Current Topics in Membranes and Transport, Volume 10

Current Topics in Membranes and Transport Volume 10

Advisory Board

1. S . Edelman Alvin Essig Franklin M . Harold James D. Jamieson Anthony Martonosi Shmuel Razin Martin Rodbell Aser Rothstein Stanley G. Schultz

Contributors

Ernest0 Carafoli Ta-Min Chang Martin Crompton A. A. Eddy E . A. Evans Hugo G . Ferreira R. M . Hochmuth Virgilio L. Lew David M . Neville,]r.

Current Topics in Membranes and Transport VOLUME 10

Membrane Properties: Mechanical Aspects, Receptors, Energetics and Calcium-Dependence of Transport

Edited by Felix Bronner Department of Oral Biology Unioersity of Connecticut Health Center Farmington, Connecticut and

Arnort Kleinzeller Depurtment of Physiology Unioersity of Pennsylvania School of Medicine Philadelphia, Pennsylvania

1978

Academic Press

New York

Son Francisco

London

A Subsidiury of Harcourt Brace Jooanouich, Publishers

COPYRIGHT @ 1978, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING P n o r o c o P Y , RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING F R O M THE PUBLISHER.

ACADEMIC PRESS, INC. 111 Fifth Avenue, New

York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NW1 IDX

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 70-1

ISBN 0-12-153310-7 PRIN'IED IN THE UNITED STATES OF AMERICA

17091

List of Contributors, vii Preface, ix Mechanochemical Properties of Membranes E. A. EVANS AND R. M. HOCHMUTH I. 11. 111. IV. V.

Introduction, 1 Intrinsic Membrane Forces and Moments, 4 Membrane Elasticity and Free Energy Storage, 6 Membrane Viscosity and Fluidity, 41 Summary,58 Symbols, 59 References, 61

Receptor-Mediated Protein Transport into Cells. Entry Mechanisms for Toxins, Hormones, Antibodies, Viruses, Lysoromal Hydrolases, Asialoglycoproteinc, and Carrier Proteins DAVID M. NEVILLE, JR. AND TA-MIN CHANG I. 11. 111. IV. V. VI . VII. VIII. IX. X.

Introduction, 66 Toxins, 68 Carrier Proteins, 101 Asialoglycoproteins, 11 1 Fibroblast Lysosomal Hydrolases, 115 Antibodies, 116 Viruses, 120 Growth Factors and Hormones, 122 Summary, 131 Pharmacological Implications of Receptor-Mediated Protein Transport, 137 References, 139

The Regulation of lntracellular Calcium ERNEST0 CARAFOLI AND MARTIN CROMPTON I. Introduction, 151 11. The Chemical Basis o f t h e Biological “Fitness” of C P , 153 111. The Influence of Ca2+on the Molecular Architechire and Functional Properties of Biological Membranes, 157 IV. Intracellular Concentrations of Caz+,160 V. General Considerations on the Regulation of Intracellular C 3 + , 164 VI. The Transport of Ca2+across Plasma Membranes, 166 V

vi

CONTENTS

VII. VIII. IX. X.

The Transport of Ca2+by Sarcoplasmic and Endoplasniic Reticnlmn, 173 The Transport of Ca2+by Mitochondria, 183 The Transcellular Transport of Ca2+,193 Conclusions, 195 References. 197

Calcium Transport and the Properties of a Calcium-Activated Potassium Channel in Red Cell Membranes VIRGIL10 L. LEW AND HUGO G. FERREIRA I. Introduction, 218 11. The Ca-Sensitive K Channel in Red Cells, 224 111. Ca Transport, 234

IV. V. VI. VII.

The Gating Mechanism, 247 Pharmacological Effects, 256 The Movement of K and the Nature of the Permeability Mechanism, 265 Conclusions, 270 References, 271

Proton-Dependent Solute Transport in Microorganisms A. A. EDDY

I. Introduction, 280 11. Principles of Gradient Coupling, 282 111. Cation Transport, 290

IV. V. VI. VII. VIII. IX.

Carbohydrate Transport, 304 Amino Acid Absorption in Fungi, 320 Amino Acid Transport in Bacteria, 327 Sodium-Dependent Systems, 335 Miscellaneous Compounds, 338 General Conclusions, 343 References, 346

Subject Index, 361 Contents of Previous Volumes, 365

List of Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland (151)

Ernesto Comfoli, Laboratory

of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland (65)

Ta-Min Chang, Section on Biophysical Chemistry, Laboratory

Martin Cmnpton, Laboratory of Biochemistry, Swiss Federal Institute

of Technology

(ETH), Zurich, Switzerland (151)

of Biochemistry, University of Manchester Institute of Science and Technology, Manchester, England (279)

A. A. Eddy, Department

E. A. Evans, Department

of Biomedical Engineering, Duke University, Durham,

North Carolina (1) Hugo 0. Ferreim,* Physiological Laboratory, Cambridge University, Cambridge, Eng-

land (217) R. M. Hochmuth, Department of Chemical Engineering, Washington University, St.

Louis, Missouri (1) Virgilio 1. Lew, Physiological Laboratory, Cambridge University, Cambridge, England

(217) David M. Neville, Jr., Section on Biophysical Chemistry, Laboratory of Neurochemistry,

National Institute of Mental Health, Bethesda, Maryland (65)

* Permanent address: Grupo de Biofisica, Instituto Gulbenkian d e Ciencia, Oeiras, Portugal. vii

This Page Intentionally Left Blank

Preface In the Preface to the first volume of this series in 1970, the Editors, the Advisory Board, and the Publishers of Current Topics in Membrunes and Trunsport, having recognized the crucial role of membranes in cellular function, committed themselves to publish scholarly reviews of significant work in the field. This tenth volume offers an occasion to evaluate the expectations of those involved. In the past eight years, the field has witnessed an explosive growth to the extent that only few areas of biology have not felt its impact. The hope of the Editors has been justified in that several milestones have been passed on the road from a mostly phenomenological description of the underlying processes towards their understanding at the molecular level. Judging from the broad range of comments from our readers, w e trust that the 48 authoritative reviews published so far in this series have contributed to an evaluation of the basic problems and to mapping further experimental and conceptual adventures. These critical reviews have covered many areas of interest. If major gaps still exist, it is often because potential authors could not be persuaded to review a field. Much of the success of this series has been due to the Advisory Board’s help in identifying authors and emerging fields of interest and to the critical assistance of the many external referees who helped us get the manuscripts ready for publication. This tenth anniversary issue continues the traditions of past volumes. Membranes are reviewed in terms of their mechanochemical properties and their receptor proteins; and the pivotal intracellular role of calcium is analyzed. The last two chapters deal with transport, one with the calcium-activated channel of the red cell membrane, the other with proton-dependent microbial solute transport. I n the future we plan to issue not only broad-based volumes like the present one, but also topic-oriented volumes with guest editors sharing our burden. Volume 11, to be published later this year, will be the first in our guest-edited issues and will deal with glycoproteins. We hope our readers, sharing our excitement at the future of our field, will want to join us and the others directly involved in wishing Current Topics in Membranes and Transport a Happy Anniversary.

FELIXBRONNER

ARNOSTKLEINZELLER ix

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Mechanochemical Properties of Membranes E . A . EVANS AND R . M . H O C H M U T H Department of Biomedical Engineering Duke University Durham, North Carolina and Department of Chemical Engineering Washington University S t . Louis, Missouri

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . 11. Intrinsic Membrane Forces and Moments . . . . . . . . . . . . 111. Membrane Elasticity and Free Energy Storage . . . . . . . . . . . . . A. Thermodynamics and Mechanics . . . . . . . . . . . . . . . . . B. Compressibility: Volume, Area, Thickness . . . . . . . . . . . . . C. Rigidity: Resistance to In-Plane Shear or Extension . . . . . . . . . D. Curvature Elasticity: Bending Resistance and Chemically Induced Moments. . . . . . . . . . . . . . . . . . . . . . . IV. Membrane Viscositv and Fluidity, . . . . . . . . . . . . . . . A. Internal Dissipation and Coefficients of Surface Viscosity . . . . . . B. Fluidity, Particle Diffusion, and Membrane Viscosities . . . . . . . V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Symbols. . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

1 4

6 6 15 22 29 41 42

52 58 59 61

INTRODUCTION

The significance of the membrane barrier surrounding a cell in the transport of ions and larger molecules to the cell’s interior has long been recognized and has attracted much attention. However, the structural characteristics of the membrane provide security for the interior cytoplasm; these characteristics have been studied seriously only during the recent decade. The importance of membrane mechanical properties in cell viability is obvious, and defects or breakdowns in these properties can b e catastrophic for the cell. In addition, the mechanical properties are determined by the molecular organization 1

2

E. A. EVANS AND R. M. HOCHMUTH

of the membrane and thereby provide a measure of the intrinsic molecular structure. The first mechanical experiments on biological cell membranes were performed in the 1930s,,using sea urchin eggs (Cole, 1932) and subsequently nucleated red cells (Norris, 1939). It appeared to these early experimentalists that the membrane was a composite material made up of a bilayer of amphiphilic molecules called lipids plus additional material, presumed to be protein, that provided structural rigidity and support for the membrane (Seifriz, 1926; Norris, 1939). In recent years, biochemists and ultrastructuralists have established a similar picture of plasma membrane molecular arrangement; Singer and Nicolson (1972) summarized the evidence and presented a conceptual view of the membrane as a “fluid mosaic” with mobility restricted to the plane of the membrane. In addition, the work of Marchesi et al. (1969) established the existence of a protein, spectrin, which is associated with the cytoplasmic membrane face. Incorporating spectrin as a structural component with the fluid mosaic, Steck (1974), Elgsaeter and Branton (1974), and Singer (1974) brought the conceptual model back into agreement with the 1930 postulate. A schematic view of such a membrane composite is shown in Fig. 1(from Evans and €loch-

FIG.1. Symbolic representation of the red cell membrane as a solid-liquid composite. The structural backbone from which the membrane obtains its solid properties is shown as a random matrix on the underside (cytoplasmic side) of the lipid bilayerintegral protein mosaic. (Taken from Evans and Hochmuth, 1977.)

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

3

muth, 1977). For cell membranes more complicated than that of the red cell, additional structure exists in association with the membrane, e.g., connective tissue, cytoplasmic elements, and so on. Consequently, the membrane composite appears to be made up of both solid and liquidlike elements including intramembranous particles perhaps “floating” in the amphiphilic bilayer and interacting with the solidlike elements. Definitive evidence about the solid or liquid aspects of the membrane is provided only by direct measurement of the intrinsic material response to an applied force. Mechanical experiments, when properly analyzed, yield the appropriate material properties, e.g., elastic moduli and coefficients of viscosity, for the membrane. Such properties characterize the structure as a continuum material; in other words, the scale in time and space over which these properties are measured must include enough molecules so that the eccentric behavior of an individual molecule is averaged out. I n biological membranes, the material can be considered only as a continuum in the two dimensions describing the membrane surface; the membrane retains its molecular character in the third dimension (thickness). The material is therefore anisotropic in three dimensions but can be isotropic in the surface plane. Because the fluctuation in a material property is inversely proportional to the square root of the number of molecules in the material element (Landau and Lifshitz, 1969, Ch. 12), the scale of the area of a membrane element must b e on the order of 0.1 p m or greater in order to be considered a continuum (assuming a surface area per molecule of 100 A’ and a 1%fluctuation in the property). Consequently, the intrinsic material forces and moments created in response to applied extrinsic forces can be considered as distributed per unit length along imaginary edges of the membrane surface; the material forces perpendicular to the surface are distributed per unit area. In this article, we intend to describe briefly the intrinsic material forces, deformation, and rate of deformation of the membrane as a continuum. From thermodynamic principles, we develop the relationship between the intrinsic forces and the deformation and rate of deformation. This provides the basis for evaluating past experimental studies on membrane mechanics, leading us to a first-order appraisal of the constitutive relations between the intensive material parameters. In turn, the appraisal provides a complementary structural view of the membrane and perhaps indicates new areas for experimental and theoretical investigation. The presentation is not designed to be a comprehensive literature survey; on the contrary, we have selected references that w e feel have had a strong influence on the developments in

E. A. EVANS AND R. M. HOCHMUTH

4

membrane mechanics. Since the vast majority of experimental work has been done using red cell membranes and amphiphilic monolayer and bilayer systems, we restrict our discussion to these membrane systems.

II. INTRINSIC MEMBRANE FORCES AND MOMENTS

We consider the membrane as a thin strata consisting of a few molecular layers. Each monomolecular layer can essentially oppose only forces that act in the plane of the layer. A monomolecular layer primarily supports in-plane forces, because the energy produced by molecular moments is negligibly small until the surface radii of curvature approach molecular dimensions. The distribution of these forces per unit Iength in the surface, e.g., dynes per centimeter, is illustrated by creating a free-body diagram, i.e., cutting the surface (Fig. 2); the cut exposes the distributed force acting along the edge. The force per unit length along the edge can be decomposed into two parts, normal and tangent to the edge. The force resultants are called the tension T , and shear resultant T,, respectively. For a thin multilayered membrane, additional resultants are produced in the membrane plane (Fig. 3). Strongly associated layers produce couples of force resultants between layers; these create a moment resultant M acting on the exposed edge. Because the couples are formed by small interlayer distances (e.g., on the order of molecular dimensions), the moment resultant usually contributes negligibly to the membrane equilibrium in response to external forces. There are

.

J FIG. 2. Free-body diagram for a monomolecular layer. The applied forces (which are equal and opposite) that can be supported by the monomolecular layer are shown by the heavy arrows. The force resultants are distributed per unit length along the cut made in the layer and are shown by the light arrows. Here T , is the shear resultant and T, is the (normal) tension acting within the membrane at the location of the cut. The cgs units of T , and T, are dynes per centimeter.

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

M

c

QT

1

Tr Tn

1

5

MomentRedtant Transverm Resultant Shear

Force Resultant

-..-&

*'

x'

Shear Resultant Tension

FIG.3. Free-body diagram for a thin multilayered membrane in which the adjacent layers are strongly associated (i.e., coupled). Here, in addition to the shear resultant T , and tension T , shown in Fig. 2, the membrane can have a moment resultant per unit length M (cgs units of dyne-centimeters per centimeter) and a transverse shear resultant QT (cgs units of dynes per centimeter).

important situations, however, in which the moment resultant is the dominant response to the external forces. Moment resultants have units of dyne-centimeters per centimeter, dynes, or ergs per centimeter in the cgs system. I n addition to the moment resultant, a transverse shear resultant QTcan exist, which acts to cut the membrane normal to its plane. The transverse shear is specified directly by the surface gradient of the moment resultant. The transverse shear resultant has cgs units of dynes per centimeter or ergs per square centimeter. Normal forces acting on opposite faces of the membrane are distributed as a normal stress u3, illustrated in Fig. 4. The normal stress is an intrinsic force per unit surface area and is given in cgs units as dynes per square centimeter. For thin membranes, the normal stress is uniform across the membrane; in other words, each layer of the membrane exerts the same normal force per unit area on the adjacent layer. Therefore the normal stress is determined by the parts of the extrinsic normal forces on the two membrane faces which are equal and opposite in direction. If normal forces act on the membrane faces which are not equal and in opposition, the difference must be balanced by projections of the membrane tensions T , in the direction normal to the membrane surface (as in the law of Laplace) plus any contribution from the transverse shear QT.This normal force balance is one of three equations of force equilibrium for the membrane element; the

6

E. A. EVANS A N D R. M. HOCHMUTH

t3

J

TI

+03

FIG.4. Free-body diagram of the normal force per unit area u3and the principal membrane tensions TI and T z acting on an element of the membrane. A principal axis system is chosen such that the shear resultants T , along the edges are zero.

other two are the balance of forces in the two directions tangent to the membrane surface. Including the sums of moments acting on the membrane element, these equations specify the equilibrium of any thin shell and must be accounted for in the analysis of mechanical experiments. Flugge (1973) gives an excellent treatment of shell theory. Our discussion does not explicitly involve the equations of equilibrium but instead focuses on the relation of the force and moment resultants to the material deformation and time rate of deformation. These constitutive relations represent the reversible (elastic) and irreversible (viscous) response of the membrane material to applied forces. Fung (1966) first discussed the equations of equilibrium of thin shells as applied to a biological cell membrane (the mammalian red cell). Fung considered the effects of small bending moments on cell shape and the limited ability of the cell to change shape without local stretching of the membrane. This discussion was based on the extensive developments in classical mechanics concerning thin-walled shells. The article by Fung clearly pointed out the major oversimplifications in previous analyses of membrane mechanical experiments, such as the inappropriate use of the law of Laplace for soap films to define mechanical equilibrium except in simple situations.

111.

MEMBRANE ELASTICITY A N D FREE ENERGY STORAGE

A. Thermodynamics and Mechanics

Elastic deformations are synonymous with completely reversible and recoverable changes in the geometry of the membrane; in other words, the free energy stored by the material when work is done on it

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

7

by external forces can b e totally recovered. When the external forces are removed, the material assumes its initial shape as if the material possessed a “memory.” For a reversible process, the free energy changes in the material are directly related to the material deformation and are time-independent. The intrinsic forces in the material are given by the partial derivatives of the free energy density in the material with respect to the independent parameters that characterize the deformation (Landau and Lifshitz, 1970; Prager, 1961). 1. INTENSIVE DEFORMATION

In the process of membrane deformation, we assume that the membrane material system is closed; in other words, there is no exchange of material or molecules between the membrane and its environment. This is true only on a limited time scale, because the membrane can be remodeled by cellular processes or b y exchanging lipids and proteins with the extracellular surroundings. In general, these exchange processes take place over long time periods compared with the mechanical experiments performed on the membrane, and therefore our assumption is reasonable. The result is that any deformation can be related to intensive changes in membrane geometry. For the case of a thin membrane which is isotropic in the surface plane but anisotropic with respect to the thickness, deformation of a membrane material element can be decomposed into three parts: (1) a change in thickness represented by the fractional change in thickness e3, (2) uniform area dilation or condensation given by the fractional change in area a,(3) extension of the material in the surface plane at constant area represented by the extension ratio i. [Because we consider only the practical situations in which the surface radii of curvature are much larger than a characteristic molecular dimension, we can neglect the transverse shear strain (which represents rotation of a molecular axis relative to the surface normal) and presume that all displacements occur along the normal to the surface and in the plane of the surface. In deformations of liquid crystals, the transverse shear strain cannot be neglected.] Figure 5 schematically illustrates each type of deformation; the intensive deformation parameters are determined from relative changes in element geometry: (1) T h e fractional change in membrane thickness: €3

= x3/u3 - 1

(2) The fractional change in membrane element area:

8

E. A. EVANS A N D R. M. HOCHMUTH

31

a2

FIG.5. The three principal deformations of a thin-membrane material element: (1) the fractional change in thickness eg at constant surface area, (2) the fractional increase in area a at constant thickness, and (3) the extension of the membrane at constant surface area and constant thickness.

(3) The extension ratio for the material element at constant element area: j;

=a a1

for

XlXZ = -1

alaz

The element dimensions in the initial and instantaneous (deformed) states are given by a and x, respectively, with the subscripts corresponding to the material axes. Figure 6 shows how a general deformation in the plane of the surface can b e accomplished by first changing the area of the element, followed by extension at constant area. Extension of a material element in the plane of the surface at constant element area involves progressive shear deformation of the surface along lines +'45" to the principal axis of extension. Fung and Tong (1968) initiated the use of large-deformation analysis in biomembrane mechanics; subsequently Skalak et al. (1973) and Evans (1973a) utilized the analysis of large deformations in conjunction with the membrane anisotropy to develop elastic constitutive relations from free energy potential functions.

2. INTENSIVEFORCES

The coordinate system shown in Fig, 5 is coincident with the principal axes of the element deformation; i.e., the element dimensions

9

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

change

AREA

\

EXTENSION

FIG.6. The deformation, at constant thickness, of a two-dimensional, square material element into a rectangular strip. First the area is increased (the square is “dilated”) from L,2 to L2 = ( 1 + a)L,Z, and then the square is extended at constant area into a rectangular strip with length and width dimensions of AL x A-~J!,. The dashed arrows at 45” show the lines of maximum shear in the surface.

are simply extended or compressed along these directions. The force resultants that produce the extension or compression of the element dimensions act along these directions as well; the force resultants in the principal axes system are shown in Fig. 4. The principal force resultants include the two principal tensions TI and T2plus the normal stress cr3. The principal tensions, furthermore, are made up of two independent parts: (1)the mean or isotropic tension T and (2) the deviatoric or maximum shear resultant T,:

TI = T

+ T,

Tz = T

-

T,

The decomposition is illustrated in Fig. 7. Note in Fig. 7 that rotating the element coordinate system by 45” about the surface normal gives maximum shear plus isotropic tension. The isotropic tension acts equally in all surface directions at a specific location (independent of rotation of the surface coordinates); it is associated with uniform dilation or condensation of the surface area. The deviatoric part, the maximum shear resultant, acts at k45” to the principal axes and is responsible for in-plane shear deformation or flow of surface material. For example, a free surface like a soap film supports only an isotropic tension, called surface tension, in static equilibrium; a shear resultant produces flow of the soap film in the surface, which is resisted only by the viscous, dissipative force. Consequently, the ability to sustain a shear resultant T , in static equilibrium is the characteristic of a solid or rigid membrane.

10

E. A. EVANS A N D R. M. HOCHMUTH

1" 4 TI Isotropic Tension

Deviotor

-0R-

FIG.7. Decomposition of the two principal membrane tensions T , and TZinto an isotropic tension t and a deviator or shear resultant T,. A 45" rotation of the element coordinate system shows that the deviator is in fact equal to the maximum shear resultant.

3. DIFFERENTIAL WORK

The differential work dW done on a material element is the infinitesimal displacement of each force resultant times the appropriate element dimension. Using the independent force resultants, this can be simply expressed in terms of the intensive deformation parameters:

or

where A. and A are the initial and instantaneous area of the material element, respectively, and d o is the initial thickness of the membrane element shown as u3 in Fig. 5. Equation ( 2 ) states that the work done in deforming the material element is the superposition of work contributions from uniformly increasing or decreasing the surface area plus the extension or shear deformation of the element at constant surface area plus the expansion or compression of the membrane element thickness. Throughout the development, we assume that the fractional changes in volume, area, and thickness of material elements are small, but that the extension in the surface plane may be large. This assumption is valid for the red cell membrane and amphiphilic bilayer systems with which we are primarily concerned. Therefore the frac-

MECHANOCHEMICAL PROPERTIES

11

OF MEMBRANES

tional change in volume is given b y the sum of the small fractional changes in element area and thickness:

(34

u=a+c3

The differential change in volume of the membrane element is

dV

(da + dc,)A,d"

=

dV

=

(3b)

dvA,do

The contribution of the work produced b y the internal hydrostatic pressure p times the change in membrane element volume dV is implicit in Eq. ( 2 ) .The following identity gives this implicit relationship: T d a A0

+

~3

- p dV

dE3 d&

+ (T + pdo) d a A0

+ ( r 3 + P ) dc3 d& 4. ELASTICFREEENERGY For elastic membrane deformations at constant temperature, the differential work done on the membrane is equal to the differential change in the Helmholtz free energy (dF),:

dW = (dF)T (4) This is obtained from the first law of thermodynamics which states that the differential change in total energy dE of the material equals the heat exchange SQ from the surroundings to the material plus the work SW done on the material b y the surroundings:

dE = SQ

+ SW

If the process is reversible and isothermal, the heat exchange and work are exact differentials. For a reversible process, the heat exchange divided by the material temperature is equal to the change in entropy dS of the material: dQ =

T

dS

Therefore, at constant temperature, the first law for the reversible process is written

dW

=

d(E

-

TS)T

which is Eq. (4),defining the Helmholtz free-energy function. Since we are dealing with a closed membrane system which is a continuum

12

E. A. EVANS AND R. M. HOCHMUTH

in the two dimensions of its surface, we can define an intensive free energy density F which is the free energy per unit surface area:

(dF)T = (&),A,

=

dW

The free energy density is independent of the extent of the membrane surface. For the reversible process of elastic deformation, the free energy density is a conservative potential that can be considered analytic in the intensive deformation parameters; this is expressed mathematically by the chain rule of differentiation taken with respect to linearly independent parameters:

(The partial derivatives are performed with respect to a single variable, holding constant the other variables as well as the temperature.) The independent, intensive deformation variables (u , 5, and P ) are defined by u = a + E 3 5 = a - E 3

p

= i(P

+

i-2)

-

1

These variables are chosen because they are linearly independent and rotationally invariant in the membrane plane. The first variable is recognized as the fractional change in volume; the second variable is the deviation between the fractional change in area and the fractional change in membrane thickness. The third variable is a quadratic form in the material extension ratio K at constant area; this is selected because the variable must be invariant to rotation of the material axes in the membrane plane (i.e., the membrane material is isotropic in the membrane plane). Using the intensive variables (v, a, e3, and X) in Eq. ( 5 )specifies the elastic free energy density change associated with the membrane deformation; the free energy change is produced by a series of independent operations:

1. The volume is uniformly increased or decreased, holding the shape of the material element constant. 2. Then the area and thickness are altered, holding the volume and shape of the element constant.

13

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

3. Finally the shape of the element is extended or contracted, holding constant the volume and surface area (likewise the thickness). Equation ( 5 ) is written in terms of the intensive geometric variables ( u , a, e3, i) in the following manner:

(5b) Since an isothermal, reversible process is path-independent, this sequence of deformations is a valid decomposition of the free energy change for general thin membrane deformations (noting the restriction discussed in Section 111, A, 1).Holding the membrane element volume constant in the second operation implies that the fractional change in membrane area is equal and opposite to the fractional change in membrane thickness. Equation ( 3 )verifies that a fractional change in area at constant volume dii is minus the fractional change in membrane thickness -dZ3 at constant volume:

dii

+

dg3

=0

Therefore the deviation at constant volume d i is given by either

dg

= 2dii

or

d i = -2dZ3

and the partial derivative (dF/dg)T,,,p has equivalent expressions:

and

5. MECHANOCHEMICALEQUATIONSOF STATE With these relations in mind, the elastic free energy changes produced by material deformation can be directly related to the intrinsic membrane forces; the relations are determined from Eq. (5) and the expression for differential work, Eq. ( 2 ) . These equations represent isotherms of the mechanochemical equations of state for the membrane:

14

E. A. EVANS AND R. M. HOCHMUTH

(7) (8) (9) An elastic modulus is associated with each of these relations; Eqs. (6), (7), and (9) yield moduli of compressibility for fractional changes in volume, area, and thickness at constant temperature. Equation (8) determines the constitutive relation between the shear force resultant and extensional or shear deformation; a membrane elastic shear modulus characterizes this relationship and the rigidity of the membrane surface. The thermodynamic approach used to arrive at these equations is classical, and its use in three dimensionally isotropic materials has been extensively developed by Prager (1961) and Green and Adkins (1970), for example. However, specialization of the methodology to the anisotropic molecular strata of membranes is recent (initiated by Skalak et al., 1973; Evans, 1973a) and has been further developed to include membrane thermoelasticity (temperature dependence plus decomposition of the free energy into internal energy and entropy contributions, Evans and Waugh, 1977). [A monograph by Evans and Skalak (1978) entitled Mechanics and Thermodynamics of Biomembranes is in preparation and will provide an extensive tutorial on this subject.] It is apparent from Eqs. (7) and (9) that the hydrostatic pressure couples the stress resultant in the thickness direction to the membrane isotropic tension acting in the membrane plane. I n many situations¶ the hydrostatic pressure can be eliminated by a boundary condition. For example, if no appreciable compression or stress of the membrane occurs in the thickness direction,

which gives the following isotropic tension relation:

However, if the membrane is subject to compression or stress in the thickness direction but is free of in-plane membrane tensions,

15

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

which gives the stress resultant in the thickness direction:

This relation is in terms of a derivative with respect to the fractional change in area at constant volume, but it also can be written equivalently in terms of the fractional change in thickness at constant volume: u3

=

& ($)

3 T.u.0

6. Compressibility: Volume, Area, Thickness

As developed above, the intrinsic membrane forces are related to the partial derivatives of the elastic free energy density with respect to specific intensive deformation variables. In turn, elastic moduli or material properties that characterize the relationship between intrinsic forces and deformation are defined b y the derivatives of the forces with respect to deformation parameters. The three compressibility moduli may be functions of the deformation parameters and are therefore defined local to the equilibrium state, i.e., for small deformations relative to the equilibrium state. 1. ELASTICCONSTANT FOR

VOLUME CHANGES

We begin with the modulus of volumetric compressibility K B , sometimes referred to as the bulk modulus. It is defined by

or

for d v 0, where all conjugate deformation parameters are again held constant (we do not give the full list, as it is implicitly assumed). The hydrostatic pressure is given by the first-order relation (an isotherm of the bulk equation of state):

The common method of measuring the volumetric compressibility modulus is volume dilatometry; the method is designed to measure

E. A. EVANS AND R. M. HOCHMUTH

16

extremely small changes in volume produced by changes in the total system pressure. No measurements of volumetric compressibility have been -made on biological cell membranes. However, Ponder (1971) discusses early work on the unusual effects of large atmospheric pressures on the shape of red cells (at 1500-1700 atm red cells form spheres), which are a strongly temperature-dependent. The strong temperature dependence indicates that perhaps the membrane lipids are experiencing a phase transition (ordered hydrocarbon chains). Recently, Srinivasan et al. (1974) used volume dilatometry on hydrated amphiphilic bilayers, Their data indicate a volumetric modulus of compressibility on the order of 101o-lO1ldyn/cm2, i.e., on the same order as that of most “incompressible” liquids. As we show, membranes are two to three orders of magnitude less compressible in volume than in area or thickness; consequently, we can assume that the membrane is volumetrically incompressible. With this idea in mind, it is apparent that equal fractional changes in area per molecule or membrane thickness produce equivalent free energy density changes, as in Eqs. (10) and (11). Therefore the modulus of area compressibility is directly related to the modulus of compressibility for membrane thickness.

2. ELASTICCONSTANT

FOR

THICKNESS CHANGES

The elastic thickness modulus of compressibility K 3 is defined b y

or

ford&, + 0 in the absence of membrane isotropic tension. The stress resultant in the thickness direction is given by the first-order elastic relation ~3

= K323

(15)

assuming no stresses are initially present. Again, it has not been possible to measure the thickness compressibility of biological membranes, but considerable effort has been made to measure the K3 of amphiphilic bilayer systems. With the use of a variable voltage differential across the bilayer membrane, electrocompression of the membrane is produced. By measuring the mem-

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

17

brane capacitance, fractional changes in membrane thickness can be calculated. Crowley (1973) and White (1974) performed these experiments on black lipid films which are lipid bilayers spread with a small amount of organic solvent (the organic solvent primarily exists in small lenses and at the boundary torus). They concluded that the thickness compressibility modulus K 3 was on the order of lo6 dyn/cm2. Subsequently, Requena et al. (1975)showed that the change in boundary condition at the bilayer intersection with the solventcontaining regions (e.g., lenses) could account for the capacitance change without a change in bilayer thickness. From Eqs. (7) and (9), with a negligible elastic contribution (i.e., ( @ / d i 3 ) , = 0), the stress resultant in thickness is proportional to the change in isotropic tension at the boundary: m3

ATo - -&-

This is obtained by eliminating the hydrostatic pressure. The isotropic tension T oat the boundary is produced by the surface tension of the free exchange of organic solvent and lipids with the lens and torus regions (Evans and Simon, 1975). Therefore the reduction in membrane capacitance is the result of solvent separating out of the membrane into the lens and torus regions, causing a change in surface tension. Furthermore, Requena et d. (1975) concluded that the rapid, transient thickness change (of less than 1%) observed in these experiments, as well as similar small changes seen in membrane bilayers essentially free of solvent (White et al., 1976), represented an elastic compression resistance. Recently, Alvarez and Latorre (1978) made precise measurements of bilayer thickness changes in response to applied voltages. These results indicate a modulus K 3 on the order of 10' dyn/cm2, substantially smaller than the volumetric compressibility modulus.

3. ELASTICCONSTANT FOR AREA CHANCES

The elastic area compressibility modulus K , is defined by

or

K,

= T)$(

18

E. A. EVANS AND R. M. HOCHMUTH

for dtr + 0, assuming that the stress resultant in the thickness direction is negligible. The first-order elastic relation between isotropic tension and fractional changes in area per molecule at constant volume is given b y

f

=

Katr

+ fo

assuming that the membrane initially possesses a tension the membrane is nearly incompressible by volume,

(17)

To. Because

and the thickness elastic modulus is simply related to the area elastic modulus : K3

=

1 pa

Therefore measurement of the area elastic constant gives the thickness modulus when divided by the effective membrane thickness. (Note: The membrane surface modulus has units of dynes per centimeter or force per unit length like the force resultants in the membrane; for a membrane on the order of cm thick and with the previous estimate of lo* dyn/cm2 for Kt,we expect the area elastic constant to be on the order of 100 dyn/cm.) The area elastic modulus is determined by producing isotropic tension and measuring the uniform fractional increase in membrane area while keeping the shape of the membrane constant. Two types of experiments have been used on red cell membranes: 1. Swelling and lysis of osmotically swollen red cell spherocytes. 2. Micropipet aspiration of osmotically preswollen red cells.

The first type of experiment was analyzed by Katchalsky et al. (1960). Even though these investigators did not recognize the particularity of the elastic modulus they produced, they gave a value on the order of 10 dyn/cm (actually given as 2 x lo7 dyn/cm2 for Kudo, with do = 6 x cm). However, as these workers stated, this was a preliminary indirect calculation with many susceptible variables. Four years later, Rand (1964) used a micropipet to aspirate nearly spherical red cells; this produces essentially isotropic tension and area dilation as shown schematically in Fig. 8. Rand was unable to measure the fractional change in area of the membrane, so he used an estimate of about 40% obtained from photomicrographs of hemolyzing red cells as an upper bound and the lower bound of 8%given by Katchalsky et al.

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

I

I

19

kotroDic Tension

FIG.8. An experimental technique for producing an isotropic tension in the membrane of a red cell. Here a micropipet aspirates a portion of the membrane of a nearly spherical cell until the cell becomes spherical. This produces an isotropic tension throughout the membrane, except near the mouth of the micropipet. Further aspiration increases the area of the membrane and the isotropic tension within the membrane.

(1960). For a 10-6-cm-thick membrane, Rand estimated K,do to be on the order of 107-108 dyn/cm2 or 10-100 dyn/cm for the area elastic modulus. Recently, Evans et al. (1976)used the micropipet aspiration technique of Rand, coupled with detection of the cell projection movement in the micropipet, to determine the fractional increase in membrane area assuming that the cell volume remains constant. Figure 9 shows a red cell under suction in the micropipet at two different suction pressures; the movement of the cell projection in the micropipet is proportional to its increase in area. Figure 9 also contains a plot of the isotropic tension versus the fractional increase in membrane area for a single cell. The average value for the membrane area elastic modulus was found to be 290 dyn/cm (k50 dyn/cm standard deviation) measured at 25°C. Subsequently, Evans and Waugh (1977) analyzed the effects of osmotic and hydrostatic pressure forces across the red cell membrane. It was found that about one-third of the movement of the cell projection in the pipet tip was due to the reversible movement of water out of the red cell; consequently, the area elastic modulus was corrected to be 450 dyn/cm at 25°C instead of the original value of 300 dyn/cm. This gives a modulus of thickness compressibility on the order of 4.5 x lo8 dyn/cm2 for a 10-6-cm-thick membrane. No direct measurements of area compressibility in closed amphiphilic bilayer systems have been published. Measurements of the isotropic tension in a spherical bilayer film containing separated solvent regions have been reported (Pagan0 and Thompson, 1973; Tien, 1967). Again these experiments simply determine the surface tension associated with the free exchange of membrane materials with the lens and torus regions. However, it is possible to estimate the area

20

E. A. EVANS AND R. M. HOCHMUTH

15.0

1

u

'0

0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 A m Dibtion

FIG.9. Micropipet aspiration of a sphered human red blood cell. In the photograph, which is taken directly from a video screen, the suction pressure is increased from 4 mmHg to 6 mmHg. Each division on the micrometer scale equals 0.9 pm. A typical result from this kind of experiment is illustrated by the graph of membrane tension versus area expansion for a single red cell. Note the linearity.

elastic modulus from the equation of state for a single lipid monolayer at an oil-water interface (because the lipid in biological membranes exists above the order-disorder hydrocarbon chain transition and the oil-water interface most closely resembles this situation). For a constant amphiphile mass, the modulus of compressibility in a monolayer is given b y

Kam = -A

(x)Tm an - an =

where ll is the surface pressure, which is a function of area per molecule (Davies and Rideal, 1961). The Langmuir trough technique is used to determine the reduction in oil-water surface tension as a function of the area per molecule occupied by the monolayer molecules. The surface tension decreases as the area per molecule decreases. The amount of reduction in the surface tension is defined as the surface pressure produced by ampbiphile-amphiphile interactions. Bilayer surface pressure is idealized as the sum of the surface pressure in two monolayers :

(3 m

K,

=

2K," = - 2

T

21

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

The change in isotropic tension is the negative of the change in surface pressure for a closed membrane system (Evans and Waugh,

1977): AT=

-An

Therefore the mechanochemical equation of state (isotropic tension versus fractional area change) is directly related to the internal equation of state (surface pressure versus fractional area change). I n the disordered hydrocarbon chain state, the monolayer equation of state can be approximated by a two-dimensional Van der Waal's gas equation (Davies and Rideal, 1961):

II"'(A - A,)

=

2kT

where A is the area per molecule, A, is the excluded area per molecule, and k is Boltzmann's constant. Therefore the area compressibility modulus is modeled by

K,

=

ZA.IT" A -A,

For a lecithin monolayer at an oil-water interface above the transition temperature, a value of 38 hiz per molecule forA, gives a good approximation to experimental data (Taylor et al., 1973). Hydrated lecithin bilayers at 300 K exhibit an area per molecule of about 60 hi' as determined from x-ray diffraction. This gives an expected monolayer surface pressure of 35 dyn/cm and an area compressibility modulus of 180-190 dyn/cm for a bilayer. [Some preliminary measurements have been made on large lecithin single bilayer vesicles (5- to 10-pm diameter) using micropipet aspiration, giving an area modulus near the expected range (data to b e published by R. Waugh, E. Evans, and S. Simon).] For a 5 x lo-' cm thick bilayer, the elastic modulus K 3 for thickness changes is on the order of lo8 dyn/cm2. In summary, red cell and amphiphilic bilayer membranes are characterized by two independent moduli of compressibility: (1)a modulus of volumetric or bulk compressibility KB, and (2) a modulus for area compressibility K , or the equivalent modulus for thickness compressibility K 3 = K,/d,. Table I summarizes the experimental data available for these properties. The compatability of isotropic tension and electrocompression experiments with bilayer equations of state determined by Langmuir trough techniques is apparent from the comparable orders of magnitude determined from each type of experiment listed in Table I. It is clear that the pdV work associated with the volumetric compressibility is negligible in mechanical experiments, pro-

22

E. A. EVANS AND R. M. HOCHMUTH

TABLE I

ELASTICMODULI OF

COMPRESSIBILIT~

Volumetric, KB

1O1O-10" dyn/cm2 (volume dilatometry of lipid bilayers; Srinivasan et al., 1974)

-

(-100 dyn/cm)

100-450 dyn/cm (Rand, 1964; Evans and Waugh, 1977; micropipette aspiration of swollen red cells) 180 dyn/cm (bilayer equation of state deduced from monolayer data)

lo8 dyn/cm* (Requena et al., 1975; electrocompression of lipid bilayer films; Alvarez and Latorre, 1978) ( -lo8 dyn/cmz)

(-lo8 dyn/cmz)

' The values in parentheses are estimates made using the experimental measurements from the adjacent column. vided large hydrostatic pressures are not involved; i.e., the membrane can be considered volumetrically incompressible. The elastic compressibility moduli are common to solid and liquid membrane materials; in Section 111, C, we investigate rigidity-the difference between a membrane surface as a two-dimensional solid material as opposed to a two-dimensional liquid (e.g., a fluid mosaic). C. Rigidity: Resistance to In-Plane Shear or Extension

In-plane shear of a lipid bilayer is illustrated in Fig. 10. Because the lipid bilayer behaves as a two-dimensional liquid when the hydrocarbon chains are disordered, the bilayer membrane flows irreversibly in the plane of the surface. However, red cell membranes can sustain shear force resultants without flow and also exhibit elastic, recoverable membrane extensions. As previously shown (Fig. 7) the shear resultant is always present when the principal membrane tensions TI and T2are unequal; it acts along surface lines 245" to the principal axes.

23

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

FIG. 10. In-plane shear of a two-dimensional, lipid bilayer. Since the structural backbone shown in Fig. 1 has been omitted from this figure, the bilayer behaves as a two-dimensional j u i d . Therefore a relation between the in-plane shear and the resulting in-plane rate of deformation is characterized by a two-dimensional coefficient of viscosity.

T , = TI

-

T2

2

For instance, in a simple extension with T 2 = 0, the shear resultant is half the uniaxial tension T J 2 . Consequently, the resistance to extension of membrane material elements at constant element area is the resistance to membrane shear. Previously, it was shown that the membrane shear resultant is related to the change in free energy density with respect to extensional deformation, quantitatively represented by p = +(X2 X-') - 1. Recalling Eq. (8),

+

we note that the shear resultant is proportional to a quadratic function of the extension ratio, at constant area. This function is the largedeformation shear strain at constant area:

[In this form, it is the Langrangian shear strain (measured relative to the initial, undeformed system); however, at constant element area, the Eulerian shear strain (measured relative to the instantaneous, deformed system) is simply minus the Langrangian shear strain: e , = - E , . Such distinctions are in general important for large deformations (Prager, 1961).]For small extensional deformations, Eq. (19) reduces to the fractional change in element length - 1) commonly recognized as strain in linear elasticity. However, membranes can un-

(x

24

E. A. EVANS AND R. M. HOCHMUTH

dergo large recoverable shear deformations at constant area; we must consider the general form. The shear resultant is written

T, = 2

($1

E,

and therefore a membrane elastic shear modulus is defined b y

[Note: The shear modulus used in earlier developments, e.g., Evans (1973a), Evans and Hochmuth (1977), and Waugh and Evans (1976), includes the factor of 2 from Eq. @).I The shear modulus is obtained directly without taking an additional partial derivative, because the intensive deformation parameter /3 is quadratic in the extension ratios. The parameter /3 is a measure of the eccentricity or radius of gyration squared of molecular complexes as the complexes are extended at constant area per molecule. The first-order elastic constitutive relation or mechanochemical equation of state isotherm is given by

T,

= 2pes

(21)

It is apparent that solid materials require this additional equation of state, complementary to isotropic state equations. As mentioned above, lipid bilayers do not exhibit surface shear rigidity above the order-disorder transition temperature; below this temperature, when the hydrocarbon chains are ordered, amphiphilic bilayers (and monolayers) can support small shear resultants (Davies and Rideal, 1961). However, biological membranes contain lipids in the natural state, which are above the transition temperature; therefore the lipid bilayer components of membranes do not contribute to the surface shear rigidity or solid character. However, most cell membranes have associated material that can provide structural rigidity and support, e.g., the spectrin of red cell membranes. The only way to assess the ability to support in-plane shear is to measure directly the membrane resistance to static extension or shear deformation. Three types of experiments have been used to produce extensional deformations in red cell membranes:

1. High shear stress applied to cell suspensions such as in a viscometer 2. Fluid shear deformation of cells attached to glass substrates 3. Micropipet aspiration of flaccid, unswollen cells.

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

25

The high shear stress deformation of cells in suspension has been of no use in determining the membrane elastic shear modulus, because the distribution of forces acting on the membrane is not known and it is not certain that the membrane is statically deformed (it may be moving, e.g., like a tank tread). The second technique was developed by Hochmuth and Mohandas (1972) to determine the membrane elastic constant for extensional deformation, using red cells attached to glass in a flow channel (Fig. 11).Using linear elastic approximation, Hochmuth et al. (1973)and Hochmuth and Mohandas (1972) determined the quotient of the shear modulus divided by the membrane thickness p/doto be on the order of lo4 dyn/cm2 for a 10-6-cm-thick membrane. The membrane surface shear modulus from these calculations is on the order of dyn/cm. Using a large-deformation analysis including the nonuniform distribution of material extension in the cell membrane, Evans (1973b) predicted the shapes observed by Hochmuth and associates (Fig. 11)and calculated a membrane shear modulus of dyn/cm from their data. Micropipet aspiration of flaccid, unswollen red cells is illustrated in Fig. 12. Rand and Burton (1964) first used this technique on red cell disks; they suggested that the negative pressure required to aspirate a cell membrane projection was associated with membrane “stiffness,” perhaps bending stiffness. T h e inappropriate use of the law of Laplace provided no analytical insight into the origin of the progressive increase in negative pressure required to lengthen the projection. Subsequently, numerous investigators used micropipet aspiration as a qualitative technique to determine membrane deformability (e.g., LaCelle, 1969, 1970; Leblond, 1972). Evans (1973b) analyzed the extensional deformation produced by the pipet, assuming the area of the membrane was not required to increase. In this situation, the extension ratio at the entrance to the pipet is approximately

x, - 4where D is the length of the cell projection in the pipet and R , is the pipet radius. As illustrated in Fig. 12 and as is apparent from the extension ratio relation, progressive increases in the aspirated cell projection produce increases in material extension (shear deformation). The membrane shear resultant for such deformations is approximately [from Eqs. (8) and (20)l

which approaches a linear relationship between projection length and

E. A.

26

I

I

I

EVANS AND

I

R.

