i d . 73, 145- 155. Geletyuk. V. I.. and Kazachenko, V. N. ( 1985). Single chloride channels in molluscan nrurones: Multiplicity of the conductancc states. J . Mcmhr. B i d . 86, 9- I S . Giraldez, F., and Sepulveda, P. V. ( 1987). Changes in the apparent permeability of Necturus enterocytes during the $odium-coupled transport of alamine. Biodiim. Rinphvs. Acta 898, 248-252. Giraldcz, F., Scpulvcda, F. V.. and Shcppard, D. N. (1988). An outwardly rectifying Clk-selective channel in isolated Nccrrrrus enterocytes. J . P h y i o l . (Londnti) 407, 108P (abstr.). Gbgclcin, H. (1988). Chloride channels in epithelia. Biochirn. Riophvs. Actu 947, 521 -547. GOgelein, H.. and Greger, R . (1986). A voltage-dependent ionic channel in the basolateral membranc of late proximal tubules ofthc rabbit hidncy. F'//rrugrrsArch. 407 (Suppl. 2),S142-SI48. GOgelcin, H., Schlatter, E.. and Grcger, R. (1987). The "sindl" conductance chloridc channcl in the luniinal nienihrane of the rectal gland of the dogfish (Squalrt,s aranrhias). Pjueger.y Arch. 409, 112-125. Gray. R . . and Johnston. D. (198.5). Rectification of single CABA-gated chloride channels in adult hippocainpal neurons. J . Nuirrophvuiol. 54, 134- 142. Gray, M. A., Greenwell, J. R., and Argent, B. E. (1988). Secretin-rcgulatcd chloride channel on the apical plasma membrane of pancreatic duct cells. J . Membr. Riol. 105, 131 142. Gray, P. T. A , , Bevan, S . , and Ritchie, J. M. (1984). High conductance anion-selective channels in rat cultured Schwann cells. Proc. R . Soc. London B 221, 395-409. Grcger, R . (1989). Ion transport mechanisms in thick ascending limb of Hcnlc's loop of mammalian nephron. Phwiol. Rn,. 65, 760-797. Greger. R., and Schlatter, E. (1983). Properties of the basolateral membrane of the cortical thick ascending limb of Henle's loop of rabbit kidney: A model for secondary active chloridc transport. Pfluegrrx Arch. 396, 325-374. Greger, R . , Schlatter, E..and Gogelcin. H. ( l 9 X S ) . Chloride channels in the apical cell membrane ot' the rectal gland "induced" by CAMP. F'//uvgcrs Arch. 403, 446-448. Greger. R., Schlatter, E . . and Gtigelein, H . (1987). Chloride channels in the luminal membrane of thc rectal gland of the dogtish (Squulus wcrrithicrs): Properties of the "larger" conductance channel. Pfluqers Arch. 409, 114- 121. Halm. D. R.. and Frizzell, R. A . (198X). Ion permeation through the apical memhrane chltrride channel in a secretory epithelial cell. FASEB J . 2, A1283 (dbstr.). Halm, D. R.. Krasny, K. J . , J r . , and Frizzell, R. A. (1984). Electrophysiology of Hounder intcstinal mucosa: I . Conductance properties of the cellular and paracellular pathways. J . Gen. Physiol. 85, 843-864. Halm, D. R . , Rechkemmer, G . R., Schoumacher, R. A,, and Frizzell, R. A. (1988). Apical membrane chloride channels in a colonic cell line activated by secretory agonists. A m . J. Phvsiol. 254, C505-C5 I I . -
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Hamill, 0. P., Bormann, J . , and Sakmann, B. (1983). Activation of multiple-conductance state chloride channels in spinal ncurones by glycine and GABA. Nature (London) 305, 805-808. Hanrahan, J. W., Alles, W. P., and Lewis. S. A. (1985). Single anion-selective channels in basolatcral membrane of a mammalian tight epithelium. Proc. Natl. Acad. Sci. U . S . A . 82, 7791-7795. Harck, A. F., and Larsen. E. H. (1986). Concentration dependence of halide fluxes and selectivity of the anion pathway in toad skin. Actu Phvsiol. Scand. 128, 289-304. Hayslett, J . P., Gogelein, H.. Kunzeirndnn, K., and Greger, R. (1987). Characteristics of apical chloride channels in human colon cells (HT,,). F‘flr4egers Arch. 410, 487-494. Hille, B. (1984). “Ionic Channels of Excitable Membranes.” Sinauer, Sunderland, Massachusetts. Hoffmann, E. K. (1987). Volume regulation in cultured cells. Curr. Top. Memhr. Transp. 30, 125-180. Hudson, R . L., and Schultz, S. G. (1988). Sodium-coupled glycine uptake by Ehrlich ascites tumor cells results in an increase in cell volume and plasma membrane channel activities. Proc. Natl. Acad. Sci. U.S.A. 85, 279-283. Hwang, T.-C., Lu, L., Zeitlin, P. L., Greunert, D. C., Huganir, R . C., and Guggino. W. B. (1989). CI channels in CF: Lack of activation by protein kinase C and CAMP-dependent protein kinase. Science 244, 135I 1353. Inoue, I . (1985). Voltage-depcndent chloride conductance of the squid axon membrane and its blockade by some disulfonic stilbene derivatives. J . Gen. fhysiol. 85, 5 19-537. Kanemasa, T , Banba, K., and Kasai, M. (1987). Voltage-gated anion channel of the electric organ of Nurke japonica incorporated into planar bilayers. J . Biochem. (Tokyo) 101, 1025- 1032. Koefoed-Johnscn, V., and Ussing, H. H. (1958). The nature of the frog skin potential. Acta Phvsiol. Scand. 42, 298-308. Kolb, H. A,, Brown, C. D. A , , and Murer. H. (198.5). Identification of a voltage-dependent anion channcl in the apical membrane of a chloride-secretory epithclium (MDCK). fjuegers Arch. 403, 262 - 265. Kolesnikov, S . S . . Lyubarsky, A. L., and Fesenko. E. E. (1984). Single anion channels of frog rod plasma membrane. Vision Res. 24, 1295 - 1300. Krasne, S. (1978). Ion selectivity in membrane permeation. I n “Physiology of Membrane Disorders” (T. E. Andreoli, J. F. Hoffman, and D. D. Fanestil, eds.). pp. 217-241. Plenum, Ncw York. Krouse, M. E., Schneider, C . T., and Gagc. P. W. (1986).A large anion-selective channel has seven conductance levels. Nuture (London) 319, 58-60. Krouse, M. E.. Hagiwara. G . . Lewiston, N. J.. and Wine, J. J. (1987). Ion channel activity in apical membranes of cultured sweat gland secretory cells. f e d . Pulmon.. Suppl. 1, I14 (abstr.). Lambert, I. H. (1987). Effect of arachadonic acid, fatty acids. prostaglandins and leukotrienes on volume regulation in Ehrlich ascites tumor cells. J . Memhr. B i d . 98, 207-221. Landry, D. W., Reitman, M . , Cragoe, E. J . , Jr., and Al-Awqati, Q. (1987). Epithelial chloride channels: Development of inhibitory ligands. J . Gen. Phvsiol. 90,779-798. Larsen, €3. H.. Ussing, H. H., and Spring, K. R. (1987). Ion transport by mitochondrial-rich cells in toad skin. J . Mrmhr. B i d . 99, 25-40. Li, M., McCann, J. D., Liedtke, C. M., Nairn, A. C., Greengard, P., and Welsh, M. J . (1988). Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic tibrosis airway epithelium. Nature (London) 331, 358-360. Li, M., McCann. J. D., Anderson, M. P., Clancy, J . P., Liedtke, C. M., Nairn, A. C., Greengard, P., and Welsh, M. J. (1989). Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia. Science 244, 1353- 1356. Llano, I . , Marty, A., and Tanguy. J. (1987). Dependence of intracellular effects of GTPyS and inositol trisphosphate on cell membrane potential and on external calcium ions. ffluegers Arch. 409,499-506. -
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Marty, A., Tan. Y. P., and Trdutniann. A. (1984). Three types ofcalcium-dependent channel in rat lacrimal glands. J. Ph.v.~iol.(London) 357, 293-325. Miller, C. ( 1982). Open-state substructure of single chloride channels from Torpedo electroplax. Philos. Trons. X. Sol.. London B 299, 401 -4 I I . Millcr, C . , and White, M. M. (1980). A voltage-dependent chloride conductance channel from Torpedo clcctroplax membrane. Ann. N . Y . Arad. Sci. 341, 534-551. Nelson, D. J., Tang, J. M.. and Palmer. L. G . (1984). Singlc-channel recordings of apical mcrnbrane chloridc conductance in A6 epithelial cells. J. Memhr. B i d . 80, 81 -X9. Novak, I., and Greger, R. (1988). Properties of' the luminal membrane of isolated perfused rat pancreatic ducts. Effect of cyclic AMP and blockers of chloride transport. Pfluegcrs Arch. 41 I , 546- 553. Oberleithner, H.. Kittcr. M., I m g , F.. and Guggino. W. (19x3). Anthracene-9-carboxylic acid inhibits renal chloride reahsorption. Yflurgrrs Arch. 398, 172- 174. Palfrey, H. C., and Grecngard, P. ( 1981). Hormone-sensitive ion transport systems in erythrocytes as models for epithelial ion pathways. Ann. N.Y. At&. Sci. 372, 291-008. Perkins, F. M . , and Handler, J. S. (1981). Transport properties of toad kidncy cpithclia in culture. Am. J. Phvsiol. 241, C154-CI59. Petersen, K . , and Reuss, L. (1983). Cyclic-AMP induced chloride permeability in the apical rnernbrane of Necturus gallbladder epithelium. J . Gun. Phyiol. 81, 705-729. Randrianiampita, C., Chanson, M., and Trautmann, A. (1988). Calcium and secretagogue-induccd conductanccs in rat exocrine pancreas. f/luuxers Arch. 41 I , 53-57. Reinhardt, R., Bridges, R. J., Rummel, W., and Lindemann, B. (1987). Properties of an anionsclcctive channel from rat colonic cntcrocytes plasma membranes reconstituted into planar phospholipid bilaycrs. J. Memhr. Riol. 95, 47-54. Rcuss, L. ( 1989). Ion transport across gallbladder epithelium. P h y i o l . Rev. 69, 503-545. Reuss, L . , Constantin. J. L., and Bazile. I. E. ( 1987). Diphenylamine-2-carboxylate blocks CIHCO, exchangc in Necturus gallbladder epithclium. Ant. J . Phvsiol. 253, C79-C89. Sato, K . , and Sato, F. (1981). Role of calcium in cholinergic and adrcncrgic mechanisms of sweat secretion. q / / u q r r s Arch. 390, 49-53. Schein. S. J., Colonibini. M.. and Finkelstein, A. (1976). Rcconstitution in planar lipid bilayers of' a voltage-dependent anion-selectivc channel obtained from Ptrrumrcium mitochondria. J . Memhr. B i d . 30, 99- 120. Schniid, A., Gogelein, H., Kemrner, T. P , and Schultz, I. (1988). Anion channels in giant liposomes made of cndoplasmic reliculuni vesicles from rat exocrine pancreas. J . Memhr. Riol. 104, 275-282. Schneidcr, G . T., Cook. D. I., Cage, P. W., and Young, J. A. (1985). Voltage sensitive, highconductance chloride channels in the luminal membrane of cultured pulmonary alvcolar (type 11) cclls. F'/lur,qers Arch. 404, 354-357. Schotield, P. R., Darlison. M. G., Fujita, N., Burt. D. R . , Stephenson, F. A , , Rodriguez, H., Rhee, L. M.. Ramachandrdn, J . , Rcale, V., Clencorse, T. A , , Sccburg. P., and Barnard. E. A. (1987). Sequence and functional expression of the GABA, receptor shows a ligand-gated receptor super-family. Nufitre (London) 328, 221 -227. Schounrachcr, R . A.. Shoemaker, R. L., Halm. D. R., Tallant. E. A,, Wallace. R. W., and Frizzcll, R. A. (1987a). Phosphorylation fails to activate chloride channels from cystic tihrosis airway cells. Nuturc (Londun) 330, 752-754. Schoumacher, R . A . , Shoemaker, R. L., and Frizzcll. R . A. (1987b). Abnormal regulation of apical membrane chloride channcls in sweat gland sccretory cells in cystic librosis. Frd. Proc., Fed. Am. Soc. Exp. B i d . 46, 1272 (abstr.). Schmid, S. M . , and Fri7.7~11,R . A . ( 1988). Expression of the cystic Schoumacher, R. A., Ram, .I., tihrosis defect in cancer cclls cultured from a cystic tibrosis patient with pancreatic adenocarcinonia. Prd. Pulmon. Suppl. 2, 109 (abstr.).