M. HOCHMUTH

1

FIG. 11. Illustration of the agreement between a theoretical prediction (line drawing at top; Evans, 1973b) and an experimental result (photograph at bottom; Hochmuth and Mohandas, 1972). The two theoretical analyses (Evans, 1973a,b; Skalak et al., 1973) of the Hochmuth and Mohandas experiment (fluid shear deformation of pointattached red blood cells) are based on the hypothesis that the red cell membrane behaves as a two-dimensional highly elastic material (e.g., hyperelastic solid). q,is the fluid shear stress which deforms the red cell, Ro is the undeformed red cell radius, and p is the two-dimensional shear modulus of elasticity.

FIG.12. The micropipet aspiration ofa flaccid biconcave cell. Here the surface area is constant during this (shear) deformation (contrast this figure with Fig. 8). The resistance of a flaccid cell to aspiration is characterized by the shear modulus p. The maximum shear resultant in the membrane T, is given by half the difference between the two principal membrane tensions TI and T2.

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

27

shear resultant. Solving the equations of equilibrium, .Evans (197313) developed a relation between the negative pressure in the micropipet and the length of the aspirated projection; with the theoretical prediction, the experimental data give the membrane shear modulus. Figure 13 is an example of a single experiment taken from Waugh and Evans (1976).The important results are: (1)Again the membrane shear modulus was found to be dyn/cm [as in Hochmuth’s experiments

AP . RP/ 2 fl

FIG. 13. Micropipet aspiration of a flaccid opossum red blood cell (Waugh and Evans, 1976). Each division on the photograph, which is taken directly from a video screen, equals 0.9 pm. The experimental results (solid circles) illustrate the linear relationship between the extrinsic membrane deformation D / R , and the characteristic membrane tension AP R , divided by the two-dimensional shear modulus p . The solid line in the figure is from the analysis of Evans (1973b).D is the length of cell projection in the pipet; R , is the radius of the pipet, and AP is the pressure drop between the surroundings and the interior of the pipet.

28

E. A. EVANS AND

R. M. HOCHMUTH

TABLE I1 ELASTICSURFACESHEARMODULUS P

Red cell membrane

Lipid bilayer (above hydrocarbon chain phase transition temperature)

lo-’ dyn/cm (Hochmuth et al., 1973; Hochmuth and Mohandas, 1972; fluid shear deformation of glass-attached cells-human) lo-* dyn/cm (Evans, 1973b; Waugh and Evans, 1976; micropipet aspiration of flaccid red cells-mammalian) 10-Idyn/cm (Waughand Evans, 1976; nucleated red cells using micropipet technique)

(Hochmuth and Mohandas, 1972; Hochmuth et al., 1973)l; (2) the shear modulus appears to be constant up to extension ratios of 3 or 4; (3) the red cell membrane is able to undergo large, elastic extensions (hyperelastic) at constant membrane area. For extension ratios greater than 3 : 1 or 4 : 1, the membrane yields and begins to deform in an irreversible plastic manner (Evans and LaCelle, 1975). With use of the micropipet technique, the membrane shear moduli of nucleated red cells (from amphibians, fish, birds, and reptiles) were found to be an order of magnitude larger than that of the mammalian red cell (Waugh and Evans, 1976). Table 11 gives the elastic shear modulus for red cell membranes determined by the two techniques we have discussed. The shear modulus is an intrinsic membrane property, verified by the observation that the two different experiments give the same result. Also, the shear modulus is observed to be four orders of magnitude smaller than the elastic area compressibility modulus; this implies that the membrane can b e considered a two-dimensionally incompressible material (i.e., it maintains a constant area) when subject to shear deformation without large isotropic tensions. Red cells can easily swell from biconcave disks to almost spheres; further swelling of the spherical cell is difficult and results in lysis. Therefore osmotic fragility tests give primarily an indication of the surface area to volume distribution of red cells and no information about mechanical properties. T h e fact that the shear modulus is not zero shows that the red cell membrane has a solid character which cannot be attributed to the lipid bilayer component. Consequently, additional membrane structure as illustrated in Fig. 1 must exist to give the membrane rigidity; measurement of the shear elastic modulus provides a direct method for “probing”

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

29

this component of membrane structure (Evans and Hochmuth, 1977). The low shear modulus and hyperelasticity of the red cell membrane certainly cannot be considered representative of cell membranes possessing additional connective tissue. Essentially no work has been done with membrane mechanical experiments other than on red cell and amphiphilic bilayer membranes, because of the difficulty in differentiating between membrane and cytoplasmic contributions. (The red cell interior is normally liquid.) Rapoport (1973) has published work on the extension of muscle sarcolemma with the muscle fiber interior removed; considerable connective tissue remains, and the elastic moduli reflect the strong contribution of these elements. D. Curvature Elasticity: Bending Resistance and Chemically Induced Moments

As mentioned previously, curvature elasticity or bending resistance is essentially negligible in a monolayer until the radii of curvature for the surface layer are on the order of molecular dimensions; then of course the continuum hypothesis is no longer valid. So we have considered membrane deformations that involve radii of curvature which are much greater than the membrane thickness. (Even a radius of curvature of 0.1 pm is approximately 10 times greater than the membrane thickness.) Composite membrane structures introduce the possibility of coupled interactions between the layers of the composite. These interactions are usually second-order compared with the energy changes produced by the force resultants and displacements previously discussed. The surface deformation of the whole membrane will be equally experienced by each layer if the surface radii of curvature are much larger than the distance between the monomolecular layers of the composite. However, between each layer, there can be small relative deformations (e.g., curvature can produce inner layer compression and outer layer expansion with no net deformation of a membrane element). Consequently, we must consider bending resistance and moments produced by changes in curvature of the membrane composite. Curvature elastic effects are produced by the variations in deformation variables of each layer in the composite strata relative to the mean deformation of the whole membrane (such as expansion or compression of one layer relative to another induced by curving the membrane). Fung (1966) pointed out that bending resistance is most often negligible compared with membrane force resultants (tension), which is indeed the case for large deformations produced b y micropipet aspiration or fluid shear deformation of glassattached red cells. However, Fung also noted that many situations

30

E. A. EVANS AND R. M. HOCHMUTH

arise where force resultants are small or do not contribute to the local membrane stability; here small bending moments are essential. An example is the shape change of the red cell during intermediate states of osmostic swelling before the spherical state is reached (Zarda, 1974; Zarda et al., 1977). Not only can curvature induce elastic free energy variations in a stratified membrane composite, but the reverse can occur: Free energy variations produced in the various layers by environmental changes can induce curvature. The strong effects of chemical environment on the shape of red cell membranes has long been recognized. The red cell shape spectrum ranges from a cup-shaped cell (stomatocyte) to a normal biconcave disk (discocyte) to a tightly crenated sphere (echinocyte). Extensive observations of the specifics of the chemical agents involved have been reported (Bessis, 1973). The reversible shape changes were first described by Hamburger (1895). Ponder (1971) in the 1930s accumulated a vast amount of information concerning the multiplicity of agents and conditions that produce these transformations. The biconcave disk is considered normal, or the natural state, because it is observed under normal physiological conditions in blood samples from healthy individuals. In addition, micropipet aspiration experiments indicate that the membrane is essentially unstressed in the biconcave state, However, the extraordinary smoothness, symmetry, and beauty of the normal red cell discocyte has stimulated considerable interest and study. The minimum curvature shape for the surface, with an area about 40%in excess of the area required for a sphere of the same cell volume, led Canham (1970) and Lew (1972)to postulate that the surface is a minimum bending energy configuration. Similar ideas caused Helfrich (1973) to create and extensively investigate “curvature elastic energy” as a membrane elastic constitutive relation. Alterations in the membrane curvature produced by changes in chemical equilibrium were considered initially for bilayer membranes by Evans (1974) and have been generalized to N-layer systems (Evans and Skalak, 1978). We do not discuss the origin of the red cell shape as a disk or any other shape, but we briefly develop the thermodynamic basis for curvature elasticity.

1. CURVATURE ELASTICENERGYOR BENDINGENERGY Relative deformations between layers caused by curvature changes are not easily determined. If the layers are uncoupled so that layers can slip or slide relative to each other, little or no deformation variations will occur. However, the bending of rigidly coupled layers

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

31

causes the expansion of outer layers relative to inner layers (in the sense of the outward normal of a convex surface), which is proportional to the layer separation. The maximum curvature elastic energy storage or bending resistance is represented by the rigidly coupled membrane strata. Therefore we investigate this case to specify the upper bound for the curvature or bending elastic modulus. Figure 14 illustrates the principal radii of curvature for a two-layer surface with a distance h between the layers. With the convex layers shown, the relative expansion of the outer surface with respect to the inner layer is apparent. These small relative deformations give rise to variations in elastic free energy density (8FJTbetween the layers (the 2th layer is defined b y the subscript 2). The differential of the free energy density variation is given by

which has been obtained from the analytical relation for the free energy density, Eq. ( 5 ) ,and the assumption that the material is volumetrically incompressible. The variations in the intensive deformation parameters 8iil and SP, are measured relative to a hypothetical surface

I FIG.14. The convex curvature of a two-layer surface separated by a distance h . Note the expansion of the outer layer (identified by the outward normal n) relative to the inner layer. The principal radii of cuivature are given by R , and R2.

32

E. A. EVANS A N D R. M. HOCHMUTH

called the neutral surface. This surface is the centroid location where the total force resultants or principal tensions act. The variation in total free energy density is given b y the sum over all N layers:

This is the free energy density associated with changes in membrane curvature. Since we are considering membrane systems with small shear moduli (lipid bilayer and red cell membranes), the first term involving the relative area changes between layers dominates and we can neglect the shear rigidity (see Evans and Skalak, 1978, for further discussion). Therefore the curvature or bending free energy density is approximated by

For radii of curvature much larger than the distance h, of an individual layer from the centroidal surface, the area variations are approximated by

6&1 = hl(C1 + C,)

(25)

Cl and C2are the changes in principal curvatures describing the surface (Fig. 14) away from the natural curvature state: C1=A($)

and

C2=A(k)

The partial derivative of the free energy density of each layer with respect to the fractional change in area is the isotropic tension in that layer [Eq. (lo)]:

The first-order constitutive relation [Eq. (17)1, relates the isotropic tension to the fractional area change in the particular layer:

F1

K1(&+ 6tuJ

Wb) where ii is the fractional change in area of the centroid surface and K , is the area compressibility modulus of the Zth layer. i=

Equations (24) through (26) give the variation in elastic free energy density of the membrane produced by changes in curvature as two sums:

33

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

Kzhl,is zero because it defines the location of the

The first sum, Z

centroid surface (Evans, 1974; Evans and Skalak, 1978);conceptually, the centroid is the location of the center of force for plane deformation with zero moment; i.e.,

The result is that the curvature elastic energy density is proportional to the square of the total curvature: d(SF)T=

(C Klh:)

d(C, + CZ)'

1

or

(27) (8F)T =

@(cl+ CZ)'

where B is the bending or curvature elastic modulus. Equation (27) is analogous to the curvature elasticity equation developed independently b y Helfrich (1973); here we have demonstrated the relation between the curvature elastic modulus and the relative area compressibilities of individual layers in a membrane composite. For instance, a bilayer membrane has a curvature or bending elastic modulus given b y =

KlKZ h2 ( K , + K,)

where h is the separation distance and K1 and K z are the area compressibility moduli for the two layers (Evans, 1974). Likewise, a trilayer membrane yields

B = h,2K,Kz

+ hz2KzK,+ (h, + h2)2K1K3 K1 + K z + K3

where the separation distance between layers 1 and 2 is h l , and between layers 2 and 3 is h,. This can be expanded indefinitely. The important features are that the compressibility resistances of the layers act in parallel (similar to electric resistors); curvature produces compression in the inner layers and tension in the outer layers. Thus there is a moment resultant or force resultant couple acting in the membrane as shown in Fig. 3. Remember, Eq. (28) is the upper bound expected for the membrane curvature elastic modulus.

34

E. A. EVANS AND R. M. HOCHMUTH

The differential work per unit area produced by the rotation of the moment resultants M i and M z acting along the edges of a membrane material element is given by

-dw - - M1 dC, + M z dC, A

(29)

Therefore the moment resultants can be obtained by the partial derivatives of the free energy density variation with respect to principal curvature changes :

These equations give the elastic constitutive relation

M1 = M z

=

B(C1

+ C,)

(31)

as determined using the free energy variation, Eq. (27).The moment resultants are proportional to changes in curvature; consequently, the elastic modulus B is called a measure of the bending stiffness or rigidity. For a lecithin bilayer, we expect each layer to have an area compressibility modulus of 80 dyn/cm; the layer separation distance is about 3 x lo-' cm. Therefore we anticipate the bending or curvature elastic constant to be on the order of

B = -h2Km -3 x 2

erg (dyn-cm)

providing that the amphiphilic layers are restricted such that no slip can occur between them (analogous to rigid coupling). Recently, Servuss et al. (1976)used thermal bending fluctuations of long, cylindrical tubes formed by single lecithin bilayers to calculate the curvature elastic modulus B of the lecithin bilayer; they determined the modulus to be 2.3 X lo-', erg at 22°C (Table 111). This agreement with the above prediction is excellent, indicating that such experiments can be used to determine membrane area compressibility moduli. Obviously, the bending of long, cylindrical tubes that are capped at the ends essentially limits slip or relative movement between layers to a minimum. However, a cell membrane subject to local curvature changes may permit substantial relative movement between layers,

35

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

TABLE I11 CURVATURE OR

BENDING ELASTICMODULUS B

Red cell membrane

Lipid bilayer

erg (estimated from area compressibility data, see text; Evans and Skalak, 1978) -1-3 x erg (analysis of the red cell membrane “flicker” phenomenon, as noted by Servuss et al., 1976)

2.3 x erg (bending of lecithin bilayer cylinder; Servuss et al., 1976)

-5 x

-3 x erg (estimated for coupled bilayer from area compressibility data, see text; Evans and Skalak, 1978)

such that little curvature elastic resistance is encountered. For instance, consider a trilamellar model for the red cell membrane: an uncoupled, lipid bilayer with a layer of spectrin strongly associated with the inner layer. In this case, the red cell membrane exhibits a curvature elastic modulus, B 5 x erg, that is an order of magnitude lower, assuming the spectrin layer center is lo-? cm from the lower lipid layer center. T h e bending rigidity results entirely from the coupling between the spectrin and the inner amphiphilic layer. erg have been obServuss et ul. (1976)note that values of 1 - 3 x tained for the red cell membrane based on analysis of the “flicker” phenomenon. It is of significant interest to evaluate the relative magnitudes of curvature elasticity and shear rigidity produced in forming a membrane projection (e.g., the initial “hemispherical bump” in a micropipet aspiration experiment). T h e comparison indicates the approximate radius of curvature below which curvature elastic effects are paramount and above which shear rigidity is dominant. For such a comparison, we take a flat disk and produce a hemispherical cap at constant area with a radius of R,. Because the area of the disk is equal to the area of the hemisphere,

-

rro2= 2rR,2

the extension ratio at the hemisphere base is given by

where ro is the radius of the disk and X is the principal extension ratio along the meridian of the hemisphere at the base. T h e extension ratio is identically one at the pole of the hemisphere; therefore the area

36

E. A . EVANS A N D R. M. HOCHMUTH

mean deformation parameter characterizing shear ( /3) is approximately two-thirds of the value at the equator:

Therefore the free energy density representing shear deformation at constant area is

The curvature free energy density variation is given by

The ratio of the two free energy density contributions is of the order

-

-

For B 5 x dyn/cm, this ratio depends on the erg and p radius of curvature of the hemisphere:

I n order to neglect the curvature free energy contribution (bending resistance), the radius of the hemisphere has to satisfy the condition Rs > 2 x cm. The curvature elastic free energy contribution is cm (4 pm). This perabout 20% for a radius of curvature of 0.5 x centage decreases as the material extension ratio increases, e.g., as the red cell membrane projection extends into the micropipet. For the red cell membrane and changes in curvature on the order of lo4cm-I, the material shear rigidity is dominant for extension ratios of 1.1 or greater. However, for red cell deformations like osmotic sphering where the material stretch is negligible over large regions of the cell membrane, the bending rigidity is the dominant energy mechanism (Zarda, 1974; Zarda et al., 1977).

INDUCEDCURVATURE 2. CHEMICALLY We have discussed the elastic free energy variation produced by mechanical bending of the membrane strata; the assumption was made that the material properties remain constant over the time period of the experiment. I n general, this is appropriate; but many sit-

M E C H A N O C H EMlCAL PROPERTIES OF MEMBRANES

37

uations arise in biology in which the external environment or chemical equilibrium of the membrane is altered. Changing the chemical state of the membrane changes its equilibrium free energy state and disturbs the natural force resultant distribution in the membrane. Such chemical alterations may induce resultants or produce deformations in the membrane, because the constituent layers of the composite attempt to condense or expand relative to one another as they approach the new equilibrium state. The elastic free energy differential must include an additional term to account for the change in chemical equilibrium; this term is given by

c N

6yl d ( G

+ 6G1)

(32)

1=1

where 6y, is the variation in interfacial free energy density due to changes in the equilibrium state. Equation (32) separates into two contributions: a change in the free energy density of the centroid surface given by N

and an addition to the free energy density variation, Eq. (23) or (27), given by N

I= 1

The fractional area variations produced b y curvature in coupled layers give an induced free energy variation expressed b y

This equation demonstrates that the membrane tends to approach the new, altered equilibrium state by changing curvature; in other words, there is a chemically induced moment contribution (Evans, 1974; Evans and Skalak, 1978). T h e moment resultants induced by the chemical free energy change [determined by applying Eqs. (30) to (34)l are given by

I=1

which we define by the symbol sity [Eq. (27)l becomes

r. T h e curvature elastic energy den-

38

E. A. EVANS AND R. M. HOCHMUTH

If we neglect shear resistance and externally applied forces, a new equilibrium state can be achieved by altering the curvature of the membrane to minimize the elastic free energy density:

This defines a new equilibrium curvature (Evans, 1974, Evans and Skalak, 1978):

co = ( C , + C,)O = - r

(37)

Therefore the curvature elastic free energy density can be expressed in the form

B

( 8 F ) T = 2 ( C , + c, - CJ2

(38)

when the chemically induced curvature is used as a reference state. This is the counterpart of the “spontaneous” curvature parameter introduced by Helhich (1974). Assuming that the area compressibility resistances of the membrane layers dominate the curvature elastic modulus, the induced moment for a bilayer membrane is given b y

and the induced curvature is

The subscripts are numbered starting with the inner layer and moving outward along the direction of the surface normal. The effect is evident from Eqs. (39) and (40).For example, reducing the free energy of the outer layer (Sy, < 0) results in an expansion of the outer layer relative to the inner layer, inducing a positive curvature change, and vice versa. As mentioned previously, uncoupled monomolecular layers do not produce local moment resultants proportional to curvature because the layers are free to shear or slip relative to one another. If the extremities of the membrane composite strata are constrained or closed in a cellular envelope, there will be a variation in free energy

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

39

due to differences in the total free energy of the individual layers, integrated over the whole membrane surface. This can be represented symbolically by the average over the surface:

if it is assumed that the membrane properties are homogeneous over the whole surface. Because the layers are uncoupled, the variation in area between the layers and the neutral surface is uniform over the surface. Therefore (ah?) = ( 6hJ2

and ((8%)

=

B

( c ,+ c 2 + r ( c ,+ cd

which is the average total free energy variation due to membrane curvature for uncoupled layers. This relation must be minimized in conjunction with the total free energy density over the entire membrane surface to obtain equilibrium. No direct experimental measurements have been made to evaluate the magnitude of chemically induced moments. However, as mentioned in the introduction to this section, extensive observations of reversible curvature changes induced by changes in chemical environment have been made, e.g., the crenation of red cell disks to form echinocytes. Therefore it is of interest to estimate the free energy density change (in ergs per square centimeter) required to produce a spicule, say, with 1-pm radius. By choosing a radius of cm, we can neglect the bending rigidity but must balance the dominant shear elastic energy against the chemical changes:

From our previous calculation, we obtained

( A F J~2

x 10-~ erg/cm2

Therefore (AFc)T

- R, 2r = ( 2 x io4)rergs/cmZ

This implies that

r - - lo-'

erg/cm

40

E. A. EVANS AND R. M. HOCHMUTH

The induced moment coefficient r is given by different expressions for either (1) a free energy change in the inner spectrin layer, or (2) a free energy change in, say, the outer lipid layer:

-

=

%

[K3(h1

+ h,) + K,hl]

where K K1 + K z + K 3 , the total membrane area compressibility modulus. We use values of K 300 dyn/cm, K , = K 3 = 80 dyn/cm, hl lo-' cm, and h2 3 x lo-' cm to represent the trilamellar model of the red cell material composite. For the inner spectrin layer change, case (l),the induced moment and change in interfacial free energy density is

-

-

-

r - - tiyl (1 x

10-7)

giving 6y,

- 1 erg/cmz (dyn/cm)

For the red cell membrane, the number of moles per square centimeter of network material (spectrin) is about 0.5 x lo-', mole/cm2. Therefore this change is equivalent to 2 x 10l2 ergs/mole or 48 kcal/mole of spectrin molecules. For the outer lipid layer change, the induced moment and change in interfacial free energy density is

r - ay3 (3 x

- Sy3

-3 X

10-7)

lo-' erg/cm2 (dyn/cm)

The chemical change 6y3 is only of the interfacial free energy density of the water-hydrocarbon interaction of the outer layer, 40 dyn/cm (Evans and Waugh, 1977). In calories per mole of phospholipid, this is only a 20 cal/mole change out of 2 kcal/mole total. From these two estimates, it is apparent that small changes in the outer surface free energy density are required to begin crenation of the membrane; however, it seems that free energy changes in the spectrin layer have to be of the same magnitude as the hydrolysis of ATP to ADP ( - - 12 kcal/mole). Indeed, it has been suggested (Weed et al., 1969) that ATP depletion of red cells affects spectrin in such a way (presumably condensing the material) as to crenate red cells. Weed et aZ. (1969) showed that ATP depletion is correlated with echinocyte transformations; however, the intrinsic mechanism is diffi-

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

41

cult to establish. Recently, Sheetz and Singer (1975) postulated a membrane “couple” to account for red cell shape changes but provided no mechanical or thermodynamic basis for their hypothesis. Their proposal differs in that they assume that material from the aqueous environment is taken up preferentially by each of the amphiphilic layers. In this case, the system is not closed; it is necessary to incorporate the enthalpy of mixing of the material with the amphiphilic layers. It is clear from the simple example already discussed that the enthalpy difference between layers is very small.

IV.

MEMBRANE VISCOSITY AND FLUIDITY

Our consideration of elastic material behavior of a membrane was based entirely on reversible thermodynamics, i.e., conservative free energy potentials. For membrane deformations produced at a slow rate with no permanent material alteration due to the membrane forces, elastic constitutive relations are reasonable representations of the material character of the membrane. However, if the rate of deformation increases, thermodynamically irreversible processes become evident which result from internal “friction” and heat dissipation within the membrane. Irreversible processes create nonconservative forces that depend on the rate of deformation as well as on the instantaneous deformation alone. But if no permanent (i.e., plastic) deformation of the membrane material occurs, the membrane forces will relax to the elastic, conservative level, approximating equilibrium. The time dependence of the force relaxation is determined by the rate of viscous dissipation in the material. In the absence of applied forces, the material returns to its initial shape. This is simple viscoelastic behavior and does not involve irrecoverable (plastic) material changes; by comparison, prolonged exposure to applied forces usually results in plastic flow, which represents permanent material changes. If the magnitude of the applied forces is sufficiently large, the material will begin plastic flow immediately, with the work per unit time or mechanical power produced by the applied forces being dissipated as heat in the material. For a perfect liquid, there is no force resultant threshold or yield level; flow commences in response to any shear force resultant or stress. The constitutive relations between the nonconservative, frictional force resultants and rates of deformation are characterized by coefficients of membrane viscosity. Viscosity represents the rate at which energy is dissipated in the material in propor-

42

E. A . EVANS A N D R. M. HOCHMUTH

tion to the square of the rate of deformation. We use a first-order, irreversible thermodynamic approach to demonstrate the modes of entropy production and heat generation produced by different intensive rates of deformation; this yields the independent coefficients of viscosity. Clearly, in discussing viscosity and membrane thermodynamics, we are again treating the membrane as a two-dimensional continuum. Consequently, viscosity coefficients are to be determined directly by mechanical experiments involving macroscopic regions of the surface and are continuum properties. Viscosity is a measure of the fluidity of a substance, like the elastic shear modulus is a measure of its rigidity. However, “fluidity” and “rigidity” are qualitative terms which must be carefully handled; they are not substitutes for the viscosity of a liquid or the shear modulus of a solid, Furthermore, in membrane biology, these qualitative descriptors have become especially illusory. For instance, experimentalists measure kinetic or thermal motions of molecules in the membrane; these measurements, coupled with classical kinetic theory, are used to calculate membrane viscosity and to measure membrane fluidity. Certainly, diffusive motions of molecules are related to the coefficients of viscosity of the membrane as a continuum, but the relationship must be carefully evaluated. We compare the coefficients of viscosity measured by direct continuum mechanical experiments with the viscosities deduced from diffusion measurements.

A. Internal Dissipation and Coefficients of Surface Viscosity

1. IRREVERSIBLE THERMODYNAMICS The first law of thermodynamics relates the change in total material energy dE to the exchange of heat i3Q and work done on the material 6W:

dE

= i3Q

+ 6W

Because the total energy is conserved, a cyclic process results in

§SQ

=

- §6W

demonstrating that the irreversible heat loss during an isothermal cyclic process is provided by additional work in the cycle, called hysteresis. If we assume that the irreversible process is totally entropic, in other words, that only heat is generated by nonzero rates of defor-

43

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

mation, we can write the first law for either a reversible or irreversible process at constant temperature:

+ 6W dE = dQ + dW dE

=

SQ

(irreversible) (reversible)

(41)

Therefore (SQ - dQ)

+ (SW - dW) = 0

We can define the irrecoverable work awlRR done on the system, which is lost as heat transfer to the environment:

For an isothermal process, the exact differentials specify the reversible, elastic material behavior previously examined, and Eq. (42) represents the internal heat dissipation and mechanical losses. The loss of mechanical power is proportional to the rate of production of internal entropy which is eventually exchanged with the environment as heat. The mechanical power is specified by the work per unit time performed by the material force resultants. Without normal stresses, (u3= 0), Eq. (2) gives the mechanical power relation

aw

-=

at

( . i .a;-; + 2 T S - ) aAl n at at

L

(43)

The mechanical power dissipated in the material as heat is proportional to the time rate of generation of the material entropy density s :

1 aQIRR- 1 aWtRR s=-----TA a t TA at

(44)

The heat exchange is opposite in sign to the rate of entropy production, because it is lost to the environment. T h e mechanical power loss is produced by the nonconservative or “frictional” part of the force resultants; if we consider only the nonconservative force resultants, Eqs. (43) and (44) will give 1 A

awlRR at

(45)

which must be a positive definite quantity (i 5 0) as originally stated by Clausius. In 1931, Onsager postulated phenomenological equations relating

44

E. A. EVANS AND R. M. HOCHMUTH

the rate of entropy production s to the scalar product of “flows” X, and “conjugate forces” J,:

as the first-order approximation to irreversible processes (Onsager, 193la, b). The conjugate force is linearly related to the flows b y coupling coefficients L,:

Consequently, the entropy production is quadratic in the flows:

s

=

2 2 Lp,JqXp P

P

For a continuum material, the flows are the time rates of deformation, which describe the intrinsic time rate of change in distances between material points (Prager, 1961). A membrane, which is isotropic in the two dimensions of its surface and volumetrically incompressible, is characterized by two independent rates of deformation (Evans and Skalak, 1978): (1)the time rate of change in the uniform area dilation or condensation of membrane a&/at and (2) the time rate of change in the material extension or shear deformation at constant area (slat) (In A). Since these two rates of deformation are linearly independent, the rate of entropy production per unit membrane area is given by i = L11 + 2L22 a I n X

; (g)2

(at)

(46)

(The factors of&and 2 appear because products of tensor quantities are involved.) This implies that the nonconservative force resultants are given by TLll

2. COEFFICIENTSOF SURFACEVISCOSITY The phenomenological coefficients are therefore associated with specific coefficients of viscosity, defined as:

45

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

with units of dyne-seconds per centimeter or surface poise. [To provide a sense of perspective toward surface viscosity, a 10-A (1 x lo-' cm) thick layer of liquid like water or an organic solvent such as decane with a viscosity of dyn-sec/cm2 (poise) would exhibit a pseudo surface viscosity of (1 x 10-7)(10-2)dyn-sec/cm or 1 x low9 surface poise, if constrained to flow in two dimensions. Olive oil has a viscosity on the order of 1 poise and thus gives a surface viscosity of 1 x lov7dyn-sec/cm for a lo-A-thick layer.] The constitutive relations for the nonconservative, frictional force resultants are now determined by these viscosity coefficients:

acu

T=K-

at

a In i T , = 27)-

(49)

at

The coefficient

K represents viscous energy dissipation produced by finite rates of area dilation or condensation; because the membrane is volumetrically incompressible, the area dilation viscosity also specifies the coefficient of viscosity for finite time rates of change in membrane thickness. The latter constitutive relation, b y analogy to the elastic case, is given by

where the viscosity coefficient is K/do. The other viscosity coefficient 7) is the surface shear viscosity for energy dissipation produced b y time-dependent shear deformation in the membrane plane; this is the commonly recognized surface viscosity appropriate to the surface shear 245" to the direction of extension (Fig. 10). If the membrane behaves as a simple viscoelastic solid, the constitutive relations will b e the superposition of elastic and viscous components :

46

E. A. EVANS AND R. M. HOCHMUTH

These equations specify the time-dependent behavior of membrane force resultants and are characterized by relaxation time constants for each mode of deformation. The time scale for relaxation and recovery of area dilation or thickness changes is given by rU=

K/K,

seconds

and the time scale for extension or stretch recovery is

77 r A= E.L

seconds

It is emphasized that membranes are characterized by at least two surface viscosity coefficients, one for area dilation and the other for surface shear; there can be other coefficients for more complicated situations. In general, as we have seen previously, the area compressibility is so small that most membrane deformations are in-plane extension or shear; therefore the surface shear viscosity is of primary importance in time-dependent membrane deformation.

3. DIRECT VISCOSITYMEASUREMENTS-INSOLUBLE MONOLAYERS Extensive work has been done by surface chemists on measurement of the surface shear viscosity of insoluble monolayers (Davies and Rideal, 1961). Beginning with Harkins and Meyers (1937), these researchers have shown that the shear viscosity of insoluble monolayers of fatty acids above the transition temperature for the condensation of the hydrocarbon chains is in the range of 10-4-10-3 dyn-sec/cm or surface poise (Table IV). Consequently, we anticipate that the lipid bilayer component of biological membranes is characterized by this viscosity range; however, these values greatly exceed the surface viscosities derived from lateral diffusion measurements (as we discuss in Section IVYA, 4).A crucial and important observation has been made by surface chemists: The surface viscosity of an insoluble monolayer is strongly dependent on the area occupied by a molecule in the layer; the shear viscosity increases as the area per molecule decreases. Above the transition temperature, the surface pressure exhibits little or no effect from changes in the hydrocarbon chain length (Taylor et al., 1973); consequently, the strong area-per-molecule dependence of

47

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

TABLE IV

SURFACE VISCOSITIES Extension or shear, 7

Area dilation,

K

-lo-' dyn-sec/cm (estimated from hydrocarbon interior viscosity, see text)

Red cell membrane

Lipid bilayer (above transition temperature)

- 10-4-10-3dyn-sec/cm

-lo-' dyn-sec/cm (calculated from visco(as calculated from microelastic relaxation experiviscosity of 1poise for the ments on mammalian red hydrocarbon interior, cell membrane; Evans assuming a thickness on and Hochmuth, 1976a; the order of lo-' cm) Waugh and Evans, 1976) -10-~-10-~ dyn-sec/cm -10-3-10-2 dyn-sec/cm (derived from measure(viscoelastic relaxation ments oflateral diffusion of nucleated red cell in solvent-free lipid membranes Waugh and bilayers using the twoEvans, 1976) dimensional mobility relation of Saffman, 1977; Fahey and Webb, 1977; Po0 and Cone, 1974) dyn-sec/cm (plastic -10-4-10-3 dyn-sec/cm (based on measurements flow of human red cell of surface viscosity in membrane; Hochmuth et al., 1976) monolayers; Harkins and Meyers, 1937; Davies and Rideal, 1961)

the shear viscosity appears to be primarily associated with the kinetic interaction of the polar head group region of the amphiphile and not with the hydrocarbon tails. The bilayer component of membranes exists in a relatively condensed state with disordered chains above the transition temperature; therefore we expect the bilayer surface to behave like a two-dimensional liquid and not a gas. This is consistent with the concept of liquid viscosities being proportional to the free (unoccupied) volume (Hildebrand, 1971). In the case of a twodimensional liquid, the surface viscosity is proportional to the free (unoccupied) area per molecule. Clearly, if the surface viscosity of a lipid layer is determined by the polar head group region of the amphiphiles, the free area per molecule will increase in proportion to the area per molecule in the surface. By analogy to the surface viscosity of a three-dimensionally isotropic liquid (Hildebrand, 1971),the surface viscosity is given b y

E.

48

A. EVANS A N D R. M. HOCHMUTH

7 - C/(A - A o ) where C is an intrinsic constant and (A - A o ) is the free area. We suggest that the methods and analysis used in the study of ordinary liquids are appropriate to two-dimensional fluids like a lipid monolayer or bilayer. From the examples in Section 111, A, 2, we see that surface viscosities in the range 10-4-10-3 dyn-sec/cm for thin layers correspond to those of extremely viscous materials (like waxes and butter, for instance, and not to those of olive oil and organic solvents like n-decane). I n addition, adsorbed films of proteins and polymers exhibit shear viscosities on the order of 10-3-10-2 dyn-sec/cm, for film densities of lo-' gm/cm2 (Davies and Rideal, 1961; Joly, 1964). The surface density of the spectrin protein associated with the cytoplasmic face of the red cell membrane is also on the order of lo-' gm/cm2. As we discuss next, the measured red cell membrane shear viscosity is also in the range of 10-3-10-2 dyn-sec/cm.

4. DIRECTVISCOSITYMEASUREMENTS-RED CELL MEMBRANES Little attention has been given to the time response of biological membrane deformations. Viscoelastic models for red cell lysis were considered by Rand (1964) and Katchalsky et al. (1960).However, the unusually large surface viscosity of 10-103 dyn-sec/cm deduced from the micropipet and osmotic lysis experiments of Rand and Katchalsky et al. do not represent a viscous flow process but actually result from the temporal dependence of the lytic phenomenon itself with no observable surface rate of deformation. Viscoelastic relaxation times on the order of lo-' second were observed by Hoeber and Hochmuth (1970)for red cells expelled from large micropipets. Recently, Waugh and Evans (1976) performed viscoelastic relaxation experiments on both nucleated (Fig. 15) and nonnucleated red cells from various vertebrates. Mammalian (nonnucleated) cell membranes exhibit relaxation times on the order of 10-'-10-2 second; whereas the cell membranes of other vertebrates relax more slowly, 2-4 x lo-' second. As demonstrated previously [Eq. (51)], the relaxation time for surface extensional deformations is given by Th

= V/P

Therefore it is possible to estimate the membrane shear viscosity when the membrane behaves as a viscoelastic solid by 7-

TAP

MECHANOCHEMICAL PROPERTIES

OF MEMBRANES

49

FIG.15. Photographs taken directly from a video screen, which illustrate the viscoelastic relaxation of a portion of a frog erythrocyte membrane (Waugh and Evans, 1976). Initially, a membrane “bump” is aspirated into a micropipet and then quickly released. Note the disappearance (i.e., relaxation) of the “bump.”

Recalling that the red cell membrane shear modulus is on the order of 10” dyn/cm, we can use the measured relaxation times to calculate the shear viscosity:

77

- 10-4-10-3 dyn-sec/cm

(surface poise)

which characterizes the energy dissipation by shear for the solid membrane (Table IV). In addition, it has been shown that a surface viscosity of this magnitude dominates membrane relaxation and that the cytoplasmic plus extracellular fluid dissipations are negligible by comparison (Evans and Hochmuth, 1976a). When a red cell membrane is subjected to shear force resultants T , greater than 2-8 x dyn/cm, the membrane commences irrecoverable plastic flow. The plastic constitutive behavior is represented by the inequalities:

(1) T , < T s

(viscoelastic behavior, Eq. 51)

50

(2) T , > f,

E. A. EVANS AND R. M. HOCHMUTH

(liquid behavior)

(The net shear resultant is proportional to the rate of deformation: T,

-

f, = 27)-a ath i

where f, is the yield shear resultant and 7) is the surface shear viscosity coefficient for plastic flow.) The yield shear resultant f, was calculated by Evans and Hochmuth (1976b) from data originally reported by Hochmuth et aZ. (1973) for microfilaments of a red cell membrane; the microfilaments are produced by fluid shear deformation of glass-attached red cells. The microfilament or membrane “tether” pulled from the cell membrane is attached to the glass as shown in Fig. 16 (Evans and Hochmuth, 1977). The rate of growth of the membrane “tether” is determined by

FIG.16. A red cell membrane “tether” plastically pulled from a human red cell by a fluid shear force. The fluid shear force produces a membrane shear resultant T , which exceeds the limit for viscoelastic behavior. Thus the membrane deforms (flows) plastically. It behaves as a viscoplastic material (Evans and Hochmuth, 1976b). Original scanning electron micrograph furnished by Dr. J. R. Williamson, Department of Pathology, Washington University.

MECHANOCHEMICAL PROPERTIES

OF MEMBRANES

51

the membrane shear viscosity r) characterizing plastic flow (Evans and Hochmuth, 197613). The membrane shear viscosity in plastic flow was calculated to be dyn-sec/cm from preliminary data by Hochmuth et al. (1973) and again by Hochmuth et al. (1976). This value, r)

- lop2dyn-sec/cm

represents the energy dissipation in the red cell membrane when it is flowing as a liquid or plastic (Table IV). It is emphasized that both shear viscosity values, one from viscoelastic relaxation and the other from plastic flow, are properties of the total membrane composite: lipid bilayer plus integral and peripheral proteins (Fig. 1).Comparing surface poise the red cell membrane viscosity in plastic flow of with the lipid bilayer viscosity ( 10-4-10-3 surface poise) estimated from monolayer work demonstrates that the lipid bilayer is essentially “along for the ride” and that the dynamic behavior of the red cell membrane in plastic flow is most likely controlled by the peripheral protein component, spectrin. It is an interesting, even if circumstantial, observation that adsorbed protein layers with similar surface gm/cm2) like spectrin exhibit surface viscosities in the densities ( same range as that of the red cell membrane. We have not discussed the other coefficient of viscosity K which represents energy dissipation in the membrane produced by finite rates of area or thickness change. No direct measurement of this viscosity coefficient has been made. However, we can make a hypothetical calculation as a rough estimate of the value. For a lipid bilayer, uniform expansion of the bilayer or corresponding thickness changes produce internal shear of the hydrocarbon chains; if the polar head group interactions are nearly reversible (elastic) for uniform area dilation or condensation (no shear), the energy dissipation primarily occurs in the hydrocarbon interior. Therefore an approximation to the surface area viscosity K is the three-dimensional shear viscosity ijHCof the hydrocarbon interior (with units of poise) times the bilayer thickness do: K

-

ijHCd0

The three-dimensional viscosity of the hydrocarbon interior is referred to as the microviscosity and has been measured by fluoresence polarization (Azzi, 1975), as well as other techniques. It is found to be on the order of 1 poise. Therefore the area viscosity is on the order of K

-

dyn-sec/cm (surface poise)

for a bilayer on the order of

cm in thickness.

52

E. A. EVANS AND

R. M. HOCHMUTH

The area viscosity is an important determinant in the relaxation time of elastic area or thickness deformations; we recall that ra = K / K a

Since the area elastic compressibility modulus is about 10’ dyn/cm, our approximate calculation indicates relaxation times on the order of 7,

- 10-Bsecond

which is very fast for elastic area or thickness changes. However, such times were measured in Brillouin scattering experiments, e.g., experiments with smectic liquid crystal systems (Liao et al., 1973). Electrocompression of lipid bilayers (White, 1974; Requena et al., 1975) involves small changes in bilayer thickness produced by the rapid application of voltage differences across the membrane; the shortest experimental times used are about 10-7-10-s second. These times appear to be too long, indicating that the ability to measure elastic area changes with electrocompression of bilayers can be seriously questioned. 8. Fluidity, Particle Diffusion, and Membrane Viscosities

Since the pioneering experiments of Hubbell and McConnell

(1968,1969) and Frye and Edidin (1970), evidence that the lipid component of biological membranes is in a fluid state has been accumulating rapidly. In particular, the translational mobility of “large” (relative to the dimensions of lipid) markers in the plane of the membrane (Frye and Edidin, 1970; Edidin and Fambrough, 1973; Po0 and Cone, 1974; Liebman and Entine, 1974) has been measured. Translational mobility is only one of the mechanisms of thermal motion in membranes (Edidin, 1974), but it specifically characterizes lateral diffusion in the membrane plane and thereby is related to surface viscosity. Surface viscosity is a continuum property that relates the shear force resultant to the rate of shear deformation v, (i.e., flow of the surface):

a hi

T , = 2qV, = 27) at

(49)

It is apparent from the units of surface viscosity (dyne-seconds per centimeter, poise-centimeters, or grams per second) when compared with units of three-dimensional liquid viscosity (dyne-seconds per square centimeter, grams per centimeter per second, or poise) that the two types of coefficients characterize different dissipative mechanisms. Therefore attempting to relate the surface viscosity of a mem-

53

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

brane to the viscosity of ordinary liquids by dividing b y the “equivalent” membrane thickness can be erroneous and definitely obscures the nature of the anistropic fluid behavior (i.e., the two-dimensional flow of the surface). Nevertheless, this has been the traditional approach when dealing with lateral diffusion of membrane-bound particles. Here the Einstein kinetic relation for the diffusion of a particle in a three-dimensional medium has been applied to membrane diffusion in order to estimate membrane viscosity. Thus

D = kTb

(52)

where D is the measured diffusion constant (in square centimeters per second), k is Boltzmann’s constant (1.4 x erg/K), T is the absolute temperature in kelvins, and b is the mobility (Landau and Lifshitz, 1959). The specific error has been to introduce the Stokes relation for the mobility of a spherical particle in a three-dimensionally isotropic medium. I n this case, the mobility is

b

=

1/(6~?/R)

(53)

where 3 is the equivalent ordinary liquid viscosity (poise) and R is the particle radius. From the lateral diffusion measurement and Eqs. (52) and (53), the equivalent viscosity has been calculated to be 1 poise (dyn-sec/cm2) (Po0 and Cone, 1974). A pseudo membrane surface viscosity is given by 71

- ?/do

which is on the order of lo-’ surface poise (dyn-sedcm). Immediately, we are suspicious, because the direct measurements of surface vissurface poise) in the same state are cosity for monolayers (10-~-10-~ several orders of magnitude greater than this deduced value. As we show, only a small part of the discrepancy results from the inappropriate use of the Stokes relation for mobility.