8 . CHLORIDE CHANNELS IN EPITHELIAL CELLS
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Schwarze, W., and Kolb, H. (1984). Voltage-dependent kinetics of an anionic channel of large unit conductance in macrophages and myotube membranes. ~ f l u e g e r sArch. 402, 281 -291. Segal, Y . , and Reuss, L. (1989). Chloride channels in cyclic AMP stimulated gallbladder epithelium. FASEB J . 3, A862 (abstr.). Shoemaker, R. L., Frizzell, R . A , , Dwyer, T. M., and Farley. J. M. (1986). Single chloride channel currents from canine tracheal epithelial cells. Biochim. Biophvs. Acia 858, 235-242. Smith, P. L. (1985). Electrolyte transport by alkaline gland of little skate, Ruju erinacea. Am. J. Physiol. 248, R346- R352. Smith, P. L., and Field, M. (1980). 1n Litro antisccretory effects of trifluoperazine and other neuroleptics in rabbit and human small intcstine. Gustroenterologv 78, 1545- 1554. Soejima, M., and Kokubun, S . (1988). Single anion-selective channels and its ion selectivity in the vascular smooth muscle cell. Pfluegers Arch. 411, 304-31 I . Sorgato, M. C., Keller, B. U., and Stuhmer, W. (1987). Patch-clamping of the inner mitochondria1 membrane reveals a voltage-dependent ion channel. Nuiure (London) 330,498-500. Stetson, D. L., Beauwens, R . , Palmisano, J., Mitchell, P. P., and Steinmetz, P. R. ( 1985). A doublemembrane model for urinary bicarbonate secretion. Am. J. Physiol. 249, F546-FS52. Tanifuji, M., Sokabe, M., and Kasai, M. (1987). An anion channel of sarcoplasmic reticulum incorporated into planar lipid bilayers: Single channel behavior and conductance properties. J. Membr. B i d . 99, 103- I 1 I . Ubl, J., Murer, H., and Kolb, H. (1988). Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells. J . Membr. B i d . 104, 223-232. Valdivia, H. H., Dubinsky, W. P., and Coronado. R. (1988). Reconstitution and phosphorylation of chloride channels from airway epithelium membranes. Science 242, 1441 - 1444. Voute, C. L., and Meier. W. (1978). The mitochondrial-rich cell of frog skin as hormone-sensitive . 151- 165. “shunt-path.” J . Mcmbr. B i ~ l 405, Wangemann, P., Wittner, M., DiStefano, A , , Englert, H. C., Lang, H. J . , Schlatter, E., and Greger, R. (1986). Chloride-channel blockers in the thick ascending limb of the loop of Henle: Structure activity relationship. Pfluegers Arch. 407, S128-SI41. Welsh, M. (l986a). An apical-membrane chloride channel in human tracheal epithelium. Science 232, 1648- 1650. Welsh, M. J. (l986b). Single apical membrane anion channels in primary cultures of canine tracheal epithclium. Pfluegers Arch. 407 (Suppl. 2). SI 16-SI22. Welsh, M. J. (1987). Effect of phorbol esters and calcium ionophore on chloride secretion in canine tracheal epithelium. Am. J . Phvsiol. 253, C828-C834. Welsh, M. J., and Liedtke, C. M. (1986). Chloride and potassium channels in cystic fibrosis airway epithelia. Nature (London) 322, 467-470. Welsh, M. J . , Smith, P. L., and Frizzell, R . A. (1983). Chloride secretion by canine trachea epithelium: 111. Membrane resistances and electromotive forces. J . Membr. B i d . 71, 209218. Weymer, A , , Huott, P., Liu, W., McRoberts, J. A, and Dharmsathaphorn, K. (1985). Chloride secretory mechanism induced by prostaglandin E, in a colonic epithelial cell line. J . Clin. Invest. 76, 1828- 1836. White, M. M., and Miller, C. (1979). A voltage-gated anion channel from the electric organ of Torpedo cdfornicu. J . Biol. Chem. 254, 10161- 10166. White, M. M., and Miller, C . (1981). Chloride permeability of membrane vesicles isolated from Torpedo cdij‘ornica electroplax. Biophvs. J . 35, 455-462. Widdicombe, J. H., and Welsh, M. J. (1980). Anion selectivity of the chloride-transport process in dog tracheal epithelium. Am. J . Phvsiol. 239, CI 12-CI 17. Willunisen, N. J . , and Larsen, E. H. ( 1986). Membrane potentials and intracellular chloride activity of toad skin epithelium in relation to activation and deactivation of the transepithelial chloride conductance. J . Mcmbr. B i d . 94. 173- 190.
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Woll. K . H., and Ncuincke, B. (1987). Conductance properties and voltage dependence of an anion channel i n amphibian skeletal muscle. P f l i t e p w Arch. 410,641 -647. Woll. K. H., Lcibowiti. M. D., Neurncke, B . . and Hille, B. (1987). A high-conductance anion channel in adult amphibian skeletal muscle. p/hieger.v Arch. 410,632-640. Wurrell, R. T.,Butt. A. G . , Cliff, W. H., and Frizzell, R . A. (1989).A volume-sensitive chlorideconductance in human colonic cell line T84.Am. J . P h p i o l . 256, CI I I I-CI 119. Wright, E. M., and Diamond, J. M. (1977). Anion selectivity in biological systems. Physiul. Rev. 57, IOY- 156. Yanianioto, D., and Suzuki, N. (1987). Blockagc of chloride channels by HEPES buffer. P r o r . R . Suc. Lunilon U 230, 93- 100.
NOTE him>I N PROOF. Since writing this review, several notable developments have occurred in this rapidly expanding field. (I)A 40 pS CI channel with ohmic conductance that is activated by CAMP has heen recorded from the baSOlaterd1 inemhrane of thick ascending limh cells (Paulais, M., and Teulun, J . (1990). CAMP-Activated CI channel in thc basolateral membrane ofthe thick ascending limb of the mousc kidney. J . Memhr. B i d . 113, 253-260).Activation by cAMP and blockade by diphenylainine-2-carboxylateis consistent with the known CI conductance properties of this memy ~ . heen ,) estimated for the outwardly brane. (2)The relative bicarbonate conductancc ( ~ , , ~ ~ , , / has rectified CI channel in T84 cells and in the pancreatic duct cell line, PANC-I (Tabcharani. J. A , , Jensen, T.J., Riordan, J. R . , and Hanrdhdn, J. W. (1989).Bicarbonate permability ofthe outwardly at negative voltages was 0.35, rectifying anion channel. J . Mernhr. B i d . 112, 109- 122).yHc.,,,/ycI but conductance ratios greater than I wcre ohscrved at positive voltages with high external pH, indicating that this channel could contribute to HCO, secretion. ( 3 ) The biophysical properties of a 20 pS CI channel in airway cells have bccn characterized (Duszyk, M . , French, A. S.. and Man, S. F. P. (1'9'901. The 20 pS C1 channel of the human airway epithelium. Bioph.v.s. J . 57, 223-230) and claimed to bc activated by PKA in normal, but not CF, excised membrane patches (Duszyk, M., and French, A. S. (1989). Cystic fihrosis affects C1 and Na channels in human airway epithelia. Con. J . Pkvsiol. Phormocd. 67, 1362- 1365).(4) A CAMP-stimulated 5 pS CI channel was detected in apical membranes of thyroid cells (Champigny, C i . . Verrier, B., Gerard, C . , Manchamp, J . , and Ixdunski, M. (1990). Small conductance CI channels in thc apical membrane of thyroid cells. FEBS Left. 259, 263-268).This channcl is similar to that reported hy Gray ef ul. (1988) for pancreatic duct. (5) Using whole-cell recordings, CI conductances with different biophysical properties were activated by cAMP and Ca in T84 cells, implying that different CI channels are responsible for the CAMP- and Ca-activated CI conductances (Clilf, W.'H.. and Frizzell, R. A. (1990). Separate CI conductances activated hy CAMP and Ca in CI secreting epithelial cells. Proc. N o t / . Arad. Sci. U.S.A. 87, 493-4960),Interestingly, the properties of the outwardly rectified CI channcl are difficult to reconcilc with the whole-ccll conductance activatcd by CAMP, but !it well with that activated by Ca.
CURKENT TOPICS IN MEMBRANbS AND TRANSPORT. VOLIJMt 17
Chapter 9 Reconstitution of Epithelial Ion Channels ROBERT J . BRIDGES A N D DALE J . BENOS Department of Ph,vsiology arid t3iophysic.s T ~ Universir?, K of Alabama (11 Birmitigkrrrn Birmingham. A/ahwnci 352Y4
I . Introduction Isolation and Purilication of Specihc Membrane Vesicle Populations Bilayer Techniques Used in Reconstitution Ion Channel Incorporation into Planar Lipid Bilayera Reconstitution of Ion Channels from Epithelia into Planar Phospholipid Bilaycrs A . Epithelial Sodium Ion Channels B. Epithelial Chloride Ion Channels C. Epithelial Potassium Ion Channels VI. Conclusions References 11. 111. IV. V.
1. INTRODUCTION
In recent years, there has been a widespread appreciation of the central role that ion channels play in the life of a cell. Many physiological functions, such as the conduction of nerve and inuscle impulses, pacemaker activity, hormone secretion, fertilization, and epithelial salt and water absorption and secretion, require appropriate and exquisitely timed changes in ion channel-mediated conductances (Hodgkin and Huxley, 1952; Dawson et a / . , 1982; Miyazaki and Igusa, 1982; Dale and DeFelice, 1984; Dubinsky and Oxford, 1984; Matteson and Armstrong, 1984; DeFelice e t a / ., 1986). With the myriad of different transporters existing in a cell, that is, pumps, carriers, and channels, ion channels catalyze by far the “simplest” type of transport, namely, diffusion down gradients of electrochemical potential energy. One major inherent characteristic of ion channels that has, in the past, limited their study is their high transport rate; ion channels can easily conduct lo6- 10nions/sec. As such, a cell does not need very many of these entities to accomplish a significant rate of movement of ions. For 283
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example, a typical nerve cell body may contain 1000 Na' channels, and a typical epithelial cell may have 100 to 1000 Na' and/or CI channels. Contrast these numbers with those of the active-transport Na ,K+-ATPase pump, wherein a cell might contain anywhere from 100,000 to 10 million units. Special electrical measuring systems have therefore been dcvised to record current flow through single ion channels. Also, intricate techniques have evolved in order to fish these proteinaceous ion channels from the dilute sea of lipid constituting the plasma membrane to accomplish the seeniingly formidable task o f studying the molecular properties of these elusive members of the cellular transportation system. The development of these techniques to study channels and their high transport rate now permits a detailed kinetic and pharmacological evaluation of siriglr ion channels at the molecular level to be undertakcn. Investigators interested in pumps, cotransportcrs. and countertransporters, on the other hand, are still relegated to macroscopic or population studies because of the low turnover rates of these transport systems. The most compelling evidence for the existence of ion channels in membranes has come from the direct recording of quanta1 current jumps produced by the random opening and closing of individual transport proteins (Neher and Sakmann. 1976). Single-channel activity has been measured in one of two ways: either by the patch-clamp technique (see Chapters 9 and lo), or in planar lipid bilayer membranes into which ion channel-forming proteins have been reconstituted (Latorre and Alvarez, 19x1; Latorre and Millcr, 1983; Miller, 1983a.b; Latorre and Benos, 1985; Darszon, 1986; Coronado, 1986; Montal, 1987). These techniques allow direct and quantitative study of single-channel properties in an environment of essentially controllable elcctrical and chemical composition. The advantages of the bilayer technique, as opposed to the patch-clamp technique, are several: ( I ) the accessibility to the solutions bathing both sides of the channel enables one to control and vary easily the composition of both solutions, including the actual conducting ion and the addition of inhibitors or activators; (2) the possibility of perfusing both sides; (3) the control o f the nurnbcr of channels present in the bilayer; and (4) the ability to define the lipids surrounding the channel. The principal disadvantage of the bilayer technique is that channelcontaining membrane vesicles from some cell types fuse at only very low rates. Although there are several ways of enhancing fusion and hence channel incorporation, the study of epithelial channels has proved to be difficult due to low fusion rates. For example, incorporation of epithelial ion channels requires anywhere from 30 min to 8 hr, and in only a small percentage of the attempts (<10%) has a channel successfully incorporated. In this chapter, we will focus o n the reconstitution of ion channels, emphasizing both functional and biochemical approaches. By functional reconstitution, we mean the direct transfer of an ion channel from its native mcnibrane into a planar lipid bilayer, whereas biochemical reconstitution refers to the detergent +
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extraction and purification of an ion channel and its subsequent reinsertion back into a defined lipid environment. We will summarize the experimental approaches used in the isolation of ion channels and discuss in detail planar lipid bilayer techniques used in reconstitution studies, as well as provide a description of the channel incorporation process. We will discuss the advantages and disadvantages of each method using specific examples of channels studied in each fashion. We next turn to a consideration of reconstitution studies of Na', CI -, and K + channels from epithelial tissues. We conclude by giving our projections and expectations of the future of the reconstitution field. It is our goal to provide an article complementary to the primary research literature so that students and investigators alike will have an overview of the steps necessary to undertake ion channel reconstitution studies. An excellent source book to which the reader is referred is that edited by Miller (1986).