1. LATERALDIFFUSION AND MOBILITY Recognizing that particle motion is restricted to the plane of the membrane and that momentum can be transferred from the lipid surface to the aqueous surroundings, Saffman and Delbriick (1975) and Saffman (1976) developed an approximate relation for the mobility of a particle in a two-dimensional fluid:

b - - 1 n1 -

4Tr)

r)

r)a

E. A. EVANS A N D R. M. HOCHMUTH

54

where ijmis the viscosity of the environmental aqueous solution (- low2poise). I n Saffman's analysis (1976),he uses the product of an equivalent, three-dimensional viscosity times the membrane thickness, in place of the surface viscosity q ; however, it is proper to introduce the surface viscosity to represent the anisotropic fluid character of the membrane. For the lateral diffusion of rhodopsin in the photoreceptor membrane of the frog and mud puppy, Po0 and Cone (1974)and Liebman and Entine (1974)report a diffusion coefficient in the range of 2-4 x cm2 per second. The initially half-bleached photoreceptors were about cm in diameter. The diffusion constants were calculated using the measured half-time t l l z for the spread of rhodopsin. Fahey and Webb (1977)used fluorescence photobleaching recovery methods to measure the lateral diffusion of a lipidlike dye marker, 3,3'-dioctadecylindocarbocyanineiodide, in hydrated multilayers of lipids above the phase transition temperature. They reported values on the order of lo-*cm2 per second for the lipidlike probe. The question we pose now is: What value for surface viscosity do we obtain using the lateral diffusion measurements and the appropriately two-dimensional mobility relation of Saffman (1976)?A surface viscosity of surface poise and a particle radius of 2 x lo-' cm (corresponding to that of the rhodopsin particle) give a lateral diffusion constant on the order of

-

D 2.8 x cm2 per second using Eqs. (52)and (54).This value correlates well with the observations of the photoreceptor membrane experiments (Po0 and Cone, 1974).A surface viscosity of 0.35 x surface poise provides a lateral diffusion constant comparable to the measurements on hydrated lipid multilayers (Fahey and Webb, 1977). Clearly, the values of 10-s-10-5surface poise calculated from lateral diffusion constants differ significantly from the range of 10-4-10-3surface poise measured for the surface viscosity of monolayers. This incompatibility indicates that our experimental and theoretical techniques must b e carefully reviewed and checked; in general, the resolution of such anomalies has provided major progress in understanding the associated problem. For example, note that the anisotropic fluid character contributes an order of magnitude to the mobility in comparison with the StokesEinstein relation

- 10

Wl7/7)wR)

It is clear that constraining the marker particle to movement in two dimensions significantly decreases fluid resistance to the motion over

MECHANOCHEM1CAL PROPERTIES OF MEMBRANES

55

that of the three-dimensional situation. Therefore the lateral diffusion is commensurately increased, and the calculation of surface viscosity from an experimentally determined diffusion coefficient must include the anisotropic correction (a factor of about 10 in the rhodopsin experiment). At the other extreme, a surface viscosity of lo-? surface poise corresponding to the equivalent liquid viscosity of 1 poise does not correlate with lateral diffusion measurements, since

D

- 130 x lop9cm2 per second

from Eqs. (52) and (54).The equivalent liquid viscosity of 1 poise is the usually accepted value of microviscosity for membranes, e.g., as obtained from fluorescence polarization (Azzi, 1975), rotational diffusion (Cone, 1972), and permeability measurements (Solomon, 1974). Clearly, these microviscosity values are not related to the surface shear viscosity characterizing the resistance to flow of membrane surface. The microviscosity represents the approximate average fluid behavior of the hydrocarbon chain interior.

2. ROTATIONALDIFFUSION AND MOBILITY Confusion of surface viscosity and microviscosity has been enhanced b y the involvement of rotational diffusion measurements. For instance, ignoring the previous measurements of surface viscosity in amphiphilic monolayers, Saffman and Delbriick (1975) reached the opposite conclusion, namely, that the equivalent viscosity of 1 poise (surface viscosity on the order of lo-' dyn-sec/cm) correlates both rotational and lateral diffusion experiments. This was determined by evaluating the ratio of the respective diffusion constants. Consider the rotational diffusion constant DR. Saffman and Delbriick (1975) give the rotational diffusion constant DR =

kTbR

(55)

where the rotational mobility bRis

bR = 1/(4~r7) &RZ) in terms of an equivalent liquid viscosity. If the surface viscosity is used, Eq. (56) is expressed as

bR = 1/(4rrqR2)

(57)

Using the values of surface viscosity calculated from lateral diffusion measurements, we obtain, from Eqs. (55) and (57), a range of the rotational diffusion constant:

56

E. A. EVANS A N D R. M. HOCHMUTH

DR

- 8-24 x lo3 per second

which is a factor of about 3 to 4 smaller than the measured range of the rotational diffusion constant, 2-10 x lo4 sec-' from Cone (1972). However, if we use the equivalent liquid viscosity often quoted for the membrane, 1poise, i.e., lo-' surface poise, Eqs. (55) and (56) give a rotational diffusion constant on the order of

DR

- 80 x 104 per second

which is significantly in excess of the experimental range. But, the ratio of the lateral diffusion constant to the rotational diffusion is very nearly the same for both types of calculations:

D/DR

- 3-4

D/DR

-2

X

X

(using a surface viscosity of 0.35-1.0 dyn-sec/cm calculated from lateral diffusion measurements) (using a surface viscosity of 1 x lo-' dynsec/cm equivalent to a liquid viscosity of 1 poise)

Obviously, this ratio does not discriminate between the surface viscosity choices.

3. CONCLUSIONS The important observation is that the continuum property measured directly for surface shear viscosity of amphiphilic monolayers, 10-4-10-3 dyn-sec/cm, is about two orders of magnitude greater than the surface viscosity determined from lateral diffusion experiments using Eqs. (52) and (54).These two approaches must be brought into closer agreement: (1) if we are to use the measurements of lateral diffusion as determinants of the fluidity of large, continuous regions of the membrane, or (2) if we are to use surface viscosity values as representative of molecular mobility in the membrane surface. As discussed previously, the strong dependence on area per molecule indicates that the surface viscosity in shear and, thereby, the lateral diffusion constant should be primarily dominated by the collisional interactions of the polar head groups of the amphiphiles, not those of the hydrocarbon chains. However, collisions of the hydrocarbon chains with the large particle may play a controlling role in rotational diffusion, giving a lower apparent value for surface viscosity. Certainly, it is not unlikely that the kinetic interactions which determine the lateral and rotational Brownian motion are different, because

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

57

the lipid bilayer is anisotropic and the amphiphiles are heterogeneous molecules. Consequently, rotational diffusion may not be related directly to the lateral diffusion in this anisotropic material; it may be that the hydrocarbon chain fluidity primarily determines the rotational diffusion, whereas the two-dimensional fluidity of the surface polar head groups determines the surface viscosity and the lateral diffusion. Other complications should be considered; for instance, does the particle penetrate one or both layers of the bilayer? Many times there is additional material strongly associated with the lower amphiphilic layer, e.g., spectrin in red cell membranes. Here, the lower layer may not be free to slip at the polar head group-cytoplasm interface (e.g., when there is very large subsurface viscosity or yield shear for the cytoplasm). In this case, the fluid resistance is augmented by the drag of the particle moving through the restricted amphiphiles of the lower lipid layer. Therefore the diffusion is limited by the frictional (viscous) resistance between the end of the particle and the fixed cytoplasmic interface, and the movement in the upper half of the bilayer becomes unimportant. In this situation, the diffusion constant is very small, giving a gross overestimate of lipid layer surface viscosity. From consideration of surface viscosity and lateral diffusion, it is apparent that the calculation of surface viscosity from lateral diffusion measurements of large particles in bilayers and other membranes is complex. Using the (three-dimensional) Stokes-Einstein equation for the diffusion constant can result in about an order of magnitude underestimate in the value for the surface viscosity compared with that calculated from the two-dimensional mobility relation (Saffman, 1976). In addition, the diffusion of a “nearly transmembrane” particle can b e greatly retarded by the presence of a highly viscous subsurface structure at the cytoplasmic interface. In fact, it is tempting to speculate that something like this may have occurred in the diffusion measurements of Peters et al. (1974). In their experiment, they obtained a diffusion constant two to three orders of magnitude less than values obtained for the diffusion of rhodopsin in photoreceptor membranes. As discussed previously, viscoplastic flow of membrane material (Evans and Hochmuth, 197613) yields a surface viscosity of the total membrane structure on the order of dyn-sec/cm, which is three orders of magnitude greater than the values of surface viscosity calculated from lateral diffusion experiments. The large value for total membrane surface viscosity is consistent with the concept that a subsurface matrix dominates the membrane response to the rate of deformation in plastic flow. The lower value for surface viscosity, 10-4-10-3

58

E. A. EVANS A N D R. M. HOCHMUTH

dyn-sec/cm, obtained from viscoelastic relaxation of red cell membrane in the solid state may be partially associated with the bilayer component of the membrane as well as the spectrin structure. V.

SUMMARY

Thin biological membranes are most simply characterized by four independent elastic moduli and two coefficients of surface viscosity. The elastic constants include: (1) an elastic modulus for volume compressibility, 101o-lO1ldyn/cm2; (2) an elastic modulus for area compressibility (at constant volume), 102 dyn/cm; (3) a surface elastic shear modulus for recoverable extension (at constant area), dyn/cm for the red cell membrane and -0 for lipid bilayer membranes; and (4) a curvature or bending elastic modulus, 10-13-10-12 dyn-cm (erg). Assumed to be volumetrically incompressible, membranes also exhibit a very large resistance to fractional changes in area per molecule or membrane thickness. On the contrary, red cell membranes have a low resistance to shear or extensional deformations, and these membranes are capable of large elastic extensions. Even though red cell membrane shear rigidity is small, the red cell membrane is a solid material for shear resultants less than the yield shear, 2-8 x 10+ dyn/cm; above the yield point, red cell membranes begin plastic flow, resulting in permanent deformation. The lipid bilayer, however, has no shear rigidity (above the transition temperature) and thus is a twodimensional liquid. The red cell membrane shear elastic energy storage is in general much greater than the curvature or bending energy. However, for a lipid bilayer and in situations in which membrane force resultants contribute neglibibly, bending rigidity can determine the mechanical stability. The coefficients of viscosity for the membrane represent: (1) the energy dissipation produced by finite rates of membrane area expansion or condensation, dyn-sec/cm (estimate); and (2) the energy dissipation resulting from finite rates of extension or shear in the membrane plane, 10-4-10-2 dyn-sec/cm for the red cell membrane and 10-5-10-3 dyn-sec/cm for lipid bilayer membranes. Associated structural material, e.g., red cell membrane spectrin, or connective tissue strongly influences the membrane dynamic behavior in extension or shear deformation. Continuum mechanical properties are related to the molecular structure and therefore provide direct assessment of the material arrangement. In conjunction with thermodynamic considerations, mechanochemical equations of state and material thermodynamic relations can be investigated. The major difficulties are associated with

-

-

-

-

-

-

-

MECHANOCHEMICAL PROPERTIES

OF MEMBRANES

59

the mechanical experiments and analyses. In many cases, forces on the order of 10-9-10-g dyn have to be produced, controlled, and detected; also, observation of the experiment is often limited by microscope resolution or can only be indirectly performed. On top of this, mathematical complexity can obscure the “physics” and make the analysis almost intractable. Experiments must be carefully designed to measure the independent material properties of the membrane. It is clear from Tables I -1V that there is a significant requirement for more experimental data.

SYMBOLS Geometry and Deformation Undeformed membrane length, width, and thickness Deformed membrane length, width, and thickness Membrane thickness in an initial state (do = u 3 ) Membrane area in initial and deformed state Membrane area per molecule and excluded area per molecule Membrane volume Membrane extension ratio at constant area An independent deformation parameter given as a quadratic function of the extension ratio A mean value for P Large-deformation shear strain [es = f ( k 2 - I-’)] Fractional change in membrane thickness and fractional change in membrane thickness at constant volume Fractional change in membrane area and fractional change in membrane area at constant volume The deviation between the fractional change in membrane area and the fractional change in membrane thickness, and the same deviation at constant volume Fractional change in membrane volume Length of cell projection (“tongue”) as it is aspirated into a pipet Radius Radius of pipet Radius of red cell Radius of hemisphere Radius of disk Principal radii of curvature Changes in the principal radii of curvature away from the natural curvature state Equilibrium curvature in the natural state [C, = ( C , + C& The distance between two monolayers A subscript which refers to the Ith monolayer, i.e., h,, at, P l , and so on Tensions, Shear Resultants, and Stresses Normal membrane tension in the plane of the membrane T” T, Maximum shear resultant

60

T T,,TP M QT m3

70

E. A. EVANS AND R. M. HOCHMUTH

Isotropic tension Principal membrane tensions (membrane tensions along the two principal axes) Moment resultant Transverse shear resultant (perpendicular to the plane of the membrane) Normal stress (perpendicular to the plane of the membrane) Fluid shear stress at the surface

Thermodynamics E Total energy of the membrane F,B Helmholtz free energy and free energy per unit surface area (free energy density: fi = F/Ao) AF,, A F , The change in free energy density due to shear deformation and curvature, respectively r The induced moment coefficient, i.e., the moment resultants induced by a chemical free energy change [Eq. (35)l Interfacial free energy density of a monolayer (subscript 1 refers to the first Y monolayer, 2 the second, and so on) A conjugate force and flow in the Onsager equations I,, XP. Coupling coefficients between the flows XQand the forces], LPQ k Boltzmann's constant Hydrostatic pressure and the reference hydrostatic pressure p , Po P Membrane surface pressure ,lfn Surface pressure in a monolayer Heat transfer to the membrane 0 S Entropy s Rate of entropy production T Temperature (as a subscript, T denotes an isothermal process) W Work done on the membrane Elastic and Viscous Constants Bending elastic modulus (dyn-cm) Particle mobility in translation and rotation (dyn-sec/cm)-' Diffusion constant and rotational diffusion constant (cm2/second) Membrane surface viscosity (shear viscosity in two dimensions) (dyn-sec/cm) A three-dimensional liquid viscosity (dyn-sec/cm2) Three-dimensional viscosities of water and hydrocarbons (dyn-sec/cm2) Area compressibility modulus of a membrane (dyn/cm) Thickness compressibility modulus of a lipid bilayer (dyn/cm2) Area compressibility modulus of a monolayer (dyn/cm) Area compressibility modulus of the Ith monolayer (dyn/cm) Bulk (three-dimensional) compressibility modulus (dyn/cm') A two-dimensional area viscosity (a resistance to the rate of change in area) (dyn-sec/cm) The Onsager coupling coefficient related to the area dilation viscosity K The Onsager coupling coefficient related to the shear viscosity 7 The membrane shear modulus of elasticity (dyn/cm) The time scale for area relaxation (seconds) The time scale for shear relaxation at constant area (seconds) Time (seconds)

MECHANOCHEMICAL PROPERTIES OF MEMBRANES

61

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Phase changes and mosaic formation in single and mixed phospholipid monolayers at the oil-water interface. Biochirn. Biophys. Actn 323, 157-160. Tien, H. T. (1967). Black lipid membranes in aqueous media: Interfacial free energy measurements and effect of surfactants on film formation and stability. J. Phys. Chein. 71,3395-3401. Waugh, R., and Evans, E. A. (1976).Viscoelastic properties of erythrocyte membranes of different vertebrate animals. Microoasc. Res. 12, 291 -304. Weed, R. I., LaCelle, P. L., and Merrill, E. W. (1969). Metabolic dependence of red cell deformability. J. Clin. Invest. 48, 795-809. White, S. H. (1974). Comments on “Electrical breakdown of bimolecular lipid membranes as an electromechanical instability.” Biophys. J. 14, 155-158. White, S. H., Petersen, D. C., Simon, S., and Yafuso, M. (1976).Formation ofplanar bilayer membranes from lipid monolayers. Biophys. J. 16, 481-489. Zarda, P. R. (1974). Large Deformations of an Elastic Shell in a Viscous Fluid. Ph.D. Thesis, Columbia University, New York. Zarda, P. R., Chien, S., and Skalak, R. (1977).Elastic deformations of red blood cells.]. Biornech. 10,211.

Receptor-Mediated Protein Transport into Cells. Entry Mechanisms for Toxins. Hormones. Antibodies. Viruses. lysosomal Hydrolases. Asialoglycoproteins. and Carrier Proteins DAVID M . NEVILLE. J R. AND TA-MIN CHANG Section on Biophysical Chemistry Lobo ra tory of Neu rochemistry National Institute of Mental Health Bethesda. Maryland

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 68 I1 . Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . DiphtheriaToxin . . . . . . . . . . . . . . . . . . . . . . . . 68 B . Abrin and Ricin . . . . . . . . . . . . . . . . . . . . . . . . 76 80 C . Tetanus Toxin . . . . . . . . . . . . . . . . . . . . . . . . . 86 D . Botulinum Toxin . . . . . . . . . . . . . . . . . . . . . . . . 91 E . Cholera Toxin . . . . . . . . . . . . . . . . . . . . . . . . . F . Colicins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 101 111. Carrier Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Transcobalamin I1 . . . . . . . . . . . . . . . . . . . . . . . 101 B . Transferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 C. Low-Density Lipoprotein . . . . . . . . . . . . . . . . . . . . 107 111 IV . Asialoglycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . A . Structural Requirements for Transport . . . . . . . . . . . . . . 111 B . Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 C . Pinocytotic Mechanism? . . . . . . . . . . . . . . . . . . . . 114 V . Fibroblast Lysosoinal Hydrolases . . . . . . . . . . . . . . . . . . 115 VI . Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 A . Maternal-to-Young Transfer . . . . . . . . . . . . . . . . . . . 116 B . Transport into IInmunological Cells . . . . . . . . . . . . . . . 119 C . Retrograde Axonal Transport . . . . . . . . . . . . . . . . . . 120 120 VII . Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Evidence for Receptor-Mediated Entry . . . . . . . . . . . . . 120 B . General Transport Mechanisms . . . . . . . . . . . . . . . . . 121 VIII . Growth Factors and Hormones . . . . . . . . . . . . . . . . . . . 122 A . Nerve Growth Factor . . . . . . . . . . . . . . . . . . . . . . 122 B . Glycoprotein Hormones . . . . . . . . . . . . . . . . . . . . . 126 65

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C. Lactogenic Hormones . . . . . . . . . . . . . . . . . . . . . . D. I n s u l i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Epidermal Growth Factor . . . . . . . . . . . . . . . . . . . . IX. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Intracellular Localization Following Transport . . . . . . . . . B. Mechanisms of Transport . . . . . . . . . . . . . . . . . . . . C. Unique Functions of Receptor-Mediated Protein Transport . . . X. Pharmacological Implications of Receptor-Mediated Protein Transport References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . .

129 130 131 131 131 132 135 137 139

I. INTRODUCTION

Many cells are capable of transp0rtin.g certain proteins from the extracellular environment to an intracellular. compartment by a process which has an initial obligatory binding step to a cell surface receptor. The process is distinct from bulk fluid pinocytosis which does not require receptor binding. We refer to this process as receptor-mediated protein transport. Receptor-mediated protein transport is widespread in nature and is responsible for a variety of biological effects. Several examples of proteins transported by this process are transcobalamin I1 (TC 11)-cobalamin complex, immunoglobulins, desialylated glycoproteins, nerve growth factor (NGF),and a variety of bacterial and plant seed toxins. A common feature of each example is the high degree of cell type specificity present in the transport process. Cells lacking the specific receptor are incapable of transport. A major difference among the examples is the ultimate localization of the transported protein. Some proteins are transported through the cell to another extracellular compartment. Others localize in the lysosomes, while a few exert profound effects within the cell cytosol compartment. Consideration of these facts leads to the posing of several questions. Do all cell surface receptors participate in receptormediated transport, or is entry mediated by a class of receptors with unique properties? Are receptors divisible into certain classes which mediate entry to particular intracellular compartments? Of the receptors which mediate entry is the binding process sufficient to initiate entry, or is a more complex interaction between the receptor and the transported protein required? These questions are posed as a first step in constructing simple models of receptor-mediated protein transport. In the following sections we discuss the available data on a variety of receptor-mediated protein transport systems and attempt to answer these questions. The formal literature search for this article was completed in December 1976, although a few 1977 papers have been included. In

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order to keep the size manageable other reviews or well-referenced research papers are sometimes cited instead of the original reports. This review is limited to examples of protein transport into cells which have been demonstrated to occur b y receptor-mediated processes and which cause a change in the physiological state of the cell or organism. We have attempted to include all such examples which have been described but undoubtedly have missed some. The article is written primarily for experimentalists interested in one or more areas of receptor-mediated protein transport. Therefore pertinent experimental details and numbers derived from experiments are given. The diverse nature of this material and the presumed diversity of the readership has impelled us to include background physiology and pathology for many subject areas. These factors have produced a lengthy review. However, each section is complete within itself, and readers may choose from the table of contents any desired subject or ordering of subjects. The particular focus of this article is the result of our interest in utilizing receptor-mediated protein entry processes for pharmacological purposes, by the construction of artificial hybrid proteins. These concepts are discussed in Section X. Beyond a simple understanding of the transport process and its attendant specificity and compartmentalization is the broader question of the utility of the process to the organism. Carrying vitamin B12into a human cell interior is easily grasped as a useful event, but carrying diphtheria toxin is not. We consider the possibility that toxin transport systems are used for other purposes which have so far escaped detection. A further question for consideration is the utility humans can make of receptor-mediated protein transport. Because of the high degree of cell type specificity present in the process, and the profound effects of certain enzymes after transport to the cell cytosol, we have suggested that a whole new class of cell type-specific pharmacological reagents could be constructed which utilize this process (Chang and Neville, 1977). Attempts to construct such reagents by the formation of artificial protein hybrids are analyzed in terms of the present knowledge of receptor-mediated protein transport systems. Many receptor-mediated protein transport systems are operative at extremely low extracellular protein concentrations and transport minute quantities of protein, in the range of tens of molecules per hour. Were it not for the profound intracellular toxicity of some of these proteins, such systems would escape detection. However, since toxicity is a quantifiable end point in the process, it provides investigators with a means of studying these low-capacity transport

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systems. It is not surprising then that various bacterial and seed toxins constitute the best known examples of receptor-mediated transport. Of these diphtheria was the first to be studied.

II. TOXINS A. Diphtheria Toxin

1. GENERALCHARACTERISTICS Human diphtheria is characterized by an upper respiratory infection which progresses to severe localized necrosis, forming a pseudomembrane which may cause death by suffocation. Profound muscle weakness and lethargy also occur, and death is often ascribed to heart failure. An exotoxin was identified as the causative agent in 1888. It is secreted by Corynebacterium diphtheriae lysogenic for or infected with bacteriophage p carrying the tox+ gene. The toxin is easily purified and may account for 5% of the total bacterial protein synthesized. The purified toxin is a 62,000-dalton protein. Twenty-five nanograms injected into a guinea pig causes lethargy and muscle weakness in 3-4 days and death within 5 days. Only certain animal species are sensitive to diphtheria toxin. Humans, rabbits, chickens, and guinea pigs are equally sensitive on a weight basis. Mice and rats are resistant, requiring over 1000 times the dose before toxicity is noted. Today we have an almost complete understanding of the pathogenesis of this disease in molecular terms. The one remaining gap concerns the transport process. We outline the present state of knowledge as briefly as possible and suggest that the reader consult the reviews of Collier (1975) and Pappenheimer and Gill (1973) for a fuller appreciation of how the mystery of this disease process was unraveled.

2. INTRACELLULAR SITE OF ACTION In 1959 Strauss and Hendee discovered that the toxicity of diphtheria toxin toward intact sensitive cells was due to an inhibition of protein synthesis. Following exposure to the toxin there is a lag period followed by an exponential fall in protein synthetic rate as a function of time. The higher the toxin concentration, the shorter the lag period up to a limiting lag period which depends on cell type and incubation conditions (Uchida et al., 1973). Cell-free protein synthetic systems reconstituted from ribosomes

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and various cell cytosol soluble fractions were later tested. Diphtheria toxin was inhibitory in these systems, and a cofactor requirement was found which proved to be NAD+ (Collier and Pappenheimer, 1964). Shortly thereafter Honjo and co-workers (Honjo et al., 1968) showed that diphtheria toxin was an enzyme which catalytically inactivated elongation factor 2 (EF-2), a necessary soluble protein component of the protein synthetic machinery. Inactivation was accomplished by transferring 1 mole of adenosine diphosphoribose from NAD+ to 1 mole of EF-2: N A D + + EF-2 (active)

.

diphtheria toxin

.ADPR-EF-2

+ nicotinamide + H+

(inactive)

The cause of the extreme toxicity of diphtheria toxin was apparent. There are only about 1.2 molecules of EF-2 per ribosome, and the turnover of EF-2 is slow. The equilibrium of the ADP-ribosylation reaction under intracellular conditions is such that the reaction goes virtually to completion (Collier, 1975). Calculations reveal that a steady-state concentration of only a single diphtheria toxin molecule per cell is required to inactivate the cell's supply of EF-2 within 1 day. Since the cellula,r EF-2 concentration is not normally rate limiting for protein synthesis, the dose-dependent segment of the lag period represents the time taken to reduce the EF-2 to a rate-limiting concentration (Pappenheimer and Gill, 1973). In the intact cell EF-2 cycles between the cell cytosol and its ribosome-binding site. Studies had shown that ribosome-bound EF-2 was immune to inactivation by toxin (Collier, 1975). This established the cell cytosol as the site of action of diphtheria toxin. A mechanism was then needed to transport diphtheria toxin from the extracellular fluid across the cell membrane to the cell cytosol.

3. STRUCTURAL

AND

FUNCTIONAL INTERRELATIONSHIPS

Knowledge of the mechanism of entry of diphtheria toxin into the cell cytosol was greatly increased by elucidation of the structural and functional relationships of the toxin molecule. It was discovered that diphtheria toxin was susceptible to site-specific proteolytic cleavage and reduction by thiols, generating two fragments of 39,000 daltons (fragment B) and 21,000 daltons (fragment A) (Gill and Dinius, 1971; Collier and Kandel, 1971). The B fragment was devoid of enzymic activity, while the A fragment was highly active. T h e separated fragments showed no toxicity toward intact cells. By mixing A and B frag-

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DAVID M. NEVILLE, JR. AND TA-MIN CHANG

ments and removing thiol, toxicity toward cells was reestablished. The hypothesis was advanced that only the A fragment was needed within the cytosol to produce toxicity and that the B fragment was somehow involved in the entry mechanism. To test this hypothesis Uchida and co-workers (Uchida et al., 1973) studied various purified nontoxic proteins which are serologically related to diphtheria toxin (CRM proteins) and are obtained from culture filtrates of C. diphtheriae lysogenized with phages which are mutant in the toxin structural gene (tox-CRM+P).Two nontoxic CRM proteins, CRMIOand CRM,,, were found which contained normal A fragments, as judged by enzymatic activity and electrophoretic mobility, and which lacked large regions of the N-terminal portion of the B fragment. When the altered B fragment was replaced by a normal B fragment or one from CRMlWby forming the hybrid protein &5B197 or Ad5Bwildtype, 55% of the wild-type toxicity returned. Clearly the B fragment performed a necessary function in cell intoxication which was separate from the enzymatic activity of the A fragment. Further studies showed that the interaction of the B fragment with the cell occurred via receptors. Ittelson and Gill (1973) demonstrated that nontoxic CRM19,which contained an enzymatically inactive A fragment and a normal B fragment competitively blocked the toxicity of wild-type toxin toward HeLa cells with K , = lo-* M ,Saturation kinetics of toxin-promoted inhibition of protein synthesis were also noted with saturation at 5 x lo-’ M toxin (Uchida et al., 1973).The phenomenon of saturation and competitive inhibition requires the presence of a finite number of saturable binding sites, and such sites are called receptors. [Throughout this review we use the term “membrane receptor” in a phenomenonological sense, as originally defined by Langley (1905)to describe a process which can be explained by binding that exhibits saturation and competition with related substances. Thus receptor binding can be approximated by the term

(or a sum of such terms), where B1 is the amount bound, n1 is the number of sites, K1 is the equilibrium affinity constant, and F is the free ligand concentration. Nonspecific binding refers to nonsaturable binding which over the concentration range specified can be represented by B2 = K2F (Neville, 1974).1 B-Chain toxin receptors appear to be localized to the external surface of the cell membrane. When cells are exposed to toxin, toxin is

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71

rapidly bound to them. The binding step precedes the transport step. The dissection of these two events was made possible by the discovery of Kim and Groman (1965) that the presence of ammonium ion blocks the toxicity of toxin toward cells but does not affect the binding step. The binding is sufficiently strong to resist washing of the cells with buffer. Cells can be treated with toxin in the presence of 4 x M NH4+, washed, and reincubated with and without NH4+.Toxicity develops only in cells washed free of NH4+. However, when the washing out of NH4+is preceded by a 30-minute exposure to antitoxin antibody, the cells are protected from toxicity (Duncan and Groman, 1969; Ivins et al., 1975). Antitoxin antibody is without a protective effect when added after toxin in the absence of NH4+ and presumably cannot cross the membrane to reach the cytosol. The conclusion is that the antitoxin antibody is neutralized at the toxin-binding site on the external surface of the membrane. T h e presumption is that this binding site is identical to the receptor inferred from competition and saturation experiments.

4. THE TRANSPORT PROCESS

The mechanism of transport of diphtheria toxin has been investigated by following the uptake of 1251-labeleddiphtheria toxin by tissue-cultured cells. Bonventre et al. (1975) observed the uptake b y a sensitive cell line, HEp-2, and a resistant line, L929, using concentrations of diphtheria toxin in the range of M . The resistant cell line had a higher uptake than the sensitive line. The presence of poly-Lornithine at 10 pg/ml doubled uptake in the sensitive line. However, subsequent experiments showed that poly-L-ornithine inhibits the toxicity of diphtheria toxin. Uptake studied in the presence and absence of 4 x M ammonium chloride, which fully protected the sensitive line from toxicity, revealed no differences in the rate of uptake. Since ammonium ion does not block binding yet must block transport to the cytosol compartment of active fragment A, it can be concluded that 90% or more of the uptake observed in these experiments is localized to a compartment other than the cell cytosol, and that this transport process is not related to toxicity. This uptake decreases with time, a situation encountered with other examples of presumed adsorptive or receptor-mediated pinocytosis (Steinman et a1., 1974). [Pinocytosis is the process by which cells take up fluid from the external medium in bulk by the internalization of small portions of plasma membrane. As Fawcett (1965) points out, a variety of different kinds of surface activity lead to this result. Several examples are the

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M. NEVILLE, JR. AND TA-MIN C H A N G

invagination of microscopically visible tubes in the ameba, the development of apical canaliculi of submicroscopic size in the absorptive epithelium of the kidney, and the formation of minute vesicles just below the surface membrane of a variety of cells. The last-mentioned process is subdivided into two distinct morphologies, vesicles formed from smooth-surfaced membrane and vesicles formed from specialized membrane. The specialized membrane often exhibits an indentation in which both surfaces are thickened. These are called coated regions, and the vesicles so derived are called coated vesicles (Roth and Porter, 1964; Fawcett, 1965). The terms “macropinocytosis” and “micropinocytosis” were originally defined by the resolution of the light microscope. Many investigators use these terms in a relative sense without an absolute size cutoff. Uptake of solubilized macromolecules by cells undergoing pinocytosis has shown that certain proteins are taken up in direct proportion to the fluid engulfed. This uptake process is called bulk fluid pinocytosis. Other proteins are taken up in excess of the fluid engulfed, and uptake exhibits saturation and competition with analogs. The latter process is explained by receptors on the surface membrane, which concentrate the macromolecule (Steinman et al., 1974). This process is called adsorptive pinocytosis or receptor-mediated pinocytosis, as distinguished from bulk fluid pinocytosis.] Uptake of lZ5I-labeleddiphtheria toxin has been studied as a function of concentration over the range 5 x 10-9-10-7 M b y Boquet and Pappenheimer (1976). These experiments were performed using HeLa cells at 30°C in the presence and absence of excess unlabeled diphtheria toxin. At each concentration point the uptake of lz5Ilabeled toxin at 1 hour was greater in the absence of excess unlabeled toxin. In an attempt to delineate the number of receptor sites, points obtained in the presence of excess unlabeled toxin have been fitted with a straight line, whereas a curved line has been used to fit the points obtained in the absence of unlabeled toxin. Points obtained by subtracting cold plus hot points from hot points have been fitted to a hyperbolic function. This is called specific binding. The number of receptor sites per cell calculated from the point of saturation is 3500. Considering the scatter of the data and the forcing of the fit, it is unlikely that these data can be interpreted as providing a direct demonstration of saturable binding sites with a K of 10“M-’ (which agrees with the competition experiments using cytotoxicity as an end point.) In addition it is not clear in these experiments how much of the observed uptake at 1 hour represents binding and how much constitutes transport into a compartment which is not in equilibrium with the external media. However, the data do show that a portion of the

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toxin uptake occurs b y a saturable process. Saturable uptake was not found in a toxin-insensitive cell line. It is tempting to consider this finding as support for the proposition that insensitive cell lines lack the specific cell surface receptor. However, it is not known whether or not the saturable uptake measured in the sensitive cell line is the process productive of toxicity. Until this relationship can be established, the above proposition remains untested. Another type of experiment has been performed in an attempt to delineate the events occurring subsequent to the binding step. We have already mentioned experiments which have shown that the nontoxic cross-reacting protein CRMle7 can compete with toxin for entry into HeLa cells. These results were interpreted as indicating the presence of specific receptors interacting with toxin with a reversible equilibrium. Boquet and Pappenheimer (1976) were unable to obtain unequivocal evidence for a reversible equilibrium from direct uptake measurements at low temperatures or by using chase experiments with excess unlabeled toxin added after the addition of labeled toxin. These investigators postulated an initial rapid reversible interaction between toxin and receptor followed by a slow irreversible process. M was incubated with To test this hypothesis CRM,,, at 4 x HeLa cells for various intervals, followed b y the addition of M toxin. A time-dependent protection against toxicity was noted, and after 1 hour of preincubation with CRM almost complete protection was achieved. Thus CRMls7 appeared to block the toxin entry sites. This conclusion appears valid, but the irreversible nature of the process may simply be due to a very slow rate of dissociation of CRM,,, from its receptor. Without knowledge of the forward rate constant of association and the rate constant of dissociation it is impossible to interpret whether this experiment dealt with a single-step binding process or with a subsequent irreversible process which occurred after binding. Further insights into the mechanism of the binding and transport of diphtheria toxin have been provided by studying materials which block cytotoxicity. These experiments are performed by incubating cells with toxin plus another agent, followed by washing out the toxin and the agent. At this point the cells are divided into two groups, one of which is treated with a diphtheria antitoxin wash. In this way the action of a protective agent can be determined to occur at the binding site or during the transport step. Toxin bound but not transported by the action of a protective agent is neutralized by antitoxin. Agents interfering with binding do not require antitoxin treatment to elicit their protective effect during the cytotoxic assay. I n this way, Duncan and Groman (1969) showed that cations were essential for the binding

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DAVID M. NEVILLE, JR. AND TA-MIN CHANG

of diphtheria toxin to HeLa cells. The effectiveness of cations in mediating binding was related to their charge. Cells incubated in isoosmolar sucrose were completely protected against toxin and 0.1 M sodium ion was required to achieve significant toxicity. Calcium at M was more effective, and M aluminum could replace calcium. In a similar manner, a pH value above 9.5 was also found to inhibit binding. Binding was not inhibited by a l-hour exposure to 1% trypsin, 1% chymotrypsin, pronase, lysozyme, neuraminidase, or hyaluronidase. Metabolic inhibitors of respiration, cyanide, and 2,4dinitrophenol failed to protect against cytotoxicity. However, M sodium fluoride gave complete protection. Under these conditions glycolysis fell 60%.Iodoacetate, another glycolytic inhibitor, gave no protection at 1.3 x M . Higher concentrations were toxic to cells, and glycolysis was unfortunately not measured at the utilized concentration. Cells not washed with antitoxin, which had been pretreated with sodium fluoride, showed complete toxicity, indicating that fluoride blocked a step subsequent to binding. Protection by fluoride has been confirmed by Ivins et al, (1975) and Middlebrook and Dorland (1977a), but the mechanism of protection is not known. Fluoride may work by inhibiting the formation of ATP through the glycolytic pathway. To be effective, ATP production through respiration could occur only at low levels. Since cellular ATP levels were not measured in these studies, the problem remains unsolved. Fluoride is also known to be a potent inhibitor of a variety of phosphatases. Using the cell line HEp-2, Ivins et al. (1975)found that cyanide or dinitrophenol gave partial protection against diphtheria toxin. Again, ATP levels were not measured. Sodium arsenite also gave partial protection, as did poly-L-ornithine and cytocholasin B. Sulfhydryl reagents such as p-chloromercuriphenylsulfonic acid and dithiothreitol were without effect. Middlebrook and Dorland (1977a) carried out an interesting comparative study on the effect of a variety of agents that protect against diphtheria toxin and the toxin from Pseudomonas aeruginosa, utilizing HeLa and HEp-2 cell lines. This study is particularly interesting, since Pseudomonas toxin has been found to have the same site of action as diphtheria toxin. Iglewski and Kabat (1975) showed that P . aeruginosa toxin catalyzed the transfer of ADP-ribose from NAD+ to EF-2. Since both toxins have the same intracellular site of action and utilize the same enzymic reaction, differences among protective agents reflect differences in transport or binding. Of a variety of agents tested, none protected against Pseudomonas toxin. In addition to the agents previously found to protect against diphtheria toxin, Middle-

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75

brook and Dorland (1977a) also noted almost complete protection by 10 p M ruthenium red. The anesthetic agents procaine hydrochloride and lidocaine also gave significant protection. Whether the protection achieved by ruthenium red is a consequence of inhibition of calcium-magnesium-dependent ATPase or by nonspecific blocking of multivalent cationic binding sites remains to be determined. Middlebrook and Dorland (1977a) tested all their protective agents in the cell-free EF-2 ADP-ribosylation system. Only salicyclic acid and procaine affected the reaction in a manner which could explain their protective effects. The differential protection afforded against diphtheria toxin toxicity b y ammonium ion, ruthenium red, and sodium fluoride, in contrast to the situation with Pseudomonas toxin, is best explained by different transport processes for these two toxins. It also appears from the studies of Middlebrook and Dorland (1977b) that these two toxins have different receptor specificities. These investigators determined the tissue culture median lethal dose for both toxins in 21 different cell lines. For diphtheria toxin the tissue culture median lethal dose varied from 0.02 ng/ml to greater than 103 ng/ml. For Pseudomonas toxin the range was 0.1 ng/ml to 300 ng/ml. For diphtheria toxin a general species hierarchy was seen, monkeys being the most sensitive, followed by hamsters and humans, with rats and mice being very insensitive. No species hierarchy was determined for Pseudornonas toxin, and in general there was little correlation between the sensitivities toward the two toxins, Pseudomonas having high toxicity for some mouse and rat cell lines. These data are particularly welcome, since most data of this sort used for comparative purposes have been obtained in various laboratories under various conditions. Middlebrook and Dorland (1977~) addressed themselves to the problems of performing quantitative comparative cytotoxic studies utilizing these toxins. In particular they found that most serum used in tissue culture displayed various degrees of inhibition toward the toxins and that fetal calf serum was preferred for the studies. The large difference in sensitivity between mouse cell lines and human cell lines toward diphtheria toxin has been used by Creagan et al. (1975)to localize the human chromosome responsible for diphtheria toxin sensitivity. These investigators studied the sensitivity toward diphtheria toxin for a variety of hybridized cell lines. Hybridization between mouse and human lines was achieved by treating cells with inactivated Sendai virus. Chromosomal distribution was studied in all lines, and a correlation was achieved between the presence of human chromosome 5 and hybrid cell sensitivity toward diphtheria toxin.

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Whether human chromosome 5 carries information for the receptor, the transport system, or both remains to be determined. Various models for toxin transport to the cytosol are considered in Section IX,B, B. Abrin and Ricin

1. GENERALPROPERTIESAND STRUCTURES Abrin and ricin are two extremely toxic proteins present in the seeds of the taxonomically unrelated species Abrus precatorius and Ricinus communis. These toxins are quite similar to each other in structure and mechanism of action and also share similarities in general structural features and mechanism of action with diphtheria toxin. Abrin and ricin both inhibit protein synthesis in eukaryotic cells following a dose-dependent lag period. The site of action of abrin and ricin is the 60s ribosome. These proteins are both two-chain structures of MW -60,000, the two chains being held together by a disulfide bond. Separated purified chains are nontoxic toward eukaryotic cells. For both proteins the higher-MW chain, 34,000 for ricin and 35,000 for abrin, is known as the B chain and is involvtd in the binding process. The A chain, 32,000 for ricin and 30,000 for abrin, catalytically inactivates 60s ribosomes in cell-free systems (Refsnes et al., 1974). Seeds of Abrus and Ricinus also contain two hemagglutinins known as Abrus and Ricinus agglutinin, which are related to the toxic proteins. These agglutinins are capable of agglutinating erythrocytes of human blood groups A, B, and 0. The MW of each agglutinin is approximately double the toxin MW, 120,000. Immunological studies indicate that the agglutinins contain two B chains indistinguishable from the toxin B chains. In addition, each agglutinin contains two chains similar but not identical to toxin A chains. In the case of Ricinus agglutinin, the A chains lack certain antigenic determinants present on the A chains of Ricinus toxin. In addition, the agglutinin A chains appear to be approximately 1000-3000 daltons smaller than the A chains of Ricinus toxin (Pappenheimer et al., 1974).

2. BINDING SITES The binding properties of abrin and ricin have been extensively studied. The receptor site presumably contains lactose or galactose, since serum proteins containing nonreducing terminal galactose residues interfere with the binding. Direct binding of lactose to ricin has

RECEPTOR-MEDIATED PROTEIN TRANSPORT INTO CELLS

77

been demonstrated. The binding constant is approximately 105 M-', and there is one site per ricin molecule. The Ricinus agglutinin molecule contains two lactose binding sites (Olsnes et al., 1974). The binding affinity for HeLa cells and erythrocytes is higher, with equilibrium association constants reported at 3 x 10s M-' for both ricin and abrin. Binding is rapid at both 0 and 37°C and is reversible. Reciprocal transformation of the binding isotherms over a concentration range of 10-'o-10-8 M is linear, indicating a single class of receptor sites. There are approximately 3 x lo7 sites per HeLa cell (Sandvig et al., 1976). The minimum concentrations of abrin and ricin producing inhibition of protein synthesis have been detected at 0.2 ng/ml for abrin and 2 ng/ml for ricin. For abrin this calculates to a molar concentration of 3.4 pM. At this concentration only about 0.01% of the total sites are occupied. The binding sites for ricin and Ricinus agglutinin are similar in that each substance competes to a considerable extent for the same sites (Nicolson et al., 1974). However, some differences may exist, since although ricin completely competed for Ricinus agglutinin, the agglutinin did not completely compete for all bound ricin. However, these studies were not performed as equilibrium competition experiments and contained a wash step separating the two incubations. Olsnes et a l . (1976) showed that the purified B chain of ricin can compete for HeLa cell binding sites with intact ricin and intact abrin. These investigators report a binding constant of ricin B chain essentially equal to that of intact ricin. However, the competition data do not indicate equal affinities. This is probably due to the lack of knowledge of the amount of binding of iodinated ricin to the cells which is nonspecific. Purified ricin B chain also protects against toxicity against ricin when present in a 200-fold weight excess. Protection by B-chain ricin against toxicity for abrin under the same conditions appears to be of borderline significance. Since ricin B chain appears to compete to some extent with the binding sites for both abrin and ricin but protects against toxicity only for ricin, all the binding sites revealed b y these techniques may not be involved in the transport process of the toxin to the cell interior. When ricin and abrin are bound to HeLa cells at 3TC, a fraction of the toxins becomes irreversibly bound as a function of time. This is determined by washing cells in 0.1 M lactose at various time points. The fractional amount becoming irreversibly bound for both toxins is 2 x per minute. Abrin and ricin bound to cells at 37°C enter an eclipse phase similar to that seen with diphtheria, cholera, tetanus, and botulinum toxins. Toxin antisera is effective in preventing toxicity only when added to the cells immediately or only shortly after addition of the toxin (Refsnes et al., 1974).

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3. INTRACELLULAR MECHANISM OF ACTION When abrin and ricin in concentrations ranging from 1 ng/ml to 10 pg/ml are incubated with HeLa cells, the kinetics of inactivation of protein synthesis are found to be similar to those seen with diphtheria toxin. The rate of protein synthesis falls (following a lag period) exponentially with time. For abrin and ricin the rate of fall increases with increasing concentration up to a limiting concentration between 1 and 10 pg/ml. This is about the expected saturating level for a binding constant of 108 M-'. Studies utilizing cell-free protein synthetic systems have shown that abrin and ricin inactivate the 60s ribosome. Ribosomes exposed to these toxins have reduced ability to bind aminoacyl-tRNA, reduced GTPase activity, and reduced affinity for binding EF-2 (Benson et al., 1975; Montanaro et al., 1975; Carrasco et al., 1975). The ability of abrin and ricin to inactivate 60s ribosomes increases 50- to 100-fold after exposure of the toxins to reducing agents. Evidently, as is the case with diphtheria toxin, the B chain in the intact molecule blocks the enzymic activity of the A chain (Olsnes et al., 1976). Olsnes and co-workers (Olsnes et al., 1976) noted in the cellfree protein synthetic system that a lag period following the introduction of intact ricin and abrin is due to the time taken to separate the A and B chains. Chain reduction was demonstrated directly by sodium dodecyl sulfate (SDS) gel electrophoresis of iodinated toxin. When purified abrin and ricin A chains are used, there is a linear relationship between the number of ribosomes inactivated per minute and the toxin A concentration. The inactivation rate increases with temperature, and the estimated activation energy is 10.6 kcal/mole. The turnover number for both abrin and ricin A chains is approximately 1500 ribosomes per minute. Interestingly, the A chain of Ricinus agglutinin had the same Michaelis constant, 0.1-0.2 p M , as abrin and ricin A chains and a turnover number of 100 ribosomes per minute (Olsnes et al., 1976). The Ricinus agglutinin used in this study was purified by a method yielding a very low toxicity for Ricinus agglutinin, 1/2300 compared to ricin (Olsnes and Pihl, 1973). Thus Ricinus agglutinin is 150-fold more active in a cell-free assay than in an intact cell assay. The B chains for ricin and Ricinus agglutinin have been found to be indistinguishable (Pappenheimer et al., 1974). The B chains are believed to mediate entry of the active A chains. The discrepancy raises interesting questions. It is possible that having a B chain which binds is not sufficient for entry. The relationship between the B and A chain may not be in a necessary specific configuration. It is also possible that bivalent B chains do not gain entry. Another possibility is that the configuration of the B chain with respect to the A chain is not sufficient to

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inhibit inactivation of the A chain within the cytosol. Thus, although we know that a configurational change must take place after reduction between the A and B chains to achieve activation of enzymatic activity, the B chain may stay associated with the A chain and protect against degradation. It is also possible that the very low i n vivo toxicity of Ricinus agglutinin reflects a very high degradation rate of the A chain within the cytosol, which is due to a structural difference within the A chain. These differences were noted above. This hypothesis places the A chain of Ricinus agglutinin in a category similar to that of the CRM17eA-chain mutant reported by Uchida et d. (1973).Th'is mutant of diphtheria toxin exhibits 9% of the wild-type fragment-A enzymatic activity and only 0.3%of the whole-animal toxicity. Fragment B of CRM,,, is normal. When HeLa cells are exposed to CRM,,, in saturating quantities ( M ) the rate of inactivation of protein synthesis M wild-type toxin. Cells treated with is equivalent to that of 5 x CRM,,, for 3 hours can be rescued b y washing, treating with antitoxin, and resuspending in toxin-free media. The protein synthetic rate continues to fall for another 9 hours and then climbs to the initial toxinfree value. However, it is impossible to rescue cells treated with 5 x M wild-type toxin. Pappenheimer and Gill (1973) interpret this experiment as indicating that CRM176has a more rapid half-time of degradation within the cytosol than wild-type toxin A. This explains the ability to rescue CRMl16 and the discrepancy between the much higher enzymatic activity of CRM,,, A chain and its in vivo toxicity. Recently, Etinger and Goldberg (1977) described an ATP-dependent proteolytic system operating within reticulocytes, which selectively degrades abnormal proteins. Systems such as this one may play a role in degrading exogenous proteins transported to the cell cytosol.

4. THE TRANSPORT PROCESS The mechanism of the transport of abrin and ricin into the cell interior which leads to toxicity is unknown. Nicolson (1974) demonstrated that ricin is taken u p and encased within pinocytotic vacuoles using ferritin-labeled ricin. However, the relationship between this process and the process which produces toxicity is unclear. Studies with metabolic inhibitors and inhibitors of various transport systems have not been reported for abrin and ricin. However, Olsnes and co-workers (Olsnes et al., 1973) have reported experiments which are highly relevant to the pertinent questions of receptor-mediated transport. These investigators purified A and B chains of ricin and then proceeded to reform the various hybrid species. They found that ricin A-abrin B

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and ricin B-abrin A were both cytotoxic, as judged by the LD,, dose for mice, In this study reconstituted abrin LD,, was 0.08 pg, and the reconstituted ricin LD5o was 0.2 pg. The hybrid abrin A-ricin B had an LD5o of 0.15 pg, and the hybrid ricin A-abrin B had an LDSoof 0.3 pg. It is interesting to note that the increased toxicity of abrin over that of ricin appears to be determined b y the A chain. This is in keeping with our previous discussion that the half-time of degradation of the A chain within the cell may be a major factor in governing cytotoxicity. The significance of this experiment is in part dependent upon whether or not the B chains of abrin and ricin utilize the same transport system. Ifdifferent transport systems are utilized, then Olsnes and co-workers (Olsnes et al., 1973) have shown that competent B chains can be recombined with A chains from other molecules and that these A chains gain entry, directed solely by the B-chain transport system. This generalization from their results is somewhat weakened because of the similarity between the abrin and ricin B and A chains. However, these chains are distinct immunologically, even if they have similar receptor binding sites and similar enzymatic activities. Antisera directed against ricin or abrin could not protect for the hybrid toxins. An indication that the two transport systems are different was obtained when it was shown that ricin B chain could block ricin toxicity without having a significant effect on abrin toxicity in HeLa cells (Olsnes et al., 1976). Olsnes and co-workers (Olsnes et al., 1973) were unable to form hybrids of ricin and diphtheria toxin. Their methodology involved dialysis of the reduced chains and presumably involved autoxidation. Hybrid formation between toxins and other putative B chains is discussed in Section X. C. Tetanus Toxin

EXPERIMENTALTOXICITY Tetanus toxin is a potent protein neurotoxin made by Clostridia tetani. In most cases bacterial entry to the body occurs through minor trauma, although in 20%of the cases no wound can be detected. The disease is characterized by muscle rigidity and reflex spasms severe enough to cause compression fractures of the spine and loss of pulmonary ventilation. Death is often the result of respiratory failure and may be induced by laryngeal spasm. The presenting symptoms are usually trismus (lockjaw) and stiffness of the neck. Rigidity of facial, pharyngeal, thoracic, abdominal, arm, and leg muscles follows. Reflex spasms increase in frequency until they follow one another in rapid 1.

CLINICAL AND

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succession. This pattern (Adams, 1971) constitutes generalized tetanus and can be produced in animals by the intravenous or subcutaneous inoculation of the purified toxin. For mice the LD50 is 2.5 ng/kg (Habermann and Dimpfel, 1973).A more limited disease called localized tetanus can be produced by injecting a smaller dose intramuscularly. After a lag period (dose-dependent), rigidity and spasm result which are confined to the groups of muscles innervated by the local motor neuron. Localized tetanus is a rare clinical entity (Habermann and Dimpfel, 1973). The site of action of tetanus toxin appears to be the pre- and postsynaptic junctions of the motor neurons located in the spinal cord and brain stem. Inhibitory reflexes in these areas are impaired or abolished (Curtis, 1971).The evidence indicates that tetanus toxin reaches these sites by first being fixed at the motor neuron end terminal and then being transported into the axon and up the axosplasm (Curtis, 1971). Since axonal transport has a relatively constant rate from nerve to nerve, toxin fixed at one time reaches the motor synaptic junction at different times, depending on the length of the motor neuron. The fifth cranial nerve, being the shortest, is the first to malfunction. This explanation of the clinical syndrome has been given credence for some time but was put on a firm basis by the experiments of Habermann and co-workers who prepared biologically active tetanus toxin labeled with lZ5I (Habermann and Dimpfel; 1973, Wellhoner et al.,

1973). When tetanus toxin is injected intravenously into mice at low dosage (3.5 ng/kg), there is a long symptomless period, and death does not ensue until 72-96 hours. With increasing dosage these time periods decrease until a limit is reached at 10 pg. Increasing the dosage beyond this point fails to kill animals sooner than 2 hours, or to produce signs of intoxication sooner than 45 minutes. Zacks and Sheff (1971), who investigated this phenomenon, reasoned that the minimum lag period represented the time needed for the toxin-promoted exhaustion of a crucial biochemical and that this process was rate limiting. By performing similar experiments with goldfish they showed that increasing the body temperature shortened the survival time. The relationship was log/linear and yielded a Qlo value of 4.2. These investigators calculate a corresponding activation energy for this process of 27 kcal/mole. The situation seen with tetanus toxin is likely to be analogous to that for diphtheria toxin. Both show a dosedependent lag period with saturation kinetics. I n the case of diphtheria toxin the saturation phenomenon is due to the receptor-mediated transport process. The lag period is the time required to exhaust the

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cell of EF-2 catalytically, a process having a large activation energy. No information is available on the biochemical process affected by tetanus toxin. However, we believe it reasonable to propose that the effect is enzymatic and that the activity resides in a fragment of the tetanus toxin molecule. Habermann and Dimpfel (1973) estimated that the spinal column concentration of tetanus toxin in an intoxicated anM . It is difficult to conceive of a nonenimal is in the range of zymatic inactivating process operative at this concentration. 2. RECEPTORS There are other similarities between the processes of intoxication by tetanus and by diphtheria toxin. In both cases antitoxin has minimal effectiveness when administered after the toxin, indicating rapid fixation of the toxin by the tissue (Zacks and Sheff, 1971). This fixation of tetanus toxin can be demonstrated in vitro by suspending nervous tissue in a solution of tetanus toxin, removing the tissue, and noting a decline in the toxicity of the solution-a phenomenon described by Wassermann and Takaki (1898). More recently Habermann (1973) developed a radioreceptor assay for tetanus toxin utilizing 1251-labeled tetanus toxin and brain homogenate. When the homogenate is iricubated with tracer in the presence of increasing amounts of cold toxin, a typical competition-displacement curve is seen. This indicates that binding of the toxin involves a finite number of saturable sites (receptors) on the brain tissue. When a tracer concentration of 8 ng/ml was used, the one-half displacement point occurred at about 40 ng/ml of cold toxin. Using a toxin MW of 160,000, we calculated an apparent equilibrium affinity constant at a half-displacement of 4 x lo9 A4-l. Van Heyningen (1973) showed that the receptor materials in brain and spinal cord are certain gangliosides containing sialidase-sensitive bonds. Gangliosides with the structure GGnSSLC or SGGnSSLC can bind tetanus toxin at low concentrations of both tetanus (50 ng/ml) and ganglioside (100 ng/ml) (van Heyningen and Mellanby, 1973). [The convention used for abbreviating gangliosides is that of McCluer (1970): G, galactose; Gn, N-acetylgalactosamine; L, lactose; S, sialic acid; C, ceramide.] Hydrolysis of the sialyl residue of GGnSSLC to GGnSLC renders the product inert toward tetanus toxin but capable of deactivating cholera toxin. Fixation of tetanus toxin by GGnSSLC does not deactivate the toxin, and complexed mixtures injected into mice are toxic. Although gangliosides can complex tetanus and cholera toxin, it is possible that the naturally occurring receptor which mediates toxicity is more complex.

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3. STRUCTURE

The structure of tetanus toxin has been investigated by Matsuda and Yoneda (1974). Toxin isolated from extracts of bacteria (intracellular toxin) has a MW of 160,000, which is unchanged by treatment with thiol. Intracellular toxin contains a trypsin-sensitive region spanned by a disulfide bond. Mild trypsinization creates a two-chained structure linked b y a disulfide bond. This material appears identical to material isolated from bacterial filtrates (extracellular toxin). Reduction of extracellular toxin or trypsin-nicked intracellular toxin with thiol in the presence of urea generates two subunits known as the a fragment (53,000 daltons) and the /3 fragment (107,000 daltons). The separated chains show no toxicity. Remixing in equimolar ratios followed by the removal of urea and dithiothreitol (DTT) by dialysis restores 100%of the toxicity (Matsuda and Yoneda, 1976). On SDS gel this material has a MW of 160,000, and only traces of the subunits are seen. Van Heyningen (1976) recently showed that only the /3 subunit binds to the ganglioside SGGnSSLC. The similarities to diphtheria toxin are striking. These similarities led us to inquire whether or not the tetanus toxin gene resides in a bacteriophage as is the case with diphtheria toxin and botulinum toxin. Prescott and Altenbern (1967) reported that all seven strains of C . tetani tested by induction with mitomycin C displayed lysis, presumptive evidence for lysogeny. However, phage plaques were not found. I n two strains phage particles were detected by electron microscopy. After induction with mitomycin C, the ratio of toxin production to total protein production was constant, indicating that the toxin gene was not translated at an increased rate. This might constitute evidence against the toxin gene being a phage gene. However, the toxin gene is under regulatory control involving the iron concentration (Mueller and Miller, 1954; Largier, 1956) (another similarity to diphtheria toxin), and the site of the regulatory gene, phage or host, is unknown. 4. RETROGRADE AXONAL TRANSPORT

Tetanus toxin reaches its site of action within the central nervous system by retrograde axoplasm transport from the peripheral motor terminals. This phenomenon can be observed by assaying nerve segments, ventral root segments, and cord ventral gray areas at various times either for toxicity in a mouse assay (Kryzhanovsky, 1973) or for radioactivity utilizing 1251-labeled tetanus toxin (Habermann and Dimpfel, 1973). Twenty-four hours after the injection of toxin into the

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DAVID M. NEVILLE, JR. AND TA-MIN CHANG

gastrocnemius muscle a steep gradient, 50-fold higher at the periphery, is found along the ipsilateral sciatic nerve. The ventral gray segments at L7 and S1 contain high concentrations of label. No gradient is observed in the contralateral nerve. Contralateral L7 and S1 ventral segments contain low concentrations of label, as do ipsilateral segments L6 and S2. Most of the transported label resides in the ventral roots of the spinal cord segments supplying the injected muscle (Wellhoner et al., 1973). This type of specificity was not seen when tetanus antitoxin was given intravenously and simultaneously with labeled. toxin or when Nalz5I was substituted for 1251-labeledtetanus toxin. A similar time-dependent appearance of labeled toxin within the motor gray areas of the brain stem and spinal cord was observed after intravenous injection of labeled toxin which induced generalized tetanus. These areas concentrated toxin before the onset of symptoms. Forebrain and cerebellum gray areas (devoid of lower motor neurons) did not concentrate toxin, although homogenates from these areas can bind toxin. It was concluded that the blood-brain barrier was impermeable to toxin, and that retrograde transport through the motor neuron explains generalized as well as localized tetanus (Habermann and Dimpfel, 1973).

5. INHIBITION

OF

NEUROTRANSMITTER RELEASE

The muscle rigidity and spasm produced b y tetanus toxin results from the loss of inhibitory reflexes impinging on the lower motor neuron. By the use of microrecordings and microinjection techniques with single cells, this loss of inhibition has been found to be the result of decreased secretion of the inhibitory neurotransmitters glycine and y-aminobutyric acid (GABA). Renshaw cells are the inhibitory interneurons of the recurrent motoneuron axon collateral pathway. By recording directly from these cells, the inhibition produced by hind paw stimulation or the local administration of glycine or GABA was observed before and after the local injection of tetanus toxin (Curtis and DeGroot, 1968). Twenty minutes after tetanus toxin administration the inhibition following hind paw stimulation decreased. Fifty minutes after tetanus toxin injection afferent inhibition was abolished. However, the neurons were still sensitive to the inhibitory neurotransmitters. Local administration of glycine and GABA produced inhibition. Since the levels of neurotransmitters in the spinal cords of toxin-treated animals were found unchanged (Osborne and Bradford, 1973), a failure in transmitter release rather than synthesis was postulated. Other workers have reported similar findings (Kano and Ishikawa, 1972; Curtis et al., 1973).

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Osborne and Bradford (1973) observed the stimulated release of neurotransmitters from synaptosomes prepared from spinal cords. These synaptosome preparations maintained respiration and concentration gradients of potassium and amino acids during the course of the experiments. Stimulation by raising the external potassium or electrical pulses in the presence of calcium provoked increases in respiration and neurotransmitter release. Synaptosomes prepared from animals intoxicated with tetanus toxin showed reduced release of neurotransmitters following electrical stimulation (glycine, 70% inhibition; GABA, 44% inhibition). Toxin added directly to synaptosomes ( lo4 mouse MLD per 5 ml) was without effect. The total duration of these experiments consisted of a 30-minute preincubation and a 10minute stimulation. Tetanus toxin in high dosage and acting over long periods of time appears to block release of the excitatory neurotransmitter acetylcholine. These effects are often masked clinically by rigidity and spasms; however, flaccid paralysis has been reported in tetanus, as well as alterations in the autonomic nervous system (Curtis, 1971). The best documented study is on the effect of intraocularly injected toxin on the sphincter pupillary muscle. The resulting dilatation was unresponsive to oculomotor nerve stimulation but was responsive to adrenergic stimulation or to local administration of acetylcholine (Curtis, 1971). In this respect tetanus toxin is similar to botulinum toxin. Mellanby (Mellanby et d., 1973) has commented on this and other similarities. In Section II,D we consider possible molecular mechanisms of action for these two toxins which inhibit neurotransmitter release. 6. TRANSSYNAPTIC MIGRATION

The inhibitory neurotransmitter blockade produced by tetanus toxin places the main site of action presynaptic to the motor neuron. Yet convincing evidence demonstrates that the toxin is taken up at the motor neuron end terminals and transported to the cell bodies and synaptic regions of the motor neurons prior to the onset of toxin action. Taken in conjunction, these two facts indicate that the toxin, once reaching the synaptic region in the motor neuron, is transported across the synapse into the presynaptic region where it interferes with inhibitory neurotransmitter release. Schwab and Thoenen (1976), using '251-labeled tetanus toxin, performed an electron microscopic, autoradiographic, morphometric study on the distribution of labeled toxin in the spinal gray areas after injection (tetanic rigidity became visible 12-13 hours after injection). Most of the labeled synaptic terminals were afferent to the motor neurons. These workers conclude that

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these observations strongly favor the assumption of a transsynaptic migration of tetanus toxin. It is known that synaptic membranes show the highest binding affinity of any tissue fragments of the spinal cord. It is not possible with present techniques to determine whether these receptors are present on both post- and presynaptic membranes. The evidence for transsynaptic migration indicates that tetanus toxin probably remains intact as a two-chain disulfide-linked structure during axonal transport and transsynaptic migration. The argument is as follows. The receptor binding function has been linked to the B chain of the toxin. The same binding function is presumably used at the presynaptic membrane, hence dissociation cannot take place before the toxin gains entry through the presynaptic membrane. Habermann et al. (1973) performed gel filtration in SDS (Sephadex G-200) and SDS gel electrophoresis on spinal cords obtained from rats intoxicated by 1251-labeledtetanus toxin. Most of the label moved coincident with marker tetanus. If the dissociation of A and B chains had occurred on entry, as has been postulated for diphtheria toxin (Boquet and Pappenheimer, 1976) and cholera toxin (Gill and King, 1975),this result would not have been obtained. D. Botulinum Toxin

1. STRUCTURE-ACTIVITYRELATIONSHIPS Botulinum toxin is a potent neurotoxin causing flaccid paralysis. Unlike most protein toxins which are active systemically, this toxin can be absorbed from the gastrointestinal tract. The estimated human oral lethal dose lies between 0.5 and 5 pg (Koenig, 1971). The basic structure of the toxin is similar to that of diphtheria and tetanus toxin. The toxin is secreted as a single polypeptide chain of about 150,000 daltons. A tryptin-sensitive region spanned by a disulfide bond is present, and following proteolytic nicking and reduction two chains of 100,000 and 50,000 daltons are obtained. These are referred to in the literature as heavy and light chains. However, in keeping with the scheme and nomenclature for other toxins we refer to the 100,000dalton chain as B and the 50,000-dalton chain as A (DasGupta and Sugiyama, 1976). Neither chain alone is active (Kozaki and Sakaguchi, 1975).Attempts to reoxidize the structure and regenerate activity have not been reported. Botulinum toxin is made by Clostridium botulinum, and six distinct strains (A, B, C, D, E, and F) are known, as defined by antigenic differences in the toxin. Strains A, B, E, and F are associated with human

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disease, while C and D produce botulism exclusively in animals (Koenig, 1971). These strains can be interconverted by curing the lysogenized phage and reinfecting from phage produced by an alternate strain (Inoue and Iida, 1968). The toxin is thus a phage gene product, like diphtheria toxin. Toxins A, B, E, and F are believed to contain a common antigen (Kozaki et al., 1975).However, a detailed immunological analysis of the B and A chains of the six toxins has not been reported. Botulinum toxin is rapidly fixed to tissues like tetanus toxin through a neuraminidase-sensitive material. Studies on the binding of separated A and B chains have not yet been reported but, presumably, as with diphtheria and tetanus toxins, binding activity resides in the heavier B chain. Since antitoxins generated from toxins A, B, C, D, E, and F show no cross-protection, w e presume that the binding chains are antigenically distinct. I n keeping with our remarks on tetanus toxin we imagine that the A chain is an enzyme active in the intracellular milieu. Botulinum toxins B and A differ from tetanus and diphtheria toxin in that they are found in culture filtrates in association with high-MW proteins which are nontoxic. The type-A toxin complex has a MW of 1 million and can be crystallized in this form. The association is weak and can be broken by chromatography with DEAE-Sephadex (Kozaki et al., 1975). The nontoxic fragment is a hemagglutinin but apparently plays no role in animal toxicity. Unlike diphtheria and tetanus toxin, botulinum toxins require proteolytic nicking to achieve full animal toxicity. DasGupta and Sugiyama (1976) showed that unnicked type-E toxin undergoes a 29-fold increase in activity following nicking. Since the sensitivity of detecting nicked species in the unnicked starting material was not given, it is possible that the activation by nicking is actually much greater. This feature is reminiscent of the nicking required to activate diphtheria toxin in the cell-free assay system, where unnicked, unreduced toxin is devoid of activity (Collier, 1975).To account for the full animal toxicity of unnicked diphtheria toxin it has been postulated that nicking occurs at the cell membrane or after entry. Evidently this process in mice is limited for botulinum toxins B, E, and F (DasGupta and Sugiyama, 1976).

2. EXPERIMENTAL TOXICITY Intramuscular injection of limited amounts of botulinum toxin produces a localized flaccid paralysis in the injected muscle. A larger intramuscular dosage causes generalized flaccid paralysis, as does sub-

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cutaneous, intravenous, or oral administration. A portion of the injected material is rapidly fixed to tissue in a compartment not accessible to specific antitoxin. However, some toxin continues to circulate, and the definitive diagnosis is made by injecting 1 ml of a suspected patient’s serum into mice, a subgroup of which has already received univalent antitoxins A, B, E, and F (Koenig, 1971).

3. RECEPTORSAND TRANSPORT Receptors for botulinum type-A toxin have been demonstrated in synaptosomes where 1251-labeledtoxin binds and competition with cold toxin is observed (Habermann, 1974). Treatment with neuraminidase virtually abolishes binding. Toxin binding to a variety of gangliosides could not be demonstrated. With the use of 1251-labeledtoxin, specific binding to the neuromuscular junction of mouse diaphragm has been reported (Hirakawa and Kitamura, 1975).The presence of receptors for botulinum toxin appears to show species specificity, as is the case with diphtheria toxin. Rats are quite resistant to botulinum toxin B, requiring 500 times the A-toxin dose for similar effects. Guinea pigs, however, show equal sensitivity to both toxins (Burgen et al., 1949). It is possible that not all humans have a receptor for botulinum toxin. There are two reports of finding toxic levels of botulinum toxin in the serum of individuals known to have consumed contaminated foods, yet these individuals remained free of toxic symptoms (Koenig, 1971). Direct evidence that botulinum toxin once bound enters the motor neuron in an active form comes from the studies of Habermann (1974), Wiegand et a l . (1976), and Wiegand and Wellhoner (1974). These investigators, using 1251-labeled toxin, demonstrated retrograde axon transport from the motor neuron of the injected muscle to the ipsilatera1 spinal cord half-segments of the motor neuron. In addition, they obtained evidence that the toxin reduced synaptic transmission from recurrent motor axon collaterals to Renshaw cells in this animal preparation. Although central effects of botulinum intoxication are not the predominant symptomatology, they are present in clinical descriptions of the disease (Koenig, 1971).

4.

INHIBITION OF

NEUROTRANSMITTER RELEASE

I n a study on isolated rat diaphragms, Burgen et al. (1949) reported the basic facts of botulinum intoxication. The toxin produces a blockade at the neuromuscular junction after a dose-dependent lag

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period. A minimum lag period of 15-30 minutes was always present, no matter how high the dose. The lag period could be lengthened for a given dose by lowering the temperature. A restudy in frogs indicated a high Qlo consistent with the activation energy required to break a covalent chemical bond. Nerve conduction was not altered. The neuromuscular junction maintained its normal sensitivity to acetylcholine, and the output of acetylcholine following nerve stimulation was reduced in intoxicated preparations. The conclusion, supported in rich detail since then, was that botulinum toxin interfered with release of the excitatory neurotransmitter acetylcholine. Burgen et al. (1949) also noted the similarity between the dose-dependent lag period in diphtheria, tetanus, and botulinum toxins.

5. BIOCHEMISTRY O F NEUROTRANSMITTER RELEASE Since both botulinum toxin and tetanus toxin appear to act by blocking neurotransmitter release, it should be useful to explore what is known about the biochemical details of neurotransmitter release. Acetylcholine is believed to be synthesized in the region of the synaptic terminals and then packaged into synaptic vesicles. Neurotransmitter release occurs by exocytosis of the synaptic vesicle or the contents of the vesicle. This process occurs at a low frequency in a random manner independent of neural conduction. It gives rise to miniature end plate potentials of a constant amplitude. The consistency of the amplitude has led to the postulate that neurotransmitter release is a quantum phenomenon and that each vesicle contains a fixed amount of neurotransmitter (Drachman, 1971). Calcium ion is known to be involved in neurotransmitter release. It has been postulated that botulinum toxin in some way interferes with calcium metabolism, and this could explain the toxin’s effect. However, both Drachman (1971) and Simpson (1971) concluded that all the available data were not consistent with this hypothesis. Recently, however, Lundh et a l . (1976) demonstrated the restoration of acetylcholine release in botulinum-poisoned skeletal muscle. Using the calcium ionophore A23187 they restored miniature end plate potentials to their normal frequency when the motor end plate was perfused with 15 mM calcium. Similarly, when calcium ions in excess of normal were allowed to enter the nerve terminal by use of the calcium ionophore, neuromuscular transmission was restored. The same effect was achieved by using tetraethylammonium, which prolongs the duration of the nerve terminal action potential and thereby increases the amount of calcium entering the terminal. These results obtained on

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the extensor digitorum longus muscle of male rats were interpreted as indicating that the botulinum toxin-poisoned muscle contained an intact neurotransmitter release mechanism which required higher than usual levels of intracellular calcium to produce activation. The relationship between neurotransmitter release, calcium ion, and contractile proteins was explored by Berl et al. (1973) and Puszkin and Kochwa (1974). These investigators reported that brain actin was found in synaptosomal membrane-enriched fractions, whereas myosin molecules were found on synaptic vesicles. They proposed that the release of neurotransmitters resulted from an interaction of vesicle, myosin, and synaptosomal membrane actin. This proposed mechanism involves contact between vesicles and presynaptic membranes, with the formation of actomyosin that contracts in the presence of calcium ion and ATP. The contractile force would induce a change in the synaptic vesicles, with release of neurotransmitters into the synaptic clefs. Puszkin and Kochwa (1974) isolated from brain synaptic membranes a protein fraction with the properties of an actin-troponintropomyosin complex. This protein fraction, when incubated with synaptic vesicles, induces the release of preloaded 14C-glutamatefrom the vesicles in the presence of calcium. In the absence of calcium, glutamate release is reduced by a factor of 5. Glutamate release evoked by the protein complex plus calcium is associated with an increase in magnesium-ATPase activity. When purified muscle actin is substituted for the protein complex isolated from brain synaptic membranes, magnesium-ATPase activity increases, and glutamate release occurs both in the presence and absence of calcium. Addition of the protein complex from the brain synaptic membranes restores the inhibition of glutamate release and magnesium-ATPase activity in the absence of calcium. These investigators conclude that, as in the case of muscle, the relaxing proteins from brain seem to be part of a calcium receptor. Because the blockage of neurotransmitter release induced by either tetanus or botulinum toxin involves a dose-dependent lag period, we believe that the site of action of these toxins occurs at a step preceding neurotransmitter release from the synaptic vesicle, and that this step is initially not rate limiting but becomes rate limiting due to the exhaustion of a component through the enzymatic activity of the toxin. It appears to us that the calcium-binding proteins which confer calcium specificity on the neurotransmitter release in the systems of Puszkin and Kochwa are a likely candidate for enzymatic inactivation b y the A chains of tetanus and botulinum toxin. It further appears that these cell-free systems may serve as an effective assay

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system for the activity of the isolated A chains of these toxins. If such activity could be demonstrated, the transfer of a labeled cofactor to the proteins involved could pinpoint the actual substrate of the proposed toxin enzyme. E. Cholera Toxin

1. EXPERIMENTALTOXICITY AND BIOCHEMICALMECHANISM Cholera is a disease characterized by watery diarrhea in which the stool production may reach 20 liters per day. This leads to severe dehydration, circulatory collapse, and death. The entity causing this disease is a toxin elaborated b y the bacterium Vibrio cholerae. The toxin has been purified, and the experimental disease can be produced in animals by injecting the toxin into the small bowel. Quantitative studies can be performed b y forming loops of small bowel sealed with ligatures and cannulated at each end. In this manner toxin and other substances can be introduced for varying periods of time, washed out, and the loop refilled with water and electrolytes. At various times water and electrolyte can be removed for sampling purposes. T h e results of such studies show that after a 10-minute contact with cholera toxin the normal transport of water and electrolyte from the bowel lumen to the bloodstream is reversed following a lag period of approximately 40 minutes. The effect is half-maximal at 1b hours, maximal at 3 hours, falls to half-maximal at 24 hours, and requires 48 hours to reach baseline values. Intestinal adenylate cyclase activity also rises after a 40-minute lag period, and these values parallel the water and electrolyte changes throughout the time course. Other agents which stimulate intestinal adenylate cyclase, such as prostaglandins and theophylline, also cause this type of alteration in intestinal water and electrolyte transport. The disease is therefore caused b y the ability of the toxin to produce an abnormally high and prolonged stimulation of intestinal adenylate cyclase activity (Guerrant et al., 1972; Finkelstein, 1973, 1975). When naturally occurring stimulators of adenylate cyclase interact with cells, the stimulation does not last much longer than the duration of the stimuli. In the case of cholera toxin stimulation of intestinal adenylate cyclase activity, the stimulation lasts for a time period equal to the turnover of the intestinal epithelium (Finkelstein, 1973). Naturally occurring substances which stimulate adenylate cyclase activity do so without a lag period.

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2. TOXINSTRUCTURE AND RECEPTOR BINDING The subunit structure of cholera toxin has been described (Finkelstein et al., 1974; Mendez et al., 1975). The toxin is an 84,000-dalton protein and consists of an A and a B subunit held together with noncovalent forces. The A subunit has a MW of 28,000 daltons and consists of two polypeptide chains of 22,000 daltons and 7500 daltons linked by a single disulfide bond. These chains are known, respectively, as the Al and A2 chains. The B-subunit MW is 56,000 daltons, and dissociating reagents reduce this MW to a single 9000-dalton species. The subunit structure of cholera toxin appears to be ABs. Each B monomer subunit contains a single disulfide bond, but no interchain disulfide bonds exist within the B subunit. Studies of the binding of cholera toxin to cells have been performed by several investigators. These studies were prompted by the finding that a sialidase-resistant monosialyl ganglioside bound to cholera toxin and inactivated its biological activity (van Heyningen, 1973). Using 1251-labeledcholera toxin and fat cells, saturable binding was observed with an equilibrium dissociation constant in the range of 2.5 x M (Cuatrecasas, 1973). Holmgren and co-workers (Holmgren et al., 1975) obtained evidence that the ganglioside GMl or GGnSLC is the biological receptor in intestinal tissue. These workers demonstrated a relationship among the endogenous GMl concentration, the number of binding sites for cholera toxin, and the sensitivity of the intestinal mucosa to the biological activity of the toxin. These investigators also demonstrated the incorporation of exogenously added GMlinto intestinal mucosa cells and found this to be associated with both an increased number of binding sites and an increased sensitivity to cholera toxin. Similar studies have been reported for fat cells (Cuatrecasas, 1973). However, it is not clear whether added GMlincreases the sensitivity of the fat cell to cholera toxin or whether it simply alters the time course of the stimulatory effect (Kanfer et al., 1976). Recently, Moss et al. (1976a) demonstrated that a mouse fibroblast line which had lost its native capacity to synthesize GMl could be restored to responsiveness to cholera toxin by the addition of exogenous GMl. These studies argue for the case that GMl is the native receptor for cholera toxin in these cell types. This generalization has been questioned b y Kanfer et al. (1976), Donta (1976), and King et al. (1976). The last-mentioned investigators added GM1to pigeon erythrocytes and studied the enhancement of adenylate cyclase activity. They observed that at least 90% of the exogenously added toxin-binding sites were nonproductive of cyclase stimulation. It is possible that productive toxin binding sites require the association of GMl with another membrane component.

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3. EVIDENCEFOR TRANSPORT The binding activity of cholera toxin appears to be associated with the B chain. Cholera toxin B subunit, also known as choleragenoid, can block the action of cholera toxin when instilled with toxin into experimental animals (Finkelstein, 1973). The competitive nature of this effect was shown by Gill and King (1975), who incubated pigeon erythrocytes in the presence of 1pg/ml cholera toxin and various concentrations of B subunit. Adenylate cyclase activity fell with increasing B-subunit concentration, and 50% inhibition occurred at approximately 3 pg/ml of B subunit. A considerable body of evidence argues for the case that cholera toxin must be first transported through the plasma membrane before it can activate adenylate cyclase. Several facts led to the formulation of this hypothesis. First, cholera toxin is highly effective in stimulating adenylate cyclase when applied to the mucosal surface of the intestine. It is only minimally effective when injected into the bloodstream. However, adenylate cyclase is not concentrated in the brush border of the intestinal epithelium; rather it is believed to be concentrated on the blood front of the cell. I n order for cholera toxin bound to the brush border to gain proximity to adenylate cyclase it either has to be transported into the cell interior or transported around the cell membrane through mobile receptors. Once toxin is transported within the membrane to the vicinity of the adenylate cyclase it still has to interact with the cyclase molecule, whose active site is localized to the internal surface of the plasma membrane. Activation of cyclase by toxin does not occur via cyclase hormonal receptors, since toxin-treated cells do not lose their hormonal cyclase stimulation. More direct evidence for toxin transport has been obtained by observing that toxin-treated cells or tissues are accessible to the neutralizing effects of antitoxin antibodies for only short periods of time. This was observed in vivo utilizing the system of isolated intestinal loops and in vitro utilizing pigeon erythrocytes. Antitoxin added within 30 seconds after cholera toxin prevented adenylate cyclase stimulation. Antitoxin added 10 minutes after the toxin had no effect on the cyclase stimulation by cholera toxin, and 10- to 15-fold levels of stimulation were seen. Fifty percent inhibition of the toxin-stimulated cyclase activity was observed with addition at 24 minutes. An identical phenomenon is seen in the case of tetanus, botulinum, and diphtheria toxin interaction with cells. I n the case of diphtheria toxin the inference is quite strong that this eclipse period represents transport of the toxin into the interior of the cell. This is because the substrate for the toxin’s enzymatic activity is known to reside within the interior of the cell. The inference that the eclipse phase represents transport for chol-

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era toxin is less strong, since the primary site of action of the toxin is unknown. However, it is difficult to construct a model for the eclipse period which does not involve changing the relative position of the toxin molecule with respect to the surrounding membrane. This process appears to involve crossing a high-energy barrier, since the molecule is no longer accessible to antibodies present in the media bathing the cell. Therefore, on both energetic and structural grounds it appears that during the eclipse period the toxin has moved from one phase to another, and in our view this constitutes transport. Whether this transport takes place to the interior of the cell cytoplasm or simply to the interior of the plasma membrane remains to be determined.

4.

SIGNIFICANCE OF THE

LAG PERIOD

Following the application of cholera toxin to tissues or cells, there is a lag period lasting approximately 30 minutes before the rise in adenylate cyclase activity is seen. .4 similar lag is observed in diphtheria toxin, tetanus toxin, and botulinum toxin effects. However, in the case of these toxins, the lag period is dose-dependent. In addition, the temperature dependence of the lag period is high, and the Qlo.is in the range of that required for breakage of a covalent bond. The usual explanation for a dose-dependent lag period involves the postulation of a preceding step in the reaction process, which is not initially rate limiting but becomes rate limiting as a result of application of the reagent in question. In the case of diphtheria toxin this postulate was shown to be accurate. The observable in this case was the rate of protein synthesis, while the substrate for the enzymatic activity ofthe toxin was EF-2. Under normal conditions the concentration of EF-2 is not rate limiting. Thus the lag period, particularly the long lag period seen at low concentrations of toxin, is a function of the time it takes to reduce the EF-2 concentration to a rate-limiting value. It should be pointed out that long lag periods due to this process are seen only when the rate of turnover of the substrate in question is long compared to the observation period. The lag periods associated with cholera toxin do not show the marked dose dependency exhibited by diphtheria, tetanus, and botulinum toxin, and no dependency may in fact exist. Studies on pigeon erythrocytes reported by Gill and King (1975) failed to show a dose-dependent lag period. However, these studies were not designed to answer this question, and more data points at early times would be desirable. Studies on fat cells reported by Kanfer and co-workers (Kanfer et al., 1976) measure the rate of toxin-stimulated glycerol release. The half-maximal glycerol release

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occurs earlier at high toxin dosage; however, the curves are sigmoidal in shape and nonparallel, and no obvious difference in the initial lag can be detected. An alternative explanation of a lag period which does not display dose dependency is that it represents the time required to transport the toxin from its binding site to the compartment where it displays its activity. This explanation does not rule out the possibility of a preexisting step (before adenylate cyclase) where the toxin exerts its effect. This step may simply have a rapid turnover which is short compared to the lag period. The hypothesis that the lag period represents the time required to transport toxin from the binding site on the plasma membrane to its intracellular site of action has been proposed (Parkinson et al., 1972). Recently, Gill and King (1975) showed that lysates of pigeon erythrocytes are sensitive to the stimulation of adenylate cyclase b y cholera toxin. The kinetics of this system differ from those of the intact cell system in that no lag period is present and that the maximal rates of cyclase activation are much higher than in the intact cell preparation. These investigators believe that the absence of a lag period in the broken cell system is due to the lack of a requirement for transport through the membrane in this system.

5. IS

THE

A,

CHAIN AN

ENZYME?

Recently, Gill and King (1975) showe'd, utilizing the pigeon erythrocyte broken cell system, that the A, chain of cholera toxin is active in stimulating adenylate cyclase. Cholera toxin was dissociated into B monomeric subunits and an A subunit by S D S and purified on gels. Toxin subjected to prior treatment with DTT was also run, and the A2 chain was dissociated from the A subunit. Both the A subunit and the A, chain were effective in stimulating adenylate cyclase activity more than 10-fold in the broken cell pigeon erythrocyte system. The intact B8 subunit was inactive. It had been previously shown with intact cell systems and tissue systems that purified B and A fragments were inactive. These results are then similar to the general findings observed with the A and B chains of diphtheria toxin, abrin, and ricin. The question then arose whether or not the A, fragment of cholera toxin was an enzyme, as is the case with diphtheria toxin. A requirement for N A D as a cofactor was demonstrated by Gill and King, which is consistent with the enzymatic hypothesis. When the increasing amounts of A, chain are added to lysed pigeon erythrocytes, the rate of cyclase activation increases, which is also consistent with the presence of an enzyme. Gill and King (1975) also estimated that 10 to 50 copies of a subunit

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per erythrocyte volume give the same rate of cyclase activation in lysates as saturating amounts of toxin in the intact cell system. This number is less than the estimated number of cyclase molecules per cell (several hundred to 1000)and implies some type of amplification. Most biological amplification systems involve an enzyme at some step. However, if adenylate cyclase is the substrate for the A, chain, the ratio of A, chains per erythrocyte to total cyclase molecules per erythrocyte gives a very low presumptive turnover number (Gill and King, 1975). Recently, Moss and co-workers (Moss et al., 197613) demonstrated NADase activity of cholera toxin A subunit. I n addition, they showed that in the presence of NAD the cholera A subunit catalyzes the ADPribosylation of arginine.

F.

Colicins

1. GENERALCHARACTERISTICS Colicins are bacteriocidal proteins synthesized by certain strains of enteric bacteria, including Escherichia coli, and are active against other strains of the same or related bacterial species (Nomura, 1967). Sensitivity or resistance to a colicin is determined by the presence or absence of a receptor for the colicin on the outer membrane of the bacterium. Various colicins have the same receptor specificity, and these are grouped under a common letter. Thus colicins E2 and E3, although different, utilize the same receptor and are bacteriocidal toward all strains of bacteria carrying this receptor, with the exception of strains producing the homologous colicin. Escherichia coli carrying the E receptor contain about 250 receptor sites (Bradbeer et al., 1976).Strains of E . coli producing a colicin are said to be colicinogenic. Colicinogenic cells carry a plasmid, a small circular DNA called colicinogenic factor, which confers the ability to produce the colicin and also the immunity for the corresponding colicin in that particular strain. Immunity differs from resistance, since immune cells retain receptors and adsorb homologous colicins. However, colicinogenic cells are sensitive to very high concentrations of homologous colicin.

2. INTRACELLULAR SITE

OF

ACTION

Colicins E2 and E3 have their site of action within the bacterial cell cytoplasm. Colicin E3 produces a specific cleavage of 16s rRNA

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within the 30s subunit at a point approximately 50 nucleotides away from the 3’ end. This cleavage inactivates protein synthesis. This process has been shown to be catalytic (Sidikaro and Nomura, 1974). Ribosomes from eukaryotic cells are equally sensitive to colicin E 3 (Turnowsky et al., 1973).The cell-free assay of colicin E3 activity involves incubation of colicin with 70s ribosomes at 37°C for 45 minutes, following which the components necessary for assay of poly-U-directed polyphenylalanine synthesis are added. Colicin E 2 is an endonuclease with broad specificity toward a variety of DNAs. When covalently closed circular DNA is used as a substrate, open circles appear as the initial product. With higher concentrations of colicin E 2 small linear fragments are formed. Agarose gel electrophoresis serves as a cell-free assay system for colicin E 2 activity (Schaller and Nomura, 1976). Colicin E, is a single-chain 62,000-dalton protein. No detectable carbohydrate is present. The protein contains a single sulfhydryl group (Glick et al., 1972). There are no reports in the literature of attempts to separate the enzymatic activity and the binding activity of the colicin molecule by limited proteolysis. Both colicins E 2 and E 3 are secreted from bacteria in tight association with a 9500-dalton peptide (Jakes and Zinder, 1974; Sidikaro and Nomura, 1974; Schaller and Nomura, 1976). This peptide confers immunity to the strain secreting the colicin and is known as the immunity protein. Immunity is conferred by blocking the enzymatic activity of the colicin. This has been observed in the cell-free assay systems for colicins E2 and E3. The blockage is reversible, since in the presence of dissociating agents such as 6 A4 guanidine hydrochloride the colicin can be separated from the immunity protein. The increase in activity is on the order of 10-fold. Sensitive strains of bacteria (noncolicinogenic) are equally as sensitive to colicin complexed with immunity protein as purified colicin. The conclusion from this result is that the immunity protein does not cross the plasma membrane in an active form. It is inferred that immunity is conferred on a colicinogenic strain by a high concentration of immunity protein within the bacterial cytoplasm. In diphtheria toxin and in the seed toxins abrin and ricin the B chain performs two functions. It binds the toxin to the cell surface receptor, and it inhibits the enzymatic activity of the toxin until chain separation occurs. In colicins E 2 and E3 the immunity protein serves only the latter function of inhibiting enzymatic activity. It would be interesting to determine if these separate activities in the B chain of diphtheria toxin can be separated. Two early termination mutants in the B chain have been reported which lack binding ability, and in CRM,, enzy-

9%

DAVID M. NEVILLE, JR. AND TA-MIN CHANG

matic activity is present prior to reduction (Pappenheimer and Gill, 1973). Investigations of point mutations within the B chain might be of interest.