II. ISOLATION AND PURIFICATION OF SPECIFIC MEMBRANE VESICLE POPULATIONS The incorporation into model membranes of ion channels from biological membranes is an area that has flourished in the past decade. Planar lipid bilayer and vesicle reconstitution techniques allow for the detailed electrical examination of channels that could not have been accomplished by other more conventional techniques, such as patch clamp (Hamill, et ul. 1981). Once a channel is reconstituted and as soon as its functionality is established independent of its native membrane, an unequivocal demonstration of the minimal number of protein components required for channel physiological activity can be done. Isolation of individual ion-conducting proteins from biological membranes and their functional reconstitution into planar lipid bilayer membranes has been achieved using many different systems, including sarcoplasmic reticulum (Miller, 1978), transverse tubules (Latorre et d., 1982). axon membranes (Coronado et a / ., 1984), mitochondria (Krueger et ul., 1983), brain (Furman et ul., 1986; Keller et d . , 1986; Hanke ef ul., 1984a; Hartshorne et a/., 1985), and more recently, epithelia (Sariban-Sohraby et ul., 1984; Olans et u l . , 1984; Keinhardt et a / . , 1987; Bridges e t a / . , 1989b; Zweifach and Lewis, 1988). The initial step in this functional reconstitution approach necessitates the development of techniques that yield a relatively pure native membrane vesicle population enriched in the channel-forming protein of interest. The current methods employed by investigators to purify a specific cellular membrane vesicle fraction depend upon differences in membrane vesicle size, density, or electrophoretic mobility. These isolation techniques make heavy use of differential and density gradient centrifugation, or free-flow electrophoresis. The degree of separation is usually assessed by measuring the activities of enzymes associated with a particular membrane
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fraction. For example, in many epithelial cells, Na' ,K +-ATPaseactivity is restricted to basolateral cell membranes but alkaline phosphatase is often used as an apical or luminal cell marker. Unfortunately, these methods lead only to a partial purification because sufficient differences in the physical properties of the vesicles and distribution of marker enzymes do not exist in most cell types to permit the isolation of a pure membrane fraction. Moreover, current methods generally require large amounts o f starting material, provide a low yield of the final fraction, and are time consuming and labor intensive. An important goal, then, is to develop and refine efficient techniques to effect the isolation and purification of purer membrane vesicle populations. One such technique that has been used, for example, for the isolation of essentially pure epithelial Na' channels (Benos 1'1 ul., 1986) and T lymphocytes (Hcllstrom cf trl., 1976). is affinity chromatography. Affinity chromatography has been used in the separation and purification of soluble biological nioleculcs for niore than two decadcs. The principle of affinity chromatography involvcs the specific and reversible absorption of a molecule (e.g., protein) to be isolated to a binding substance (e.g., antibody) immobilized on an insoluble solid support rriatrix (e.g., Sepharose). Purification by affinity chrornatography is often several orders of magnitude better than conventional biochemical procedures, it is rapid, and the recovery of active material is very high. Recently, the technique of affinity chroniatogriphy has been applied successfully to the separation of specific cell types (e.g., Hellstrom et id., 1976). Affinity chromatography of cells exploits differences in the molecules, often proteins, expressed on the outer membrane surfaces of different cells. Thus, if a molecule specific to a given cell type can be identified, it is possible to separate these cells from a heterogeneous population of cells by application of this technique. Moreover, with a polarized cell such as an epithelial cell, if a molecule specific to a given membrane domain (e.g.. apical or basolateral) can be identified, it should be possible to separate this membrane domain from the hetcrogeneous population of membranes after cell homogenization. One means of obtaining the expression of highly antigenic membrane proteins in the apical or basolateral domains of epithelial cells is suggested by the use of enveloped RNA viruses. Epithelial cells infected with enveloped RNA viruses have been extensively used in plasma membrane protein-sorting experiments (Kodriguez-Boulan, 1983). These viruses assemble and bud with a striking polarity. For example, influenza, Sendai, and simian virus 5 bud from the apical surface and vesicular stomatis virus (VSV) and several type C retroviruses bud from thc basolateral membrane surface. Polarized viral budding is determined by the asymmetric distribution of the viral envelope glycoproteins. Studies have shown that each virus retains the same budding polarity in several different epithelial cell lines grown in culture. Nonepithelial o r isolated epithelial cells grown in suspension culture show budding over their entirc membrane surface. However, epithelial cells do not have to be grown to confluency; simply the attach-
287
9. EPITHELIAL ION CHANNEL RECONSTITUTION
ment of an epithelial cell, but not a nonepithelial cell, to a substrate or another epithelial cell is sufficient to trigger polarized viral budding. It should thus be possible to infect epithelial cells grown in culture with a virus that buds from either the apical or basolateral membrane surface. Antibodies to the viral glycoproteins could then be used to isolate apical membrane vesicles by immunoaffinity chromatography, as outlined in Fig. 1 . The isolation of an extremely enriched plasma membrane fraction would be achieved. Of course, the influence of these viruses on the functional expression of ion channels in infected cells and on bilayer electrical properties would have to be assessed in independent experiments. However, preliminary experiments conducted in our labora-
/
Monolayer
1
KCI
Protein ASepohorose 6M
ProtelnA
KCI
+
*
Incubate
1 1
Elute
+
Incubate
1 Elute
Antibody (I0 Apical ( A ) Basolateral (El) FIG. I . Method for isolation of apical membrancs from T84 human colonic tumor cells using immunoaftinity chromatography and virus glycoproteins. Confluent monolayers of T84 cells grown on permeable supports are infected with inlluenz~virus, an apical membrane budding virus. Antibodies to the viral glycoproteins expressed in the apical membranes are added after a 12-hr incubation period. Homogcnates of scraped cells are then placed on a protein A-Sepharose 6MB aftinity column. Because only the apical mcmbranes will have viral glycoproteins, and thus be antibody attached, only the apical membranes will bind to the protein A aftinity column. After elution of nonapical membranes, excess protein A is added to cause the competitive elution of the apical membranes.
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ROBERT J. BRIDGES AND DALE J. BENOS
tory dcnionstrate that the influenza virus WSN does not significantly influence the expression of the secretory CI- channel in T84 colonic cell monolayers grown on permeable supports nor are bilayer characteristics significantly altered.
111. BILAYER TECHNIQUES USED IN RECONSTITUTION Ion channels can be transfcrred from their native membrane into planar lipid bilayers by a variety of techniques. The planar phospholipid bilayer membrane was introduced as a technique in the early 1960s by Mueller and colleagues (Mueller rt a / . , 1962; Mueller and Rudin, 1968). The methods for forming planar bilayers are relatively straightforward and the reader is referred to Alvarez et al. (1985), Latorrc and Benos (1985), and Darszon (1986) for details. In the original painting method, a solution of phospholipid micelles in a hydrocarbon solvent (usually n-decane or squalene) is spread across a hole (0.2-0.3 mm in diameter) drilled in a plastic or Teflon partition separating two aqueous compartments (Fig. 2). Thc spreading is accomplished by “painting” the mixture across the hole using a small glass rod, a brush, or a lipid bubble formed at the end of a hollow glass capillary. The film is initially several micrometers thick, but with time thins spontaneously to a bilayer configuration. The forces involved in this thinning proccss (namely, Plateau-Gibbs border suction and London-Van der Waals attraction) are cloquently discussed in a study by White ( 1986). The excess hydrocarbon solvent eventually (the time depending upon the lipids) drains away, with the phospholipids arranging themselves into a bimolecular leaflet. This lipid arrangement was expcrimentally established using both optical (Tien, 1967) and electrical (Hanai et u / . , 1965; White, 1970) procedures. Typically, the bare bilayer conductances are less than 10 nS/cm2, at least two orders of magnitude lower than the conductance of human or amphibian red blood cell membranes and some five orders of magnitude less than that of most nerve and muscle cell membranes (Hodgkin and Huxley, 1952; Lassen and Sten-Knudsen, 1968; Lassen, 1972). Thus, single ion channel events can be studied in a pure phospholipid membrane of very low background conductance. For example, single-channel conductance limits of 0.4 pS for nionazomycin (Bamberg and Janko, 1976) and 360 pS for maxi Ca?+-activatedK ’ channels (Latorre and Miller, 1983) in 100 mM univalent electrolyte solution have been measured in lipid bilayer menibranes. There are, however, instrumentation problems limiting the resolution of channel conductances. Using very small membranes (-7 X 10 cm?), Hanke et al. (1981) reported that single-channel conductance can be resolved with a current-duration product of 2 X 1 0 ~ ~A’ ” sec. For example, if a single K’ or Na+ channel has a conductance of 4 pS at 100 mV and a mean opcn time of 1 msec (Latorre and Miller, 1983; Keller rt a / . , 1986), the currentduration product is on the order of 4 x A * sec, barely at the limit of resolution of the system.
289
9. EPITHELIAL ION CHANNEL RECONSTITUTION
m
Q 6
0
/
/’
/
FIG. 2. Method of solvent-containing planar lipid bilayer formation: painted bilayers. The lipid solution is applied with a “brush,” such as a fire-polished glass capillary tube, or is directly pipetted onto the septum hole separating the Ruid-tilled cis and trans compartments. The thick lipid drop thins spontaneously to form a bilaycr, which can be monitored as an increase in capacitance (top). We have found polystyrene and polyvinylchloride are useful septum materials. Bilayer formation and stability are facilitated when the septum hole is pretreated with lipid and dried with a stream of N,. [After White (19861.1
Montal and Mueller (1972) later devised a way to produce planar phospholipid bilayer membranes virtually free of hydrocarbon solvent. These membranes are termed folded or solvent-free bilayers. The development of this technique was prompted in large measure by the scientific objections raised about having hydrocarbons in the membrane. One problem with solvent-containing membranes is that they are susceptible to electrocompression, i.e., the thickness of the bilayer decreases with increasing electric field strength because the hydrocarbon is in effect squeezed out of the membrane by the applied transmembrane voltage. As depicted in Fig. 3 , solvent-free bilayers can be made by first forming lipid monolayers at an air/water interface on each of two solutions separated by a thin Teflon partition. Monolayers can be made from pure phospholipids (Fig. 3A) or assembled from a suspension of vesicles in the subphase (Fig. 3B). Initially the levels of the aqueous/air interfaces are below the aperture in the partition (0.050.5 mm diameter). A lipid solution of any composition is made up in a volatile solvent (usually n-pentane or chloroform) and a small amount (10 pi of a
290
ROBERT J. BRIDGES AND DALE J. BENOS
A
B
FIG. 3 . Folded planar bilayers. (A) Monolayen made from exogenous lipids. Phospholipid monolayers can be spread. using N,-dried, purified lipids, followed by addition of channel-containing vesicles to one aqueous subphase. The planar hilayer is then formed by raising the levels of each subphase hy injection of more solution. (B)Monolayers madc from preformed or native vesicles. Both compartnicnta of a bilayer chamber arc filled with vesicle suspensions without (clcar symbols) or with (solid symbols) ion channels. Note that the vesicles containing channel proteins are added only to onc coniparmiCnt. Monolayers will self-assemble at the air-water interface. Monolayers are thcn “folded” to form a planar bilayer.
10 nig/ml solution per I cm’) is placed upon the surface of the electrolyte solution. After 5-10 min (to allow for the evaporation of the solvent), a planar bilayer is constructed by raising the levels of each subphase by injection of new solution. As each solution level is incrcased, the surface phospholipid monolayers “fold” against the water/Teflon interface. At the aperture the monolayers contact each other to form a bilayer membrane. To increase membrane stability, thc aperture is usually pretreated with squalene or hexadecanc in pentane 10.5% (v/v)], hence the term “virtually solvent free.” The two advantages of this approach are that the bilayer is more physiological in that it contains little hydrocarbon solvent, and each leaflet can be composed of different phospholipids (see c.g., Hall and Latorre, 1976), more closely mimicking natural membranes. A third method in use to study ion channels in planar bilayers is the so-called tip-dip method (Fig. 4). In this method the bilayer is formed at the tip of a patchclamp electrode (Coronado and Latorre, 1983; Suarez-lsla P I cil., 198.3; Wilmsen et a / . . 198.3; Hanke ut d., 1984b). As in the foldcd-membrane technique, a monolayer of phospholipid is first spread at an air/water interface after immersion in the solution of a saline-filled patch electrode. After the solvent has evaporated, the pipet is taken out of the solution into the air, taking with it a monolayer film of absorbed phospholipid. Reinsertion of the pipet into the same (or different) phospholipid monolayer solution results in the formation of a stable bilayer membrane. This technique has the advantage of not requiring the presence of alkane solvents for bilayer formation. The small area of these membranes also
291
9. EPITHELIAL ION CHANNEL RECONSTITUTION
FIG.4. The tip-dip method to form planar bilayers at the end of a patch-clamp electrode. A monolayer of phospholipid is lirst spread at the air-water interface, after immersion of a saline-tilled patch pipet in the solution (left). The pipet is removed from the solution, taking with it an adsorbed film of phospholipid (middle). Reentry of the pipet into the same (or different) phospholipid monolayer solution produces a stable bilayer membrane (right). Channels can bc incorporated either hy making channel-containing monolayers (see Fig. 3 ) or by adding channel-containing vesicles to the aqueous subphase (right) and allowing fusion to proceed.
significantly increases the signal-to-noise ratio compared to painted or folded bilayer membranes (Sigworth, 1983), hence, current amplitudes of 0. I pA at I kHz can easily be resolved. The main disadvantage of the technique is that the channel protein briefly contacts the air phase. Patch pipets have also been used to study biocheniically purified ion channels that have been reconstituted into large (>I0 p m diameter) liposomes. A patch of liposomal membrane containing an ion channel is transferred to the tip of a glass pipet by suction. Examples of ion channels that have been studied in this way include electroplax C1- channels (Tank er a / ., 1982), acetylcholine receptor channel (Suarez-lsla et cil., 1983), voltage-dependent Na’ channels (Correa et ul., 1988), and “degraded” epithelial K +-Ba channels (Zweifach and Lewis, 1988). +
IV. ION CHANNEL INCORPORATIONINTO PLANAR LIPID BILAYERS Ion channels can be transferred from their native membranes into planar lipid bilayers by a variety of techniques. The first and perhaps most widely used technique is the fusion method first implemented by Miller and Racker (1976) and by Miller et (11. (1976). These investigators reported that three experimental conditions must be met to achieve appreciable fusion of native membrane vesicles to solvent-containing planar bilayers: ( I ) an osmotic gradient across the planar bilayer, the vesicle-containing (cis) side being hyperosmotic; (b) millimolar concentrations of calcium in the cis electrolyte solution; and (3) the presence of negatively charged phospholipids in the planar bilayer. However, many exceptions to these initially defined requirements exist (Miller, 1983b; Latorre and Benos, 1985). Native membrane vesicles containing ion channels can be fused to painted, solvent-free, and tip-dip bilayers. Table 1 presents a selected
292
ROBERT J. BRIDGES AND DALE J. BENOS TABLE I SOMEEXAMW 1,s OF ION CHANNELS INCORPORATED
Method busion
Folded bilayers
Origin
I N T O I.IPID Bii A Y ~ RMI:MBRANI.S
Channel type and conductance
Ionic conditions
Rat hrain
Na
'
Cultured A6 renal epithcliuni Transverse tubule
Na
' (4-80
Sarcoplasmic reticulum
K
140 pS)
0.1 M KCI
Sarcoplasmic 1-eticuluiii
CI (I00 ps)
0.1 M KCI
Rat cdonic cpitheliuin
CI- (50 p s )
Synthetic peptidc
Na
Lobster axon
K
Rat olfactory epithelium Torpedo clcc tropla x
K * (60 pS)
(30 pS) pS)
K ' (230 pS)
'
(
' ( 2 0 pS)
' ( 8 5 pS)
Cation~c(28 pS)
0 . 5 A4 NaCl 0.2 M NaCl 0.1 M KCI
Refercnce
Krueger
01 n l . (19x3) Sarihan-Sohraby e/ NI. ( 1984) I.atorrc Y I a / . ( 1982) Coronado and Latorre ( 1983) Tanifuli cr trl (1987) Rcinhiirdt C'I 01. (1987)
0 S M NaCl
Diki et ol. (19x8)
0 5 M KCI/ 0 I M KCI" 0.07 M NaCli I)02 M KCI 0.3 M NaCl
Coronado P I rrl.
(1984) Vodyanoy and Murphy (19x3) Monfal el trl. (19x4)
Tip-dip
Rabbit urinary hladdcr
K
TotyJcdockCtr(1pktX
Lohster axon
ACh channcl. cationic (40 pS) K (XS pS)
Cardiac zarcolerria
CI (70 PS)
+
(20 pS)
0 . 2 M KCl
Zwcifach and
0.45 M KCI
Montal el rrl ( i w ) Coronado and Liitorrc (19x3) Coronado and Latorrc (19831
LCWlS
0. I M KCl/ 0.05 M KCI 0.1 M KCli
0.05 M Comhination fusion/ plpel technique
Dog brain
Na ' (45 pS)
(1988)
2 M
"Cisitranc conccntrations
compilation of different ion channels incorporated into planar lipid bilayers using either of these different techniques. Another wily to fuse vcsicles to bilayers was reported by Hanke ('1 ul. (198 1) in their studies of the acetylcholine (ACh) receptor channel. These investigators were able to fuse membrane fragments rich in ACh receptor from ~ O r p ~ delecO troplax to planar bilayers constructed from synthetic mixed-chain phosphatidyl-
9. EPITHELIAL ION CHANNEL RECONSTITUTION
293
choline. Fusion occurred several degrees below the liquid solid-phase transition temperature (30°C). A clear advantage of the fusion technique employed in these experiments is that it avoids the use of solvent-containing bilayers, and that it never exposes a channel formed from integral membrane proteins to an air phase, as is the case when using folded bilayers. In this work, these investigators reported that although the presence of calcium in the aqueous solutions or of negatively charged lipids is not a prerequisite for fusion, an osmotic gradient is necessary for high rates of fusion. The same conclusions are also true when using solvent-containing membranes. For example, Latorre et a/. (1982) and Olans et a / . (1984) have observed that high rates of incorporation of either the t-tubule calcium-activated K' channel or the A6 apical membrane Na' channel into neutral phosphatidylethanolamine (PE) or charged phosphatidylethanolamine/ phosphatidylserine (PEIPS) membranes can be obtained in virtually calcium-free solutions as long as an osmotic gradient is established across the planar bilayers. In solvent-containing bilayers, fusion can proceed, albeit at a very slow pace, even in the absence of an osmotic gradient across the planar bilayer. However, it appears that an osmotic gradient is an absolute requirement for fusion when using solvent-free, folded membranes (see Alvarez et a / . , 1985). The effects of osmotic gradients as a requirement for fusion with planar bilayers have been discussed by Cohen and co-workers (Cohen et d.,1980, 1982; Cohen, 1986). These authors concluded that the osmotic swelling of the phospholipid vesicles absorbed to the planar bilayer causes fusion. When using the fusion method, incorporation proceeds either in packages of many channels (Miller, 1978; White and Miller, 1979) or in a one-to-one fashion (Latorre et a l . , 1982). The simplest explanation for these phenomena is that the membrane vesicles either contain many channels or one or no channels. For practical considerations, it is convenient for statistical analysis of channel records to have only a single channel in the membrane. Hanke and Miller (1983) reported incorporation of single CI - channels into solvent-free membranes, if the Torpedo electroplax vesicles are sonicated for about 2 min. It is possible that sonication reduces vesicle size and hence diminishes the average number of channels per vesicle. In our hands, freezing and thawing vesicles usually results in an increased rate of channel incorporation, presumably due to the larger size of vesicles (see e.g., Anholt er d . , 1981). It should be pointed o u t that Horn and Lange (1983) have presented a method for statistical analysis of current fluctuations measured from bilayers containing more than one channel. We would like to emphasize that we have been discussing the fusion process in a rather imprecise way. The mechanism of fusion of membrane vesicles to planar lipid bilayers and the reasons why membrane vesicles from some cell types fuse more readily than others are only poorly understood (see Miller, 1986). Fusion is thought to proceed in two steps: ( I j attachment of the membrane vesicle to the planar bilayer, a stage where both bilayers still coexist, and (2) the actual fusion of the two membranes. As we have said, the former stage is thought
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ROBERT J. BRIDGES AND DALE J. BENOS
Attachment
Fusion
F I G . 5 . Channel incorporation from memhrane vesicles and prcfornied planar lipid bilaycrs. C'hannel incorporation is thought to proceed i n two stages. attachmcnt 0 1 tlic vcsicle to the bilaycr and fusion of the two bilayers. Attachment is promoted by the presence of C a ? ' and/or negatively charged lipids or the addition of polyvalcrit cationic proteins such as lihroncctin or polylysinc. Once attached to the bilaycr, the vesicle can sensc the iiiiposcd osmotic gradient. swell. and fuse with the bilayer. Current across the hilaycr is iiionitored and an incorporation detected as a sudden jump in current (top). IAlter White (19X6).1
to be promoted by the presence of negatively charged lipids and the addition of millimolar quantities of divalent cations, whereas the latter is promoted by the swelling of the vesicle, usually by establishing an osmotic gradient to cause destabilization of the vesicle membrane (Cohen, 1986) (see Fig. 5). However, it has not been shown that the channels incorporated into solvent-containing membranes [except for mitochondria1 porin (see Cohen et al.. 1982)1 are actually transferred from the native vesicle into thc planar bilayer by a process of fusion. The evidence for fusion is only indirect in that the experimental conditions known to increase the rate of fusion (Cohen et al., 1980, 1982; Cohen, 1986) also increase the rate of channel incorporation. The main message that we would like to convey is that the optimal conditions for incorporation of ion channels from vesicles to planar bilayers must be determined empirically for each preparation. When incorporating ion channels into planar bilaycrs, there are several potential difficulties that can arise. These problems and some suggestions are discussed in the following paragraphs. 1. Membrane vesicles from some cell types do not fuse or do so with such a low probability that channels are not observed. Unfortunately, plasma membrane vesicles from epithelial cells are one of these difficult types. We have utilized and are developing various ways to enhance the rate of channel incorporation of
9. EPITHELIAL ION CHANNEL RECONSTITUTION
295
epithelial ion channels. One means we have utilized has been to increase the concentration of vesicular proteins. Because only small quantities of epithelial membrane material can be obtained, it was necessary to reduce the chamber compartment volume. Thus, by reducing the cis volume from 3 ml, as required in the chamber design employed in many bilayer laboratories, to 300 pl, much higher protein concentrations can be utilized without necessitating the expenditure of precious membrane material. We have also made use of fibronectin and polylysine (Young and Young, 1984; Gad and Eytan, 1983) to promote membrane vesicle attachment and hence fusion and channel incorporation. In addition to employing these attachment-promoting proteins, we are currently developing means of utilizing fusogenic proteins. Enveloped animal viruses possess exceptionally potent membrane fusion activities triggered and catalyzed by two specific glycoproteins in their membrane or envelope (White et d . , 1983). One of these glycoproteins, the recognition protein, is responsible for the attachment of the virus to the host cell membrane. In several cases, this protein binds to sialic acid residues of host cell gangliosides or glycoproteins. The second viral glycoprotein, the fusion protein, is responsible for the actual fusion of the host cell membrane and the viral membrane, leading to the introduction of the viral nucleocapsid into the cell cytosol. The detailed mechanism by which the fusion protein promotes membrane fusion is still unknown, and with some viruses, both attachment and fusion are mediated by a single glycoprotein [e.g., the hemagglutinin (HA) protein of influenza virus]. Viral envelope glycoproteins have been shown to promote cell-cell, Iiposome-liposome, cell-liposome, and, in one study, liposome-bilayer fusion (for a discussion, see White ef nl., 1983). Also, another series of fusion proteins (chromobindin proteins) have been identified (Creutz et al., 1987). These proteins mediate the adsorption of exocytotic vesicles to the plasma membrane of chromaffin cells in the presence of Ca2+,and thus increase fusion rates. It would seem reasonable, then, to employ fusogenic proteins to help in the incorporation of ion channels contained in native or artificial vesicles into planar lipid bilayers. Despite the apparent feasibility of this approach, we are unaware of any report involving the use of viral glycoproteins or other fusogenic proteins to promote the fusion of native cell membrane vesicles to planar bilayers. The scheme with which we propose to use envelope viruses or purified envelope glycoproteins, to aid in the fusion of native membrane vesicles into bilayers, is illustrated in Fig. 6. It will be important to assess whether channel properties using viral proteins to promote channel incorporation are comparable to those already established in patch-clamp and bilayer studies. 2. Fusion of vesicles containing several channels or multiple fusions may occur, making single-channel analysis difficult. As we have said, the former condition may be ameliorated by sonication of the vesicles to reduce their size, and thus possibly decrease the number of channels per vesicle. The latter difficulty can possibly be overcome by decreasing the amount of vesicle material added to the cis compartment. Moreover, we have observed that it is possible to reduce
296
ROBERT J. BRIDGES AND DALE J. BENOS
PLB
Apical ( A ) Virus Protein (A) Fic;. 6 . Three alternative approacheg to the use of virus glycoproteins to proniote apical membrane fuusion to planar lipid bilayers (PLB). (a) Apical membrane vesicles from cultured cpithelial cells with viral glycoproteins are isolated by immunoaflinity chromatography, as described in Fig. 1, and added to a normal PLB. (h) Planar lipid bilayers are prepared to include viral glycoproteins. (c) Virus particles or their purilicd fusogenic proteins arc added direclly to the cis solution and rcact with the vesicles and PLB.
the number of channels in a bilayer, often to a single channel, by applying additional lipid or simply touching the bilayer with a tire-polished glass capillary tube. Andersen and colleagues (Green et al., 1987) have described a similar technique, referred to as “punching,” to reduce the number of channels in a multichannel-containing bilayer. Essentially, one must reach a compromise between waiting long times at low protein concentration in the hope of incorporating a single channel, or waiting a short time at higher protein concentration, but running the risk of multichannel incorporations. 3 . Channels other than the channel of interest may appear either alone or together with the channel of concern. The simplest solution to this problem is to paint a new membrane or to set up a new chamber. Although this is certainly a simple task, it is one requiring a great deal of discipline. It is psychologically quite difficult to destroy a bilayer containing a single channel, even if it is a channel you were not intending to study. A somewhat less stressful solution is to establish experimental conditions under which some of these channels will not conduct current (impermeant salts, specific inhibitors, etc.). The preparation of pure membrane fractions, as discussed above, will also aid in maximizing the probability of incorporating the channel of interest. There is another additional potential problem that is inherent to all reconstitution studies. This problem is that the properties of the channel may be altered as a result of membrane vesicle isolation or incorporation into an artificial membrane. Thus, it is important to ensure, preferably by an independent measurement, that the properties of the channel have not been altered. Miller (1978), in studying a K+-selective channel from sarcoplasmic reticulum in solventcontaining planar bilayers, has shown that the conduction characteristics of this channel are not influenced by the presence of organic solvent in the bilayer, inasmuch as the channel displays comparable properties in nominally solvent-
297
9. EPITHELIAL ION CHANNEL RECONSTITUTION
free planar bilayers (Labarca et a / ., 1980). Furthermore, a K + conduction pathway is formed in liposomes constructed from detergent extracts of sarcoplasmic reticulum (Latorre et al., 1982). More recently, Garcia and Miller (1984), by measuring TI fluxes into sarcoplasmic reticulum vesicles, have concluded that these conduction sites show the same properties as the K channel incorporated into planar bilayers. Specifically, they demonstrated that the ionic selectivity, conductance, and channel blockade by quaternary ammonium ions are not modified by transfer of the channel from the vesicle to the planar membrane. Purified voltage-sensitive Na' channel proteins reconstituted in planar lipid bilayers also display the same voltage dependence, neurotoxin sensitivity, and ion specificity associated with Na' channels in their native membranes (see Montal, 1987). As described below, the epithelial secretory CI - channel incorporated into planar lipid bilayers from native membrane vesicles has the same identifying biophysical properties as obtained with on-cell and excised membrane patches from intact epithelia. These three examples demonstrate it is possible to isolate and incorporate channels into artificial membranes without altering the channel properties. The preservation of channel properties must, however, be established for each new channel. +
+
V. RECONSTITUTION OF ION CHANNELS FROM EPITHELIA INTO PLANAR PHOSPHOLIPID BILAYERS Epithelia are sheets of multicellular and, therefore, multimembraneous tissues that separate fluid-filled cavities (e.g., kidney tubules, salivary and sweat ducts, and intestine) from the interstitial compartment. Epithelia secrete or absorb fluids by transporting ions from the interstitium into these spaces, or vice versa. By nature, an epithelial cell is polarized, i.e., the membrane facing the lumen, or cavity, has different permeability characteristics than the basolateral, or interstitialfacing, membranes. The concerted action of these distinct membrane domains accomplishes what neither membrane alone can do, namely, the net transepithelial movement of salt and water. In many epithelia, Na' is the dominant ion absorbed from the lumen into the interstitial space. In these tissues, Na' first enters the cell down its electrochemical potential energy gradient through a pathway (an ion channel) sensitive to blockage by the diuretic drug, amiloride (Benos, 1986; Garty and Benos, 1988). For charge balance, C1- must follow Na' either through paracellular pathways or through separate ion channels in the apical membrane. The basolateral membrane contains many copies of the Na+-K+-activated ATPase, which actively transports 3Na' ions out of the tissue in exchange for 2K+ ions. Potassium is also recycled across this membrane by entering the cells passively through a barium-inhibitable ion channel. Other epithelia secrete fluid as a result of active C1- transport. Tissues falling into this
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ROBERT J. BRIDGES AND DALE J. BENOS