3. SHARED TRANSPORT SYSTEMS

a. Colicins B , D , 1, and V, Complexed Zron, and T Bacteriophages. Direct studies of colicin transport into bacteria have been hampered by the minute quantities of intracellular colicin required to elicit bacteriocidal effects. The same problem occurs in direct studies of bacterial toxin transport into eukaryotic cells. However, considerable evidence concerning colicin transport is available from genetic manipulations. The striking feature to emerge from these studies is that the colicins utilize physiological transport mechanisms of low-molecular weight metabolites. Even more striking is the fact that these same transport mechanisms or parts of them are utilized by certain bacteriophages. In E . coli K-12, iron transport systems have been found to be associated with colicin transport systems. One of the iron transport systems transports iron chelated by enterochelin. Enterochelin is a cyclic trimer of 2,3-dihydroxybenzoylserine.Enterochelin is made by the bacterium and secreted into the external environment where it chelates iron tightly. This chelate is then transported via a transport system. The first step in transport involves the binding of the ferrienterochelin to a cell surface receptor. Ferrienterochelin is then transported actively into the cell interior. This process is inhibited by 2,4-dinitrophenol or azide. Several workers noted that various mutants ofE. coli K-12 that are resistant to colicins B and D also hyperseCrete enterochelin. This led to investigation of the relationship between ferrienterochelin transport and the colicins (Pugsley and Reeves, 1976a). By performing binding studies in the presence of dinitrophenol, binding could be separated from the uptake step. Ferrienterochelin receptors were found to be increased by a factor of 7 when the bacteria were grown in iron-poor media. This culture condition was also associated with a 10- to 20-fold increase in two outer membrane polypeptide components in the 100,000-MWrange. These polypeptides appear to be related to the receptor function and are cvidently derepressed by iron starvation. These observations suggest that colicins B and D and ferrienterochelin share a common receptor. The fact that ferrienterochelin can block the bacteriocidal action of colicin B and D supports this hypothesis (Davies and Reeves, 1975). In addition, colicins B and D can block ferrienterochelin binding to the

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bacterial outer membrane. One-half displacement occurs at about 75 pg/ml of colicin D. With a MW of 50,000, this represents a colicin D concentration of 1.5p M . Thus the colicin appears to have a somewhat higher affinity for the receptor than ferrienterochelin. Pugsley and Reeves (1976a) investigated four classes of E . coli K-12 colicin Bresistant mutants. All four mutant classes, cbt, exbC, exbB, and tonB, were defective in the uptake of ferrienterochelin. Ferrienterochelin binding was demonstrated in outer membranes in the exbB, erbC, and tonB mutants but was missing in the cbt mutant. The cbt mutant has been reported to show colicin binding, and the tonB mutant is defective in all other forms of chelated iron transport which in E . coli involves complexes with ferrichrome, citrate, and rhodotorulic acid. All these mutants show increases in the two membrane polypeptides when grown under iron starvation conditions. The complete relationship between the receptor functions, transport functions, and these mutations remains to be determined. Pugsley and Reeves (1976b) recently reported the purification of colicin B and D receptor activities following solubilization of the outer bacterial membrane with Triton X-100. The fractions containing the highest purification, which amounted to a 40-fold increase in binding activity toward colicins B and D, contained two protein peaks corresponding to the protein species which are increased in the outer membrane under conditions of iron sta.rvation. Hancock and Braun (1976) have described mutants in E . coli K-12 which are defective in ferrienterochelin uptake, which they call feu mutants. ThefeuA mutant is resistant to colicins I and V. In strain VR-42, three proteins of MW 83,000, 81,000, and 74,000, residing in the outer membranes, markedly increase under iron starvation conditions. These proteins may be analagous to the proteins previously described by Pugsley and Reeves in their strain P1552. In contrast to the mutants report by Pugsley and Reeves thefeuA mutant in strain VR-42 lacks one of the iron starvation-derepressed proteins. This protein is presumably the colicin I receptor and functions in enterochelin-mediated iron transport. These investigators also partially purified the iron-derepressed proteins after solubilization in Triton X-100. Another class of E . coli K-12 mutants defective in ferrichromemediated iron transport has been described (Hancock and Braun, 1976). These mutants, known as tonA, lack an 85,000-daIton polypeptide in the outer membrane. Ferrichrome protects sensitive cells against the bactericidal activity of colicin M. I n addition, it protects sensitive cells against phages T5, T1, and $30. The tonA polypeptide

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is probably the receptor for ferrichrome, T5, and colicin M (McIntosh and Earhart, 1976). Manning and Reeves (1976) described a group of mutants known as t s x mutants which are resistant to T6-like bacteriophages and to colicin K. They demonstrated that these mutants lack an outer membrane protein of MW 32,000. These mutants are unable to adsorb either the bacteriophages or the colicin, and it is suggested that t s x protein is the receptor for both the T6 phages and colicin K. b. E Colicins, Vitamin Biz, and Phage BF23. Bradbeer et al. (1976) studied the vitamin B12transport system in E. coli. They demonstrated that the E. coli outer envelope normally contains about 250 B12 receptors and that these receptors function both in Blz transport and as receptors for E colicins (DiMasi et al., 1,973).Bradbeer et a l . (1976) later presented convincing evidence that this same receptor is involved in the adsorption and transport of phage BF23. Blz can decrease the rate of phage adsorption 50-fold. The 50% inhibition occurs at 0.8 nM, close to the dissociation constant for B12 binding. Phage BF23 can block the transport of B12 into cells. This occurs at the binding step and, by using cell envelopes and observing the binding of B12 at various concentrations in the presence of various phage concentrations, classical competitive inhibition was observed. The K, for the phage was calculated to be 0.2 nM. These workers described a mutant btuB69 which contains only about half a receptor, on the average, per cell for both BF23 and Blz. In E. coli, bacteriophages share receptors with metabolites other than heavy metals. Hazelbauer (1975) noted that chemotaxis toward maltose is specifically defective in many strains ofE. coli carrying mutations affecting lamB, the gene coding for the outer membrane receptor for bacteriophage lambda. The receptor involved functions as a high-affinity system over the range 0.1-10 pM for maltose transport. Blockage of high-affinity maltose transport is responsible for the defect in chemotaxis. 4. SIGNIFICANCEFOR EUKARYOTIC CELLS

The finding that bacteriophages and colicins, both of which can be destructive toward bacteria, utilize physiological receptors strengthens our belief that the receptors for toxins on eukaryotic cells may also have physiological functions. To date no one has succeeded in demonstrating an adverse effect upon a eukaryotic cell, either in tissue culture or in viva, from the application of a toxin-binding chain

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protein. However, it is possible that these toxins utilize receptors involved in trace-metal transport and that adverse effects would not be demonstrated until the trace metal or other material became limiting. Such limiting conditions are not usually encountered in tissue culture or in whole-animal preparations unless they are specifically provoked. Alternatively, toxin receptors may function as receptors for protein or peptide growth factors or factors responsible for maintaining certain states of differentiation. These functions may be overlooked in tissue culture, because of the undifferentiated nature of the tissue-cultured cells under the usual logarithmic growth conditions. In whole animals it may be necessary to administer B chains of toxins over a prolonged period of time to elicit a biological effect. 111.

CARRIER PROTEINS

A. Tranrcobalamin It

1. STRUCTURE AND FUNCTION Vitamin B n or cobalamin is a water4oluble vitamin of MW 1355, containing tightly complexed cobalt. After absorption from the gut Blz is transported to all tissues, where it serves as a cofactor either as methylcobalamin or 5'-deoxyadenosylcobalamin in methyl or other group transfers involving a carbon 1,2-hydrogen shift. Uptake from the gut is a receptor-mediated process. The uptake is facilitated by intrinsic factor, a glycoprotein which forms a stable complex with BIZ. The binding of the B,,-intrinsic factor complex to receptor sites on the membranes of intestinal microvilli is the first step in the uptake process. Receptor sites for the B12-intrinsic factor complex were first identified on intestinal microvilli by Donaldson et al. (1967). In human blood B12is carried by two proteins, transcobalamin I (TC I) and T C TI. T C I carries 75%of the serum Biz., and TC I1 carries the remainder. The significance of T C I is unknown, since a congenital deficiency of T C I in humans is asymptomatic and does not result in reduced levels of Blz in tissues (Carmel and Herbert, 1969). T C I1 is required for the transport of B,2 to peripheral tissues, since a congenital lack of TC I1 leads to severe manifestations of B12deficiency (Hakami et al., 1971). TC I1 is a 40,000-dalton protein. The affinity of T C I1 for cobalamin is very high, with a reportedK, of 10" M-' (Hippe and Olesen, 1971).

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TC I1 has been found in a wide variety of mammals with similar functions. Considerable evidence indicates that B12 is transported into cells in the form of the TC II-BlZ complex. 2. TRANSPORT INTO LEUKEMIA CELLS The uptake of vitamin Blz has been extensively studied in mouse leukemia cells L1210 using 57Co-labeledBlz (DiGirolamo and Huennekens, 1975). Labeled Blz in the presence and absence of TC I1 has been incubated with washed mouse leukemia cells. Concentrations of Blz ranged between 30 and 200 pM. The cells were spun down, washed, and then counted. In the presence of TC 11, uptake increased by over 50-fold. A plot of uptake in the presence of TC I1 versus time was biphasic, with a rapid initial rate for the first 1 minute and a slower rate which leveled off to a steady state at 45 minutes. The initial rate represents the binding of TC I1 to the surface membrane receptors and can be inhibited by EDTA or reversed by EDTA. Labeled material eluted from the cells after the primary incubation period is in the form of the TC II-cobalamin complex as judged by chromatography on Sephadex G-100. The secondary process represents transport into the cell interior. This process is temperature-dependent and is inhibited by azide and 2,4-dinitrophenol. No efflux is noted from the intracellular compartment. When cells are incubated with TC II-B1z for 2 hours, washed in EDTA to remove receptor-bound label, and then homogenized in saline and subjected to sucrose density gradient fractionation, seven label peaks were found. The largest, 31-47%, was in the soluble fraction, while the next highest, 17-20%, coincided with a mitochondria1 fraction. The soluble fraction label cochromatographed with TC II-Bl2 (Rye1 et al., 1974). 3. TRANSPORT INTO LIVERAND KIDNEY

The results reported with mouse leukemia cells differ from those reported in liver and kidney (Pletsch and Coffey, 1971,1972; Newmark, 1971). In these tissues labeled cobalamin is first associated with a plasma membrane vesicular fraction and then moves to the lysosomal fraction. Smaller amounts of label in these tissues appear in the soluble fraction, and larger quantities in the soluble fraction do not appear until after 72 hours, at which time the label is associated with a macromolecule with a MW greater than lo5. In liver vitamin B,S appears to also be transported as a TC I1 complex. Lysis of the plasma membrane vesicular fraction by Triton X-100, followed by chromatography on

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RECEPTOR-MEDIATED PINOCYTOTIC TRANSPORT MODEL

3

FIG. 1. In this model high-affinity surface membrane receptors (notched squares) concentrate the specific protein at the membrane (1). Membrane invagination and pinching off result in a pinocytotic vesicle containing receptor-bound protein (2 an d 3). Vesicle fusion with a lysosome (4a) directs the protein to this compartment. When the protein is known to enter the cytosol, a second transport mechanism out of the vesicle (arrow, 4b) is required. Uptake proceeds by the continuous addition of receptor to the surface membrane. (See Sections II,A,4 and IX,B.)

Sephadex G-100 in the presence of Triton shows that the BI2 label cochromatographs with TC II-Bl2. The same is true of the lysosomal label at early time points, but lower-MW B12label appears at the later time points. These data have given rise to the following model of Biz transport (see Fig. 1). Binding occurs via TC II-BI2 complex. The complex is internalized by receptor-mediated pinocytosis. Fusion of pinocytotic vesicles with lysosomes leads to the degradation of TC 11, freeing Blz. B12 is then bound to a second intracellular binding protein.

4. THE PINOCYTOTICMODEL

The preceding model does not appear to be applicable to L1210 leukemia cells, because these cells exhibit high initial concentrations of soluble B,, as compared to the particulate fractions. In addition, the kinetic data derived b y DiGirolamo and Huennekens (1975) for mouse leukemia cells is inconsistent with receptor-mediated pinocytosis unless receptor clustering at pinocytotic sites is invoked. From

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double reciprocal plots these investigators calculated V,,, for the secondary transport process as 0.4 pmoles per minute per los cells. The number of receptor sites for the primary process was found to be 400 sites per cell. If these sites are evenly distributed, then pinocytosis of one receptor requires pinocytosis of 1/400 of the membrane. The maximum velocity of transport calculated in units of molecules per cell per hour is 14,400.To pinocytose this number of sites requires turning over the entire membrane 36 times in 1 hour. This figure is much higher than the reported rates of membrane turnover. Fibroblast surface membrane turnover has been reported as 30% per hour (Steinman et al., 1974). Receptor clustering identified by morphological techniques has also been described (see Section 1117C,3). It appears then that B12 is transported into leukemia cells, liver, and kidney as TC II-B12 complex. This is further supported by disappearance curves from serum of TC II-B12 labeled in both the B12and TC 11. The half-times of disappearance from the serum are identical, being 1.5 hours for both molecules of the complex (Schneider et al., 1976). The mechanism of transport in mouse leukemia cells does not appear to occur via the pinocytotic model a in Fig. 1, since at early time points TC II-B12 is not lysosome-associated. If the pinocytotic model is correct, a mechanism for escape from the lysosome before degradation ensues is required, as diagramed in Fig. 1, step 4b. Transport in liver and kidney appears to occur via pinocytosis in the studies cited. Whether or not a nonpinocytotic transport mechanism is present in liver or kidney remains to be determined. Fiedler-Nagy et al. (1975) studied the binding of TC II-B12 complex to isolated rat liver plasma membranes. Their affinity constant for the binding is in the same range as that reported for mouse leukemia cells (5.5 x lo9 A4-l). Binding isotherms gave a linear Scatchard plot with 7.2 x 1O'O sites per milligram of membrane protein. This appears to be even less than the number of sites observed on mouse leukemia cells. We calculate, assuming the cell membrane contributes 2% of the total cellular pro tein (Neville, 1976), 1300 x 1O'O sites per milligram of protein for mouse leukemia cells. With such a low number of specific receptor sites it is difficult to see how receptor-mediated pinocytosis could incorporate B12 into liver unless receptor clustering at pinocytotic sites is postulated (see Sections II1,C and IV,C). However, the experiments in liver and kidney were done at saturating concentrations of TC II-BI2, and nonspecific binding processes may bind considerably more material than specific receptor sites. It would be interesting to repeat the experiments of Pletsch and Coffey under nonsaturating receptor conditions. This may require a replacement transfusion uti-

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lizing serum depleted of TC II-BIz. Uptake of TC II-Bl2 has also been studied in human fibroblasts (Rosenberg et al., 1975). These studies do not further reveal the mechanism of transport but do show the role of a second intracellular binding protein for BIZ.After incubation for 76 hours most of the labeled cobalamin present is bound to a protein of MW 120,000. A mutant fibroblast line unable to synthesize cobalamin coenzymes showed no defect in transport but was unable to retain high intracellular concentrations of cobalamin at late times. The mutant line was found to be deficient in the intracellular binding protein. 6. tranrferrin

1. STRUCTURE

AND

FUNCTION

Transferrin is the major iron-binding protein of the serum and serves to distribute iron to the peripheral tissues. Transferrin is a glycoprotein of MW 78,000. Iron uptake from transferrin has been extensively studied in reticulocytes and nucleated erythroid cells from bone marrow. The uptake of Iz5I-transferrin and 59Feby these cells proceeds by a biphasic process displaying an initial rapid uptake which is independent of temperature and a slower uptake rate which is highly temperature-dependent. The phenomenon appears similar to that seen with Blz uptake, and the rapid initial uptake is believed to represent binding to receptors, while the slower rate represents transport into the cell. When uptake of transferrin labeled with iodine in the protein component and radioactive iron is studied in nucleated erythroid cells as a function of time, iron uptake increases linearly over a 45-minute period. lZ5I-Transferrinuptake rises rapidly and then levels off, net uptake ceasing at 7 minutes (Kailis and Morgan, 1974). These studies and many others of a similar nature have led to the following scheme. Transferrin containing iron is initially bound to a cell surface receptor. The transferrin-iron complex is transferred into the cell interior where the iron is removed. Transferrin is returned to the extracellular medium in an intact and functional state. Data derived from intact animal preparations support this hypothesis. Labeled iron bound to transferrin is rapidly cleared from the serum with a half-life on the order of 60-90 minutes. The half-life of transferrin, however, is between 8 and 10 days (Awai and Brown, 1963). Nonenzymatic dissociation of iron from transferrin cannot explain the differences in half-life, since the affinity of iron for transferrin is reported at 1V1M-I (Aasa et al., 1963).

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2. RECEPTORS Evidence that receptors are involved in transferrin uptake comes from two types of data, Transferrin uptake has been shown to be a saturable process in bone marrow erythroid cells. The uptake rate falls one-half when the transferrin concentration is increased from 40 pM to 100 pM (Kailis and Morgan, 1974). The initial uptake of doubly labeled transferrin can be reduced 10-fold b y short incubations with pronase or trypsin. Reticulocytes exposed to doubly labeled transferrin at 0°C and then incubated with trypsin release transferrin into the medium containing the same specific activity of iron and 1251as in the incubating media. If, however, a 37°C incubation follows the initial 0°C incubation, treatment with trypsin fails to release significant amounts of transferrin. At 37°C both labels in transferrin enter a compartment not accessible to trypsin (Hemmaplardh and Morgan, 1976).

3. TRANSPORT The details of the entry process have been investigated by Fielding and Speyer (1974),utilizing timed and chaser experiments. After incubation with labeled transferrin, reticulocytes are homogenized and fractionated into a cytosol and membrane fraction. The membrane fraction is dissolved in Triton X-100 and chromatographs on Sepharose with a MW of 230,000. This complex presumably represents the transferrin cell membrane-receptor complex. At later time points transferrin is found in the cytosol compartment. Transferrin within the cytosol compartment chromatographs with a MW of 95,000 (Sly et al., 1975). This presumably represents a complex with an intracellular binding protein of MW 20,000. By increasing the external transferrin concentration, cytosol transferrin appears at MW 78,000. This process presumably represents saturation of the intracellular carrier protein. The mechanism of transferrin transport is unknown. Although pinocytosis has been proposed, no direct or strong inferential evidence to support this proposal exists. The uptake of transferrin is highly temperature-dependent, and the activation energy of the forward rate constant has been calculated at approximately 20 kcal/mole (Kailis and Morgan, 1974). Inhibitors of oxidative metabolism depress the uptake of the temperature-dependent transport process. Inhibitors of microtubules also depress the uptake process (Hemmaplardh et d., 1974). A major difficulty for the pinocytotic mechanism is to explain how transferrin escapes degradation in lysosomes. Studies on fibroblasts have shown that virtually all pinocytotic vesicles fuse with lyso-

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somes (Hubbard and Cohn, 1975; Steinman et al., 1974). T h e situation may b e different with reticulocytes and nucleated erythroid cells. C. low-Density lipoprotein

1. STRUCTURE AND FUNCTION In the absence of dietary cholesterol the liver synthesizes more than 90% of the total daily cholesterol requirement (Dietschy and Wilson, 1970), yet the peripheral tissues, such as fibroblasts, skin cells, and aortic media cells are capable of high rates of cholesterol synthesis. Therefore a regulatory mechanism must exist linking these disparate sites of synthesis (Brown and Goldstein, 1976a). Cholesterol is transported in the serum tightly bound to proteins. The major cholesterol-carrying protein in human serum is known as low-density lipoprotein (LDL). LDL is a high-MW lipoprotein (2-3.5 x lo6) carrying a core of neutral lipid, mainly esterified cholesterol. The core is surrounded by a polar coat that contains phospholipid, free cholesterol, and a 500,000-dalton protein known as apoprotein B (Goldstein and Brown, 1977). LDL carries, generally on a weight basis, a ratio of cholesterol to protein of 1.6: 1, 70% of which is esterified. Goldstein and Brown (1976) and co-workers showed in a series of articles that LDL in addition to being a carrier protein functions as a regulator of peripheral cholesterol synthesis, and that this process is receptormediated. Fibroblasts grown in tissue culture in the absence of LDL exhibit profound changes in cholesierol metabolism when LDL is added to the medium: (1)The cellular cholesterol pool is expanded, (2) cholesterol synthesis is suppressed because of a reduction in the activity of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG CoA reductase), ( 3 ) cholesterol esterification is activated, and (4) cell surface LDL receptor number is reduced. All these effects were achieved in the absence of LDL by adding cholesterol to the medium in the presence of ethanol. Evidently, cholesterol itself initiated these feedback mechanisms when it gained entry to the cell, and LDL was the physiological vehicle for cholesterol entry. Fibroblasts obtained from patients suffering from the disease familial hypercholesterolemia exhibited high rates of cholesterol synthesis which were unchanged by the addition of LDL. However, these cells returned to normal levels of synthesis after cholesterolethanol was placed in the medium. The mutation apparently involved a failure in LDL-mediated cholesterol entry.

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2. FIBROBLAST RECEPTORS AND TRANSPORT Binding studies were performed on normal cultured human fibroblasts utilizing lZ5I-labeledLDL. Saturable binding was detected, half-saturation occurring in the region of 1pg/ml and complete saturation occurring in the range of 10-20 pg/ml of lz5I-labeledLDL at 4°C. When fibroblasts were incubated with 1251-labeledLDL at 37"C, uptake was noted, and degradation products in the form of trichloroacetic acid (TCA)-soluble iodine accumulated in the cell culture medium. Degradation products increased at a linear rate with time and, when near saturating conditions of LDL were used, the rate of degradation was found to be 180 ng of LDL per hour per milligram of fibroblast protein (Basu et al., 1976). Hydrolysis of cholesterol esters paralleled the rate of protein degradation. Both processes were inhibited 90%by chloroquine. Since chloroquine is believed to inhibit lysosoma1 hydrolases, the site of degradation was presumed to be the lysosome. When the same type of study was repeated on fibroblasts from cell lines carrying the homozygous familial hypercholesterolemia mutation, binding at 4°C was decreased 50-fold and was linear with concentration, indicating the absence of detectable high-affinity binding sites. At 37°C uptake was decreased by more than a factor of 10, and no detectable degradation products in the medium were noted. Thus the overproduction of cholesterol seen in familial hypercholesterolemia appears to result from an absence of feedback inhibition, which is the consequence of a defect in the receptor for LDL required for cholesterol transport into the cell. 3. CLUSTERED RECEPTORS AT COATED REGIONS

The localization of LIjL receptors on the plasma membrane of fibroblasts has been studied utilizing a ferritin-conjugated LDL (Anderson et al., 1976). The striking feature to emerge from this study is that the receptors are not uniformly distributed but rather are clustered at thickened indentations in the membrane which appear to be sites in the early stages of pinocytosis. Such sites are known as coated regions (see Fig. 2). Seventy percent of all receptors visualized were at these sites which accounted for only 1.4% of the total membrane surface area. Controlled studies on cells carrying the homozygous mutation for familial hypercholesterolemia failed to show binding sites. The ferritin technique revealed between one-half and one-quarter the number of sites detected by the 1251-labeledLDL-binding technique. This clustering of sites does not occur by a process similar to that seen

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CLUSTERED RECEPTOR-MEDIATED PINOCYTOTIC TRANSPORT MODEL

,Bound Protein

J 1

Lysosome

3

2m ez3 A

Coated Vesicle

Secondarv Lysosome

FIG.2 . In this model receptors are clustered at specialized small areas of the surface membrane known as coated regions which are predestined to become sites for pinocytosis. Uptake proceeds as in Fig. 1. Clustering allows for a high ratio of receptormediated uptake to membrane internalized. As shown here, clustering is promoted by a second insertion protein or a second specialized region on a single polypeptide chain receptor (solid circles) which has a high affinity for the coated regions. (See Sections II1,C and IX,B. (Redrawn from Anderson et ul., 1977b.)

in lymphocytes following binding of antiimmunoglobulin, since the LDL-binding studies are performed at 4"C, a temperature which does not permit transmembrane receptor migration. By performing LDL-ferritin binding to human fibroblasts at 4"C, followed b y rapid warming to 37"C, sequential quantitative analysis of the internalization process was performed (Anderson et d.,1977a). Within 10 minutes, 98% of the LDL-ferritin bound to coated regions was internalized, predominantly into coated pinocytotic vesicles formed b y the invagination and pinching off of the coated membrane regions. With increasing time the coated vesicles were observed to migrate through the cytoplasm, to lose their cytoplasmic coat, and to fuse with either primary or secondary lysosomes (see Fig. 2). LDLferritin cores initially bound to noncoated regions of the membrane also became reduced in number with time. Pinocytosis from these regions also occurred, but these vesicles did not contain LDL-ferritin. These findings raise the interesting possibility that LDL receptors can migrate from noncoated regions of the membrane to become inserted and concentrated in coated regions. This proposition receives further support from studies of a second type of mutation producing familial

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hypercholesterolemia. This mutant (J. D.) described by Brown and Goldstein (1976b)is unable to internalize LDL like the other familial hypercholesterolemic mutation, yet binding of LDL displays normal kinetics and number of sites. Evidently two alleles are required for internalization, one specifying binding and a second related to internalization (see Fig. 2). Two findings indicate that the second allele may serve to insert LDL receptors at coated regions: (1)J. D. cells form coated pinocytotic vesicles like normal cells, and (2) although the kinetics of binding and receptor number are normal in J. D. cells, LDL-ferritin cores are not found in coated regions; rather they are distributed diffusely over the surface membrane (Anderson et al., 1977b). Clustering of sites seems to be necessary to explain the relatively high rate of LDL uptake if receptor-mediated pinocytosis is the only process operating. The rate of LDL internalized and degraded is 180 ng per hour per milligram of fibroblast protein, whereas the amount bound is half this quantity. This indicates that the plasma membrane must turn over two times each hour. Reported rates for fibroblast plasma membrane turnover are one-quarter of the membrane per hour (Steinman et al., 1974; Hubbard and Cohn, 1975).Hence the uptake of LDL appears to be eight times more rapid than the maximum possible rate calculated for receptor-mediated pinocytosis assuming a uniform receptor distribution. The maximum rate of protein uptake into fibroblasts by nonreceptor-mediated pinocytosis (bulk fluid pinocytosis) has been ascertained by Steinman and co-workers (Steinman et al., 1974) utilizing studies with horseradish peroxidase. This type of uptake is nonsaturable and is linearly related to the protein concentration in the external medium. The rate is 100 ng/mg of cell protein per hour for external protein of 1 mg/ml. In the uptake studies of LDL cited above, the LDL concentration was in the range of 5 pg/ml. This would lead to a non-receptor-mediated uptake of 0.5 ng/mg of cell protein per hour. This figure is 400-fold less than the uptake found in normal fibroblasts but is in the range of uptake reported for the mutant fibroblasts. Therefore the data are consistent with two modes of uptake, clustered receptor-mediated pinocytosis occurring in normal cells and bulk fluid pinocytosis occurring at a much lower level in both normal and mutant cells. Since bulk fluid pinocytosis uptake of a soluble macromolecule is linearly related to the macromolecular concentration, normal levels of LDL protein uptake can be achieved in mutant cells b y raising the LDL concentration to the appropriate level. The surprising result obtained is that this maneuver fails to increase the cholesterol pool, and consequently abnormal levels of synthesis and es-

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terification are unchanged (Goldstein and Brown, 1976).The failure of the cell to accumulate cholesterol acquired by this uptake route points up the specificity of the uptake process in defining the final physiological result. Recently, Basu et a l . (1976) found it possible to achieve uptake of LDL by fibroblasts lacking LDL receptors by incorporating positive charges into the LDL. This was done b y mixing LDL with N , N dimethyl-1,3-propanediamine.Cationized LDL, when incubated with mutant fibroblasts, induced a fall in HMG CoA reductase. In addition, labeled degradation products of the cationized LDL were found to accumulate in the medium at a rate approaching that for normal fibroblasts and native LDL. These workers believe that the cationized LDL was bound to receptors present on the surface membrane for cationic groups, pinocytosed, and delivered to the lysosome and hydrolyzed, similar to the proposed scheme for native LDL. It is not known whether the cation receptors are diffusely distributed or localized to coated regions; however, cationized ferritin is bound diffusely. The end result of introducing cholesterol via cationized LDL into cells lacking LDL receptors is a marked accumulation of cellular cholesterol in the form of large lipid droplets. Presumably this is because the receptors involved (now cationic receptors) are not reduced in concentration (down-regulated) as the cholesterol pool increases, as would occur with LDL receptors (Goldstein and Brown, 1977). The concept that down-regulation of receptors, subsequent to specific ligand binding, serves to stabilize an intracellular pool of the ligand or a fragment of the ligand may have general applicability. Down-regulation of receptors was first reported for the insulininsulin receptor system (Neville et al., 1973; Gavin et al., 1974); however, the physiological consequences have not been fully explored. IV.

ASIALOGLYCOPROTEINS

A. Structural Requirements for Transport

Desialylated glycoproteins are rapidly cleared from the circulation

by a receptor-mediated transport process. This process has been studied in detail by Ashwell and Morel1 (1974) and collaborators. The initial studies were performed with ceruloplasmin. The half-life of native ceruloplasmin in the rabbit was known to be approximately 56 hours. However, when the terminal sialic acid residues of this glycoprotein were removed with neuraminidase, the injected protein was

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found to clear from the rabbit serum with a half-life of approximately 3 minutes. The removal of terminal sialic acid left galactose residues as the terminal sugar on this glycoprotein. Rapid clearance of glycoprotein was found to be dependent upon terminal galactose. Treatment of asialoceruloplasmin with galactose oxidase or 0-galactosidase resulted in prolongation of the half-time of survival by a factor of 10 over that of the desialylated glycoprotein. The rapid disappearance of asialoceruloplasmin from the serum was found to be accompanied by an equally rapid accumulation of labeled protein within the liver. Less than 1% of the labeled protein was found in kidneys, spleen, lungs, and heart combined. By oxidizing asialoceruloplasmin, followed by a reduction of the resultant aldehyde derivative with tritiated borohydride, tritiated asialoceruloplasmin was prepared. A second label was introduced by using 64Cu.Cellular distribution within the liver was monitored by serial histoautoradiography. Tritium was located exclusively in the parenchymal cells. The initial uptake of doubly labeled asialoceruloplasmin by the liver occurred via the intact molecule. Six minutes after doubly labeled asialoceruloplasmin was injected into a rat, 75% of the dose was recovered from the liver with no change in the ratio of tritium to copper. The intracellular site of transport was found to be the lysosome. Sucrose density gradient fractionation of liver homogenates revealed that labeled immunoprecipitable asialoceruloplasmin migrated together with the lysosomal marker enzymes P-galactosidase and acid phosphatase. At late time points copper was shown to be cleaved from the protein, and the protein underwent catabolism within the lysosome. Further studies showed that the exposure of any two galactosyl residues was sufficient to achieve prompt removal of the glycoprotein from the plasma. The phenomenon of rapid clearance of desialylated ceruloplasmin from the plasma was found to be general for a wide variety of asialoglycoproteins. Clearance curves followed a first-order process, and half-times varied from 3 minutes for asialoceruloplasmin to 40 minutes for asialothyroglobulin. B. Receptors

The receptor nature of this process was demonstrated in vivo by showing that the rapid first-order clearance of a given desialylated glycoprotein could be blocked by higher doses of another glycoprotein or higher doses of the same unlabeled glycoprotein. The receptors were found to be localized to the cell surface membrane by performing binding studies on isolated purified plasma membrane preparations. The binding studies indicated a close parallel between the in vitro

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binding system and the in vivo clearance system. Rank orders for binding affinity and clearance for various asialoglycoproteins were identical. In addition, the binding system also showed requirements for the asialo derivative and for an intact galactose residue. Competitive binding was demonstrated, and the 50% inhibition level spanned several factors of 10 for a variety of asialoglycoproteins. The highest-affinity asialoglycoprotein was orosomucoid. Saturation of specific binding sites occurred at 4 ng/ml. This corresponds to an equilibrium dissociation constant of 10-l0 M . The maximal binding capacity was estimated to be 7.5 pmoles of asialoorosomucoid per milligram of membrane protein (Pricer and Ashwell, 1971). The receptor site on the plasma membrane was also shown to be a glycoprotein. Terminal sialic acid on this glycoprotein was required for the binding process. Removal of the terminal sialic acid by neuraminidase treatment of the plasma membranes resulted in a diminution of binding. Resialylation was performed by incubating membranes with CMP14C-sialicacid, and binding was restored proportional to the incorporation of sialic acid residues. Binding was also shown to be dependent upon the presence of calcium ion. Recently, Kawasaki and Ashwell (1976a) isolated the receptor binding protein and studied its properties. The glycoprotein exhibits concentration-dependent self-association. The smallest oligomer with asialoglycoprotein-bindingproperties appears to be 2.6 x lo5 daltons. Following extensive treatment with SDS, two subunits are found in a ratio of 1:2 with MWs of 48,000 and 40,000. Two glycopeptidex have been isolated from the binding glycoprotein and have been sequenced. The terminal residues for glycopeptide 1 are sialic acid, galactose, and N-acetylglucosamine (Kawasaki and Ashwell, 1976b). Recently, Pricer and Ashwell (1976) examined the subcellular distribution of the hepatic binding protein. Initially it was thought that the binding protein was restricted to a plasma membrane localization. However, with the experience gained in isolation of the binding protein using Triton X-100, the application of this technique to subcellular fractions revealed considerable amounts of binding protein present in the Golgi apparatus, smooth endoplasmic reticulum, and lysosomes. These investigators raise the question whether or not this localization reflects a biosynthetic cycle originating in the endoplasmic reticulum, progressing to the Golgi apparatus and on to the plasma membrane and then back to the lysosomes. In the last step from plasma membrane to lysosome the asialoglycoprotein-binding protein may function as a stable shuttle. However, no real evidence exists concerning the mechanism of transport from the cell surface re-

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ceptor into the lysosome. Regardless of the mechanism the critical information in this cell-ligand system is in specific carbohydrate structures. Ashwell and Morell (1977) believe that these same structures may provide the critical determinants for certain cell-cell and subcellular organelle interactions. C. Pinocytotic Mechanism?

It may appear at first glance that the transport process occurs by pinocytosis of receptor-bound asialoglycoprotein. This seems likely, since pinocytotic vesicles are known to fuse with lysosomes. However, it appears to us that the rates of receptor-mediated pinocytosis judged to exist are insufficient to transport asialoglycoproteins at the rates determined by Morell et al. (1971)unless a marked degree of receptor clustering occurs. The following calculation is instructive. The maximum rate of pinocytosis in fibroblasts has been determined by Steinman et a l , (1974) as 25% of the plasma membrane per hour. Pinocytotic rates do not appear to be higher than this in liver, as judged b y plasma membrane turnover data (Schimke, 1969). We therefore can calculate the amount of plasma membrane internalized by the liver per hour as follows. We assume a 7-gm liver for a 200-gm rate, which is 20% protein. A generous estimate for the amount of plasma membrane protein per total liver protein is 2% (Neville, 1976).This gives 28 mg of plasma membrane protein and, assuming one-quarter internalization per hour, gives 7 mg of plasma membrane internalized per hour. Ashwell and Morell (1974) have determined that there are 7.4 pmoles of asialoglycoprotein receptor per milligram of plasma membrane protein. With the assumption of an even distribution of receptors, this leads to a maximum rate of receptor internalization of 52.5 pmoles per hour. If each receptor internalizes one molecule of ceruloplasmin, 7.8 mg of ceruloplasmin can be internalized per hour by this process. Morell et al. (1971) have reported plasma clearance data for ceruloplasmin after the injection of 9.3 mg of ceruloplasmin into a 200-gm rat. The half-time of clearance is about 3 minutes, and the process appears to be first-order. Dividing the logarithm of 2 b y the half-time of clearance gives a first-order rate constant of 0.23 per minute. The maximum transport rate is the first-order rate constant times the initial load, giving a figure of 4.1 mg per hour, almost 500 times the rate calculated on the basis of a receptor-mediated pinocytotic process. The same calculation performed on orosomucoid data yields an even greater discrepancy (2000 times). Receptor clustering at the site of pinocytosis could raise the rate for the pinocytotic process. Such clus-

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tering may b e induced by binding of the asialoglycoprotein to the receptor in a manner similar to that reported for IgG binding to lymphocytes. However, such clustering does not achieve the degree of segregation of receptor relative to other membrane components required for this process (Gonatas et al., 1976). Clustered receptor-mediated pinocytotic models (see Sections III,C,3 and IX,B) imply a much higher rate of turnover for the receptor than for components of the membrane at large, unless mechanisms exist to extract receptors from pinocytotic vesicles and reinsert them in the plasma membrane. Widely discrepant turnovers of membrane proteins analyzed b y SDS gel electrophoresis have not yet been reported (Tweto and Doyle, 1976). In any case, the clustering hypothesis is open to test. If clustering does not occur, then a nonpinocytotic mechanism of transport exists, operating from the membrane receptor site to the lysosome. V.

FIBROBLAST LYSOSOMAL HYDROLASES

Considerable evidence indicates that fibroblast lysosomal glycosidases and sulfatases gain entry to the lysosome after first being excreted extracellularly. Uptake from the extracellular compartment proceeds through a receptor-mediated process. The secretionrecapture hypothesis and its experimental foundation have recently been reviewed b y Neufeld and co-workers (Neufeld e t al., 1977). Much of the evidence comes from studies of inherited mucopolysaccharide storage diseases which result in the lysosomal accumulation of dermatan sulfate or heparin sulfate. Two general types of diseases exist. One type, exemplified by Hurler’s syndrome, is characterized b y a deficiency in one of a group of enzymes necessary to cleave the variety of linkages found in these polymers of uronic acid and sulfated hexosamine. In Hurler’s syndrome the deficient enzyme is a - ~ iduronidase. In the second type several enzymes are deficient within the lysosome but are present and active in the extracellular fluid. I-Cell disease is an example of this type of disorder (Neufeld et al., 1975). When Hurler fibroblasts are grown with normal cells or with media conditioned by normal cells, the abnormal accumulation of polysaccharide is reversed. The corrective factor found in the media proved to be a form of a-L-iduronidase. As much as 25% of the added a - ~ iduronidase was taken up in 2 days. Uptake of active enzyme was demonstrated to be a saturable process, with one-half saturation occurring at lov9 M (Neufeld et al., 1977). Not all samples of a - ~ iduronidase were capable of rapid, saturable uptake. High-uptake

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DAVID M. NEVILLE, JR. AND TA-MIN CHANG

forms of enzyme were converted to low-uptake forms by treatment with dilute periodate. This conversion appeared to destroy the uptake recognition factor. Six other lysosomal glycosidases and sulfatases were found to exist in both high- and low-uptake forms. Kaplan and co-workers (Kaplan et al., 1977) showed that uptake of the high-uptake form of P-glucuronidase was competitively inhibited by mannose 6phosphate and that the high-uptake form could be converted into the low-uptake form by treatment with alkaline phosphatase. Similar findings have been reported for a-L-iduronidase (Neufeld et al., 1977). The recognition marker appears to be a phosphorylated carbohydrate residue (Kaplan et al., 1977). The hydrolytic enzymes not present in fibroblast lysosomes but secreted by I-cell fibroblasts are all low-uptake forms. I-Cell fibroblasts can internalize high-uptake enzymes with the same kinetic constants as normal cells and retain these enzymes with the same half-life. The secretion-recapture hypothesis of Neufeld and co-workers postulates for I-cell disease a single mutation in the synthesis of the common recognition marker, explaining the resulting failure of uptake and accumulation of a group of active extracellular enzymes. The mechanism of entry of lysosomal hydrolases from the extracellular environment into the lysosome is unknown, although receptormediated pinocytosis has been postulated (see Fig. 1). The pinocytotic vesicle containing receptor-bound hydrolases could then be considered either a primary lysosome (if substrate was also engulfed) or a secondary lysosome (Neufeld et al., 1977). VI.

ANTIBODIES

A. Maternal-to-Young Transfer

Active production of antibodies is lacking in newly born mammals, and these mammals receive their immunity from the transfer of yglobulins from the maternal to the fetal circulation. In the rabbit, guinea pig, rhesus monkey, human, and gray squirrel transmission of y-globulin occurs prior to birth. Goat, sheep, pig, horse, and cat receive y-globulins after birth via colostrum and milk. In the dog, mouse, and rat both processes occur (Wild, 1973). The transfer process is highly specific for certain types of globulins and also exhibits species specificity. The route for this process was shown by Brambell (1970) to be across the yolk sac splanchnopleur to the vitelline circulation (Wild,

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1973). [Toward the end of gestation the rabbit embryo is attached to the uterus via the chorioalantoic placenta. On the .opposite side, the embryo projects into the uterine lumen and is covered by a series of membranes, the outermost being the yolk sac splanchnopleure. Beneath this membrane lie the vitteline vessels which enter the embryo via the yolk sac stalk. Thus the embryo is in contact with maternal fluids through two distinct membranes and circulatory paths (Wild, 1973)l Thus labeled antibodies injected into the uterine lumen were found to reach the fetal circulation, a process that could be i’nterrupted b y ligaturing the yolk sac stalk. In order for proteins to reach the vitelline circulation they must cross the yolk sac endoderm and basement membrane, the vascular mesenchyme, and the endothelial cells of the vitelline capillaries. I n order to explain selective uptake and competition between various species of globulins, Brambell and co-workers proposed that uptake occurred via pinocytotic vesicles and was mediated by specific receptors. These receptors, originally located on the microvilli of the endodermal cells, become internalized in the pinocytotic vesicle and protect all bound proteins from degradation within the vesicle. Fusion of pinocytotic vesicles with lysosomes would result in the degradation of unbound protein within the vesicle. On reaching the basal portion of the endodermal cell, the vesicle would fuse with the basement membrane and release the bound protein (Wild, 1975). Wild (1975), using a combination of fluorescent microscopy, electron microscopy, and autoradiography observed that a wide variety of proteins becomes localized in pinocytotic vesicles within the yolk sac endoderm. No selectivity is shown in this process. Moreover, these pinocytotic vesicles, called macropinocytotic vesicles by Wild, have never been observed to fuse with the basal plasma membrane of the endodermal cell. Wild noted a smaller vesicle (0.07-0.15 pm), called a micropinocytotic vesicle, often apparently fusing with the basal plasma membrane of the endodermal cell. These vesicles are also observed to b e generated at the base of microvilli on the lumenal side of the endodermal cell. Such vesicles are so small that they seem to consist entirely of membrane, and they may contain no aqueous phase. These vesicles do not fuse with lysosomes, as do macropinocytotic vesicles. Wild believes that they may b e involved in the selective transport of globulins from the maternal to the fetal circulation. Since only approximately 12% of the protein injected into the uterine cavity is transported intact into the fetal circulation, the rest being degraded, another process must b e invoked to explain degradation. According to Wild, this would occur in the macropinocytotic vesicles which fuse

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with lysosomes. Thus macropinocytotic vesicles would contain receptor-bound protein and soluble protein originally present in the extracellular environment, and both would eventually be catabolized. Mi'cropinocytotic vesicles containing no aqueous phase would contain only receptor-bound protein, and this would be transported across the endodennal cell. No direct evidence for this process has been obtained. However, receptors for human IgG and human Bence-Jones A proteins have been demonstrated using 1251-labeledproteins in a 60,000 g membrane fraction derived from human placenta (Gitlin and Gitlin, 1974). Jones and Waldmann (1972) studied the transport of proteins from the small intestine into the circulation of neonatal rats. Using tracer amounts of 1251-labeledproteins instilled into the small intestine, they demonstrated that 21 -35% of the instilled protein was transported to the circulation in a TCA-precipitable form for rabbit, human, rat, and mouse IgG. In contrast, less than 5% of the instilled amounts of human albumin, human transferrin, human IgA, human IgM, and polyvinylpyrolidone was transported to the circulation. The specificity for this transport appeared to reside in the Fc piece of IgG, since 12.6%of this material was transported to the circulation in contrast to 1.7%of the Fab piece. These investigators also demonstrated the saturability of this transport process. When 0.5 mg of unlabeled protein was instilled with the tracer, IgG transport to the circulation was reduced 50%.When unlabeled protein was instilled in amounts ranging from 1 to 8 mg, transport of the tracer was reduced 96%.The amount of total protein transported from the intestine to the circulation became constant when more than 1 mg was instilled into the intestine. The limiting protein transport during the 4-hour study period was 0.12 mg. These investigators also observed that the labeled proteins studied were bound to small intestine microvillous preparations in direct proportion to their transport rate. Tracer binding was competed for by cold protein in cases where high degrees of transport were observed. The ability of the neonatal rat jejunum to absorb functional antibodies selectively into the bloodstream is lost by the twenty-second postpartum day (Halliday, 1955). Immunoglobulin transport from the maternal circulation into milk was studied in lactating mice by Gitlin and co-workers (Gitlin et al., 1976). Lactating mothers were injected intravenously with 1311iodinated proteins, and the disappearance of counts in the mothers and the appearance of counts in nursing litters were followed by whole body counting. Over 80% of the counts recovered from the stomachs of nursing young were in a TCA-precipitable form. Trans-

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port of labeled proteins from a lactating mother to young is given as a daily turnover percent of the maternal body pool. These transmammary transfer percentages were low for mouse IgM (5.9%)and human albumin (4.6%),and below detection for human IgA. Higher values were observed for mouse IgG (30%) and human IgG (22%). The highest values were obtained with the K and A chains of Bence-Jones proteins, being 580% and 866%, respectively. Tracer transfer for IgG was inhibited 50% when excess unlabeled human IgG was injected into the maternal circulation. Maternal pools of cold IgG were increased to 90, 180, and 295 mg. Tracer transfer remained constant but, by assuming the validity of the tracer assumption, total transfer increased proportionately to the pool size ratios. This indicates that a portion of the transfer process is nonsaturable. B. Transport into immunological Cells

Taylor and co-workers (Taylor et aZ., 1971) reported that plasma membrane surface immunoglobulins of lymphocytes incubated with fluorescein-labeled antiimmunoglobulin antibodies aggregate into patches, form a polar cap, and undergo pinocytosis. This process is temperature-dependent and is inhibited by dinitrophenol, cyanide, and sodium fluoride. Cytochalasin B, which disrupts microfilaments associated with the plasma membrane, also inhibits this process (Nicolson and Poste, 1976). Taylor and co-workers (Taylor et al., 1971) proposed that the clustering and capping phenomena were prerequisites for the activation of lymphocytes. It is known that each B-type lymphocyte contains about lo5 immunoglobulin molecules as an integral component of the plasma membrane. These molecules function as receptors for antigens. Each individual lymphocyte contains a different species of immunoglobulin specific for a different antigen. When bivalent antigen interacts, the lymphocyte is triggered to proliferate and differentiate (Singer, 1974). Singer (1974) concludes that clustering and capping are not essential for lymphocyte activation and actually may inhibit the process. If this is the case, capping followed by pinocytosis may be a mechanism for ridding the cell surface of the stimulating antigen or antiimmunoglobulin antibody. The internalization of bound antiimmunoglobulin on lymphocytes has been confirmed by a quantitative autoradiographic study (Gonatas et al., 1976). The site of internalization is presumed to b e the lysosome, since most pinocytotic vesicles are observed to fuse with lysosomes in fibroblasts. The generality of this observation is, however, unknown. Lewis and co-workers (Lewis et al., 1974) have presented fractiona-

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tion data indicating that lymphocytes exposed to labeled antibody directed against transplantation antigen (anti-HL-A antibody) internalize the antibody predominantly at the nucleus. Isolated nuclei were shown to bind anti-HL-A antibody in amounts 10-fold greater than those bound by intact lymphocytes on an equal number basis. When cells were incubated with labeled anti-HL-A in tracer amounts, nuclear localization was stable over a 96-hour period. However, when the cells were exposed to large amounts of cold anti-HL-A antibody 30 minutes after exposure to labeled antibody, label disappeared from the nuclei at 72 hours. This time was correlated with the onset of blastogenesis. The localization data supplied by Lewis and co-workers (Lewis et al., 1974) can be criticized for its lack of data relating to the purity of the fractions. In addition, the nature of the nuclei-associated labeled antibody in terms of its integrity was not considered. However, these studies raise the question whether or not antibodies can be transported into cell compartments where immunological processes are operative. C. Retrograde Axonal Transport

The selective uptake and retrograde axonal transport of antibodies to dopamine P-hydroxylase was recently demonstrated by Fillenz et al. (1976). The experimental design and data obtained are similar to the reported studies with NGF (see Section VII1,A).