category include the large and small intestine, teleost gills and operculum, the cornea, and tracheal epithelia. In these tissues, active C P transport is directed from the interstitial region to the lumen so that thc direction of net ion movements is opposite to that in the case of Na+-transporting epithelia. The C1 sccreting cornea, trachea, and mammalian distal colon can also absorb Na (Frizzell et d . . 1981). The essential mechanism of net CI movement can be summarized as follows: CI - enters the cell across the basolateral membrane via a bumetanide-sensitive Na ' -Cl -K cotransport process. The Na' that enters with CI- is recycled across the basolateral membrane by the Na+-K+ pump, which also serves to maintain cell N a ' activities at low values to energize the net uptake of CI- through the cotransport system. Potassium, as in the case of Na'-transporting epithelia, is in addition recycled across this membrane through a K + channcl. Chloride lcaves the cell passively across the luminal membrane through a conductive pathway recently shown to be an ion channel (Frizzell Pf d . , 1986). Therefore, epithelia must contain ( I t lrcrst three separate ion channels: a Na' channel, a CI- channel (both located in thc apical or luminal mcmbranc), and a basolaterally located K channel. Direct evidence for the existence and proper location of each of these ion channels has come from patch-clamp cxpcrinients (Hamilton and Eaton, 1985; Hunter t t (11.. 1984; Palmer and Frindt, 1986; Frizzell ei a / . , 1986). In addition, each of these ion channels has becn obscrvcd in planar lipid bilayers (see below). +
A. Epithelial Sodium Ion Channels In order to observe and characterize amiloride-sensitive Na channels directly, apical membrane vesicles containing these channels were incorporated into solvent-containing planar lipid bilayer membranes (Sariban-Sohraby et cil. , 1984; Olans d., 1984). These vesicles were made from a cultured toad kidney cell line, A6, grown on Millipore tilters. These cells express amiloride-sensitive Na+ channel activity only when grown on permeable supports. The major evidence that Na' channels are present in the cells and in the apical membrane vesicle fraction is that amiloride blocks the :?Na+ influx into both preparations with a K , of 0.7 pM (at a Na' concentration of 110 mM). lncorporation into solvcnt-containing bilayers was achieved osmotically. An example of such single-channel activity is shown in Fig. 7. The conductance of this channel is 5 pS, but in all cxpcrimcnts thc rangc of singlc-channcl conductancc obscrved was 4-80 pS at symmetrical 200 mM NaCI, with a mean value of 35 pS. Both the open channel conductance and the probability of a channel being in the open or closed state were independent of voltage in the range f60 mV. The channels were found to be perfectly cation selective; however, the ability of these channels to discriminate between Na+ and K + was less than cxpcctcd from macroscopic +
(11
9. EPITHELIAL ION CHANNEL RECONSTITUTION
299
FIG. 7. Cultured A6 apical membrane Na channel incorporated into planar lipid (PE: PS. 7 : 3 ) bilayer. Arrows indicate the zero current level. Conditions: symmetrical 30 mM NaCI, f 5 0 mV cis to tran5, 100-Hz low-pass liltcrcd. [From Sariban-Sohraby et a / . ( 1984) with permission.] +
measurement in other epithelia. The measured permeability ratio, or conductance ratio, of Na' to K ' for these single channels was 3 : 1. A selectivity ratio for Na' versus K of at least 100: 1 or 1000: 1 has been reported for frog skin and toad bladder, respectively (for a review, see Garty and Benos, 1988). In contrast, Hamilton and Eaton ( 1985) have made observations of an amiloridesensitive Na+ channel in intact A6 cells using the patch-clamp technique. They report that the channel displayed a Na+-K' selectivity ratio o f 4 : 1, and that the block by amiloride was voltage dependent, with amiloride sensing approximately 40% of the applied electric field. Light et al. (1987) measured an amiloridesensitive channel in rat medullary collecting tubule cells with the patch-clamp technique. This cation channel (27 pS) did not discriminate between N a ' and K + . Patch-clamp studies have also shown the presence of a 25-pS amiloridesensitive cation channel with a N a + : K + selectivity of 3 : 1 in the apical membrane of the frog lens epithelium (Jacob P r ai., 1985). Amiloride interacts with the A6 Na+ channel, whether present in the cis or trans compartment of the bilayer setup. Addition of amiloride to the trans side produced rapid flickerings between the open and closed conductance states, suggestive of those observed by noise analysis in intact epithelia. Cis amiloride, however, did not produce flickering, but rather reduced the open state conductance, suggesting that the block was short-lived with respect to amplifier response time. The cis block by amiloride was concentration dependent. The single-channel open state conductance was reduced to 50% of its initial value by 0. I pM amiloride. This phenomenon is due to amplifier bandwidth limitations, i.e., the amiloride-induced blocked state is short-lived with respect to amplifier response time. The concentration dependence of the trans block has not yet been examined. Nonetheless, these characteristics of the amiloride block suggest a presence of multiple binding sites for amiloride. Olans et al. (1984) have analyzed the Na+ concentration dependence of these
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single amiloride-sensitive ion channcls in bilayers. Two extremes of channel conductance were studied. In one case, thc single open channel conductance reaches a maximum value of 4 pS and the conductance versus Na + activity curve was well described by a rectangular hyperbola with an apparent K , of 17 mM. L. G. Palmer (unpublished observations) recently reports comparable saturation ol' amiloride-sensitive Na+ channels in rat collecting tubules with a K, of approximately Is) m M . The conductance versus Na' activity curve for the other population of channels showed a maximum conductance of 44 pS and a K, of 47 m M . Both the small and large conductance channels displayed similar characteristics; namely, both were amiloride-sensitive (cis amiloride reducing open state conductance with a K , of 0. I p M ) , both were perfectly cation selective, both showed a P,;,IP, ratio of 3 , and both saturated with increasing Na'. Reconstitution studies with purified Na' channel protein arc just beginning (Benos and Sariban-Sohraby, 1987). In these studies, much variation was observed aniong the types of channels incorporated into the bilayer. Because native vesicles were used in these experiments, other channels in addition to the amiloride-sensitive Na+ channel were transferred into the bilayer. In fact, at lcast 12 discernible channels were observed in these experiments. However, no set of experimental conditions with regard to salt concentration, osmotic conditions, or Ca?+ concentration has been found for which only one typc of channel will incorporate into the bilayer. Such difficulty in defining the precise expcrimental conditions for predictable channel incorporation has made thc characterization of each of these channels very difficult. Recently, a procedure for the isolation and purification of an epithelial amiloride-sensitivc Na ' channel from fresh bovine renal papilla and A6 epithelia has been published (Benos et NI., 1986, 1987). A 5- to 10-fold enriched apical membrane fraction was first prepared by a calcium precipitation/differential centrifugation technique. The membrane proteins were detergent solubilized, chromatographed on immunobilized wheat germ agglutinin, and purified to near homogcneity by size exclusion high-performance liquid chromatography (HPLC). This protocol resulted in a greater than 5000-fold purification of the amiloride binding protein, with specific binding activities of > I100 pmol/mg protein. This purified ion channel was reconstituted into solvent-containing planar bilayers in preliminary experiments (Benos and Sariban-Sohraby, 1987). The channel displayed perfcct cation selectivity, with 10 nM bromoarniloride reducing open time probability by 30%. Barbry et a / . (1987) also report the purification of another putative epithelial Na+ channel protein from pig kidney cortex. However, this protein has bcen purified, based on f3H]phenamilspecific binding, to only 60% homogeneity and has a 250-fold lower affinity to amiloride than is expected for a Na + channel typically found in electrogenic Na +-transporting epithelia. This
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protein, when incorporated into planar bilayers, does form Na' conducting channels (C. Frelin, personal communication).
B. Epithelial Chloride ion Channels The incorporation of the epithelial secretory CI - channel into planar lipid bilayers is yet another example of a general phenomenon in bilayer studies, namely, that whatever channel one chooses to study, one ends up finding some other channel (Miller, 1987). The initial studies with membrane vesicles derived from rat colonic enterocytes were designed to investigate the colonic amiloridesensitive Na' channel. The colonic mucosa from dexamethasone-treated rats transports Na' at one of the highest rates reported for an epithelium (Bridges et a/., 1984, 1987). Morcover, aniiloride-sensitive Na+ uptake into membrane vesicles derived from colonic enterocytes is preserved after vesicle isolation and uptake is proportionally higher than reported for other epithelial vesicle preparations (Bridges et a / . . 1989a). Thus, this membrane preparation seemed to be an ideal preparation with which to initiate bilayer studies of the Na' channel. Sodium channels using this preparation can in fact be incorporated into bilayers. However, the frcquency of Na' channel incorporation is disappointingly low. Instead, the channel that appears most frequently and usually before any cation channels incorporate is a CI --conducting anion channel. Because the colonic mucosa is also a robust CI -secreting epithelium, we suggested this channel was the channel responsible for the exit of CI- across the apical membrane. Patchclamp studies have now identified an apical membrane C1- channel from several C1--secreting epithelia (see Chapter 8) with the same properties as those observed in the bilayer. The activity of this CI- channel is increased by CAMP- and Ca?+-mediated agonists that stimulate thc secretion of C1-. As the predominant apical membrane C1- channel, this channel is thought to be the secretory CI channel. The properties of the secretory CI- channel that distinguish it from the plethora of other CI- channels that have been reported in different cell types include one dominant open-state conductance, an outwardly rectified currentvoltage relation with a zero voltage slope conductance of approximately 50 pS in 150 mM NaCI, a CI --to-Na selectivity of at least 10: 1 , and a halide selectivity sequence of I > Br > CI > F (Reinhardt rt al., 1987; Frizzell, 1987). A current trace of an epithelial CI channel incorporated into a PE/PS bilayer is shown in Fig. 8. Between intermittent long-lived closed periods, the channel is predominantly open, interrupted by short-lived closed periods. The long-lived closed periods last from 100 ms to several minutes. The major variation in the open channel probability from channel to channel is caused by the variation in the frequency and duration of these long-lived closures. Between the long-lived +
~
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0-
c-*
3 PA[
-
300 mM MU150 rnM MU -30 rnV 400 Hz
3 s
300 rns
FIG. 8 . Current tracc of colonic CI- channcls incorporated into a PE/PS bilayer. A large SO-pS and sinall 4- to 6-pS anion channel can be secn. Upper tracc is a 7.5-min record at a low time resolution, illustrating thc burstlike kinetic behavior of the larger channcl. Bottom trace is the last 30 bee of the uppcr trace at a higher time resolution, illustrating the rapid opening and closing of the channel during a burst period. Conditions: 300 mM NaCl cis to 50 mM NaCl trans, - 30 mV cis to trans. 400-Hz bandwidth.
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closures, the channel displays a burstlike behavior. The open probability within a burst is greater than 90%, is stationary with time, and shows little variation from channel to channel. The open and closed times, during burst periods, can be approximated by single exponential distributions with time constants of 200 and 3 msec, respectively. The anion transport inhibitor 4,4’-isothiocyanatostilbene-2,2’-disulfonicacid (DIDS), has been shown to block irreversibly conductive C I ~transport in a variety of cell types (White and Miller, 1979; Nelson et al., 1984; Hanrahan rt al., 1985; Inoue, 1985; Bosma, 1986). We have observed that DIDS also irreversibly blocks the secretory C1- channel. Moreover, substitution of the covalently reactive isothiocyano groups of the DIDS molecule with nonreactive substitutes, as in 4,4’-dinitrostilbene-2,2’-disulfonic acid (DNDS) provides a potent reversible blocker of the secretory CI- channel (Bridges rt al., 1989b). A current trace of the channel shown in Fig. 8 after the addition of 1.5 p M DNDS to the cis side is shown in Fig. 9. DNDS caused a dramatic increase in current transitions and consequently a decrease in the open channel probability during burst periods. Kinetic analysis revealed that DNDS blockade could be explained by a linear closed-open-blocked kinetic scheme. The estimated DNDS block rate constants were k,,, = 3.2 lo7 M - ’ sec-I and k,,,, = 52 sec-’, yielding an equilibrium dissociation constant of 2. I pM. DNDS was effective only when added to the cis (or vesicle-containing) side of the bilayer. The rectification of the current-voltage relation of the CI- channel in the bilayer, when assessed at symmetric CI- concentrations, was always in the same direction so that cis-to-trans movement of CI- was greater than transto-cis movement. The direction of the rectification of the CI- channel in bilayers and the observed rectification in patch-clamp studies suggest that the channel orients in bilayers, with the cis side corresponding to the outer membrane side and the trans side to the cytoplasmic side of the channel. Therefore, the DNDS binding site is in all probability on the outer membrane side of the channel. A much smaller anion channel can also be seen in the current trace presented in Fig. 8, especially during the long-lived closed periods of the larger channel. The smaller channel has a conductance of approximately 4-6 pS. It frequently coincorporates along with, and often precedes, the incorporation of the larger CI- channel. These observations suggest that the smaller channel may also be an apical membrane channel. The biophysical properties and functional significance of the smaller anion channel remain to be investigated. An intriguing possibility is that the smaller channel is actually the larger channel in a different regulatory state. Chloride secretion in intact secretory epithelia is a nonconstitutive process that requires the continuous stimulation by secretagogues and is quickly down-regulated upon removal of the agonists. Because the colonic vesicles are prepared from unstimulated tissue, it is surprising to find active CI-
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0
.
c .
FIG. 9. Effect of DNDS on the colonic CI chdnncl. nNDS (I.5 p M ) was addcd to thc cis side o l tlir channel shown in Fig X. Upper trace is a 7 0-min record at low t h e r c d u t i o n and lower tr;icc is the last 30 stx tit' the rccord at B higher timc rcolution. Conditions ;is in Fig X . ~
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channels. How these channels remain in a conductive active state and how they are regulated will be an important area of future investigation.
C. Epithelial Potassium Ion Channels The properties of K + channels as revealed by noise analysis and patch-clamp studies are considered in Chapters 5 and 6. Potassium channels in both the apical and basolateral membranes from a wide variety of epithelial cell types have been characterized. Several different kinds of K' channels have been identified based on single-channel conductance, alkali metal cation selectivity, sensitivity to Ca?+ and voltage activation, and pharmacological profile to various blockers. Despite the apparent diversity of K + channels and their widespread distribution in epithelia, K + channels have been the subject of only a few reconstitution studies. Indeed, we are aware of only one report describing the single-channel properties of an epithelial K' channel after incorporation into planar lipid bilayers. Vodyanoy and Murphy (1983) described the properties of a chemosensitive K' channel from rat olfactory epithelium incorporated into solvent-free phosphatidylcholine bilayers. The channel had a conductance of 60 pS at 10 mV in 20 mM KCI and did not conduct Na'. Diethyl sulfide (25 nM) activated the channel, causing an increase in the mean open time from 29 to 42 msec and a 23% increase in the open channel probability. The cellular membrane origin, apical versus basolateral, of the channel is not certain because cell homogenates were used for reconstitution. Klaerke et ul. (1987b) have purified, by calmodulin affinity chromatography, a Ca?+-activatedK + channel from porcine kidney medulla. The purified protein has two subunits of 36 and 51 kDa. When reconstituted into liposomes, the protein mediates the conductive uptake of X"Rbthat is directly activated by Ca2+ and calmodulin and by CAMP-dependent protein kinase phosphorylation. The calmodulin antagonists, pimozide, calmidazolium, and trifluoperazine, inhibit the activation by calmodulin, and Ba?+ blocks the uptake of xaRbi. The channel reconstitutes into liposomes asymmetrically, with the cytoplasmic side facing outward (Klaerke el al., 1987a). Mild trypsinization activates XhRb+uptake that can no longer be stimulated by calmodulin. Trypsinization also causes a decrease in the size of the 36-kDa subunit and CAMP-dependent kinase phosphorylates the 56-KDa subunit, suggesting that these subunits are involved in the Ca?+-and CAMP-mediated regulation of this channel. In a series of exploratory experiments, we have observed that K + channels can be incorporated into bilayers when using a membrane vesicle preparation derived from rat colonic enterocytes. Compared to Na+ or CI- channels, K + channels incorporate quite easily, often incorporating before C1- channels when using KCI solutions. Two different types of K' channels were observed, a large
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20 nM Ca2+
250 nM Ca2+
100 mM KCI +30 mV 400 Hz
C-
FIG. 10. Current trace of a colonic K ' channel at 20 and 2.50 nM C ; t ? ' .In the uppcr tracc. only onc channel can be seen to open at one time. Bottom trace, Ca?+added to both the cis and trans sidcs causes the activation of the channel and thc appearance of il second channel. Conditions: 100 mM KCI cis and trans. 30 mV cis to trans. 400-Hz bandwidth.
+
150- to 190-pS channel and a smaller 35- to 50-pS channel. In biionic conditions (200 mM KCI/NaCI), neither channel conducted Na' . Both channels were activated by C a 2 + ,but only the larger channel was affected by voltage. A current trace of the larger K ' channel and its activation by Ca2+is shown in Fig. 10. In the upper trace, only one channel was apparently active. The open channel probability at 20 nM Ca?' and +30 mV was less than 108, but could be increased to >50% at i80 mV. Addition of Ca'+ to 250 nM caused the activation of the channel and the appearance of a second channel. The open channel probability at 250 nM Ca" and + 3 0 mV was increased to greater than 50%. The membrane vesicle preparation used in these experiments was only a partially purified plasma membrane fraction. Thus, both apical and basolateral membranes are present and the cellular origin of these K channels cannot be stated with certainty. +
VI. CONCLUSIONS A major development in the field of membrane transport has occurred in recent years, namely, the incorporation of native ion channels into planar phospholipid model membranes. This development makes possible an exacting quantitative study of these transporters in an environment of defined and controlled composition. The tcchniques reviewed allow for a detailed kinetic study of inaccessibly located channels that could otherwise not be done by conventional methodology. We are now in a position to modify ion channels either chemically, biochemi-
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cally, or genetically, and assess the functional consequences of such modifications in a situation independent from other complicating cellular influences. Hence, some channel structure-function relationships can now be studied at the molecular level. A paper by Oiki et ul. (1988) exemplifies the potential of this approach. The genes coding for excitable membrane N a + channels from electric organ (Noda et ul., 1984), rat brain (Noda er ul., 1986), and Drosophila (Salkoff et ul., 1987) have been cloned, and the respective cDNAs were sequenced. From the deduced polypeptide sequences of the 260-kDa subunit of the Na+ channel, various models for the tertiary structure of this polypeptide have been proposed (e.g., Greenblatt et ul., 1985). In one such model, the protein is considered to have four homologous regions, each region consisting of eight symmetric membranespanning helices. At the center, four amphipathic helices coalesce, with their hydrophilic surfaces facing each other to form an ion conduction pathway. What Oiki et ul. (1988) did was to make a 22-amino acid-long synthetic peptide corresponding to the sequence of the putative amphipathic regions, and incorporated this peptide into folded bilayer membranes. These investigators found that this 22-mer peptide was indeed able to form a cation-selective ion channel in the bilayer, although the pore was unable to discriminate between Na+ and K + and displayed no voltage sensitivity. Although distinct from the native Na+ channel, these experiments nonetheless establish the feasibility of applying protein engineering techniques toward an elucidation of the structural requirements for “complete” channel activity. The reconstitution of ion channels from epithelia is still in its infancy. To date, only two epithelial ion channel proteins have been biochemically extracted from their native membrane and purified (see Garty and Benos, 1988), and only one of these has been examined by reconstitution (Sariban-Sohraby er a l . , 1984; Olans et ul., 1984). In addition, only four other epithelial ion channels have been studied in planar bilayers (Vodyanoy and Murphy, 1983; Reinhardt et ul., 1987; P. Labarca, K . Anholt, and S. A. Simon, unpublished observations; our unpublished observations). This situation is sure to change in the coming years. Planar lipid bilayer technology is posed to play a major role in understanding the physiology of these proteins. ACKNOWLEDGMENTS We thank Ms. Jan Tidwell for cxcellent secretarial assistance in preparing this manuscript. This work was supported by NIH Grants DK37206 and DK38S I8 and Cystic Fibrosis Research Development Grants (Projects 2 and 4). REFERENCES Alvarez. O., Bcnos, D., and Latorre, R. (1985). The study of ion channels in planar lipid bilayer membranes. J . Electrophvsiol. Tech. 12, 159- 177. Anholt, K., Lindstrom, J . . and Montal, M. (1981). Stabilization of acetylcholine receptor channels
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by lipids in chulare solution and during reconstitution i n vesicles. J. H i d . Chem. 256, 4377-4387. Bamherg, E . , and Janko, K . (1976). Single channel conductance of lipid bilayer membranes in ~ . 426, 447-450. prescnce of monazoinycin. Biochirn. R r o p h ~ . Acin Harbry, P.,Chassande. O., Vigne, P.,Frclin. C., Ellory, C.. Cragoe, E. J . . Jr., and Luzdunski, M. ( 19x7). Purification and subunit structure of thc [’H]phcnaniil receptor associated with the renal apical Na+ channel. Pnx-. Nut/. Aced Scr. U.S.A. 84, 4836-4840. Benos. D. J. (1986). Amiloride-scnsitive cpithclial sodium channels. In “Ionic Channels in Cells and Model Systems” ( R . Latorre, cd.), pp. 401-420. Plenum, New Kirk. Benos, D. J . , and Sariban-Sohrahy, S. ( 1987). Isolation and purilication of the amiloride-sensitive Na channel from renal epithelia. In “Diuretics: Basic, Pharmacological, and Clinical Aspects” (V. E. Andreucci and A. Dal Canton, eds.), pp. 27-32. Nijhoff, Boston, Massachusetts. Bcnos, D. J., Saccomani, C . , Brcnner, B. M., and Sariban-Sohraby, S. (1986). Purification and characterization of the amiloridc-sensitive sodiuin channel from Ah cultured cells and bovine renal papilla. Proc. Nuil. A m d . Sci. U.S.A. 83, 8525-8529. omani, G . , and Sariban-Sohrahy, S . (1987). ‘The epithelial sodium channel. Subunit number and location of the amiloride binding site. J . Biol. Chmi. 262, 10613- 10618. Bosnia. M. M. (1986). Chloride channels in neoplastic B lyniphocytes. Sioqdiys. J. 49, 41 3a. Rridgcs. R . J . . Rumrnel. W., and Wollcnherg. P. (1984). Effects of vasopressin un electrolyte transport across isolated colon from normal and dexanicthasone-treated rats. J . Physiol. (London) 355, 11-23, Hridges, R. J.. Rummel, W., and Schreinci-, J. (1987). In vilm effects of dcxamethasonc on sodium transport acrosS rat colon. J . Phvsiol. (London) 383, 69-77. Bridges, R. J., Cragoe, E. J . . Jr., FriLzcll, R . A , , and Benos, D. J. ( I 9 X 9 a ) . Inhibition of colonic Na+ transport hy amiloride analogs. Am. J. Ptry~iol.256, Ch7-C74. Bridges, R. 1. Worrrl, R. T.. Frizzell. R. A , , and Bcnos, D. J. (1989b). Stilhene disulfonate blockade of colonic secretory C1 channels in planar lipid bilayers. A m . J. Physiol. 256, C902-C912. Cohcn, F. S . (1986). Fusion of liposomes to planar bilayers. I n “Ion Channcl Reconstitution” (C. Miller, cd.), pp. 131-139. Plenum, New York. Cohcn, F. S., Finkelstein, A,, and Zimmerbarg, J. (1980). Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II.Incorporation of a vesicular membrane marker into the planar membrane. J. Gen. Phvsrol. 75, 25 I - 270. Cohen, P.. Akahas, M., and Finkelstein, A . (1982). Osmotic swelling of phospholipid vesicles causes them to tuse with a planar phospholipid hilayer iiienihrane. Scicvic.e 217, 458-460. Coronado, R . ( 1986). Kccent advances in planar phospholipid hilaycr techniques for monitoring ion channels. A m Rm. Bioph.w. Clzem. 15, 2.50-277. Coronado, R., and Latorrc. R. (1983). Phospholipid bilayers made from monolayers on patch-clamp pipettes. Biophys. J. 43, 231 -236. Coronado. R . , Latorrc, R . . and Mautner, H. C. (19x4). Single potassium channcls with dclayed rectifier bchavior froni lobster axon membranes. Biophys. J. 45, 2x9- 299. Corrca. A. M . , Zhou. J . , and Agnew, W. S . (1988). Optimized fusion of native o r reconstituted membranes to artilicial liposomes for single channel recording. Bi<~/7hy.T.1.53, 227a. Crcutz, C. E., Zaks. W. J., Hamman, H. C . , Crane, S . , Martin, W. H . , Gould, K . L., Oddie, K . M.. and Parsons, S. J. (1987). Identification of chromaflin granule-binding proteins. Relationship of the chroniobindins to calclectlin, synhibin, and the tyrosine kinase substrates. J. B i d . Chem. 262, 1x60- 1868. Dale, R., and IMelice, L. (1984). Sperm-activated channels in ascidian oocytes. Dn.. Riol. 101, 23s - 239. Darszon, A . (1986). Planar hilayers: A powerful tool to study membrane proteins involved in ion
9. EPITHELIAL ION CHANNEL RECONSTITUTION
309
transport. I n “Methods in Enzymology” (L. Packer, ed.), Vol. 127, pp. 486-502. Academic Press, Orlando, Florida. Dawson, C. M., Atwater, I.. and Rojas, E. (1982). Potassium-induced insulin release and voltage noise measurements in single mouse islets of Langerhans. J . Membr. Eiol. 64, 33-43. DeFelice, L. J . , Dale, B., and Talvei, R. (1986). Distribution of fertilization channels in ascidian oocyte membranes. Proc. R . Soc. London B 229, 209-214. Dubinsky, J. M., and Oxford, G . S. (1984). Ionic currents in two strains of rat anterior pituitary tumor cells. J . Gen. Physiol. 83, 309-339. Frizzell. R. A. (1987). Cystic Fibr A disease of ion channels? Trends Neurosci. 10, 190- 193. Frizzell, R. A., Welsh, M. J., and h. P. L. (1981). Electrophysiology of chloride-secreting epithelia. In “Ion Transport by Epithelia” (S. G . Schultz, ed.), pp. 137- 149. Raven, New York. Frizzell, R . A , , Rechkemmer, G., and Shoemaker, R. L. (1986). Altered regulation of airway epithelial cell chloride channels in cystic fibrosis. Science 233, 558-560. Furman, R. E., Tanaka, J. C., Mueller, P., and Barchi, R. L. (1986). Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes. Proc. Nut/. Acad. Sci. U.S.A. 83, 488-492. Gad, A. E.. and Eytan, G . D. (1983). Chlorophyll probes for membrane fusion: Polymyxin Binduced fusion of liposornea. Biochim. Biopphvs. Acla 727, 170- 176. Garcia, A. M., and Miller. C. (1984). Channel-mcdiated TI+ fluxes in sarcoplasmic reticulum vesicles. Biophvs. J . 45, 49-5 I . Garty, H . , and Benos. D. J. (1988). Characteristics and regulatory mechanisms of the amilorideblockable Na’ channel. Phvsiol. Rev. 68, 309-373. Green, W. N., Weiss, L. R.,and Anderscn, 0. S. (1987). Batrachotoxin-modified sodium channels in planar lipid bilayers. J . Gen. Phvsiol. 89, 841-872. Greenblatt, R. E., Blatt, Y., and Montal, M. (1985). Thc structure of the voltage-sensitive sodium channel. Inferences derived from computcr-aided analysis of the Electrophorus electricus channel primary structure. FEBS Lett. 193, 125-134. Hall, J. E., and Latorre. R . (1976). Nonactin-K+ as a probe for membrane asymmetry. Biophys. J . 15, 99- 101, Hamill, 0. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981). Improved patchclamp techniqucs for high-resolution current recording from cells and cell-free membrane patches. PJuegers Arch. 391, 85- 100. Hamilton, K. L., and Eaton, D. C. (1985). Single-channel recordings from amiloride-sensitive epithelial sodium channel. Am. J . Physiol. 249, C200-C207. Hanai, T., Haydon, D. A., and Taylor, 1. (1965). Polar group orientation and the electrical properties of lccithin bimolecular leaflets. J . Theor. Biol. 9, 278-296. Hanke, W., and Miller, C. (1983). Single chloride channels from Torpedo electroplax: Activation by protons. J . Gen. Physiol. 82, 25-45. Hanke, W., Eibl. H . , and Boheim, G . (1981). A new method for membrane reconstitution: Fusion of protcin-containing vesicles with planar bilayer membranes below the phase transition temperature. Eiophys. Struct. Mech. 7, 131- 137. Hanke, W.. Roheim, G . , Barhanin, J . , Pauron, D., and Lazdunski, M. (1984a). Reconstitution of highly puritied saxitoxin-sensitive Na’ channels into planar lipid bilayers. EMEO J . 3, 509 -5 15. Hanke, W., Mcthfessel, C., Wilmsen, H . O., and Boheim, G. (1984b). Ion channel reconstitution into planar lipid bilaycrs on glass pipettes. Eiochem. Bioeng. J . 12, 329-339. Hanrahan, J. W., Alles. W. P., and Lewis, S. A. (1985). Single anion-selective channels in basolateral membrane of mammalian tight epithelium. Proc. Narl. Acad. Sci. U . S . A . 82, 7791-7795. Hartshorne, R. P., Keller, B. O., Talvenheimo, J. A., Catterall, W. A., and Montal, M . (1985).