VII.

VIRUSES

A. Evidence for Receptor-Mediated Entry

For many viruses the first step in the infection of a eukaryotic cell involves binding of the virus to a specific cell surface membrane receptor (Fenner et al., 1974). For influenza and polyoma viruses the receptor has been found to be a glycoprotein with a terminal sialic acid residue. Treatment of cells with neuraminidase renders them resistant to the viral infection. Competition for the cell surface membrane has been observed between adenoviruses and the type I1 fiber antigen isolated from adenoviruses. The membrane’s receptors for avian tumor viruses are genetically determined, and in chickens the receptor is controlled by a single dominant autosomal gene. Infection of cells b y picorna viruses, polio virus being an example, is limited to

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human and simian cells because only these cells carry the specific receptor. However, the adsorption of pox viruses to cells does not require a specific receptor. 8. General Transport Mechanisms

There appear to b e at least three different mechanisms for the entry of animal viruses into eukaryotic cells. In all three mechanisms the viral protein coat remains cell-associated, although the compartmentalization of the protein varies among the mechanisms. Pox viruses, which do not require specific receptors, enter the cell by pinocytosis. The pinocytotic vesicle rapidly fuses with the lysosome, forming a phagocytotic vacuole. Within this vacuole the outer membrane of the virus is rapidly degraded, and the virus is converted to a subviral particle called the core. The membrane of the vacuole then undergoes dissolution, and the core particle is released into the cytoplasm (Fenner et aZ., 1974). A second mechanism of entry is seen with adenoviruses, in which the viral particle crosses the cell membrane and appears in the cytoplasm devoid of any association with a membranous particle. Although the viral particle appears intact, density gradient centrifugation has shown that the virus has lost approximately 5% of its protein. Viral particles move rapidly to the nucleus where further loss of protein occurs and DNA is released. This process requires only 1hour (Morgan et al., 1969). Coincident with this process of entry, some adenovirus particles are also found to enter the cell in phagocytic vesicles. The ratio of the number of viral particles entering b y pinocytotic vesicles to the number entering directly varies with the cell type and the genetic strain of the virus (Chardonnet and Dales, 1970). A third mechanism of entry has been observed in Newcastle disease virus, a member of the paramyxovirus group. Entry for this group is presumed to be receptor-mediated since, like influenza viruses, paramyxoviruses have a hemagglutinin and a neuraminidase in the outer viral envelope. Evidence exists that Newcastle disease virus nucleoprotein is released into the cell cytosol following fusion of the viral envelope with the plasma membrane. The fusion process is believed to be aided b y a hemolysin carried by the paramyxovirus. Herpes virus may also enter cells by a fusion process (Fenner et al., 1974). Most of the above observations have been obtained by morphological techniques, and knowledge of the biochemistry of the transport process is meager. Evidence exists that certain viruses, notably rabies and herpes, can gain entrance to the central nervous system from the

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subcutaneous compartment by retrograde axonal transport. The reverse process, centrifugal transmission from the dorsal root ganglion cells to the skin and mucous membrane, most likely explains the mode of spread of recurrent herpes simplex and herpes zoster (Fenner et al., 1974). VIII.

GROWTH FACTORS AND HORMONES

A. Nerve Growth Factor

1. STRUCTURE AND FUNCTION

P-NGF is a protein of MW 26,500 present in the serum of all vertebrates. NGF is necessary for development of the sympathetic nervous system in young animals. The injection of antiserum directed against NGF into newborn mice results in selective and permanent destruction of 90-95% of the sympathetic nerve cells (Levi-Montalcini and Angeletti, 1968). NGF is also necessary for the maintenance of tissue-cultured sympathetic neurons in serum-free media. When dorsal root ganglions from 8-day chick embryos are incubated with NGF, neurite outgrowth is observed which is dependent upon the presence of NGF. This constitutes the usual bioassay, and the maximum response for purified preparations occurs at 0.26 nM. In the mouse the serum concentration of NGF is reported to be 0.4 nM (Hendry and Iversen, 1973). NGF is synthesized and highly concentrated in the salivary gland of the mouse and in the venom gland of vipers. The material is produced elsewhere, since extirpation of these glands does not produce a deficiency disease. Considerable evidence exists that NGF is fixed by receptors located primarily at the sympathetic nerve cell terminals, transported across the plasma membrane, and transported by retrograde axonal transport up the axon to the region of the cell body where it exerts its stimulatory effects. P-NGF is isolated from a higher-MW complex, 'IS-NGF, in the mouse submaxillary gland. It is associated with two other polypeptide chains, one of which has proteolytic activity (Baker, 1975). NGF is prepared by dissociation of 7s-NGF. It contains two identical polypeptide chains held together by noncovalent forces. Each chain contains three intrachain disulfide bonds (Stach and Shooter, 1974). The sequence of P-NGF has been compared to the sequence of human proinsulin by Frazier and co-workers (Frazier et al., 1972). These investigators conclude that there are sequence similarities and that the two proteins are evolutionarily related,.

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2. MEMBRANERECEPTORS

NGF has been successfully labeled with 1251,and binding studies with dorsal root nerve cells and membranes derived from sympathetic ganglion have revealed the receptor nature of the binding process. Eight-day chick embryo dorsal root ganglion cells contain approximately 2 x lo4 receptors per cell. The specific binding reaches halfsaturation at 0.26 nM (Herrup and Shooter, 1973). A crude vesicular membrane fraction isolated from the sympathetic ganglia of young rabbits exhibited a 10-fold increase in binding activity over that of the homogenate. An apparent equilibrium dissociation constant was calculated to be 0.2 nM. No cross-reactions with insulin or epidermal growth factor were found. Specific binding was limited to membrane preparations from sympathetic ganglia (Banerjee et al., 1973). The binding of NGF to these membranes has been found to be dependent upon calcium. Low concentrations of trypsin and phospholipase A from Vipera russelli decrease NGF binding (Banerjee et al., 1975). 3. BIOCHEMICALEFFECTS

The treatment of newborn rats with NGF enhances growth and induces differentiation in the sympathetic neurons. Thoenen and coworkers (Thoenen et al., 1971) showed that NGF stimulates the activity and concentration of two enzymes, tyrosine hydroxylase and dopamine P-hydoxylase, which are localized exclusively in adrenergic neurons and which are concerned with the synthesis of adrenergic neurotransmitters. The activity of these enzymes increases 13- to 18-fold after 10 days of treatment with NGF. The specific activity rises 4-fold. Monoamine oxidase and dopa decarboxylase activities rose 1.2and 1.5-fold, respectively. Combined extracts from controls and treated animals gave additive activities, indicating that formation of an activator or loss of an inhibitor was not responsible for the phenomenon. These investigators conclude that NGF evokes a selective induction of tyrosine hydroxylase and dopamine P-hydroxylase. These enzymes are known to be synthesized in the cell body of the sympathetic neurons and transported in an orthograde manner to the axon e n d terminals where the neurotransmitters are synthesized and packaged. 4. RETROGRADE AXONALTRANSPORT Studies on the binding of NGF to homogenates of sympathetic ganglia and dorsal root cells do not provide information on the anatomical localization of the receptor sites. The following evidence indicates that NGF specifically interacts at the end terminals. When

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1251-labeledNGF is injected intraocularly, counts are observed to rise in the ipsilateral superior cervical ganglion as a function of time, peaking at 8 hours. The number of counts is two to three times higher on the ipsilateral side compared to the contralateral noninjected side. When the same experiment is repeated with a variety of proteins, such as cytochrome c, insulin, horseradish peroxidase, or bovine albumin, counts on the contralateral and ipsilateral sides are identical and the time course follows a decay curve rather than a peak preceded by a lag period. The time required for the peak to be achieved with NGF is consistent with the known rates of retrograde axonal transport (Stockel et al., 1974). When 1251-labeledis given intravenously, there is a rise in the number of counts in the superior cervical ganglion, which is half complete at 3 hour and complete at 1hour. Counts are constant until 4 hours, when a further, larger rise takes place which again peaks at 8 hours, giving a specific activity seven times that of the blood. Other organs observed, such as heart muscle, show a simple decay curve paralleling the decay curve for blood (Stoeckel et al., 1976).It is believed that the initial, low-level rise represents uptake from the blood by the cell bodies within the superior cervical ganglion. The later, more profound rise has the same time course as retrograde axonal transport and represents this phenomenon. Most of the NGF reaching the superior cervical ganglion arrives by the retrograde route from the end terminals of the axon. This is understandable, since the ratio of the cross-sectional area of the sympathetic end terminals to the cell body has been estimated to be 100: 1(Stoeckel et al., 1976).The specificity of this process was further demonstrated by showing that NGF was not taken up and transported retrograde in the lower motor neuron, as is the case with tetanus toxin (Stockel et al., 1974).

5. INTRACELLULAR VERSUS MEMBRANESITE

OF

ACTION

Evidence that the stimulatory effect of NGF on tyrosine hydroxylase is mediated through retrograde axonal transport was obtained by Paravicini and co-workers (Paravicini et al., 1975). These investigators administered large doses of NGF and lz5I-1abeled NGF systemically to 8-day-old rats. Experimental animals had the axon to one superior cervical ganglion sectioned. At 48 hours tyrosine hydroxylase activity was assayed in the superior cervical ganglions. The stimulation of tyrosine hydroxylase activity seen on the intact side was twice that on the axotomized side. With the use of lZ5I-labeledNGF the counts were also reduced by one-half on the axotomized side. NGF appears to arrive at the superior cervical ganglia after retro-

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grade transport in a largely intact form. SDS gel electrophoresis 16 hours after NGF injection shows that labeled NGF migrates coincidentally with marker NGF. It is not known whether NGF is transported in a free form within the axoplasm or whether it is transported within a vesicle. However, fractionation of the ganglia at 16 hours reveals 18% of the NGF associated with the microsomal fraction and 36%associated with the nuclear fraction. In contrast, at 2 hours, a time at which NGF reaching the ganglion must do so independently of retrograde transport, 72% of the NGF is in the supernatant fraction and only 3% is in the microsomal fraction (Stoeckel et al., 1976). The evidence that NGF enters the axoplasm and is transported in a retrograde fashion is strong. Supporting evidence exists that part of the stimulation of tyrosine hydroxylase activity evoked by NGF occurs via axonal retrograde transport. Together these facts make a compelling argument that NGF exerts its biological activity at an intracellular site. However, it has been proposed that NGF acts on the surface membrane (Frazier et al., 1973). The evidence supporting this proposal comes from studies which show that NGF coupled to Sepharose beads is capable of displaying activity in the neurite outgrowth bioassay. The control for this experiment, which attempts to show that stimulation is not due to NGF dissociated from the Sepharose beads, has ganglia and beads in separate adjacent clots connected by media. The bead environment is thus different in the control and experimental cases. An adequate control for this experiment requires coupling of high-specific-activity labeled NGF to the Sepharose on the order of 1 mole of iodine per mole of NGF. A demonstration that dorsal root ganglion cells did not accumulate iodine would constitute adequate evidence that NGF did not leave the Sepharose phase and enter the cell. Such an experiment appears difficult to perform, necessitating coupling at the tracer level or the use of large amounts of radioactivity. This type of experiment in general has other pitfalls, most notably the fact that it is not amenable to testing by a quantitative model in which the stimulating variable is changed while the response is noted. The difficulty arises from the fact that the active growth factor or hormone is localized only at the periphery of the bead and the interaction with the cell membrane is not a simple function of either bead or cell number. Early experiments of this type with insulin have been criticized on quantitative grounds (Katzen and Vlahakes, 1973; Butcher et al., 1973). Subsequent experiments showed that solubilization of insulin from Sepharose-insulin occurred at levels sufficient to explain the stimulatory effects (Garwin and Gelehrter, 1974; Kolb et al., 1975; Davidson et d.,1973). These criti-

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cisms have never been adequately dealt with in the literature by investigators utilizing this technique.

B. Glycoprotein Hormones

1. FEATURES SHARED

WITH

CHOLERA TOXIN

The glycoprotein hormones of mammals have many functional and structural characteristics in common. These hormones evoke differentiation and differentiated responses in their target tissues b y stimulating adenylate cyclase activity. They are thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and (human) chorionic gonadotropin (hCG). The response of the target tissues is quite specific; for example, the relative activity of LH in the thyroid hormone release bioassay is less than 0.1%(Wolff et

al., 1974). These hormones consist of two dissimilar polypeptide chains held together by strong noncovalent forces. The chains have been separated and purified, and it has been possible to form artificial hybrids of the type TSH,-hCG, and hCG,-TSH,. When the hybrids are bioassayed, nearly complete activity is recovered for bioassays of psubunit-type activity. Conversely, the hybrids show little activity when assayed for a-subunit-type activity. Thus TSH,-hCG, is as effective as TSH in a T S H bioassay (Pierce et al., 1971). Separated p and a subunits lack in vivo bioactivity. Structural studies revealed that the a chains of LH and TSH had nearly identical amino acid sequences, whereas the /3 chains showed marked differences in their amino acid sequences (Liao and Pierce, 1970). Since the common feature of these hormones is cyclase stimulation and the variable feature is target specificity, it seemed reasonable that the p chains were involved in binding to the specific target organ receptors, while the a chains were involved in stimulating adenylate cyclase. On these grounds, glycoprotein hormones are similar to cholera toxin. Subsequently, it was shown that additional similarities existed. It is for this reason that glycoprotein hormones are considered in this article. At the present time it is not known whether the glycoprotein hormone or its a subunit is transported into the cell cytosol or even into the interior of the membrane as apparently happens with cholera toxin. However, we briefly consider some of the similarities between cholera toxin and glycoprotein hormones. The knowledge in this area is accumulating rapidly and has been recently reviewed (Kohn et al., 1977).

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Evidence for the simple scheme of separate functions for glycoprotein hormone subunits could not be obtained from studies of binding and adenylate cyclase stimulation using isolated bovine thyroid membranes. The p subunits of TSH and LH both bind to the TSH receptor, having 2%of the activity of TSH. Both the p subunits of TSH and LH, and LH alone, stimulated adenylate cyclase activity ranging from 2 to 8% of the stimulations achieved by TSH (Wolff et al., 1974). The investigators state that neither this binding activity nor the biological activity of the p subunits could be accounted for by TSH contamination. Either the system is more complicated than the proposed model or experimental difficulties obscure its simplicity.

2. INTERRELATIONSHIPS BETWEEN TSH, CHOLERA TOXIN, AND TETANUSTOXINRECEPTORS

The relationship between the TSH receptor and the cholera toxin receptor has been investigated by performing studies involving the binding of lZ5I-labeledTSH to thyroid plasma membranes in the presence of varying amounts of cold cholera toxin. Additions of cholera toxin in the range of 5 x loF0M to 5 x lo-' M produce a gradual increase in the amount of tracer TSH binding. Tracer binding is competed with additions of cholera toxin greater than M , and a maximal inhibition of 40% is achieved at 2 x M (Mullin et al., 1976). Thus, although competition for receptor sites is seen at high cholera toxin concentrations, cooperativity must be invoked to explain the enhancement of tracer binding at low concentrations. Further insights into this process were achieved by studying the inhibition of TSH binding by various gangliosides. The most potent inhibitor of TSH binding was GGnSSLC. It should be remembered from the previous section that this ganglioside is the most potent inhibitor of tetanus toxin binding to its receptor. The ganglioside GGnSLC was 10-fold less effective in inhibiting TSH binding. A similar situation exists with tetanus toxin. The latter ganglioside, however, is a potent inhibitor of cholera toxin binding. These gangliosides inhibit binding by complexing with the toxin and invoking conformational changes. A similar conformational change in TSH has been noted upon binding to GGnSSLC (Kohn et al., 1977). It is believed that the interaction between gangliosides and these glycoproteins reflects to some degree the interaction of the glycoproteins on the cell membrane with gangliosides or with glycoproteins having a similar structure. Thus TSH appears to have two types of ganglioside receptors differing in affinity by approximately a factor of 10. The lower-affinity receptor for TSH

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and the receptor for cholera toxin share the same site. The failure of cholera toxin to produce more than 40% inhibition of TSH binding to thyroid membranes could be explained by two separate TSH receptors, only one of which reacts with cholera toxin. Direct evidence that GGnSLC is the receptor for cholera toxin in thyroid plasma membranes has been obtained by Mullin and coworkers (1976). These investigators oxidized the terminal galactose residues in thyroid plasma membranes with galactose oxidase. Labeling of the exposed galactose residues was achieved by incubating the membranes with borotritide. Thyroid plasma membranes reacted in this fashion showed heavy labeling of GGnSLC. Other gangliosides were labeled to a minor extent. Thyroid plasma membranes containing bound cholera toxin were similarly treated with borotritide. GGnSLC labeling was markedly reduced under these conditions. However, an unexpected finding was the increased labeling of four other ganglioside components, among them SGnSSLC. The presence of bound cholera toxin enhanced the reactivity of these gangliosides. Mullin and co-workers consider this result a direct demonstration of the ability of cholera toxin when bound to its receptor to induce a conformational change within the plasma membrane. This conformational change exposes other gangliosides, notably SGnSSLC, which has the highest affinity for TSH. It appears that this experiment is a direct demonstration of the cooperative phenomena seen in binding studies with tracer TSH, cold cholera toxin, and the thyroid plasma membrane. Tetanus toxin shows the same relative binding affinities toward GGnSSLC and SGnSSLC as TSH. Ledley and co-workers (Ledley et aZ., 1977) showed that cold tetanus toxin competes with 1251-labeled TSH for receptors on thyroid plasma membranes. And conversely, cold TSH competes for 1251-labeledtetanus toxin on thyroid membranes. Thus it appears that TSH and tetanus toxin share the same receptor. The same receptor appears to be utilized by interferon (Kohn ,-' d., 1976). The relationship between the receptors defined by the ling studies and the functional activity of these proteins remains de established. It is possible that only a small proportion of the receptors produces biological activity, and that these have a more complex structure. A glycoprotein has been isolated from thyroid membranes which binds TSH (Meldolesi et al., 1977). The interrelationship between this glycoprotein, GGnSSLC, and cyclase stimulation has not yet been determined (Kohn et al., 1977). It will be of considerable interest to see if TSH can block the neurotoxicity of tetanus toxin. Kohn and co-workers (1977) have speculated that the tachycardia, la~

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bile blood pressure, and pyrexia seen in tetanus intoxication treated with curarization and positive pressure ventilation may represent thyroid storm. The previously accepted interpretation is that these symptoms are the result of inhibition of acetylcholine release within the autonomic nervous system.

3. DIFFERENCESIN TSH,

CHOLERA, AND

TETANUS TOXIN

Although TSH and tetanus toxin share the same receptors and one of these is shared by cholera toxin, the functional similarities appear to cease at this level. Cholera toxin stimulates adenylate cyclase, a stimulation requiring NAD. This was recently demonstrated in thyroid plasma membranes (Kohn et al., 1977). However, TSH does not require NAD for tlie stimulation of adenylate cyclase. In fact, NAD inhibits this stimulation. There are no reports in the literature of tetanus toxin stimulating adenylate cyclase activity. The available evidence indicates that tetanus toxin functions by inhibiting neurotransmitter release, particularly the inhibitory neurotransmitters, and that this function is accomplished within the cell cytosol (see Section 11,C). A characteristic of these toxins, which are known to have an intracellular site of action and which are transported across the membrane by a receptor-mediated process, is a dose-dependent lag period which reaches a minimum value at a saturating dose of toxin. Cholera toxin, which may penetrate only to the interior of the membrane, also has a lag period which does not appear to b e dose-dependent. The effect of TSH on nonconfluent thyroid cells in tissue culture does not exhibit an observable lag period. The addition of TSH to this system causes an increase in the mitotic index and an increase in the rate of thymidine incorporation into DNA. Thymidine incorporation is linear over a 6-hour period in untreated cells. Treatment of cells increases the slope of incorporation with time, and the extrapolated line passes through the zero-time origin (Winand and Kohn, 1975). C. Lactogenic Hormones Prolactin has been found in the milk of a variety of mammals by radioimmunoassay (McMurty and Malven, 1974). The concentrations vary between 50 and 500 ng/ml. These concentrations are equal to or somewhat higher than the concentrations found in serum. Since prolactin is synthesized only in the pituitary gland, it must cross both the vascular endothelium and the mammary gland epithelium to gain

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entry to the milk. Grosvenor and Whitworth (1976) have reported that, during an intravenous infusion of prolactin into lactating rats, the prolactin concentration in the milk rose and reached a steady-state value of 250 ng/ml. After termination of the infusion, plasma prolactin levels fell rapidly to the preinfusion level, but the milk prolactin level fell more slowly. Nolin and Witorsch (1976) have reported the presence of prolactin within the alveolar cells in milk-containing ducts of lactating rats, using histological techniques dependent on a reaction involving antiprolactin antibodies and a second chromogenic antibody. These two studies indicate that prolactin can enter mammary cells from the circulation and be transported across these cells into the milk ducts. Although prolactin receptors in mammary tissues have been documented, it is not known whether or not prolactin transport across the gland is a receptor-mediated process (McMurty and Malven, 1974). Bioassays of radioimmunoassayable milk prolactin have not been reported. D. Insulin

Insulin has many diverse actions on cells. Some actions, such as stimulation of glucose transport, can be explained by a direct effect on the membrane subsequent to receptor binding. Other actions such as inhibition of protein degradation and stimulation of DNA synthesis are intracellular events and require either a second messenger or the entry into the cell of active insulin or an active insulin fragment. Since a second messenger has not been convincingly demonstrated, the latter alternative has been proposed (Goldfine, 1977; Steiner, 1977). Cellular uptake of insulin has been demonstrated in cultured lymphocytes, and 10% of the uptake is localized to receptors found in the nuclear fraction. Uptake by this fraction is a saturable process and displays a slower time course than surface membrane uptake (Goldfine et al., 1977a). In an experiment of this type it is usually difficult to exclude an alternative interpretation-surface membrane contamination of the nuclear fraction. However, a major difference between nuclear receptors and surface membrane receptors has been reported. Surface membrane receptors avidly bind an antiinsulin antibody (isolated from an insulin-resistant patient), while nuclear receptors exhibit little affinity for the antibody (Goldfine et d., 1977b). At present the physiological significance of intracellular insulin is unknown, and a large fraction of the internalized insulin may constitute a degradative pathway (Terris and Steiner, 1976).

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E. Epidermal Growth Factor

Epidermal growth factor (EGF), a MOO-dalton mitogenic peptide is also bound to high-affinity receptors, internalized, and then degraded. However, as is the case with insulin, it is not known whether internalization, with or without degradation, is required to elicit a mitogenic response (Carpenter and Cohen, 1976). Most of the internalized EGF appears to be degraded within the lysosome, since degradation is blocked by chloroquine. Degradation is also blocked by inhibitors of metabolic energy production, various local anesthetics and, interestingly, ammonium ion (see Section 11,A74).When EGF is bound to fibroblasts at 4"C, followed by rapid warming to 37"C, the kinetics of entry can be followed by sequentially incubating cells with '251-labeledanti-EGF. The t1,2of entry is about 2 minutes, and no surface EGF accessible to antibody is found at 8 minutes (Carpenter and Cohen, 1976). Anderson et d.(1977a) point out that the kinetics of entry of human EGF into human fibroblasts are strikingly similar to those observed for LDL. These workers speculate that EGF may also enter via clustered receptors in coated regions (see Section 111,C73).

IX.

SUMMARY

A. intracellular localization Following Transport

We have reviewed the literature on several proteins which are transported from the extracellular environment to the cell interior by receptor-mediated processes. These proteins have different functional activities and different intracellular sites following transport. The proteins we have considered can be classed into various groups. Thus there are toxins, exemplified by diphtheria toxin, abrin and ricin, tetanus toxin, botulinum toxin, and cholera toxin. In addition there is the bactericidal toxin colicin. Growth factors and hormones represent a second group, and among these are NGF, glycoprotein hormones, and lactogenic hormones. TC 11, transferrin, and LDL are grouped as carrier proteins. In addition, we have considered antibodies, desialylated glycoproteins, lysosomal hydrolases, and viruses. The data on receptor-mediated protein transport can also be organized with respect to the intracellular localization of physiologically active proteins following transport. Thus diphtheria toxin, abrin and

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ricin, colicins, and some viruses are known to be physiologically active within the cytosol following transport. Other proteins are known to be active at intracellular sites. However, the specific intracellular site of action has not been determined. The site of action is believed to be extralysosomal for some of these proteins, since they are transported by retrograde axonal transport long distances away from their site of uptake and survive destruction during this process. NGF, tetanus toxin, and botulinum toxin fit into this group. Transferrin is transported into the cell and then out of the cell, without being structurally or functionally altered (except for the loss of iron), and the presumption is that the intracellular site must b e extralysosomal. The interior of the plasma membrane may be the site of action of cholera toxin, or this protein may be transported to the cytosol. Glycoprotein hormones appear to be similar to cholera toxin in some respects. Proteins transported to the lysosomes are LDL, TC 11, fibroblast lysosomal hydrolases, and desialylated glycoproteins. Some viruses are also transported into lysosomal vesicles but subsequently lyse these vesicles and escape into the cytoplasm. Finally, there is a group of proteins which are transported across the cell to another compartment. Prolactin and antibodies are both transported across mammary gland cells into the milk. Tetanus toxin appears to be transported across the presynaptic junction. Transferrin crosses the cell membrane twice and is the only reported example of bidirectionality among the proteins discussed here. The state of the proteins following transport into the cell is largely unknown, but in two cases proteins are complexed to intracellular binding proteins (TC I1 and transferrin). 8. Mechanisms of Transport

The biochemical mechanisms of transport for these proteins are largely unknown. Intuition tells us that a very large energy barrier must be exceeded to pull a large hydrophilic protein across a hydrophobic lipid bilayer. Therefore specialized processes to lower this energy barrier are expected. Receptor-mediated pinocytosis is such a process and has been postulated for many of the proteins covered in this article. This model is particularly attractive for cases in which the protein is known to be localized and then degraded in the lysosomes, since considerable evidence exists that various types of pinocytotic vesicles fuse with lysosomes (Steinman et al., 1974). The model is pictorially represented in Fig. 1, steps 1 through 4a. The receptors serve to concentrate the protein on the surface membrane so that the uptake is higher than that achievable by bulk fluid pinocytosis. Uptake pro-

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ceeds as new receptors are supplied to the surface membrane. Proteins which gain entry to the cytosol to elicit their effects, such as the toxins described in Section 11, or proteins which escape degradation, such as transferrin, require a second transport process to escape from the lysosomal compartment. This is pictorially represented in Fig. 1, step 4b. No supporting evidence for this latter model exists. Sufficient data to evaluate the receptor-mediated pinocytotic model are available for five systems: asialoglycoprotein uptake by liver, TC I1 uptake by liver and leukemic cells, LDL uptake by human fibroblasts, and E G F uptake by human fibroblasts. In each case uptake is too rapid for a model having an even distribution of membrane receptors. For these cases the model must be modified to include clustering of receptors at pinocytotic sites, as shown in Fig. 2. Very convincing evidence for this model has been obtained b y Anderson et al. (1977a) (Section III,C,3) for LDL. The model as drawn requires a second receptor protein site which serves to cluster the receptors in the coated regions, a condition required by genetic data. Two types of protein transport models not involving pinocytosis are shown in Fig. 3. Support for nonpinocytotic models comes from colicin data (Section 11,F,3), which indicate that proteins can share transport systems designed for low-MW substances such as chelated iron. The upper part of Fig. 3 shows a model in which any substance bound to the receptor enters the cell as the receptor is pulled in b y some mechanism. This is a single-interaction model, and binding is a sufficient condition for entry. In the lower part of Fig. 3 the act of binding opens a gate through which the bound protein, or a fragment of the bound protein, passes, but not the receptor. This may be a single-interaction model, the receptor undergoing a flip-flop motion through the gate, leaving the protein in the intracellular compartment. Alternatively, two interactions may be required, one with the receptor and the other with the gate. The second interaction may be negative; i.e., only proteins of a limited size or configuration can pass through the gate. Or the interaction may be positive and the gate may actually be a shuttle mechanism, accepting the protein from the receptor. No evidence for these models exists. They are presented merely as the most general types of testable models we could devise. At present, however, sufficient data to test these models are not available. As we have indicated, tests of nonpinocytotic models which provide entry to the cytosol compartment are complicated by high levels of pinocytosis going to the lysosomal compartment. Therefore, as is the case with the toxins, the more interesting transport process producing toxicity is masked by a degradative pathway.

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1

1

transport mechanism

2

1

mechanism

mechanism

FIG.3. Top: A nonpinocytotic entry model in which any substance bound to the receptor enters the cell as the receptor is pulled in by some mechanism. This is a single-interaction model; binding is a sufficient condition for entry. Bottom: The act of binding opens a gate through which the bound protein, or a fragment of the bound protein, passes, but not the receptor. This could be a single-interaction model, the receptor undergoing a flip-flop motion through the gate, leaving the protein in the intracellular compartment. Alternatively, two interactions may be required, one with the receptor and the second with the gate. Transport models involving gates have been proposed to explain the active transport of small ions (Shamoo and Goldstein, 1977).

Finally, it should be realized that there are non-receptor-mediated transport processes for high-MW polymers across epithelial surfaces. These processes are nonsaturable. The transport of polymers higher than 50,000 daltons has been observed. The amount of transport is inversely related to size and is bidirectional, and a linear relationship between log MW and log clearance has been observed (Loehry et al., 1970). A distribution of pore sizes, possibly in epithelial tight junctions, has been proposed to explain these findings (Smulders and Wright, 1971). Studies with inhibitors have provided some data for and against various transport models. Inhibitors of oxidative phosphorylation and glycolysis inhibit the transport of many of these proteins into cells. For diphtheria toxin several specific inhibitors have been found. Ammonium ion and poly-L-ornithine both inhibit transport. This raises the question whether or not this toxin shares an amine or polyamine transport system. Ruthenium red is also an inhibitor of diphtheria

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toxin transport, which suggests that calcium-magnesium-ATPase may b e involved. Cytochalasin B depresses the transport of many of these proteins, but the significance of this is not understood. It appears, however, that contractile proteins very similar to muscle actin and myosin are localized beneath the cell membrane in unique arrays, and that these arrays effect membrane receptor distribution (Ash and Singer, 1976).Whether or not these contractile proteins play a role in receptor-mediated entry processes remains to be determined. The most detailed data on protein transport across cell membranes exist for colicin transport systems which are shared by iron transport, vitamin Bl2 transport, and certain bacteriophage transport. Most of these data have been derived from genetic manipulations. It is known that at least two gene products are involved, one of which is the receptor for the specific colicin. The second gene product affects not only the specific colicin transport but a group of transport processes also. It is likely that an understanding of receptor-mediated transport in eukaryotic cells will require a genetic approach. C. Unique Functions of Receptor-Mediated Protein Transport

The requirement for an obligatory binding step to a cell surface receptor preceding transport achieves a high degree of cell type specificity for the transport process. Cells lacking the specific receptor are incapable of transporting the protein to the intracellular site. Thus asialoglycoproteins are removed from the bloodstream almost exclusively b y the liver. The uptake of NGF is largely limited to sympathetic neurons. It has been postulated in the latter case that trophic phenomena may also occur. Thus local release of NGF may stimulate neurite outgrowth along particular paths, and this process may function to establish unique neuronal connections. The degree of cell type specificity achieved by some receptor-mediated transport processes is enormous. Certain eukaryotic cells which are insensitive to diphtheria toxin are over 10,000times more insensitive than sensitive cells. This sensitivity involves the receptor-mediated transport process, since the active fragment of the toxin is totally active in broken cell preparations. The high degree of selectivity may involve other mechanisms besides transport, such as protection of the active chain from degradation. A second unique characteristic of receptor-mediated protein transport is the specificity of intracellular compartmentalization achieved. Thus asialoglycoproteins are degraded within the lysosomes, whereas transferrin escapes this fate and is transported out of the cell to func-

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tion again. NGF, after crossing the plasma membrane, is transported in a retrograde fashion and finally localized in the nucleus of the sympathetic neuron. Other proteins, notably toxins, become localized in the cell cytosol. The biochemical mechanism of intracellular compartmentalization is unknown. Few data are available to indicate whether this process is directed by the specific receptors involved or whether it is a function of a portion of the transported molecule. The specificity for compartmentalization appears to be localized to only a portion of either the transported molecule or the receptor. Rogers and Kornfield (1971) synthesized hybrid molecules of fetuin and albumin and fetuin and lysozyme. Lysozyme and albumin are not normally taken up by the liver and degraded in hepatic lysosomes. However, following coupling with fetuin and treatment with neuraminidase to remove the sialic acid residues, the hybrid molecule is processed similarly to desialylated fetuin, and the lysozyme and albumin components are degraded within the hepatic lysosomes. The remarkable similarities in the structure of diphtheria toxin, abrin, ricin, tetanus toxin, and botulinum toxin suggest that the common structural features are in some way necessary for the function of these toxins. The functions appear quite diverse, however, cytosol localization may be a common feature. A puzzling feature of toxins is how these organisms evolved and then maintained biochemical processes so highly integrated.with those of eukaryotic cells (Pappenheimer and Gill, 1973: Collier, 1975). Since a number of the toxin genes are phage-specific, evolvement could have come about by way of genetic transfer in either direction. Maintenance implies a selective advantage for both organisms. For the plant seed toxins abrin and ricin an obvious selective advantage exists, since oral ingestion of the seeds leads to death. For bacterial toxins, the death of a host under certain conditions may be a selective advantage. It is interesting to note that diphtheria toxin, botulinum toxin, and tetanus toxin genes are all regulated by the media iron content. Toxins are made in high quantity only under conditions of limiting iron. Iron is probably quite limiting for nondestructive bacteria within a vertebrate host, because of the high binding constant with which iron binds transferrin. The liberation of toxin leading to the destruction of a host places the bacterial population in a high-iron evironment as the decomposition of host iron proteins proceeds. The utility for a vertebrate organism transporting these toxins to their intracellular site of action is difficult to perceive. However, just as colicins utilize transport systems for metabolites, the same may be

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true for these toxin transport systems. Since these are low-capacity transport systems, they may have escaped detection, especially if they are involved in trace-metal transport. Another possibility is that toxin transport systems were originally derived for peptide and protein transport similar to that for NGF. There are probably many such systems in existence which have escaped detection because of the low levels of these circulating materials.

X. PHARMACOLOGICAL IMPLICATIONS OF RECEPTOR-MEDIATED PROTEIN TRANSPORT

The unique feature of receptor-mediated protein transport is that certain proteins are fixed by specific cell types and then transported to a specific intracellular site where the protein (or a fragment of the protein) exerts its biological effects. These effects can be quite profound-inhibition of protein synthesis for diphtheria toxin, abrin, and ricin, and stimulation of adenylate cyclase activity for cholera toxin. The evidence previously summarized indicates that NGF, botulinum toxin, and tetanus toxin also act intracellularly. NGF induces two key enzymes; botulinum toxin and tetanus toxin inhibit the release of stimulatory and inhibitory neurotransmitters, respectively. We have suggested (Chang and Neville, 1977), that the phenomena associated with receptor-mediated protein transport can be exploited to achieve the synthesis of an entirely new class of pharmacological reagents which would exhibit cell type-specific activities. This could be achieved by constructing hybrid- proteins, utilizing the binding chain of one protein (or the more specific receptor recognition factor when known) and the active chain of a different protein. An obvious application of such hybrids is cell type-specific cancer chemotherapy. For example, if the binding portion of NGF were hybridized with the active chain of diphtheria toxin, the resulting reagent would inhibit protein synthesis specifically in sympathetic neurons or related cell types. Such a reagent might be effective in destroying tumors of sympathetic neuronal origin, such as neuroblastomas. Metastatic lesions would be equally susceptible, as long as they carried NGF. Hybrids containing binding chains directed at tumor-specific antigens may also be effective as tumor-specific reagents. Hybrid protein reagents may have considerable application in the mental health field. It is quite likely that the high degree of differen-

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tiation exhibited by the nervous system is mirrored in receptor differences in various groupings of neural nets and nuclei within the central nervous system. By combining the active chain of tetanus toxin with the right binding chain it might be possible to depress inhibitory neurotransmitter release in particular portions of the brain. Similarly, by using the active fragment of cholera toxin, localized stimulation of adenylate cyclase might be achieved in a distribution independent of the receptors normally linked to this enzyme. Many other possibilities also exist. In order to achieve these ends, considerably more must be known about the interrelationships between receptor binding and protein transport. Do all receptors at some low level mediate entry to the cytosol compartment? Or does only a certain class of receptors with unique properties have this function? Is entry of the protein or its active chain determined uniquely by the binding chain, or does the active chain require certain properties essential for entry? The available data summarized in this article shed little light on these questions. As an initial attempt to answer some of these questions, we have devised a general method for the synthesis of disulfide-linked artificial protein hybrids in high yield (Chang and Neville, 1977; Chang et al., 1977). The method involves the reaction of an exogenously introduced alkyl thiosulfate group on one protein with a sulfhydryl group on another. This reaction is more rapid than any intraspecies competing reactions and avoids gross contamination with homopolymers, which usually occurs with conventional bifunctional cross-linking reagents (Chang and Neville, 1977). By utilizing this methodology, the hybrid human placental lactogen-SS-diphtheria toxin fragment A has been synthesized. The hybrid maintains 26% of its binding activity toward lactogenic receptors and one-third of its toxin A enzymic activity as assayed in a cell-free system. However, the hybrid is without detectable effect on the protein synthetic rate of organ-cultured lactating mammary gland explants carrying the lactogenic receptor (Chang et aZ., 1977). Although the artificial protein hybrid we have synthesized is a structural analog of diphtheria toxin with an altered binding chain specificity, it does not behave as a functional analog. The reasons for this are not yet apparent, We do not know whether or not the lactogenic receptor mediates entry to the cell cytosol where the A chain must localize to be functional. Prolactin transport into milk may occur via another intracellular compartment. Our disulfide-linked hybrid may split at the membrane binding site because of lack of protection of

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the disulfide bond, which is provided in diphtheria toxin by the diphtheria toxin-binding chain. The toxin A fragment may be inactivated in the artificial hybrid, whereas protection of the active site is probably provided by the B chain in the native toxin. Olsnes et al. (1974) constructed functional hybrids of abrin and ricin A and B chains. Although these molecules are closely related in binding and enzymic properties, they are nevertheless immunologically distinct. Further work will be required to understand the range of possibilities and limitations existing for the synthesis of functional protein hybrids which utilize receptor-mediated transport processes. REFERENCES Aasa, R., Malmstrom, B. G., Saltman, P., and VanngBrd, T. (1963).The specific binding of iron(II1) and copper(I1) to transferrin and conalbumin. Biochim. Biophys. Acta 75,203-222. Adams, E. B. (1971). The clinical effects of tetanus. In “Neuropoisons; Their Pathophysiological Actions” (L. L. Simpson, ed.), Vol. 1, pp. 213-224. Plenum, New York. Anderson, R. G. W., Goldstein, J. L., and Brown, M. S. (1976). Localization of lowdensity lipoprotein receptors on plasma membrane of normal human fibroblasts and their absence in cells from a familial hypercholesterolemia homozygote. Proc. Natl. Acad. Sci. U.S.A.73, 2434-2438. Anderson, R. G. W., Brown, M., and Goldstein, J. L. (1977a).Role of the coated endocytotic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts. Cell 10, 351-364. Anderson, R. G. W., Goldstein, J. L., and Brown, M. S. (1977b). A mutation that impairs the ability of lipoprotein receptors to localize in coated pits on the cell surface of human fibroblasts. Nature (London) 270, 695-699. Ash, J. F., and Singer, S. J. (1976). Concanavalin-A-induced transmembrane linkage of concanavalin A surface receptors to intracellular myosin containing filaments. Proc. N n t l . Acad. Sci. U.S.A.73,4575-4579. Ashwell, G., and Morell, A. G. (1974). The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adu. Enzymol. Relat Areas M o l . Biol. 41,99-128. Ashwell, G., and Morell, A. G. (1977). Membrane glycoproteins and recognition phenomena. Trends Biochem. Sci. 2, 76-78. Awai, M., and Brown, E. B. (1963). Studies of the metabolism of 1131-labeledhuman transferrin. /. Lab. Clin. Med. 61, 363-396. Baker, M. E. (1975).Molecular weight and structure of 7 S nerve growth factor protein. /. B i d . Chem. 250, 1714-1717. Banejee, S. P., Snyder, S. H., Cuatrecasas, P., and Greene, L. A. (1973). Binding of nerve growth factor receptor in sympathetic ganglia. Proc. Natl. Acad. Sci. U.S.A. 70,2519-2523. Banejee, S. P., Cuatrecasas, P., and Snyder, S. H. (1975).Nerve growth factor receptor binding. Influence of enzymes, ions and protein reagents. 1. Biol. Chem. 250, 1427-1433.