310
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Functional reconstitution of the purilicd brain sodium channel in planar lipid bilayers. Pruc N u l l . Acuil. Sci. U.S.A. 82, 240-244. Hellstrom, U., Hamrnarstrorn, S., Dillncr, M. L.. Perlnlann, H., and Perlmann, P. (1976). Thc intcractions of nonmitogenic and mitogenic lectins with T lymphocytes: Association of cellular receptor sites. Scund. J . Immrrnol. 5 , 45. Hodgkin, A . L., and Huxley, A . I;. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. J . Physiol. (London) 117, 500-544. Horn, R., and Lange. K. (19x3). Estimating kinetic constants from single channel data. Biophys. J . 43, 207-233. Hunter, M., Lopes, A. G . , Boulpacp. E. L., and Giebisch, G . H. (1984). Single channel recordings of calciuin-activated potassium channels in the apical menibranc of rabbit cortical collecting tubules. Proc. Null. Acutl. Sci. U.S.A. 81, 4237-4239. Inouc. I. ( 1985). Voltagc-dependent chloride conductance of the squid axon membrane and its blockade by some disult’onic stilbene derivatives. J . Grn. Phyiol. 85. 5 19-537. Jacob, ‘1‘. J. C . , Hangham. J . A,. and Duncan, G . (1985). Characterization of a cation channel on the apical surface of thc frog Icns cpithclium. Q.J . Exp. Physiol. 70, 403-421. Kcllcr, H . 0.. Hartshorne, R. P., Talvenhcirno, J. A , , Catterall, W. A., and Montal. M. (1986). Sodium channels in planar lipid bilayers. Channel gating kinetics of purified sodium channels modified by batrachotoxin. J . Gcn. Phvsiul. 88, 1-23. Klacrke, D.A., Karlish. S . J. D.. and Jorgensen, P. L. (1987a). Kcconstitution in phospholipid vesicles of calcium-activated potassium channel from outer renal medulla. J. Memhr. B i d . 95, 105- 1 12. Klaerke, D. A., Petersen, J., and Jorgcnsen, P. L. (1987b). Purification of Ca”-activated K ’ channcl protein on calniodulin affinity columns after detergent solubilization of luminal membranes from outer renal medulla. FEBS Ltw. 216, 21 1-216. Krucgcr, B. K.. Worley. J. F., and French, R. J . (1983). Singlc sodium channels from rat brain incorporated into planar lipid bilayer membranes. Nofirre (London) 303, 172- 175. Labarca. P., Coronado, R., and Miller, C. (1980). Thermodynamic and kinetic studies of the gating behavior or a K -sclcctive channel from the sarcoplasmic reticulum membrane. J . Gun. Ph?;.sio/.76, 397-424. nce of red cells. I n “Oxygcn Affinity of Lassen, U. V. (1972). Membrane potential and re. Hemoglobin and Red Cell Acid Base Status” ( M . Rorth and P. Astrup, eds.), pp. 291-306. Munksgaard, Copcnhagcn. Lassen, U. V.. and Sten-Knudsen, 0. (1968). llircct ~neasurementsof membrane potentials and membrane resistance of human red cells. J . f k y s i o l . (Lunclun) 195, 68 1-696. Latorre. R.. and Alvarez, 0. (1981). Voltage-dependent channels in planar lipid bilayer membranes. f‘hysiol. Ruv. 61, 77- 150. Latorre, R., and Benos. D. (1985). Reconstitution of ionic channels into lipid bilayer membranes. In “Transmembrane Signaling and Sensation” (F. Oosawa. ed.), pp. 199-213. Jpn. Sci. Soc., Tokyo. Latorre, R., and Miller, C. (1983). Conduction and selectivity in potassium channels. J . Mrrnbr. B i d . 71, 1 1-30, I.atorre, R., Vergara, C . , and Hidalgo, C . (1982). Reconstitution in planar lipid bilayers of a CaL+dependent K channel from transverse tubule membranes isolatcd from rabbit skeletal musclc. Proc. Null. Accrd. Sci. U.S.A. 79, 805 - 809. Light, D. B . . McCann, F. V., Keller. T. M . , and Stanton, B. A. (1987). Amiloride-sensitive cation channel in rat inncr medullary collecting duct. J . Gen. Physiul. 90, 28a. Mattcson, 1). R . , and Armstrong, C. M. (1984). Na+ and Ca’+ channels in a transformed line of anterior pituitary cells. J . F e n . Physial. 83, 371 -394. +
+
9. EPITHELIAL ION CHANNEL RECONSTITUTION
31 1
Miller, C. (1978). Voltage-gated cation conductance channel from fragmented sarcoplasmic reticulum: Steady-state electrical properties. J . Memhr. B i d . 40, 1-23, Miller, C. (1983a). Integral membrane channels: Studies in model membranes. Physiol. Rev. 63, 1209- 1242. Miller, C. (198%). Reconstitution of ion channels in planar bilayer membranes: A five-year progress report. Comm. Mol. Cell. Biophvs. 1, 413-428. Miller, C., ed. (1986). “Ion Channel Reconstitution.” Plenum, New York. Miller, C. (1987). Ion channel reconstitution. Why bother? In “Ionic Channels in Cells and Model Systems” (R. Latorre, ed.). pp. 257-273. Plenum, New York. Miller, C., and Racker. E. (1976). Calcium-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers. J . Membr. Biol. 30, 283-300. Miller, C., Arvan, P., Telford, J. N., and Racker, E. (1976). Ca’+-induced fusion of proteoliposomcs: Dependence on transmembrane osmotic gradient. J . Membr. Biol. 30, 271 -282. Miyazaki, S.,and Igusa, Y. (1982). Ca-mediated activation of a K current at fertilization of golden hamstcr eggs. Proc. Nail. Acud. Sci. U.S.A. 79, 931-935. Montal, M. (1987). Reconstitution of channel proteins from excitable cells in planar lipid bilayer mcmbrdnes. J . Membr. B i d . 98, 101-I 15. Montal, M., and Mueller, P. (1972). Formation of himolecular membranes from lipid monolayers 69, 3561-3566. and a study of their electrical properties. Proc. Null. Acad. Sci. U.S.A. Montal, M., Labarca, P.. Fredkin, D. R . , Suarez-lsla, B. A., and Lindstrom, J. (1984). Channel properties of the purified acetylcholine receptor from Torpedo culifornica reconstituted in planar lipid bilayer membranes. Biophvs. J . 45, 165- 174. Mueller. P., and Kudin, D. 0. (1968). Bimolecular lipid membranes: Techniques of formation, study of electrical properties. and induction of ionic gating phenomena. I n “Laboratory Techniques in Membrane Biophysics” (H. Passow and R . Stampfi, eds.), pp. 141-156. SpringerVerlag, Berlin. Mueller, P., Rudin, D. O., Tien, H. T., and Wcstcott, W. C. (1962). Reconstitution of cell membrane structure in vitro and its transformation into an excitable system. Nature (London) 194, 979-980. Neher, E., and Sakmann, B. (1976). Single-channel currents recorded from membrane of denervared frog muscle fibers. Nature (London) 260, 799-802. Nelson, D. J., Tang, J. M., and Palmer. L. G. (1984). Single channel recordings of apical membrane chloride conductance in A6 epithelial cells. J . Membr. Riol. 80, 81-89. Noda, M . , Shimizu, S . , Tanabe, T., Takai, T., Kayano, T., Ikeda, T., Takahashi, H . , Nakayama, H . , Kanaka, Y., Minamino, N., Kangawa, K . , Matsuo, H . , Raftery, M. A , , Hirose, I., Notake, M., Inayama, S . , Hayashida, H., Miyata, T., and Numa, S. (1984). Primary structure of Elecirophorus elecfricus sodium channel deduced from cDNA sequence. Nulure (London) 312, 121- 127. Noda, M.,Ikeda, T., Kayano, T., Suzuki, H . , Takeshiina, H . , Kurasaki, M., Takahashi, H., and Numa, S . (1986). Existence of distinct sodium channel messenger RNAs in rat brain. Nuiure (London) 320, 188- 192. Oiki, S . , Danho, W., and Montal, M. (1988). Channel protein engineering: Synthetic 22-mer peptide from the primary structure of the voltage-sensitive sodium channel forms ionic channels in lipid bilayers. Proc. Nail. Acud. Sci. U.S.A. 85, 2393-2397. Olans, L . , Sariban-Sohraby, S . . and Benos, D. J. (1984). Saturation behavior of single, arniloridesensitive Na’ channels in planar lipid bilayers. Biophys. J. 46, 831-835. Palmer, L. G . , and Frindt, G . (1986). Amiloride-sensitive Na+ channels from the apical membrane of the rat cortical collecting tubule. Proc. Null. Acad. Sci. U.S.A. 83, 2767-2770. Reinhardt, R., Bridges. R. J., Rummel, W., and Lindemann, B. (1987). Properties of an anion-
312
ROBERT J. BRIDGES AND DALE J. BENOS
selective channel from rat colonic enterocyte plasma membranes reconstituted into planar phospholipid bilayers. J. Membr. B i d . 95,47-54. Rodriguez-Boulan, E. (1983). Membrane biogenesis, enveloped RNA viruses and epithelial polarity. I n “Modern Cell Biology” ( B . H. Satir, ed.). pp. 119- 170. Liss, New York. Salkoll‘, L , Butler, A., Wei, A.. Scavarda, N., Giflcn, V., Ifune, C., Goodman, R . , and Mantel, G . (1987). Gcnomic organization and deduced amino acid sequence of a putative sodium channel gene in Drosuphila. Science 237,744-749. Sarihan-Sohraby, S . , Latorre, R., Burg, M . , Olans, L., and Benos, D. (1984). Amiloride-sensitive epithelial Na ‘ channels reconstituted into planar lipid bilayer membranes. Nature (London) 308, 80-82. Sigworth, P. J. (1983). Electronic design of the patch clamp. In “Single Channel Recording” (R. Sakrnann and E. Neher, eds.), pp. 3-35. Plenum, New York. Suarez-lsla, B. A , , Wan, K., Lindstrom, J., and Montal, M. (1983). Single-channel recordings from purified acetylcholine receptors reconstituted in bilayers formed at the tip of patch pipettes. Hiorhumistry 22,2319-2323. Tanifuji, M., Sokabc, M . , and Kasai, M. (1987). An anion channel of sarcoplasmic reticulum incorporated into planar lipid bilayers: Single channel behavior and conductance properties. ./. Memhr. B i d . 99, 103- I I 1 . Tank, D. W., Miller, C., and Webb, W. W. (1982). Isolated-patch recording from liposonies containing functionally reconstituted chloride channels from Turpedo electroplax. Proc. Nu//. Acud. Sci. U.S.A. 79, 7749-7754. Tien, H. T. (1967). Thickness determination and molecular organization by optical methods. J . Thror. B i d . 16,97- 110. Vudyanoy, V.,and Murphy, R. B. (1983). Single-channel Huctuations in biinolecular lipid membranes induced by rat olfactory epithelial homogenates. Science 220, 717-7 19. White. S. H. (1970). Thickness changes in lipid hilayer membranes. Biochim. Biophvs. Acru 196, 354-357. White, S. H. (1986). The physical nature of planar bilayer membranes. I n “Ion Channel Reconstitution” (C. Miller, ed.). pp. 3-35. Plenum, New York. White, M. M., and Miller, C. (1979). A voltage-gated anion channel from the electric organ of Torpedo cu/i/ornic-a.J. B i d . Churn. 254, 10161- 10166. White, J., Kielian. M., and Helenius, A. (1983). Membrane fusion proteins of enveloped aninial viruses. Q.Ruv. Biophvs. 16, 1.51- 195. Wilmsen, U., Methfessel, C., Hanke, W., and Bohcirn, C. (1983). Channel current fluctuation studies with solvent-free lipid bilayers using Ncher-Sakniann pipettes. I n “Physical Chemistry of Transmembranc Ion Motions” (C. Troyanowsky, ed.), pp. 83-90. Elsevier, Amsterdam. Young, T. M., and Young, I. D. (1984). Protein-mediated intermembrane contact of lipid vesicles with planar bilayers. Biochim. Biophvs. Acru 775, 441 -445. Zweifach, A., and I.ewis, S. A. (1988). Characterization of a partially degraded Na’ channel from urinary tract epithelium. J. Memhr. R i d . 101,49-56.