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Fiedler-Nagy, C., Rowley, G. R., Coffey, J. W., and Miller, 0. N. (1975). Binding of vitamin B,,-rat transcobalamin I1 and free vitamin BIZto plasma membranes isolated from rat liver. Br. J. Hematol. 31,311-321. Fielding, J,, and Speyer, B. E. (1974). Iron transport intermediates in human reticulocytes and the membrane binding site of iron-transferrin. Biochim. Biophys. Acta 363,387-396. Fillenz, M., Gagnon, C., Stoeckel, K., and Thoenen, H. (1976). Selective uptake and retrograde axonal transport of dopamine-P-hydroxylase antibodies in peripheral adrenergic neurons. Brain Res. 114,293-303. Finkelstein, R. A. (1973). Cholera. Crit. Rev. Microbiol. 2,553-623. Finkelstein, R. A. (1975). Observations on the cholera enterotoxin (choleragen).Jpn. J. Med. Sci. Biol. 28, 76-78. Finkelstein, R. A., Boesman, M., Neoh, S. H., LaRue, M. K., and Delaney, R. (1974). Dissociation and recombination of the subunits of the cholera enterotoxin (choleragen).J. Zmmunol. 113, 145-150. Frazier, W. A., Anyeletti, R. H., and Bradshaw, R. A. (1972). Nerve growth factor and insulin. Structural similarities indicate an evolutionary relationship reflected by physiological action. Science 176,482-488. Frazier, W. A,, Boyd, L. F., and Bradshaw, R. A. (1973). Interaction of nerve growth factor with surface membranes: Biological competence of insolubilized nerve growth factor. Proc Natl. Acad. Sci. U.S.A. 70,2931-2935. Gavin, J. R., 111, Roth, J., Neville, D. M., Jr., De Meyts, P., and Buell, D. N. (1974). Insulin-dependent regulation of insulin receptor concentrations: A direct demonstration in cell culture. Proc. Natl. Acad Sci. U.S.A. 71, 84-88. Gamin, J. L., and Gelehrter, T. D. (1974). Induction of tyrosine amino-transferase by Sepharose-insulin.Arch. Biochem. Biophys. 164,52-59. Gill, D. M., and Dinius, L. L. (1971). Observations on the structure of diphtheria toxin. J. Biol. Chem. 246,1485-1491. Gill, D. M., and King, C. (1975). The mechanism of action of cholera toxin in pigeon erythrocyte 1ysates.J. Biol. Chem. 250,6424-6432. Gitlin, J. D., and Gitlin, D. (1974). Protein binding by specific receptors on human placenta, murine placenta, and suckling murine intestine in relation to protein transport across these tissues.J. Clin. Invest. 54, 1155-1166. Gitlin, J. D., Gitlin, J. I., and Gitlin, D. (1976). Selective transfer of plasma proteins across mammary gland in lactating mouse. Am. J. Physiol. 230, 1594-1602. Click, J. M., Kerr, S. J., Gold, A. M., and Shemin, D. (1972). Multiple forms of colicin E 3 from Escherichia coli. Biochemistry 11, 1183-1188. Goldfine, I. D. (1977). Does insulin need a second messenger? Diabetes 26, 148-155. Goldfine, I. D., Smith, G. J., Wong, K. Y.,and Jones, A. L. (1977a).Cellular uptake and nuclear binding of insulin in human cultured lymphocytes: Evidence for potential intracellular sites of insulin action. Proc. Natl. Acad. Sci. U.S.A. 74, 1368-1372. Goldfine, I. D., Vigneri, R.,Cohen, D., Pliam, N. B., and Kahn, C. R. (1977b). Evidence that intracellular binding sites for insulin are immunologically distinct from those on the plasma membrane. Nature (London) 269,698-700. Goldstein, J. L., and Brown, M. S. (1976). The LDL pathway in human fibroblasts: A receptor-mediated mechanism for the regulation of cholesterol metabolism. Curr. Top. Cell. Regul. 11, 147-181. Goldstein, J. L., and Brown, M. S. (1977). The low density lipoprotein pathway and its relation to atherosclerosis. Annu. Rev. Biochem. 46,897-930. Goldstein, J. L., Brown, M. S., and Stone, N. J. (1977). Genetics of the LDL receptor:

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The Regulation of Intracellular Calcium* E R N E S T 0 CARAFOLl A N D M A R T I N C R O M P T O N Laboratory of Biochemistry Swiss Federal lnstitute of Technology ( E T H ) Zurich. Switzerland

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 I1 . The Chemical Basis of the Biological “Fitness” of Caz+ . . . . . . . . 153 111. The Influence of CaZ+on the Molecular Architecture and Functional Prop-

erties of Biological Membranes . . . . . . . . . . . . . . . . . . . IV . Intracellular Concentrations of Ca2+ . . . . . . . . . . . . . . . . . V . General Considerations on the Regulation of Intracellular Caz+ . . . . VI . The Transport of Caz+across Plasma Membranes . . . . . . . . . . . A . The Influx of Caz+into Cells . . . . . . . . . . . . . . . . . . B. The Effiux of Caz+from Cells . . . . . . . . . . . . . . . . . . VII . The Transport of Caz+by Sarcoplasmic and Endoplasmic Reticulum . . A . Sarcoplasmic Reticulum of Fast Skeletal Muscle . . . . . . . . . . . . B. Sarcoplasmic Reticulum of Cardiac and Red Skeletal Muscle C . The Release of Caz+ from the Sarcoplasmic Reticulum . . . . . . D . Endoplasmic Reticulum .................... . . . . . . . . . . . . . . . VIII . The Transport of Caz+by Mitochondria A . General Properties . . . . . . . . . . . . . . . . . . . . . . . B . The Transport Mechanism . . . . . . . . . . . . . . . . . . . C . The Capacity of Mitochondria to Accumulate Ca2+and the Reversibility of the Transport Process in V i m ; the Mechanism of CaZf Efflux from Mitochondria . . . . . . . . . . . . . . . . . . . . . . . IX . The Transcellular Transport of Ca2+ . . . . . . . . . . . . . . . . . X . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

1

157 160 164 166 166 168 173 173 178 180 182 183 183 187 189 193 195 197

INTRODUCTION

In addition to its role as a structural element. Ca2+ is uniquely important in the regulation of cell function. since it acts as a “messenger” that coordinates a multiplicity of intracellular reactions . The * Dedicated to A . L . Lehninger on the occasion of his sixtieth birthday . 151

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ERNEST0 CARAFOLI A N D MARTIN CROMPTON

number of processes known to be affected by changes in the ambient concentration of ionized Ca2+grows continually, and in all probability the list given in Table I will be far from complete in a few years’ time. The processes listed are the targets for Caz+,and the delivery of Ca2+ to these processes is controlled largely by movements across the membranes of the cell and the subcellular organelles. A natural consequence of the selection of Ca2+as an intracellular TABLE I

REACTIONS CA~+-DEPENDENT

IN

CELLS

Activation of enzyme systems Clycogenolysis (phosphorylase-b kinase) Lipases and phospholipases a-Clycerophosphate dehydrogenase Pyruvate dehydrogenase Succinate oxidation Synthesis of some phospholipids NADH dehydrogenase (plant mitochondria) Interaction of cytochrome c with the mitochondria1 membrane Light emission Decision to divide Inhibition of enzyme systems Pyruvate kinase Synthesis of some phospholipids Substrate oxidation (NADH leakage) in lung mitochondria Activation of contractile and motile systems Muscle myofihrils Cilia and flagella Microtubules and microfilaments Cytoplasmic streaming Pseudopod formation Hormonal regulation Formation and/or function of CAMP (CH, LH, TSH, MSH, PTH) Release of insulin, steroids, vasopressin, oxytocin, catecholamines, thyroxine, and progesterone Membrane-linked €unctions Excitation-secretion coupling at nerve endings Excitation-contraction coupling in muscles Exocrine secretion (pancreas, salivary glands, and HCI i n the stomach) Aggregation of platelets Action potential (nerve and muscle cells) Na+, K+,-ATPase of several membranes Tight junctions Cell contact Binding of prostaglandins to membranes

153

THE REGULATION OF INTRACELLULAR CALCIUM

messenger is the necessity for its precise regulation. A cursory inspection of the distribution of the four principal cations in the intracellular and extraceIIular spaces already underlines this point clearly (Table 11). It is evident that the intracellular concentration of Ca2+is maintained at a level much lower than the levels of the other three cations.The purpose of this article is to discuss the mechanisms that have evolved for controlling the fluxes of Ca2+ across these membranes, which thereby direct a multitude of cellular events. The discussion of the different membrane systems involved in the regulation of Ca2+is prefaced by some general considerations of the chemistry of Ca2+which may be helpful in understanding the basis of its role as a biological messenger. Then the discussion considers the structural role of Ca2+ in different membrane systems and the location and physical state of Ca2+ within cells. Next the different membrane systems involved in Caz+ regulation in vivo are discussed: the plasma membrane, the endo- and sarcoplasmic reticulum, and the mitochondria. In each case, emphasis is placed on our current knowledge of the mechanism of the transport process and on its possible physiological role. At the end of the discussion on the intracellular transport of Ca2+, its transcellular transport is also briefly considered.

II. THE CHEMICAL BASIS OF THE BIOLOGICAL "FITNESS" OF CaZf

The immediate question is: Why has Ca2+been chosen during evolution rather than other cations available in the environment? A useful way to discuss the reasons for the biological fitness of Ca2+is to comTABLE I1 THE DISTRIBUTION OF NA+, K+, CAZ+, AND MC2+IN THE ENVIRONMENT AND IN ANIMALFLUIDSO

Cation Na+

K+ CaZ+ Mg2+

Sea water (mM)

Human plasma

Mammalian cell water

(mM)

(mW

490 9.8 10 54

135-145 5.3 3.2 1.1

12-20 150 0.03-0.06 2.8

These values, which refer to total concentrations, are from Davson (l970),with the exception of those for intracellular cations, which refer to the erythrocyte and are from the work of investigators mentioned in the text.

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ERNEST0 CARAFOLI A N D MARTIN CROMPTON

pare its properties to those of the three other principal cations in living organisms. The comparison begins with a consideration of ionic radii. In this article, the discussion is based on the nonhydrated rather than the hydrated radii of the cations (Table 111). This is at variance with the traditional choice based on criteria of solution chemistry. However, consideration of the nonhydrated radii appears to be more appropriate, since analyses of metal complexes, including natural molecules involved in membrane transport, have indicated that when the metal is bound it is largely dehydrated, and that the coordination requirements are provided essentially by the ligand (e.g., see Urry, 1972; Hille, 1975; Dunitz and Dobler, 1977). In addition, interaction with dehydrated metal ions is advantageous from the standpoint of the selectivity of interaction. If the interacting metal atom were surrounded by a shell of water molecules, the selectivity of interaction would be dictated by the size of the hydrated ion and not by the other parameters discussed here. The smallness of the Mg2+ion is certainly a factor that limits the possibility of its being controlled, since it is difficult to imagine, or to build, “cavities” small enough to provide a selective fit for Mg2+ions. A M$+-selective cavity would require only six coordinating (oxygen) atoms around the central Mg2+ion, a highly improbable situation in view of the mutual repulsion of the atoms of the coordination sphere. With eight coordinating atoms, the cavity would, however, become too large to provide an optimal fit. These theoretical difficulties are reflected in the fact that so far no ionophore specific for Mg2+has been produced, and by the generally recognized fact that ligands showing a high specificity for Mg2+are very rare in biology. Additional factors in the interaction between metals and complex ligands are the charge of the ion and its polarizing power. The polarizing power, being directly proportional to the charge and inversely proportional to the radius, decreases in group IIA of the periodic table from Be2+to M$+ and Ca2+, with the result that a cation like Mgz+exerts more attraction than Ca2+ on the polarizable atoms of the coordination sphere; hence the donor atoms (oxygen, nitrogen, sulfur, and so on) tend to assume a regularly TABLE 111 THE IONIC RADII OF C A ~ +M , d + , NA+, AND Kfa

Ionic radius a

(A)

Mpz+

Ca2+

Na+

K+

0.65

0.94

0.98

1.33

From Hanzlik (1976).

THE REGULATION

OF INTRACELLULAR CALCIUM

155

spaced structure determined by the strong attraction exerted by Mg2+. The relatively weaker polarizing power of Ca2+ allows the donor atoms to maintain irregular spacings essentially determined by their mutual repulsion. Examination of the structures of some simple organic and inorganic complexes of Ca2+ and M$+, available from x-ray diffraction studies (Williams, 1975, 1976; Tables IV-VI) verifies these principles. As mentioned before, Mg2+has an optimal coordination number of 6, whereas the number for Ca2+is normally at least 6 and usually 7 or 8. As expected from the rather high polarizing power of Mg2+, however, its coordination bonds invariably are of similar length and subtend a similar angle, thus maintaining a regular octahedral geometry around the cation, whereas those of Ca2+may vary very widely; as a consequence, in the case of Ca2+irregular geometries of the coordination site are usually permitted. It is immediately evident therefore that Ca2+ is a far more versatile ligand than Mg2+ and can thus adapt to an ample choice of irregular binding sites such as those offered by complex organic molecules like proteins. It can also be predicted from these considerations that Ca2+ would be ideal in crosslinking irregular monodentate ligands, as is indeed frequently the case (Williams, 1975). An additional, important factor that restricts even further the possibility of biological interactions of Mg2+,as compared to those of Ca2+,is the tendency of the former to retain part of the hydration water when forming complexes. As a result, when presented with irregular binding sites, Mg2+can interact only with coordination centers that are compatible with its requirement for fixed bond length and angle and preserves the octahedral geometry through partial hydration (Williams, 1976). Considering these factors, one can TABLE IV SOMETYPICAL CA SALT STRUCTURES~ Ca-0 Salt

Coordination number

distance (nm)

Minimum ~~

Ca HPOI.2HzO Ca (H2PO&.H20 Ca 1,3-diphosphorylimidazole Ca dipicolinate.3HZ0 Ca Na (H,PO,), Ca tartrate.4H~O Ca (C,HSO,)Z.BH,O From Williams (1976).

0.244 0.230 0.266 0.227 0.236 0.231 0.239 0.239

Maximal variability permitted (nm)

Maximum ~

~

0.282 0.274 0.236 0.278 0.257 0.233 0.254 0.247

0.052

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ERNEST0 CARAFOLI A N D MARTIN CROMPTON

TABLE V CA

STRUCTURES OF S O M E

BIOLOGICALMOLECULES" Ca-0

Coordination nunher

Compound

7 8

Ca2+thymidylate Ca2+diphosphonate CaZ+galactose Ca2+blephavismin Ca2+behalose CaZ+arabonate

8 7

7 8

distance (nm)

Minimum

Maximum

0.230 0.240 0.235 0.235 0.235 0.245

0.265 0.260 0.255 0.245 0.255 0.250

Maximal variability permitted (nm)

0.035

From Williams (1976).

understand why the preference of ligands for Ca2+ rather than Mg2+ tends to increase as the complexity of the ligand increases (see Table VII). One factor, however, militates against the great versatility of Ca2+interaction and thus imposes a limit on its ability to interact optimally with an unlimited number of coordination sites. A coordination sphere containing also nitrogen or sulfur would have less preference for CaZ+than one containing only oxygens. This is because nitrogen and sulfur are more polarizable than oxygen; as a result, cavities containing nitrogen or sulfur in the coordination sphere may prefer the more polarizing MgZf over Ca2+. In fact, optimal Ca2+-binding sites usually contain only oxygens as the coordinating atoms, even if the rule is not absolute, e.g., in the case of EDTA (Williams, 1976). TABLE VI SOME

TYPICAL !dAGNESIUM

S A L T STRUCTURES

Mg-0

distances (nm)

Coordination number

Minimum

Maximum

Mg hexaanti-

6

0.206

-

pyrine.CI0, Mg (C,H&h.BrZ MgS203'6HzO MgS04'4H20 My (HPOJ'GH20 MRPzO, Mg (CHSCOJz.4HzO

6 6 6 6 6 6

0.216 0.205 0.204 0.200 0.200 0.200

0.212 0.209 0.212 0.211 0.210

Salt

From Williams (1976).

Maximal variability permitted (nni)

0.012

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THE REGULATION OF INTRACELLUIAR CALCIUM

TABLE VII CA'+-

A N D %lG'+-BINDING CONSTANTS"

Ligand

Binding constant for Mg2+

log,, K (lo-' moles/dm3) for CaZ+

Glycine I mido w etate Ni triloacetate E DTA EGTA Acetate Malonate Ci trate

3.4 2.9 5.3 8.9 5.4 0.8 2.8 3.2

1.4 2.6 6.4 10.7

10.7 0.7 2.S 4.8

" From Williams (197s).

It seems logical to suggest that the chemical properties of Ca2+discussed in this section may provide the basis for its role as a biological messenger. It is interesting that in many instances, particularly at the membrane level, the two cations that interact most closely are Na+ and Ca2+.Very likely, Ca2+and Na+ originally represented equivalent evolutionary choices. A common evolutionary origin, for instance, of the Na+ and Ca2+channels in the plasma membrane has been suggested (Weber, 1976). Of the factors relevant to the interaction between metals and complex ligands, one, the ionic radius, is nearly the same for the two cations, and this may well be the most important factor in the close interplay between Na+ and Ca2+.I n addition, as is the case for Ca2+,the coordination bonds of Na+ may vary greatly in length and angle (Table VIII), thus allowing irregular geometries of the coordination site. 111.

THE INFLUENCE OF CaZf ON THE MOLECULAR ARCHITECTURE AND FUNCTIONAL PROPERTIES OF BIOLOGICAL MEMBRANES

It follows from the previous discussion that Ca2+may have a role in the interactions within biological membranes, hence in their stability and function. Indeed, the literature contains several observations that point to a role of Ca2+ in membrane stability (Carafoli, 1975a). Reynolds and Trayer (1971) found that the association of several proteins with the erythrocyte membrane is influenced by Ca2+and other cations. Sandri et al. (1976) showed that the extractability of the Ca2+-bindingglycoprotein from mitochondria1 membranes decreases

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ERNEST0 CARAFOLI A N D MARTIN CROMPTON

TABLE VIII SOMETYPICALNA SALT STRUCTURES Na-0 Coordination number Na2CO3.10H20 Na ascorhate

6 6

NazPO4C3H7O2.5H20 NalP,012.4HzO N a (CH,(CHZ)&OCOz-) NaHCO, N~ICBH~O~

6 6 6

distance (nm)

Minimum

Maximum

2.31 2.41 2.26 2.28 2.26 2.4 1 2.37 2.38 2.30

2.51 2.72 2.54 2.59 2.50 2.53 2.67 2.48 2.46

Maximal variability permitted (nm)

0.031

" From Williams (1975).

in the presence of Ca2+. Similarly, Carafoli (1975a) observed that Ca2+permits penetration of the glycoprotein into phospholipid membranes. The association of cytochrome c with the inner mitochondrial membrane is also promoted by CaZ+(Azzi et al., 1975). These appear to b e specific examples of Caz+ promoting the penetration of hydrophilic proteins into nonpolar phases (Gitler and Montal, 1972). It is conceivable that Ca2+is instrumental in the association of hydrophilic (extrinsic) proteins with membranes. Somewhat related is the observation (Vinogradov et al., 1972) of the penetration of NADH into the inner mitochondrial membrane in the presence of relatively high concentrations of Ca2+(- 0.5 &). The influence of Ca2+and other inorganic cations on the stability of the red cell membrane was demonstrated by Reynolds (1972),who observed that erythrocytes subjected to hypotonic conditions form vesicles only in the presence of Ca2+.Burger et al. (1968) and Duggan and Martonosi (1970) observed that the treatment of membranes from erythrocytes and sarcoplasmic reticulum with EDTA increases their passive permeability. Evidence for an increase in cation permeability, in general revealed by an increased loss of K+, has been provided also for smooth muscle (Bulbring and Tomita, 1969), kidney cortex (Kleinzeller et al., 1968), brain cortex (Ghrdos, 1960), liver (Kalant and Hickie, 1968; Geyer et al., 1955), cardiac muscle (Reiter, 1957), and several other plant and animal cell systems (Morrill et al., 1964). Morrill et al. (1964) have concluded that extracellular Caz+is important in maintaining high intracellular K+ (and low Na+) in living systems. The

THE REGULATION OF INTRACELLULAR CALCIUM

159

effects of Ca2+may well b e complex, however; in Hela cells, for example, the absence of Ca2+from the external medium leads to the intracellular accumulation of K+ (Morrill et al., 1964). The situation is particularly complex in erythrocytes, which increase their passive permeability to K+ when suspended in media containing little Ca2+ (Lundsgaard-Hansen, 1957; Maizels, 1959; Solomon, 1960, 1968). However, in metabolically poisoned and ATP-depleted erythrocytes, extracellular Ca2+ induces a large increase in K+ permeability (the so-called GBrdos effect, GBrdos, 1958). Finally, Rorive et al. (1972) demonstrated that the plasma of kidney tubular cells alters its permeability to water when Ca2+is transported from one side of the membrane to the other. These influences of Ca2+on the structure and properties of membranes can b e viewed in relation to its chemical properties. Manery (1966) attributed effects of the type just described to the great versatility of Ca2+in accepting complex ligands. The ability of Ca2+to interact with diverse membrane components may result in membrane dehydration, and probably in conformational changes in selected membrane dornaias, thereby altering the structure and permeability of the membranes. T h e observations of Trauble and Eibl (1974) on the effects of Ca2+ on ordered + fluid transitions in bilayer membranes are also of great interest in this respect. Ca2+(and M$+) stabilize the ordered state of the phospholipid bilayer (causing an increase in the transition temperature) by charge neutralization and can thus b e used to induce the fluid -+ ordered transition at constant temperature. Interestingly, monovalent cations lower the transition temperature, thus huidizing the bilayer. Effects of this type may of course explain changes in transmembrane transport rates and have, for example, been used to explain the conformational changes occurring in the axonal membrane during excitation (Tobias, 1964; Tasaki, 1968; Adam, 1970). Williams (1975) has suggested that Ca2+may be a factor influencing membrane asymmetry. Since Ca2+ (and M$+) are distributed asymmetrically across membranes, phospholipids that have polar head groups capable of complexing Ca2+are expected to distribute preferentially in the monolayer that faces the medium containing the higher Ca2+ concentration. Other factors, however, probably complicate the prediction; in the case of erythrocytes, for example, the membrane phospholipid that complexes Ca2+ most strongly is phosphatidylserine, which is present exclusively in the inner monolayer (Verkleiy et al., 1973), facing an ambient where the concentration of Ca2+is at least 100-fold lower than that outside the cell.

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ERNEST0 CARAFOLI A N D MARTIN CROMPTON

IV.

INTRACELLULAR CONCENTRATIONS OF Ca2+

In general, the total concentration ofintracellular Ca2+is in the range 0.2-10 mmoles/liter of cell water. However, only a small fraction of this is ionized. The most comprehensive data are those available for the giant squid axon which contains 0.2-0.4 mmole Ca2+/liter of internal volume (Brinley, 1976). Hodgkin and Keynes (1957) and Baker and Crawford (1972) observed that the diffusion coefficient of 45Ca2+ injected into the axons was less than 2%of that predicted if it were all free; this indicates that the ionized Ca2+ concentration in the axoplasm is less than 10 p M . The diffusion coefficient of injected radioactive M$+, however, is far higher, so that one-third to one-half of the intracellular M&+ (7 mmoles/liter) may be ionized (Fig. 1).De Weer (1976) recently measured intracellular Mg2+ in squid axons by exploiting the stimulatory effect of this ion on the plasma membrane Na+,K+-ATPase.He concluded that, of a total M&+ concentration of 6.7 mM, between 3 and 4 mM is free. These conclusions have been strengthened considerably by the work of Baker et al. (1971) and Di Polo (1976),who injected aequorin and arsenazo I11 to determine axoplasmic ionized Ca2+and obtained pCa values of less than 6.5. The K ,

0

3000

k

20m

1

g loo0 0 Distance (mm)

FIG.1. Self-diffusion of zeMg2+ (solid circles) and 4sCaz+(open circles) in axoplasm. An axon was injected with 2 mM z8MgZ+and %a*+ and left in artificial sea water at 21°C for 510 min. It was then frozen and cut into 1-mm sections for counting. The cannula was at the left-hand end. The bar marks the nominal site of injection. The curves are calculated from the following equation

c

-c,

= 1

2

h

(erf h + x +erf-) 2fi 2

- X

6

where C is the concentration of tracer at time t, and distance x from the middle of the injected patch and C o is the concentration at t = 0; h is half the width of the injected patch. The smooth curve for 45ca2+is drawn with D = lo-' cmz/sec, and for 28Mg2+with D = 2 x 10" cmz/sec. Axon diameter, 750 pm. (From Baker and Crawford, 1972.)

THE REGULATION OF INTRACELLULAR CALCIUM

161

of the Na+-Ca2+ exchange system of the axolemma for internal Ca2+is about 0.3 pM (see Section V1,B). This low level of ionized Ca2+is maintained against a concentration of 3-10 mM ionized Ca2+in the hemolymph (Blaustein, 1974; see also Brinley, 1976). Most of the axonal Caz+ is probably sequestered by the mitochondria, since treatment of the axon with CN- increases manyfold the efflux of Caz+ from the axon; moreover, the efflux is prevented by ATP, and oligomycin inhibits the effect of ATP (Baker et al., 1971; Blaustein and Hodgkin, 1969). In erythrocytes, the total concentration of Ca2+ is 30-60 pmoles/liter (Weed et al., 1969; Valberg et al., 1965).The concentration of ionized Ca2+may b e lower yet, if the K , of the Ca2+-ATPasefor internal Ca2+ (1-4 p M ; see Table XIXI) is any reflection of the ambient Ca2+ Concentration. T h e total concentration of Ca2+ in the plasma is about 3 mmoles/liter, of which about half is ionized (Walser, 1961; Neuman and Neuman, 1958). The Ca2+ content of heart and skeletal muscle is about 2 mmoles/liter, and the content of smooth muscle (stomach, bladder, myometrium) is somewhat higher, about 8-9 mmoles/liter (Wacker and Williams, 1968). Most of this Ca2+is probably sequestered by the sarcoplasmic reticulum and mitochondria, both of which are well developed in muscle tissue. A reasonable estimate of the ionized Ca2+ in these tissues can be arrived at from the known sensitivity of myofibrils to Ca2+.From the threshold of contraction in heart and skeletal muscle one can arrive at a free Ca2+concentration between 0.1 and 1 p M , whereas 95% of maximum tension is attained with about 10 pM Ca2+(Felo et al., 1965; Ebashi and Endo, 1968; Weber, 1966; Solaro et al., 1974). The muscle fibers of the crab Maia swuinado and the barnacle Balanus nubilis exhibit similar thresholds (Portzehl et al., 1964; Hagiwara and Nakajima, 1966).In addition, the mitochondria1 and reticular uptake processes for Ca2+ and several cytosolic enzymes thought to be regulated by Ca2+are half-saturated by Ca2+in the range 0.1-10 p M ; these examples are discussed in detail in Sections VII and VIII. Direct estimates of the free CaZ+concentration in other cells are also available. With the aid of aequorin as an indicator, the free Ca2+ has been estimated in neurons of Helix aspersa (Meech and Standen, 1975), in Chironomus salivary gland cells (Rose and Loewenstein, 1976), in the photoreceptor of Limulus (Brown and Blinks, 1974), and in Physarum polycephalum cells (Ridgway and Durham, 1976) to range between 0.13 and 1.3 p M . Similar figures have been calculated for the eggs of Medaka with the aid of the calcium ionophore A23187 (Ridgway et al., 1977).

162

ERNEST0 CARAFOLI A N D MARTIN CROMPTON

The distribution of Ca2+in intracellular compartments undoubtedly varies in different cell types, with mitochondria probably playing a dominant role in axonal Ca2+ sequestration. Rose and Loewenstein (1975) have provided direct evidence that a similar situation prevails in the salivary gland of Chironornus. Visualization of the Caz+injected into the cell by monitoring the glow of aequorin with a television camera, coupled to an image intensifier, revealed that Ca2+does not move within the cell but remains near the tip of the micropipet and then disappears from the cytosol. Experiments with mitochondria1 inhibitors (cyanide, ruthenium red) showed that the constraint on the free mobility of Ca2+ within the cell is exerted by the energy-linked uptake system in the mitochondria (Fig. 2). Berridge et uZ. (1975) reached similar conclusions for the salivary glands of CaZZiphoru by microprobe analysis. This approach has been applied to other tissues also. I n smooth muscle, for example, Ba2+,a Ca2+analog, becomes concentrated in electron-dense deposits in the mitochondria close to the cell membrane (Somlyo et al., 1974). In skeletal muscle, however, Winegrad (1970), utilizing autoradiographic techniques, found that most of the CaZ+is stored in the cisternae of the sarcoplasmic reticulum. Other approaches have involved determining the Ca2+ content of purified cell fractions. In this case, there is the problem of the redistribution of Ca2+among the different cell organelles during homogen-

FIG.2. Energy-dependent Caz+diffusion restriction in the cytosol. Cells of isolated salivary glands of Chtronomus were injected with the Ca2+-sensitive luminescent protein aequorin. The light emission was viewed and recorded through a microscope with the aid of an image intensifier coupled to a television camera. The cells were impaled with microelectrodes with a 0.2-pm tip diameter; the location of the electrodes is shown in (h). Leaks of Caz+ through the electrodes are visible in the darkfield micrographs (a-f) as luminescent dots at three impalement sites. (a) Leaks in medium containing 4 mM Ca2+;(b-d) 1 , 2 , and 3 minutes after application of medium containing 4 d Caz+ and 2 mM CN-; (e) Ca*+-freemedium containing 2 mM CN-; (f) 2 minutes after return to medium (a); (g) brightfield television picture of the two aequorin-loaded cells. (From Rose and Loewenstein, 1975.)

THE REGULATION OF INTRACELLULAR CALCIUM

163

ization and fractionation of the tissue; in particular, Ca2+can be lost from organelles that undergo severe structural degradation during fractionation (e.g., the sarcoplasmic reticulum) and be taken up by organelles that remain relatively more intact (e.g., the mitochondria). In an attempt allowing for this possibility, Carafoli and Tiozzo (1967) injected radioactive S I + into rats which were then sacrificed at various times after the injection. T h e radioactive S I + distributed among the subcellular phases in a way which depended characteristically on the time elapsing between injection and death (Fig. 3). When the animal was killed immediately after the administration of radioactivity, about 60% of the total radioactivity of the homogenate was recovered in the mitochondria and only about 8% in the endoplasmic reticulum. When the animal was killed 60 minutes after the injection, the percentage of the total radioactivity of the homogenate recovered in the mitochondria decreased to only 25%, and it increased to about 15% in the fragments of endoplasmic reticulum. These data appear to be incompatible with a purely adventitious association of S I + with the cellular organelles during homogenization and fractionation and suggest the existence of subcellular pools of S I + (or Caz+) which are interchangeable. Confirming data have been provided by Thakar et al. (1973), who used ruthenium red to inhibit the uptake of

% - l

Minutes after injection d %r"

FIG.3. Association of injected radioactive Sr2+with mitochondria and endoplasmic reticulum isolated from rat liver. Rats were injected intraperitoneally with a pulse of 8aSr2+(Carafoli and Tiozzo, 1967) 5 minutes before sacrifice. The mitochondrial and endoplasmic reticulum fractions were separated from the homogenate with a conventional fractionation procedure in 0.25 M sucrose. The data underestimate somewhat the percentage of radioactivity associated with the organelle, since no correction was applied for the mitochondria and endoplasmic reticulum associated with the first low-speed sediment, which did not appear in the mitochondrial or reticulum fraction. The samples were dissolved with the aid of a detergent and counted in a lowbackground, gas flow counter. (From Carafoli and Tiozzo, 1967.)

164

ERNEST0 CARAFOLI A N D MARTIN CROMPTON

Ca2+by mitochondria. They found that the inclusion of high concentrations of ruthenium red in the homogenization medium decreased the Ca2+content of skeletal muscle mitochondria by only about 20%; this indicates that most of the Ca2+associated with isolated mitochondria is present in vivo also. The distribution of injected radioactive Ca2+among isolated subcellular fractions of rat heart has been found to be similar to that in rat liver (Patriarca and Carafoli, 1968). The amount of Ca2+ associated with isolated mitochondria varies with the tissue source. As extreme examples, mitochondria from liver (10-12 nmoles/mg of protein, Carafoli and Lehninger, 1971) and from myometrium (up to 150 nmoles/mg of protein, Malmstrom and Carafoli, 1978) (both isolated in the presence of Ca2+-chelatingagents) can be given. The endogenous Ca2+of mitochondria, even the very large pools associated with smooth muscle mitochondria, can be rapidly discharged by the addition of uncoupling agents (Carafoli, 1967; Malmstrom and Carafoli, 1978).

V.

GENERAL CONSIDERATIONS O N THE REGULATION OF INTRACELLULAR Ca2+

The role of Ca2+as a biological messenger requires that it be maintained at a very low concentration inside the cell, where most of the targets of its messenger function are found, and that it be regulated very precisely near these low levels, in synchrony with the demands of the messenger function. Some of the reasons why this precise regulation is possible have been considered in the preceding discussion. In the sections that follow, the membrane mechanisms by which this regulation is accomplished are discussed. Within the cell, however, a role may be played also by nonmembranous ligands; some of these are simple in structure (e.g., adenine nucleotides, citric acid, inorganic phosphate), while other such as Ca2+-bindingproteins are more complex [e.g., parvalbumins (Kretsinger, 1975,1977),troponin (Ebashi and Endo, 1968), spasmin (Amos et al., 1975), aequorin (Shimomura et al., 1962)l. All such ligands serve to maintain the ionized Ca2+concentration at low levels. As yet, it is not known whether or not these ligands can change the free Ca2+ concentration in response to physiological needs. If one considers the overall regulation of cellular Ca2+,it is obvious that maintenance of the intracellular Ca2+ at a concentration much lower than that in the extracellular medium must depend on Caz+ extrusion through the plasma membrane, to counteract the continuous

165

THE REGULATION OF INTRACELLULAR CALCIUM

passive influx, rather than on its sequestration within intracellular organelles. However, one might speculate that the plasma membrane is relatively less suited for the rapid regulation of cytoplasmic Caz+.This is because the membrane area of the Caz+-transportingorganelles generally much greater than that of the plasma membrane. This concept is perhaps best illustrated by the case of muscle, where most of the cell membrane area resides in the intracellular organelles which also effect the rapid Caz+movements required for contraction and relaxation. The relative areas of the plasma and organelle membranes vary widely in different cells; as extreme examples (Table IX) one can consider erythrocytes, where only the plasma membrane operates, and heart, where in addition to the sarcolemma there are two other well-developed Ca2+-transportingmembranes, namely, the mitochondria and the sarcoplasmic reticulum. Based on certain assumptions (see footnote to Table IX), one can calculate that in the heart the total membrane area available for Caz+ transport is about 12.2 m2/gm of TABLE IX

TOTALAREA OF CA~+-TRANSPORTING MEMBRANES REPRESENTATIVE CELLS"

IN SOME

Area (m2/gm tissue) Cell Erythrocyte Liver Heart

Plasma membrane

1.66 (100%) 0.55 ( 1 1.4%) 0.10 (0.8%)

Mitochondria 0 2.65 (54.8%) 10.60 (87%)

Endo- or sarcoplasmic reticulum 0 1.63 (33.7%) 1.48 (12.1%)

T h e data on erythrocytes are based on an area of 145 pm2 per erythrocyte (Davson, l970), on a hematocrit of 44.5% (Ganong, 1973), and on an erythrocyte content of 5.1 x 106/mm3 (Ganong, 1973). T h e data on liver are derived from morphometric measurements carried out by E. R . Weibel (personal communication). Only the inner mitochondrial membrane is considered, and only the smooth endoplasmic reticulum, since the outer membrane and the rough endoplasmic reticulum are not involved in the transport of Ca2+ (see text). The total area of liver cell membranes is 8.2 m2/gm of tissue. T h e data on heart plasma memlxane are derived from Winegrad and Shanes (1962). For those on heart mitochondria and sarcoplasmic reticulum, the following two assumptions have been made: ( 1 ) the inner membrane represents 50% of the total mitochondrial protein in heart; ( 2 )the ratio of protein content to area is the same in rat liver mitochondria1 inner membrane, heart mitochondrial inner membrane, and heart sarcoplasmic reticulum. T h e total mitochondrial content is 50 mg protein/gm wet weight in rat liver (Reith et nl., 1976) and 100 mg protein/gm wet weight in rat heart (Scarpa and Graziotti, 1973); the total content of t h e sarcoplasmic reticulum in heart is 7 mg protein/gm wet weight (Solar0 end Briggs, 1974).

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tissue. Of this, about 87% is provided by mitochondria, about 12% by the sarcoplasmic reticulum, and only about 1% by the sarcolemma. In liver, the situation is less extreme, although the organelles and in particular the mitochondria still provide the largest proportion of the total membrane area.

VI.

THE TRANSPORT OF Ca2+ ACROSS PLASMA MEMBRANES

A. The Influx of Ca2+ into Cells

Extracellular Ca2+ diffuses across the plasma membrane into the cell down its concentration gradient. Whether or not the activity of the Ca2+channels in the membrane is controlled, and if so, how, are open questions in most cases. It has been suggested that parathyroid hormone enhances the penetration of Ca2+ into the cells of nonexcitable tissues (Borle, 1970), whereas calcitonin depresses it (Borle, 1975). The permeation of Ca2+ across the intestinal mucosa appears to be facilitated by a hydrophilic protein able to bind Ca2+,which is synthesized under the influence of vitamin D (Wasserman and Taylor, 1966). Following the original reports (Wasserman and Taylor, 1966) the protein has been identified also in kidneys, in mammary glands, and in the uterine shell gland of the laying hen. In the chick the protein has a M.W. between 25,000 and 28,000, is strongly acidic, and binds Ca2+at two types of sites; 4 sites per mole have high affinity (& = 2 p M ) , and about 30 per mole have low affinity ( K d = 10-100 pM) (see Table X). A large body of evidence, summarized in Table X,supports involvement of the protein in the transport of Ca2+.However, direct proof of its involvement in the transport of Ca2+has not yet been obtained. In particular, there are no reports of experiments in which antibodies directed against the Ca2+-bindingprotein block the absorption of Ca2+ by isolated intestinal preparations, nor of the “reconstitution” of the transport of Ca2+ in an artificial phospholipid bilayer. However, Ca2+-binding protein can be induced in duodenal cells from vitamin-D-deficient rats in vitro; these cells also show increased Ca2+ transport (Freund and Bronner, 1975; Golub et al., 1977). In addition, some reconstitution experiments have been reported (Corradino et al., 1976). An influx of Ca2+ occurs across the plasma membrane of certain excitable tissues during excitation. Although the origin of the troponin-bound Ca2+in fast skeletal muscle is generally recognized to

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TABLE X EVIDENCEFOR THE PARTICIPATION OF CA~+-BINDINC PROTEIN (CaBP) I N THE INTESTINAL ABSORPTIONOF CAz+ The protein has been found only in tissues where extensive movements of Caz+ take place (Corradino et a l . , 1968); Taylor and Wasserman, 1874; Fullmer and Wasserman, 1973). The protein is induced hy vitamin D, the key factor in controlling t h e absorption of Ca2+in the intestine. The CaBP content is low in vitamin D-deficient animals (Ebel ct u / . , 1969). Caz+ahsorption and CaBP content increase in parallel in hens during egg-laying cycles (Bar and Hurwitz, 1973). Intestinal CaBP content and Ca2+ absorption decrease in parallel with age (Wasserman and Taylor, 1968). There is parallelism between the CaBP content and t he absorption of Ca2+in different segments of the intestine (Taylor and Wasserman, 1967). There is a correlation between the affinity of CaBP for different divalent cations and the efficiency of intestinal absorption of the same cations (Taylor and Wasserman, 1867). T h e induction of CaBP in deficient membrane preparations in oitro is paralleled b y an increase in Caz+ transport (Freund and Bronner, 1975; Golub et a l . , 1977).

be intracellular, from the sarcoplasmic reticulum, the contractile activity of cardiac muscle (Armstrong et al., 1972) decreases rapidly when the extracellular Ca2+i s removed (Bailey and Dresel, 1968; Rich and Langer, 1975; Shine et al., 1971).This indicates a requirement for Ca2+entry across the sarcolemma. I n excited cardiac fibers, following the initial rapid Na+ inward current, there is a slow, second inward current that increases and decays in 50-300msec and is considered to be carried mainly b y Ca2+ (Reuter, 1974;.Trauhvein, 1973). Integration of the slow inward current, however, shows that the influx of Ca2+ responsible for the current can be no greater than about 5 nmoles Ca2+/gmwet weight of heart (Beeler and Reuter, 1970a,b; New and Trautwein, 1972), which is barely sufficient for 10% of maximal contraction. Thus, although the slow inward current may be essential for contractions (perhaps by triggering the release of further amounts of Ca2+ from the reticulum-see Section VII,C), it is quantitatively insufficient to account for the Ca2+ requirements of contraction. The use of inhibitors has permitted the inward currents of Na+ and Ca2+to be resolved; the slow inward current is not affected when the specific Na+ channel of the sarcolemma is blocked by tetrodotoxin, lidocaine, or procaine, but is inhibited by verapramil and D-600 which block Ca2+penetration across the cardiac sarcolemma (Fleckenstein et al., 1969; Kolhardt et al., 1972; Mascher and Peper, 1969; Kolhardt, 1975; Tritthart et al., 1975).The system responsible for the increase in

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Ca2+flux during excitation has been little characterized, but its existence has been demonstrated by 45Ca2+ experiments and voltage-clamp studies (Winegrad and Shanes, 1962; Niedergerke, 1963a,b; Rougier et al., 1969; Beeler and Reuter, 1970a,b; Reuter, 1974; Trautwein, 1973).The influx of Ca2+into the cardiac cell during the action potential is stimulated by catecholamines (Grossmann and Furchgott, 1964; Reuter, 1965) and by dibutyryl CAMP(Meinertz et al., 1973). Using a sarcolemma-enriched cardiac fraction, Wollenberger et al. ( 1975) detected the CAMP-dependent phosphorylation of a 24,000 mol. wt. protein in the membrane. This phosphorylation apparently increases the affinity of the binding sites on the membrane for Ca2+. Ca2+also acts as a carrier of inward current across the plasma membrane of smooth muscle and nerve (Baker, 1975; Reuter, 1975). 6. The Efflux of Ca2+ from Cells

The plasma membrane also contains specific systems that eject Ca2+ from the cell against its electrochemical gradient. The experimental evidence available indicates the existence of two mechanisms of Ca2+ efflux. The first has been characterized in detail in erythrocytes but may be present in other cell types as well (liver, cultured L cells) and derives its energy from the splitting of ATP. The second has been identified in many tissues and derives its energy from the gradient of Na+ across the plasma membrane and perhaps from ATP as well. 1, CA~+-ATPASE

The Ca2+-ATPaseof the erythrocyte plasma membrane has been studied in considerable detail (see Schatzmann, 1975, for a review). It seems that the ATPase exists in two forms or that it has two components. One fraction, which is by far the largest, requires Mg2+ in addition to Ca2+;the other, corresponding to a small fraction of the total ATPase activity, does not require Mg2+and is actually inhibited by it (Rosenthal et aZ., 1970). According to Schatzmann (1975), this small fraction may be identical to spectrin and is not involved in the membrane transport of Ca2”. The major fraction is a protein of M.W. 130,000 (Knauf et al., 1974) and represents only about 0.02% of the total membrane protein. Experiments on resealed cells, in which the two sides of the membrane are exposed to different media, have shown that ATP is split

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only when it is inside the cell (Palet et al., 1971), i.e., on the same side of the membrane as Ca2+and Mg2+(Schatzmann, 1967). The enzyme uses Mg-ATP, rather than free ATP, as the substrate, with a K , value of about 5 x lop5(Wolf, 1970, 1972). The splitting of ATP produces a phosphorylated intermediate (Knauf et aZ., 1974; Drickamer, 1975). The K, value for Ca2+is about 1-4 pM (Table XI), and the V,,, value is between 85 and 200 pmoles of Ca2+transported per liter of cells per minute at 37°C (Ferreira and Lew, 1976; Sarkadi et al., 1977). The stoichiometry of ATP split to CaZ+transported is currently a matter of debate. Schatzmann and Vincenzi (1969) and Schatzmann (1973) obtained figures approaching 1, whereas Quist and Roufogalis (1975), Ferreira and Lew (1976), and Sarkadi et ul. (1977) report values of about 2. The enzyme is inhibited by ruthenium red (90%inhibition at 60pM; Watson et aZ., 1971) and by La3+(80-95% inhibition at 50-250 pM La3+).The latter inhibitor is effective on the external surface of the membrane (Sarkadi et al., 1977). Ronner et al. (1977) found that the ATPase shows a specific requirement for acidic phospholipids, similar to Na+,K+-ATPase (Roelofsen and Van Deenen, 1973). However, Roelofsen and Schatzmann (1977) reported that all glycerophospholipids reactivate the phospholipiddepleted enzyme. Attempts to purify this enzyme are currently underway in several laboratories (Knauf et al., 1974; Dieckvoss and Wolf, 1976; Ronner et al., 1976). With the use of a partially purified preparation (Peterson et al., 1978) reconstitution of the ATPase activity has recently been obtained in vesicular phospholipid bilayers. Lamb and Lindsay (1971) reported an ATP-driven efflux of Ca2+ from cultured L cells that was independent of the external Na+. The system responsible for extruding Ca2+ from liver cells was also reTABLE XI AFFINITY OF T H E C A 2 + , M G ~ + - A T P A S EO F T H E ERYTHROCYTE M E M B R A N E FOR C A 2 + ~~

Method

K , for internal Ca2+ ( P3.1)

Reference

~_____

Interllal CaZ+controlled with Ca2+buffers

Internal Cia2+controlled with the ionophore A12187

4 1-2 0.92 1-4 0.73- 1.02

Schatztiiann (1973) Quist and Roiifogalis (1975) Pfleger and Wolf (1975) Scharff (1976) Ferreira and Lew (1976)

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ported to depend on metabolic energy and to be independent of the concentration gradient of Na+ across the plasma membrane (Van Rossum, 1970). However, Chambaut et aZ. (1974)could not detect any Ca*+-activatedMg2+ ATPase in isolated liver plasma membranes. 2. NA+-CA~+ EXCHANGE a. Arons. Much evidence (Baker, 1976; Mullins, 1976; Blaustein; 1976)obtained over the last few years strongly indicates that the efflux of Ca2+from squid axons is driven, at least in part (and perhaps completely), by the electrochemical gradient of Na+ across the axolemma. The fluxes of Na+ and Ca2+appear to be coupled to a carrier that catalyzes an exchange between these two cations. The rate of efflux of 45Ca2+from axons is decreased in the absence of Na+ in the medium and is further decreased in the absence of external Ca2+.Hence, the total efflux of 45Ca2+probably involves exchanges with external Na+ and Ca2+,as well as a residual efflux that is apparently not coupled to the countermovement of Na+ and Ca2+.Whether or not these three components of the totaI Ca2+efflux are catalyzed by the same system is subject to debate (Baker, 1972). Conversely, external Ca2+may induce an efflux of Na+ (Baker et aZ., 1969). This implies that the carrier may operate in either direction, depending on the difference between the electrochemical gradients of Na+ and Ca2+and on the stoichiometry of the exchange. However, under conditions which permit a rapid Ca2+-Ca2+exchange, there occurs little Na+-Na+ exchange that is cardiac glycoside-insensitive (Baker et d.,1969). In internally dialyzed or CN--treated axons (ATP-free),the velocity of the Na+-dependent efflux of Ca2+displays a sigmoidal dependence on the external Na+ concentration, with half-maximal velocity at about 120 mM Na+ (Blaustein et al., 1974). The data fit quite closely a velocity equation in which the Na+ concentration is raised to the power 3, suggesting that three or more Na+ ions may be involved per cycle of exchange with Ca2+(Fig. 5). Under similar conditions, between 3 and 8 mM external Ca2+ is required to activate half-maximally Ca2+dependent Ca2+efflux (Baker and McNaughton, 1976). The stoichiometry of the exchange between external and internal Ca2+ is 1: 1 (Baker and McNaughton, 1976). The evidence from kinetic measurements that each exchange involves three or more Na+ ions implies that the exchange would be electrogenic if one Ca2+ion, and no other ions, exchanged per cycle. In fact, recent data show that the Na+-Ca2+ exchange is subject to the electric field across the membrane. The Na+-dependent efflux of Ca2+