Index
A Affinity chromatography for isolation of epithelial sodium channels, 286 Amiloride-induced noise analysis of sodium ion channels of tight epithelia, 136- 142 rate coefficients for, 136- 138 single-channel currents and densities in, 138- 142 Amiloride-sensitive macroscopic currents in sodium ion channels of tight epithelia, 142- I44 4-Aminopyridine as apical potassium ion channel blocker, 170- 171 Amphotericin B, apical membrane permeabilization by, 195- 199 Anion permeation of epithelial versus nonepithelial chloride channels, 275-276 Antibiotics, pore-forming, apical membrane permeabilization by, 195- 199 Apical membrane, permeabilization of, 194- 20 1 by detergents, 199-201 selective, by pore-forming antibiotics, 195- 199 Autocorrelation function in Fourier analysis, I3 of stochastic variable, 6 Autocorrelation time in stochastic processes, 7 Autocovariance function as temporal measure of ion channel fluctuations, 66 functional form of, 66-71 power spectral density function and, 71 -76
B Barium ion blockade of potassium ion channels, rates of, nature of permeant ion species and, 173- I75
31 3
Barium ions as apical potassium ion channel blockers, 173- 175 Basolateral membrane of epithelial cells fluctuation analysis of, 193- 194 noise from, 201 -209 channel subpopulations and, 206-209 channels and blockers of, 201 -206 potassium channel noise from, 191-209 value of, 209 in transepithelial salt transport, 192- 193 Basolateral potassium ion channels versus apical channels, 185 Biomembranes, ion conductance fluctuations in, 29-33 nonstationary noise analysis and, 3 1-33 stationary noise by spectral type and, 29-31 Blocker-induced noise in epithelia, 44-51 analysis of, problems related to, 55-57 from interaction between blockers, 47-5 1 from single blocker, 45-47 in sodium ion channels of tight epithelia, 122-126 Blockers of apical potassium ion channels, 170- 177 4-aminopyridine as, 170- 171 barium ions as, 173- 175 cadmium ions as, 171 cesium as, 170 protons as, 170 quinidine as, 170- 171 rubidium as, 170- 171 signals of, in current noise, 171- 173 tetraethylammonium as, 170- 171 voltage-dependent action of, 175- 176 channel, basolateral membrane noise and, 201-206
314
INDEX
Blockers (con/inued) of secretory chloride channels. 267-268 of sodium ion channels CDPC as, results from noise analysis with. 134-136 CGS 4270 as, results from noise analysis with, 134- I36 choicc ui, 132- 134 electroneutral and charged, noise analysis with, 130-132 rate coefficients of, apical sodium ion concentrations and, 149- 152
C Cadmium ions as apical potassium ion channel blockers, 171 Calciuni ion-aensitivc channels in amphibian epithelia, 43-44 Cation channels apical, amphibian. kinship of, 185- 186 in epithelia, search for. 233-237 identification and analysis of. 225-233 amplitude histograms in, 225-228, 229-231 duration histogram in, 228, 232-233 nonselective, in epithelia, unexpected, 237 patch clamp of, 215-242 Cesium as apical potassium ion channel blocker. 170 CGS 4270, noise analysis of sodium ion channel blockers with. results of. 134- 136 Channcl dcnsitics i n sodium ion channclb of tight epithelia, 126- 130 Channel gating as potassium ion current dcterminant, 184 Chapman-Kolmogorov equation, 8- 9 Charged sodium ion channcl blockers, noise analysis with, 130- 132 Chemical modifications of secretory chloride channels, 268- 269 Chloride ion channels epithelial, 247- 276 absorptive. 248-258 leaky, 256 regulation of. 257-258 tight. 248-256 incorporation of, into planar lipid bilaytm. 301-30s and nonepithelial, comparison of, 274276
secretory, 258 - 272 blockers of. 267-268 chemical modifications of, 268 -269 ion selectivity of, 259-263 open probability of, 266 properties of, 259-269 regulation of, 269-272 single-channel conductance of, 263266 volume-sensitive, 272- 274 in frog skin, 44 6-Chloro-3,4-diaminopyrazine~2~carboxamide (CDPC), noise analysis of sodium ion channel blockers with, results of, 134- 136 Chromatography. affinity, for isolation of epithelial sodium channels, 286 Conditional probability in stochastic processes, 7 Conductance, single-channel, of secretory chloride channels, 263-266 Conduction, physical noise in. 14- 19 diffusion, 19 fluctuation-dissipation theorem in. 14- 16 fractal. 18-19 Johnson-Nyquist description of. 16 shot, 17 Convolution, response to arbitrary driving function and, 12- I 3 Current noise, signals of potassium ion channel blockers in, 171 173 ~
D Detergents. apical niembranc perrneabikation by, 199-201 Diffusion noise in conduction, I 9 Digitonin, apical membrane permeabilization by, 199-201 Distributions in stochastic proccsses, 6
E Electrical noise in physics and biology, 3-33 Electroneutral sodium ion channcl hlackcrs, noise analysis with, 130- 132 Epithelial cells basolateral iiicmbrane of, potassium channel noise from, 191-209 chloridc channels in, 247-276: SPC ulso Chloride channels, epithelial preparation of, for patch clamping, 218-222 Epithelium(ia) cation channels in, search for, 233-237
INDEX
315
ion channels in incorporation of. into planar lipid bilayers. 291-297 noise analysis of, 115-212 reconstitution of, 283-307 bilayer techniques in, 288-291 into planar phospholipid bilayers, 297-306 sodium, incorporation of, into planar lipid bilayers, 298-301 ion transport across, potassium ion channels and, 162-163 single-channel events in, 213-312 tight, apical sodium ion channels of, 117152; see ulso Sodium ion channels of tight epithelia, apical Ergodicity in stochastic proccssa, 7-8 Expectation value of function, 5
F Fluctuation-dissipation theorem, 14- 16 Fluctuations. spontaneous; see Noise Folded bilayer technique of epithelial ion channel reconstitution, 289-290 Fourier analysis, application of, to noise probIcnis, 9- 13 autocorrelation function and, 13 convolution and, 12- 13 linear driving-point analysis and, 10- 12 spectral density and, 13 Fourier series, 9- 10 Fourier transform(s), 9- 10 of autocovariance function, power spectral density function as. 71 -76 Fractal noise in conduction, 18- 19 Fusion method of ion channel incorporation into planar lipid hilayers, 291 -297
G Gating, channel as potassium ion current determinant. 184 potassium ions in, 166- 169
H Hormone action, potassium ion channel density and, 182-184
I Impedance, linear driving-point analysis and, 10- 12
Ion channels epithelial incorporation of, into planar lipid bilayers, 29 I 297 noise analysis of, I 15-212 reconstitution of, 283-307 bilayer techniques in, 288-291 into planar phospholipid bilayers, 297- 306 fluctuations in, 61- 112 autocovariance function of, power spectral density function and. 71 -76 measurements of, determination of channel properties from, 62-76 power spectral analysis of, 62-76 single-channel, probabilistic character of, 63-66 single-channel analysis of, 77-92 comparison of, with fluctuation mcasurements, 92- I 1 I temporal measure of, autocovariance function as, 66 potassium; see Potasssiuni ion channels properties of, determination of, from fluctuation measurements, 62-76 sodium; set Sodium ion channels transepithelial noise signals from, analysis of. 37-57; see also Transepithelial nciise signals Ion conductance, fluctuations in, in biomembI'dneS, 29-33 Ion selectivity of sccretory chloride channels. 259-263 -
J Johnson-Nyquist description. 16
L Langevin equation, 15 Linear driving-point analysis, 10- 12 Lipid bilayers, planar, ion channel incorporation into, 291-297 Lorentzian function in power spectrum, 29-31 Lorentzian noise from transepithelial current, 163- 170
M Markov processes, 8-9 Membrane vesicle populations, specific, isolation and purification of, 285-288
316
INDEX
N Noise from apical potassium ion channels, 157 I87 basolateral membrane, 201 -209 channel subpopulations and, 206-20'9 channels and blockers of, 201-206 blocker-induced in epithelia. 44-51 analysis of, problems related to, 55-57 from interaction between blockers, 47-51 from single blocker, 45-47 in sodium ion channels of tight epithelia, 122-126 conduction; srr o l s o Conduction, physical noise in barium ion-induced, in discovery of potassium ion channels. 177 current. signals of potassium ion channel blockers in, 171- 173 diffusion. in conduction, 19 electrical, in physics and hiology, 3-33 fractal, in conduction, 18- 19 measuremiits and analysis technique5 for, 19 29 signal amplitude statistics in. 22-26 signal-to-noise ratio as. 19-22 spectral analysis as, 26-29 nonstationary, analysis of, in ion conductance, 31-33 phenomena producing, early observations and description of, 4 physical, in conduction. 14- 19 potassium ion channel. spontaneous, dctcction of, 163- 166 prohlems of, Fourier analysis applied to, 9-13 shot, 4 i n conduction, 17 spontaneous, absence of, in sodium ion channels of tight epithelia. 120- 121 stationary, by spectral type in ion conductance, 29 31 trdnsepithelial; see Transepithelial noisc signals Noise analysis of epithelial channels. I IS-212 of potassium ion channels history of. 158- 160
methods of, 159- 160 of sodium ion channels of tight epithelia, 117- 152 amiloride-induced rate coefficients for, 136- 138 results of, 136- 142 single-channel currents and densities in, 138- 142 with CDPC, results of, 134- I36 with CGS 4270, results of, 134-136 with electroneutral and charged blockers. 130-132 triaiiitcrene-induced. 144- 148 Nystatin, apical membrane permcabiliration by. 195, 197-199
P Patch clamp of cation channels, 215-242 data from, on single-cation channels, use and physiological relevance of, 237-242 for identification and analysis, 225-233 Patch clamping of epithelial cells cation channel identification and analysis for. 229-233 epithelial cell preparation for, 21 8-222 patch conligurations for. 222 225 patch pipet fdbrication for, 216-218 technical aspects of, 216- 233 Patch pipet fabrication, 216-218 Phospholipid bilayers, planar, reconstitution of ion channels from epithelia into, 297-306 Pipet, patch, fabrication of, 216-218 Planar lipid bilayers, ion channel incorporation into, 291-297 Planar phospholipid hilayer membrane technique of epithelial ion channel reconstitution. 288, 289 Planar phospholipid bilayers, reconstitution of ion channels from epithelia into, 297-306 Polyene antibiotics. apical mcmhrane permeabilization by, 195- 199 Potassium ion channels apical versus basolateral, 185 blockers of. 170- I77 evaluation of, methods of, 160- 162 fluctuating. "spontaneous," 163- 170 noise from, 157- 187 permcability of, influencing. 182- 184 in baso~atcra~ membrane. noise from. 191 209 value of, 209
317
INDEX chemistry of. 18 I - I82 epithelial expected, 234-235 incorporation of, into planar lipid bilayers, 305 306 unexpected, 236-237 noise analysis of history of, 158- 160 methods of, 159- 160 noise in, spontaneous, detection of. 163- 166 parameters of, microscopic. 180- 181 roles of, putative, 162- I63 selectivity of. 177- 180 unexpected, discovery of, barium ioninduced conduction noise in. 177 Potassium ion currents. determinant of, channel gating as, 184 Potassium ions long-term exposure to. potassium ion channels and, 184 para- versus transcellular pathway for, 158- 159 Power spectral analysis, 62-76 advantagesldisadvantagesof. 76 determination of channel properties from fluctuation measurements i n , 62-76 Power spectral density function as Fourier transform of autocovariance function. 71-76 Probability(ies) open, of secretory chloride channels. 266 in stochastic processes, 6-7 Probability density function of stochastic vai-iable, 5 Protons as apical potassium ion channel blockers. 170 -
Q Quinidine as apical potassium ion channel blocker. 170- I7 I
R Rubidium as apical potassium ion channel blocker. 170- I7 I S
Salt. transepithclial transport of. basolateral membrane in, 192- 193 Shot noise. 4 in conduction. 17
Signal amplitude statistics in noise analysis. 22-26 Signal-to-noise ratio in noise measurement, 19-22 Single-channel analysis of ion channel fluctuations, 77-92 advantages/disadvantages of, 91 -92 distribution of intervals in, 77-90 four-state model for, implications of, 108- I I I three-state kinetic scheme in, 104 Single-channel conductance of secretory chloride channels, 263-266 Single-channel currents in sodium ion channels of tight epithelia, 126- 130 Sodium ion channels blockers of, 130- 136; see also Blockers of sodium ion channels epithelial expected, 234 incorporation of, into planar lipid bilayers, 298-301 unexpected, 235-236 of tight epithelia, apical, 117- 152 absence of spontaneous noise in, 120- 12 I blocker-induced noise in, 122- 126 noise analysis of, I 17- 152; see also Noise analysis of sodium ion channels of tight epithelia single-channel currents and channel densities in, 126- 130 theoretical perspectives on, 118- 120 Sodium ions. concentration of, apical. dependence of blocker and spontaneous rate cocfticientb on, 149- 152 Solvent-free bilayer technique of epithelial ion channel reconstitution, 289-290 Spectral analysis of noise, 26-29 Spectral density of Huctuations in Fourier analysis, 13 Spontaneous fluctuations; .we Noise Spontaneous noise, absence of, in sodium ion channels of tight epithelia, 120- 121 Stationarity in stochastic processeh, 7 Stationary noise by spectral type in ion conductance, 29-31 Stochastic processes autocorrelation function and. 6 autocorrelation time and, 7 characterization of, 4-9 conditional probability and, 7
318
INDEX
Stochastic processes ( miiritiued) crgdicity and, 7-8 expectation value and, 5 Markov, 8-9 moments of. 5 , I probabilitics and, 6-7 probability density function and, 5 stationarify and. 7 variance and, 5
'I' Tetraethylammonium as apical potassium ion channel blocker, 170- 171 Tip-dip method of epithelial ion channel reconstitution, 290-291 Transepithelial current, Lorentzian noise from, 163- 170 Transepithelial noise signals from calcium ion-sensitive channekin amphibian, 43-44 from chloride ion channels in frog skin, 44 from ion channcls analysis of limitations of methods of, 51 -55 low-noise instrumentation in, 38 4 I
blocker-induced. 44-5 1 analysis of, problems related to. 55-57 spontaneous conipvnents of, 41 -44 from ion channels. analysis of driving force in, 52 model dependency of, 5 I peaking. frequency-dependent attenuation in, 52-55 Transepithelial salt transport. hasolateral niembrane in, 192- 193 Transition probability in stochastic processes. X 'l'riamtcrcnc-induced noise analyais of sodium ion channels of tight epithelia, 144- 148
V Variance of distrihution. 5 Voltage dependence of block on, analysis of hlockerinduced noisc and, 55-57 potassium ion channel blockers dependent on. 175-176 Volume-sensitive chloride channels, 272-274
W Wiener-Khinchine theorem, 13