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171

is inhibited by depolarization and promoted by hyperpolarization of the axolemma, suggesting that the exchange is associated with a net inward movement of positive charges (Baker and McNaughton, 1976; Blaustein et ul., 1974; Mullins and Brinley, 1975). The net charge transfer per exchange is not known. On thermodynamic grounds it appears, however, that the exchange would be in equilibrium with the known concentration gradient of Ca2+ (about 105) and Na+ (about 10) across the axolemma, and with the resting potential which is about 70 mV (negative inside) if the net charge transfer per exchange were between 1 and 2; in other words, if three or four Na+ ions were to exchange for one Ca2+ ion. Clearly, therefore, complete knowledge of the overall rection and the net charge transfer is necessary, as it permits one to know whether or not the Na+ gradient alone is sufficient to maintain the extracellular ionized Ca2+ at the low level found in the axoplasm. Interestingly, the manifest characteristics of the Na+-Ca2+ exchange in poisoned or dialyzed axons are modified by the addition of low amounts of ATP to the internal solution; dATP is also effective, but AMP, UTP, CTP, and other nucleotides are not (Di Polo, 1973b, 1974; 1976). In the presence of ATP the kinetics of the Na+ dependence are no longer clearly sigmoidal (Baker and Glitsch, 1973; Baker and McNaughton, 1976). In addition, whereas in the absence of ATP about 10-20 pM internal ionized Ca2+ is needed to provide a halfmaximal rate of Ca2+efflux, in the presence of ATP only 0.2-0.3 internal Caz+ is required for half-maximal velocity. Thus ATP seems to increase the affinity of the carrier for internal Ca2+.However, the question whether or not ATP is hydrolyzed during its action has not yet been resolved. b. Bruin. There is evidence that the movement of Ca2+ between brain slices and the external medium is influenced by the relative Na+ concentrations in the tissue and the external medium. The uptake of Ca2+b y the tissue is promoted by a decrease in the Na+ concentration of the medium or by an increased Na+ content in the tissue (Cooke and Robinson, 1971; Stahl and Swanson, 1969,1972). Na+-dependent Ca2+fluxes are also seen in the synaptosome (presynaptic nerve terminal) fraction of brain homogenates (Blaustein and Wiesmann, 1970). The efflux of internal Ca2+requires external Na+, while Ca2+influx requires internal Na+. These data are consistent with the existence of an exchange diffusion carrier for Na+ and Ca2+in the membrane. c. Secretory Tissues. There is strong evidence that Ca2+is involved in the coupling between stimulus and secretion in certain tissues (e.g., adrenal medulla, pancreas, neurohypophysis), secretion being

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triggered by an increase in the concentration of cytosolic Ca2+ (Douglas, 1968; Rubin, 1970). In the pancreas, the secretion of insulin is stimulated by a decrease in external Na+; insulin secretion is also stimulated by ouabain in the presence of external Na+. This in turn should lead to an increase in internal Na+ (Hales and Milner, 1968a,b). Similarly, the absence of external Na+ or the presence of ouabain augments the secretion of catecholamines by the adrenal medulla (Douglas and Rubin, 1961, 1963; Banks et al., 1969; Banks, 1967). These observations are consistent with an exchange of Na+ for Ca2+across the plasma membrane of these tissues. There is also evidence for a counterexchange between Na+ and Ca2+in the plasma membrane of the neurohypophysis, and it is thought to have a role in the Ca2+movements into and out of the tissue that are implicated in the release of vasopressin (Dreifuss et al., 1975). d . Muscle. Evidence for a carrier that transports Na+ and Ca2+in the plasma membrane of cardiac cells was first obtained by Wilbrandt and Koller (1948) and extended by the work of Niedergerke (Liittgau and Niedergerke, 1958; Niedergerke, 1963a,b). More recently it has become clear that the carrier catalyzes an exchange between Na+ and Ca2+ (Reuter and Seitz, 1968; Glitsch et al., 1970; Miller and Moisescu, 1976). At variance with the case of axonal membranes, kinetic data suggest that the stoichiometry of the exchange is two Na+ ions for one Ca2+ion, consistent with the proposed electroneutrality of the system (Jundt et al., 1975). The logical role of the Na+-Ca2+ exchange is to catalyze extrusion of Caz+that has entered the sarcoplasm during the plateau of the excitation phase, since no other system is known that can accomplish this. However, since the gradient of Na+ across the sarcolemma is about 10, equilibrium would be attained with a Ca2+gradient of about lo2, whereas the actual gradient is about lo4 (Reuter, 1974); i.e., an electroneutral exchange of two Na+ ions for one Ca2+ion would occur in the direction of Ca2+influx and Na+ efflux. The entry of Ca2+that occurs during the slow inward current can be inhibited by La3+,whereas under these conditions the Na+-Ca2+ exchange is not inhibited (Kutzung et al., 1973). This indicates that these events are catalyzed by different systems. If the Na+-Ca2+ exchange of the sarcolemma extrudes Ca2+,there must be either net charge transfer during the exchange, so that electric potential difference can be utilized, or some other input of energy. Regarding the latter, Jundt and Reuter (1977) recently reported that the affinity of the system for external Na+ and the maximum velocity of the exchange are both decreased by metabolic poisoning; poisoning also decreases the ATP content by about 90%. Thus ATP may be involved in Na+-

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dependent Caz+movements across the sarcolemma, as in squid axons. The activity of the system in cardiac sarcolemma is about 0.1 pmole Caz+/cm2/sec(equivalent to 0.1-1 nmole Ca2+/gmwet weight of heart per second taking the area of the sarcolemma to be 1000-10,000 cm2/gm of tissue; Reuter, personal communication). An exchange between Na+ and Ca2+ may well occur across the plasma membrane of arterial smooth muscle. Contractions are induced by a decrease in the external concentration of Na+, and there is a net influx of Ca2+ into the fiber (Bohr, 1964; Van Breemen et al., 1973; Ma and Bose, 1977). The tension developed is constant if the [Na+]2/[Ca2+]concentration ratio is maintained constant, consistent with a process in which two Na+ ions exchange for one Caz+ion (Reuter et al., 1973). In barnacle muscle fibers, the influx of Ca2+is stimulated by an increase in the Na+,JNa+,,, ratio and is accompanied by Na+ efflux (Di Polo, 1973b; Brinley, 1968). Russell and Blaustein (1974) obtained evidence that the efflux of Ca2+from these fibers occurs in exchange for external Na+ and Ca2+. VII.

THE TRANSPORT OF Ca2+ BY SARCOPLASMIC AND ENDOPLASMIC RETICULUM

A. Sarcoplasmic Reticulum of Fast Skeletal Muscle

a. Membrane System and Overall Reaction. The sarcoplasmic reticulum extends parallel to the contractile. filaments in the form of tubules which enlarge and unite into terminal cisternae near the radial invaginations of the sarcolemma [the transverse tubular (T) system]. The sarcoplasmic reticulum does not seem to open into either the T system or the sarcolemma, although the gap between the sarcoplasmic reticulum and the T system appears to be bordered by nonmembranous material (Franzini-Armstrong, 1970,1975).T h e sarcoplasmic reticulum is developed to varying degrees in different muscles. Fast muscles contain large amounts of it, and slow muscles much less. I n general, sarcoplasmic reticulum in heart muscles is even less developed than in slow muscles. The ability of sarcoplasmic reticulum in intact muscle fibers to accumulate Ca2+has been demonstrated by treating skinned fibers with oxalate to precipitate the Ca2+;under these conditions electron-dense deposits of the Ca2+ salt are visible in the terminal cisternae (Costantin et d., 1965; Podolsky et al., 1970).

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The bulk of the work done on the transport of Ca2+by sarcoplasmic reticulum has made use of the membrane fragments (or microsomes) that result from disruption of the sarcoplasmic reticulum during homogenization of the whole tissue. The fragments reseal into closed vesicles that contain the proteins of the sarcoplasmic reticulum membrane. Most of the luminal contents of the sarcoplasmic reticulum (Peachey, 1965; Franzini-Armstrong, 1970) are lost during the preparation, although matrix material is retained by some of the vesicles; this material is probably calsequestrin and a high-affinity Ca2+binding protein to be discussed in Section C (Meissner, 1975). After the pioneering observations by Ebashi (1960, 1961) and Hasselbach and Makinose (1961) on the binding of Ca2+by muscle microsomes and on the stimulation of ATPase activity by Ca2+,subsequent work established that an average of two Ca2+ions are accumulated by the vesicles per ATP molecule hydrolyzed, although the experimental values obtained are quite variable (Hasselbach and Makinose, 1963; Weber et al., 1966). Consistent with this stoichiometry, the dependence of the Ca2+-inducedATPase on the Ca2+concentration displays a Hill coefficient of 1.8 (The and Hasselbach, 1972).The process can be reversed (Barlogie et al., 1971) by imposing a concentration gradient of Ca2+across the vesicle membrane, so that the efflux of Ca2+synthesizes ATP from ADP and Pi with a stoichiometry of one ATP molecule per efflux of two Ca2+ions (Makinose and Hasselbach, 1971). b. The Reaction Mechanism. The process of Caz+transport linked to ATP hydrolysis is catalyzed by a protein that has been purified and shown to have a MW of about 100,000 (Martonosi, 1968; MacLennan, 1970; Ikemoto et al., 1971; Warren et al., 1974; Meissner et al., 1973; Deamer, 1973; Racker, 1972). It represents up to 90%of the total protein of the sarcoplasmic reticulum (Meissner et d., 1973; Deamer, 1973; Inesi, 1972; Meissner, 1975). The purified protein has been reconstituted with phospholipids into vesicles that exhibit ATP-depend,ent accumulation of Ca2+ and ATP synthesis coupled to Ca2+ efflux (Warren et al., 1974; Knowles and Racker, 1975; Racker, 1972; Racker and Eytan, 1973; Meissner and Fleischer, 1973, 1974). Studies on the interaction of Ca2+with purified ATPase have confirmed that 2 moles of Ca2+bind to 1 mole of enzyme with a high affinity (Meissner, 1973; Ikemoto, 1975; Kanazawa et al., 1971), similar to that prediced from kinetic studies which show a half-maximal velocity of Ca2+influx with 10-7.5-10--8~0 M ionized Ca2+(Weber et al., 1966; Worsfold and Peter, 1970; Makinose, 1969).Values for the maximum velocity of Ca2+influx into fast skeletal muscle microsomes lie in the range 0.4-3.6 pmole Ca2+/mg of vesicle protein per minute (Weber et al., 1966; Worsfold

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OF INTRACELLULAR CALCIUM

175

and Peter, 1970; Sreter, 1969; Martonosi and Feretos, 1964; Inesi and Scarpa, 1972). The binding of ATP to the Ca2+transport ATPase is dependent on Mg2+; this suggests that Mg-ATP is the species bound (Makinose, 1969). One ATP molecule is bound per 100,000 daltons, with a Kd value varying between 1 pM at pH 8.0 and 10 pM at p H 6.0; the affinity for ADP is somewhat lower (Meissner, 1973). A definite step forward in understanding the reaction mechanism came from the discovery that the Ca2+-ATPasebecomes phosphorylated at an intermediate stage of the reaction (Makinose, 1969; Yamamot0 and Tonomura, 1968; Inesi et ul., 1970; Martonosi, 1969). Phosphorylation by ATP displays an obligatory requirement for Caz+.This stimulation occurs over a range of concentrations similar to those required for Ca2+uptake by intact vesicles (Makinose, 1969, 1972; Inesi et al., 1970; Panet et al., 1971; Coffey et ul., 1975). Evidence suggests that the terminal group of ATP is transferred to an aspartyl residue of the protein (Degani and Boyer, 1973). The maximum level of phosphorylated intermediates ranges from 0.3 to 0.6 mole per 100,000 daltons. Microsomes can also be phosphorylated by Pi in the reverse reaction, in which Ca2+ efflux occurs down a high gradient of Ca2+ across the membrane; again, about 0.5 mole of phosphate is incorporated per 100,000 daltons (Makinose and Hasselbach, 1971; Kanazawa et ul., 1971; Deamer and Baskin, 1972). The phosphorylation reaction can be studied independently of the hydrolysis of phosphoprotein, since it occurs at a rate (20-40 sec-' at 5°C) that is one to two orders of magnitude faster than the rate of phosphoprotein hydrolysis (Martonosi, 1975). T h e phosphorylation reaction therefore is close to equilibrium during steady-state transport. The equilibrium constant of the phosphorylation reaction approaches 1 (Meissner, 1973; Panet et ul., 1971), so that energy is largely conserved within the phosphoprotein, in agreement with its acyl phosphate nature (Degani and Boyer, 1973; Yamamoto and Tonomura, 1968; Yamamoto et al., 1971). There is evidence that phosphoprotein formation occurs on the cytoplasmic side of the membrane, since treatment of microsomes with trypsin releases a fragment of 55,000 daltons that is phosphorylated by ATP (Thorley-Lawson and Green, 1973; MacLennan, 1978; Green et al., 1978); the remaining fragment seems to be buried in the membrane. The extrinsic ATPase fragment may b e identical to the 30- to 40-A projections seen on the outer surface of negatively stained vesicles (Ikemoto et d.,1968; Inesi and Asai, 1968), which are probably part of the ATPase (Sarzala et al., 1975; Hardwicke and Green, 1974). Interestingly, the 55,000 dalton

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ERNEST0 CARAFOLI AND MARTIN CROMPTON

fragment that is phosphorylated can be split by prolonged trypsin digestion into 20,000- and 30,000-dalton fragments. Only the latter can be phosphorylated by ATP, whereas the former exhibits Ca2+ ionophoric activity in artificial lipid bilayer systems (Stewart et al., 1976). MacLennan and his co-workers (1973) isolated from sarcoplasmic reticulum membranes a proteolipid having an approximate MW of 12,000 and postulated for it a role in orientating the ATPase molecule within the hydrophobic phospholipid bilayer. Using liposomes reconstituted with sarcoplasmic reticulum ATPase, Racker and Eytan (1973) demonstrated that the proteolipid markedly stimulated the transport of Ca2+.Racker (1976) has postulated that the proteolipid may function as a channel attached to the ATPase, which does not span the entire width of the membrane. The steps leading to dephosphorylation are rate limiting for the total process (Martonosi et al., 1974; Coffey et al., 1975). Presumably, transmembrane movement of Ca2+occurs at some stage, involving the portions of the enzyme which are integral to the membrane. In line with this, the rate of phosphoprotein hydrolysis may be retarded at least 80% by treating the vesicle membrane with phospholipase (whereas the rate of phosphoprotein formation is not affected; Martonosi et al., 1974). This suggests that the dephosphorylation reaction is sensitive to a phospholipid environment. There is evidence that the release of Ca2+to the interior of the vesicle occurs before dephosphorylation (Makinose, 1973). This agrees with inhibition of dephosphorylation of the purified enzyme by elevated concentrations of Ca2+, since Ca2+ can thereby stabilize the phosphoenzyme; the decay is almost completely inhibited by 10 mM Ca2+(Ikemoto, 1975). This offers an explanation for the earlier observation that the steady-state recycling of Ca2+across the microsomal membrane becomes severely inhibited by high levels of intravesicular Ca2+and is paralleled by inhibition of the Ca2+-activatedATPase activity (Weber et al., 1966; Makinose and Hasselbach, 1965; Weber, 1971). In contrast to Ca2+,Mg2+ stimulates the rate of dephosphorylation (Panet et al., 1971; Martonosi, 1969); the data of Kanazawa et al. (1971) favor the possibility that the that stimulates is intravesicular. Thus M g + may well displace Ca2+after translocation (Panet et al., 1971; Nakamura et al., 1976). Particularly incisive is the fact that the affinity of the enzyme for Ca2+ decreases upon phosphorylation (Ikemoto, 1975; Yamada and Tonomura, 1972), since this favors displacement of bound Ca2+by Mg2+. The question arises: Following the presumed displacement of internal Ca2+by internal M$+, at what stage is the Mg2+ released? It has

Me+

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177

been demonstrated that the ATPase may b e phosphorylated by inorganic phosphate in the absence of ATP or a Ca2+gradient (Masuda and De Meis, 1973; Kanazawa, 1975) in a reaction that requires Mg2+ and is inhibited by Ca2+. Microsomes also catalyze a rapid Pi-HOH exchange that requires Mg2+and is inhibited by Ca2+(Kanazawa and Boyer, 1973). Moreover, at least in vesicles reconstituted from purified ATPase and phospholipids, phosphate appears to be released on the outside of the vesicles during active uptake of Ca2+(Knowles and Racker, 1975). A scheme consistent with these results would involve the dephosphorylation and release of bound Mg2+ on the outside of the membrane and an obligatory exchange between external Ca2+and internal M$+, as suggested by Kanazawa and co-workers (1971; Kanazawa and Boyer, 1973). They proposed an influx of two Ca2+ ions in exchange for one Mg2+ ion, with a parallel efflux of two K+ ions for charge compensation. Indeed, the accumulation of Ca2+by microsomes is accompanied by a stoichiometric displacement of Mg2+and K+, although at p H values below 6.2 Ca2+exchanges mainly for H+ (Carvalho and Leo, 1967). Clearly, in order to characterize the net reaction catalyzed by the Ca2+-ATPaseit is necessary to identify the mechanism by which the countercations exit and determine whether or not the ATPase itself is in part responsible. Virtually no Ca2+ transport would take place unless the influx of positive charges were compensated for either by a simultaneous influx of anions or by an efflux of cations. c. The Binding of Accumulated C d + .Overall charge compensation by an influx of anions occurs, however, if these are added to the medium, since the microsomes are readily permeable to a variety of anions, e.g., oxalate, phosphate, pyrophosphate, and chloride (Martonosi and Feretos, 1964; Duggan and Martonosi, 1970). Indeed, since the early work of Hasselbach and Makinose (1961), many studies have employed oxalate in the reaction medium. This allows precipitation of intravesicular Ca2+when the solubility product of calcium oxalate is exceeded and thereby promotes continued Ca2+accumulation. In the absence of oxalate the apparent gradient of Ca2+ with respect to extravesicular Ca2+ amounts to about 103 (Weber et al., 1966; Sreter, 1969; Martonosi and Feretos, 1964; Fiehn and Migala, 1971; Inesi and Scarpa, 1972). When oxalate is present, the gradient in excess of the calcium oxalate precipitate also amounts to at least lo3 (Hasselbach and Makinose, 1961; Weberet al., 1966).However, Weber et al. (1966) pointed out that, if all the Ca2+accumulated in excess of oxalate were ionized, the solubility product of the internal Ca2+oxalate would b e two or three orders of magnitude too large. They proposed that most of the excess Ca2+ is bound to the membrane. The

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ERNEST0 CARAFOLI AND MARTIN CROMPTON

number of high-affinity sites (i.e., with K d = 1 p M ) in fast skeletal muscle microsomes is probably not greater than about 1.0 nmoles/mg of protein (Fiehn and Migala, 1971; Chevallier and Butow, 1971; Meissner et al., 1973), and this is attributable to the Ca2+-ATPaseitself. Thus the binding is considered to involve lower-affinity sites in equilibrium with an elevated internal concentration of ionized Ca2+ that results from its active transport into the vesicle. The binding of accumulated Ca2+ is particularly important with respect to the accumulating capacity of the sarcoplasmic reticulum, since internal Ca2+ inhibits the ATPase, as mentioned previously. Hence the isolation by MacLennan and Wong (1971) of an extrinsic protein that can bind a large amount of Ca2+with moderate affinity assumes special relevance. This water-soluble, acidic protein, called calsequestrin, has a maximum binding capacity of 43 moles of Ca2+ M (Ostwald and Macper mole, with an apparent K d of 8 X Lennan, 1974; Ostwald et al., 1974). Calsequestrin (MW 46,000) represents about 7%of the total vesicle protein and could account for the binding of about 90 nmoles of Ca2+per milligram of vesicle protein, which represents most of the medium-affinity sites present in vesicles (Fiehn and Migala, 1971; Cohen and Selinger, 1969; Chevallier and Butow, 1971).The protein seems to exhibit a high selectivity for Ca2+, since high concentrations of Mg2+are required to inhibit Ca2+binding (Ostwald et al., 1974). Recent evidence indicates that calsequestrin may be especially concentrated in the terminal cisternae of the sarcoplasmic reticulum (Meissner, 1975; Shamoo, 1978). I n addition to calsequestrin (Ostwald and MacLennan, 1974) the sarcoplasmie zetic-. ulum also contains a high-affinity Ca2+-bindingprotein (Meissner et al., 1973; Ikemoto et al., 1972). Its MW probably is in the vicinity of 55,000. It binds Ca2+with both high affinity ( 1 mole/mole, Kd = 3 pM) and low affinity (25 moles/mole, Kd = 100 (Ostwald and MacLennan, 1974). In intact vesicles, calsequestrin is protected against tryptic digestion (Stewart and MacLennan, 1974), and antibodies against it do not aggregate the vesicle (MacLennan, 1975). These findings suggest an internal location, in line with its presumed role in the binding of internal Ca2+.

a)

8. Sarcoplasrnic Reticulum of Cardiac and Red Skeletal Muscle

The general features of Ca2+accumulation by microsomes from cardiac and slow skeletal muscle appear similar to those of this process in

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179

white skeletal muscle, involving stoichiometric coupling to the hydrolysis of ATP (Carsten, 1964; Pretorius et al., 1969) and a phosphorylated intermediate (Pang and Briggs, 1973; Fanburg and Matsushita, 1973; Suko and Hasselbach, 1976). However, both the rate and extent of Ca2+uptake are usually considerably less than those observed with fast skeletal muscle microsomes (Sreter, 1969; Besch and Schwartz, 1971; Baskin and Deamer, 1969; Weber et al., 1966; Fanburg et al., 1964; Inesi et al., 1964; Fanburg and Gergely, 1965; Repke and Katz, 1969; Harigaya and Schwartz, 1969). It is likely that this is d u e partly to their lability after isolation (Weber et al., 1966; Fanburg et al., 1964; Inesi et al., 1964) and to their contamination with other membrane material (Katz and Repke, 1967a,b).Recent estimates indicate a lower content of ATPase in cardiac sarcoplasmic reticulum fractions (Suko and Hasselbach, 1976). The ability of sarcoplasmic reticulum of dog heart to accumulate Ca2+ in the tissue has been estimated recently by Solaro and Briggs (1974), who took into account the rather high losses of reticulum during standard preparative procedures. The study indicated that the reticulum of dog heart is able to bind 300-400 nmoles of Ca2+/gmwet weight of heart at 10 pM free Ca2+ and 170 nmoles Ca2+/gm wet weight at 1 pit4 free Ca2+.These values are considerably higher than the amount of Ca2+ exchanged between myofibrils and sarcoplasm during relaxation (25-100 nmoles Ca2+/gm wet weight of heart: Solaro et ul., 1974; Katz, 1970). The maximum velocity of Ca2+uptake b y cardiac microsomes is about 0.1 pmolelmg of protein per minute at 25°C (Fanburg et al., 1964; Besch and Schwartz, 1971). If the maximum content is 7 mg reticular protein pef gram of wet tissue (Solaro and Briggs, 1974), the maximum activity will b e about 0.7 pmole Ca2+/gmwet weight (calculated from data at 25°C; at 3TC, this velocity may well be doubled). This value can be compared with the requirements for the rate of removal of Ca2+from the sarcoplasm during relaxation. About 35 nmoles Ca2+/gm of tissue must be removed to relax the myofibrils from 90%activation (Solaro et al., 1974); this must occur within 200 msec, an interval that corresponds to a removal rate of at least 10 pmoles Ca2+/gmof tissue per minute. Thus the measured capability of cardiac reticulum to accumulate Ca2+falls short of the requirements for relaxation. Following the work of Entman et al. (1969) and Wray et a2. (1973),it has been reported that the rate of Ca2+uptake by cardiac and slow skeletal muscle microsomes can be approximately doubled by incubating the microsomes with ATP, CAMP, and a CAMP-dependent protein kinase from heart (Tada et al., 1974, 1975a,b; Kirchberger et al., 1974;

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ERNEST0 CARAFOLI A N D MARTIN CROMPTON

Kirchberger and Tada, 1976; Schwartz et ul., 1974, 1976). LaRaia and Morkin (1974) reported that addition of the kinase was unnecessary, since it is present in microsomal preparations together with a phosphatase. Activation depends upon phosphorylation of a serine residue of a 22,000-dalton protein that is distinct from the Ca2+-ATPase. Approximately 1.5 nmoles of phosphate per milligram of vesicle protein can be incorporated in the presence of the protein kinase. The protein appears to be absent from fast skeletal muscles. This phenomenon may bear on the response of the heart to catecholamines, which enhance myocardial contraction and relaxation and stimulate the production of CAMP. It is tempting to attribute relaxation to the action of cAMP on reticulum (Tada et al., 1975b). The picture is complicated, however, since catecholamines and cAMP analogs increase the inward flux of Ca2+across the plasma membrane (Reuter, 1974a,b) and thus potentially provide for the increased myocardial tension. C. The Release of Ca2+ from the Sarcoplasmic Reticulum

The physiological process responsible for net Ca2+movement from

the inside of the sarcoplasmic reticulum to the cytoplasm is unclear. As predicted from the accumulating capacity of microsomes, the rate of diffusion of Ca2+out of the vesicles is relatively slow; with an intravesicular concentration of 1 mM Ca2+the efflux due to diffusion is two or three orders of magnitude slower than the initial rate of ATPsupported Ca2+uptake (Vanderkooi and Martonosi, 1971; Jilka et al., 1975; Boland et al., 1975) Ca2+ can be released from the vesicle at rates similar to those of Ca2+influx by reversal of the ATP-dependent influx process in the presence of ADP, Pi, and M$+, when the extravesicular concentrations of Ca2+and ATP are maintained at low levels (Barlogie et ul., 1971). The operation of such a mechanism clearly requires that the Ca2+attain equilibrium; then, changes in the cytosolic concentrations of ATP, ADP, or Pi and the transmembrane gradient of any other related ions would cause the transmembrane gradient of Ca2+to change. However, this prediction cannot be tested at the moment, since neither the transported ions and charges nor the transmembrane potential are known. Moreover, net Ca2+efflux b y reversal of the Ca2+pump appears difficult to reconcile with the evidence available for a mechanism in which a small increase in ionized Ca2+concentration triggers release of Ca2+from the reticulum. Work with skinned skeletal muscle fibers has indicated that Ca2+release from the reticulum is induced by relatively high concentrations of free Ca2+ (10-5-10-4 M ; Ford and Po-

TABLE XI1 PROPERTIES Sarcoplasmic reticulum preparation White skeletal muscle microsomes

Reconstituted pump from white skeletal muscle microsomes Red skeletal muscle microsomes Cardiac microsomes

OF

ACTIVE CA2+ TRANSPORT I N ISOLATED SARCOPLASMIC RETICULUM

Maximal uptake (pmoles/mg protein)

V,,, (pmoles Ca/min/mg)

K, for Ca2+( p . V )

Present Absent Present Absent Absent Present Absent Absent Absent

0.17 6.0 0.25 0.14

0.4 -0.9

0.1-1.0

Present Absent Absent Present Present

1.2 0.04 0.07 0.30 0.42

Oxalate

-

1.8 1.8 3.6 2.8

0.14

0.15 0.15 0.12 0.10 -

Reference

10 3 0.03 25

Weber et (11. (1966) Weller et (I!. (1966) Sreter (1969) Sreter (1969) Inesi and Scarpa (1972) Worsfold and Peter (1970) Martonosi (1975) Fiehn and Migala (1971) Knowles and Racker (1975)

-

Sreter (1969) Sreter (1969) Besch and Schwartz (1971) Fanburg et a!. (1964) Baskin and Deamer (1969)

-

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ERNEST0 CARAFOLI A N D MARTIN CROMPTON

dolsky, 1970; 1972a,b; Endo et al., 1970). The work of Fabiato and Fabiato (1975) with skinned cardiac fibers has in addition demonstrated that Ca2+ can be released from reticulum when Ca2+ is introduced at concentrations markedly below the threshold concentration (about lo7 M ) that activate the myofibrils directly. The small changes in cytoplasmic Ca2+required to trigger Ca2+movements into and out of the cardiac reticulum may be due to the slow inward Ca2+ current and the Na+-CaZ+ exchange in the cardiac sarcolemma (see Section VI,A,B). The efflux of Ca2+from cardiac microsomes was demonstrated by Entman et al. (1972) at external concentrations of Ca2+greater than 30 pM. More recently, Katz et al. (1977) reported induction of net Ca2+efflux from skeletal muscle microsomes by increasing the extravesicular Ca2+ concentrations when the outside concentration is in the range 0.1-3 p M . Ca2+ release from skeletal muscle microsomes has also been reported b y Inesi and Malan (1976) who, however, used much higher external concentrations of CaZ+. Ca2+efflux has also been induced from skeletal muscle microsomes by mimicking the conditions used to investigate depolarizationinduced release of Ca2+ from skinned skeletal muscle fibers (Costantin and Podolsky, 1967; Endo and Thorens, 1975; Nakajima and Endo, 1973). In this case, Ca2+-loadedvesicles were incubated in the presence of a relatively large anion (methane sulfonate or proprionate), and then a smaller ion (Cl-) was added. C1- is assumed to be the more permeable of the two anions, so that a membrane potential is developed, positive outside. The procedure causes Ca2+ efflux from skeletal muscle microsomes (Inesi and Malan, 1976; Kasai and Miyamoto, 1976), but there is no evidence of such a mechanism in cardiac reticulum. The conclusions drawn from these experiments have been criticized (Meissner and McKinley, 1976) on the grounds that the procedures may cause osmotic damage to the microsomes. Nakamura and Schwartz (1970, 1972) considered the reported pH changes during the contraction-relaxation cycle (Waddell and Bates, 1969; Carter et al., 1967; Conway, 1957; Katz and Hecht, 1969) as a basis for inducing release and uptake of Ca2+ by sarcoplasmic reticulum. Successive changes in the extravesicular pH over the pH range 6.6-7.5 caused stepwise displacements of the accumulated Ca2+.The underlying mechanism is not known. D. Endoplasmic Reticulum

During recent years it has become evident that CaZ+transport activity is present also in the reticulum of nonmuscle cells, including liver, kidney, brain, salivary glands, and platelets (Otsuka et al., 1965; Ro-

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183

binson and Lust, 1968; Selinger e t al., 1970; D e Meis et al., 1970; Alonso et al., 1971; Robblee et al., 1973; Moore et al., 1974, 1975). These systems have not been characterized in as much detail, from the molecular and mechanistic standpoint, as those from the plasma membrane and the sarcoplasmic reticulum (see Section VI1,B) but are of obvious interest as possible components of the process of intracellular CaZ+regulation. A summary of the common properties and of the differences among the various preparations described is presented in Table XIII. All systems listed are energized by ATP in the presence of Mg2+ and require (with one exception), in agreement with the findings with muscle reticulum fragments, the copenetration of oxalate to trap the transported Ca2+ inside the vesicles. The exception is the brain preparation, which is not stimulated by oxalate, perhaps because of the impermeability of these microsomes to oxalate. The maximal amounts of Ca2+ bound by brain microsomes are therefore smaller than in microsomes from other sources. Ouabain, and the inhibitors of mitochondria1 energy transformations have no effect on the process. When tested, the transport activity has been found to be depressed by Na+, which may help to exclude the contribution by contaminating plasma membrane fragments (see Section V1,B). The maximal storage capacity varies from about 50 nmoles Ca2+/mgof protein in brain microsomes to over 2 pmoles in those from the parotid gland. The affinity of the system for Ca2+, when determined, has proved to be rather high, the highest affinity being shown by the liver microsomal preparation. The affinity for ATP varies with the preparation, e.g., 20 p M (Km)in salivary gland microsomes and 1.8 mM in liver microsomes. In some preparations, the maximal velocity of Ca2+ uptake has also been measured; in liver microsomes, it corresponds to slightly over 0.1 nmole Ca2+/sec(calculated from Moore et al., 1975). It has been found (Moore et al., 1975)that the ability to transport Ca2+ is more pronounced in smooth than in rough reticulum. Research on CaZ+transport by endoplasmic reticulum is relatively recent and has not yet progressed to the stage where sound hypotheses on the transport mechanism or conclusive data on the molecular components of the transport process have been adduced. It is likely, however, that new and important knowledge will rapidly accumulate in this area. VIII.

THE TRANSPORT OF Ca2+ BY MITOCHONDRIA

A. General Properties

Mitochondria from almost all sources that have been examined are able to accumulate Ca2+from the ambient medium. These tissues in-

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clude liver, heart, smooth and skeletal muscle, kidney, spleen, brain, and thyroid (Lehninger et al., 1967; Carafoli and Lehninger, 1971; Vallieres et al., 1975; Wikstrom et aZ., 1975, Makinen and Lee, 1968). The accumulation of Ca2+by respiring mitochondria was first reported by Vasington and Murphy (1961, 1962) and DeLuca and Engstrom (1961) and has subsequently beeen studied extensively (Lehninger et al., 1967; Carafoli and Crompton, 1976; Mela, 1977). In addition to energy-linked respiration, energy derived from ATP hydrolysis (Bielawsky and Lehninger, 1966) and, as discussed in Section VII1,B from ion gradients also supported Ca2+accumulation. Since the respiratory chain and energy-transducing ATPases are located in the inner mitochondrial membrane, this membrane is considered the locus of “active” Ca2+ transport. Indeed, mitochondria from which the outer membrane has been removed accumulate Ca2+ in a respirationdependent manner (Carafoli and Gazzotti, 1973).The accumulation of Ca2+is inhibited by very low concentrations of lanthanides (Mela, 1968, 1969) and by ruthenium red (Moore, 1971; Vasington et al., 1972; Reed and Bygrave, 1974);these cations, when used at these low concentrations, do not inhibit respiration and are not known to inhibit the transport of other ions. The binding of these cations to the presumed transport systems occurs with a very high affinity (Kd about lo-*M, Reed and Bygrave, 1974) and they occupy a maximum of about 0.08 nmole of carrier sites per milligram of protein in liver mitochondria (Mela and Chance, 1969; Reed and Bygrave, 1974); this number can be compared with the number of adenine nucleotide translocase units, i.e., about 0.14 nmole/mg protein (Weidemann et al., 1970). Ca2+influx exhibits saturation kinetics, and half-maximal velocity is attained with 2-70 pM ionized Ca2+ in the external medium (see Table XIV). The highest K, values are those obtained by spectrophotometric measurements using murexide or arsenazo as the Ca2+sensitive reagent; in these cases Mg2+was included in the reaction medium to minimize nonspecific binding of Ca2+by the mitochondria. However, Mg2+competes to a marked degree with Ca2+for the transport system when its concentration is several orders of magnitude higher than that of Ca2+(Sordahl, 1974; Jacobus et al., 1975; Crompton et al., 1976 b ; Akerman et aZ., 1977). Measurements made in the presence of M e + may not reflect the true affhity of the system for Ca2+.In heart mitochondria, the presence of 1 mM Mg2+ transforms the velocity-versus-Ca2+ plot from a hyperbolic to a sigmoidal function (Crompton et aZ., 1976b). A similar effect of Mg2+ on Ca2+transport by liver mitochondria has also been reported (Akerman et al., 1977), although other workers have observed sigmoidal kinetics with liver

TABLE XI11 PROPERTIES OF ACTIVE CA2+ TRANSPORT I N ISOLATED ENDOPLASMIC RETICULUM Source of endoplasinic reticulum

Energy source

M g"

Oxalate

Maximal capacity (nmoles/mg protein)

ATP

Required

N o stimulation

-50

ATP

Required

N o stimulation

-50

ATP

Required

N o stimulation

-65

50-100

ATP

Required

Stimulates

2750

-

ATP

Required

Stimulates

150

100

Kidney

ATP

Required

Stimulates

-50

20

Liver

ATP

Required

Stimulates

-400

Platelets

ATP

Required

Stimulates

-350

Brain

Salivary gland

K, for Caz+ (bU)

4.6 100

Inhibitors

Reference

SH reagents, oligomycin, amytal -

Robinson and Lust (1968)

Salyrgan

SH reagents, ol igoniycin -

Otsuka et al. (1965) DeMeis et d. (1970) Selinger et (11. ( 1970) Alonso et a!. (1971) Moore et (11. (1974) Moore et (11. (1975) Robblee et al. (1973)

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ERNEST0 CARAFOLI A N D MARTIN CROMPTON

TABLE XJV A SURVEY OF THE !dEASUREMENTS OF T H E AFFINITYOF

MITOCHONDRIA FOR C A ~ IN + DIFFERENT TISSUESO Reference Liver

50-70 2 2-3 4

8 Lucilia Hight muscle Heart

5 15 1

10-15

Myometrium

Vascular smooth muscle

10-15 25 5 17

Direct (murexide) Direct (Ca" buffers) Indirect (redox shift of cytochrome 11) Direct (Ca2+buffers, inhibitor stop) Indirect (0, consumption) Direct (Caz+buffers, inhibitor stop) Indirect (redox shift of cytochroine 11) Indirect (redox shift of cytochrome b) Direct (Ca" buffers, inhibitor stop) Direct (Ca2+electrode) Direct (murexide) Direct (Calf buffers, inhibitor stop) Direct (murexide or arsenazo)

Vinogradov and Scarpa (1973) Bygrave et a l . (1971) Carafoli and Azzi (1972) Reed and Bygrave (1975) Reynafarje and Lehninger (1973) Bygrave et al. (1975) Chance and Schoerner (1966) Jacobus et o l . (1975) Crompton et ul. (1976b) Crompton et ul. (1976b) Wikstrom et al. (1975) Malmstrom and Carafoli (1978) Vallieres et (11. (1975)

Measurements in which the affinity of heart mitochondria for Ca2+has been evaluated in the presence of excess Mg2+have not been included in this table.

mitochondria in the absence of Mg2+(Reed and Bygrave, 1975). In the latter case, the data have been interpreted to indicate that the carrier contains two binding sites for Ca2+(Spencer and Bygrave, 1973; Reed and Bygrave, 1975). The capacity of mitochondria in rat heart to accumulate Ca2+in the tissue has been estimated by Crompton et d . (1976b). T h e maximum velocity of net CaZ+influx into cardiac mitochondria is about 0.09 pmole Caz+/mg of protein per minute at 25"C, and about twice this at 38°C. Since the amount of mitochondria in 1 gm of heart corresponds to about 100 mg of protein (Scarpa and Graziotti, 1973), the maximum mitochondrial activity is about 18 pmoles Caz+/gm of tissue per minute (at 38°C). This value is higher than the requirements for relaxation (about 10 pmoles/gm of tissue per minute; from Solaro et d., 1974). However, the affinity of the mitochondrial uptake process for

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187

Ca2+becomes rather low in the presence of M$+; with 1mM M$+, for example (a concentration close to the physiological one), half-maximal velocity requires about 40 pM ionized Ca2+(Crompton et al., 1976b).

B. The Transport Mechanism

Selwyn et al. (1970) showed that Ca2+ influx can occur in the absence of respiration or ATPase activity when a diffusion potential is imposed across the inner membrane by a gradient of SCN- (which is permeant) or H+ (in the presence of an uncoupling agent to permit electrogenic proton permeation). At the same time, Scarpa and Azzone (1970) used a diffusion potential created by a gradient of K+ (in the presence of valinomycin) to drive Ca2+ movement. These observations allowed Ca2+movement to b e interpreted within chemiosmotic principles, since Ca2+appeared to move across the inner membrane in response to its electrochemical gradient, as proposed by Mitchell (1966). According to this proposal, the electric potential, negative inside, caused by respiration or ATP hydrolysis drives the purely electrophoretic influx of Ca2+. Further evidence favoring this mechanism has been provided by Rottenberg and Scarpa (1974) and Heaton and Nicholls (1976), who compared the distribution of Ca2+between mitochondria and medium in energized mitochondria with that of K+ in the presence of valinomycin. Under these conditons, K+ is assumed to attain electrochemical equilibrium according to the Nernst equation, so that the K+ distribution can b e used to determine the electric potential difference. The data were in good accordance with a purely electrophoretic migration of Ca2+,with a net charge transfer of 2 per Ca2+ions transported. However, in these experiments, the activity of Ca2+in the mitochondrial matrix is not known, since not all the Ca2+is ionized. Indeed, in the absence of simultaneous anion fluxes most of the Ca2+ taken up remains bound (Chappell et al., 1963; Gunter and Puskin, 1972; Gunter et al., 1975). With a parallel influx of anions, together with dissociable protons (e.g., acetate), less Ca2+ is bound; however, some Ca2+is invariably bound to an extent that depends on the experimental conditions (Gunter and Puskin, 1976), so that Ca2+distribution data cannot be interpreted too rigorously. The electrical difference across the inner membrane is generally considered to result from the respiration-driven (or ATP-driven) efflux of H+ (Mitchell, 1966; see also Papa, 1976). The electrochemical gradient of H+ (AbH+) is thus given by

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ERNEST0 CARAFOLI A N D MARTIN CROMPTON

An electrophoretic influx of Ca2+would tend to collapse the electrical component of the electrochemical gradient and thereby promote further respiration and H+ ejection; i.e., respiration would tend to restore A&+, but the latter would be composed of a decreased electrical term and an increased concentration term. Consistent with this, the energylinked uptake of Ca2+is accompanied by ejection of H+ into the ambient medium (Saris, 1963). However, the number of protons ejected per Ca2+ions taken up has generally been reported to be 0.8 to 1.4, i.e., about 1 (Saris, 1963; Chance, 1965; Chance and Mela, 1966a,b; Gear et al., 1967; Addanki et al., 1968; Engstrom and DeLuca, 1964; Rossi et d.,1966). This is not consisent with a purely electrophoretic movement of Ca2+since, in this case, the H+/Ca2+ratio predicted is 2. A possible complication in these experiments is that H+ backflow across the inner membrane may occur to an increasing degree with time, so that true initial rate measurements are required. Brand et a2. (1976a) introduced acetate and other permeant weak acids into the medium, so that their entry would continually dissipate the pH gradient. Under these conditions, about 2 moles of weak acid accumuIated for each Ca2+,suggesting that the actual H+/Ca2+ratio is 2. However, Reed and Bygrave (1975) provided evidence that the group responsible for the initial binding of Ca2+has a proton dissociation constant of 10-7.8and is able to bind Ca2+in the dissociated state. If it is assumed that H+ and Ca2+compete for the binding site on the inside of the membrane, this might provide an explanation for the observed stimulation of the rate of Ca2+ uptake by acetate and other anions, e.g., phosphate, that copenetrate with protons (Reed and Bygrave, 1975; Crompton et aZ., 197613). The influx of such anions would tend to prevent an increase in the matrix pH during Ca2+influx and thereby promote the dissociation of Ca2+from the carrier after transport. Reed and Bygrave (1975) incorporated this concept within a proposed mechanism of a 1: 1exchange between Ca2+and protons, involving return of the protonated carrier to the outer face of the membrane. This scheme is consistent with the observed stoichiometry of about two Ca2+ ions accumulated per pair of electrons that traverse each energy-conserving site of the respiratory chain (Chappell et al., 1963; Rossi and Lehninger, 1964; Chance, 1965; Bielawski and Lehninger, 1966), together with two protons being extruded per two electrons per coupling site (Mitchell and Moyle, 1967, 1973; Lawford and Garland,

THE REGULATION OF INTRACELLULAR CALCIUM

189

1972,1973; Leung and Hinkle, 1975; Papa, 1976). However, the equilibrium distribution of Ca2+ in energized mitochondria is quite different from that predicted from Ca2+-H+ exchange and approximates closely to that following Ca2+uniport (Heaton and Nicholls, 1976). We recall, however, that these data cannot be interpreted too rigorously. Moreover, the interpretation of uniport is not consistent with the generally reported ratio of 1 between the total moles of H + extruded during respiration-supported Ca2+influx and the moles of Ca2+accumulated. Ratios of close to 2, which are consistent with such a mechanism, have been reported (Brand et al., 1976a; Rossi et al., 1967; Wenner and Hackney, 1967). Recently, Brand et al. (1976b) reported that between three and four protons are extruded per two electrons traversing each site of the respiratory chain. In these experiments, the backflow of H+ associated with the backflow of endogenous phosphate during respirationinduced ejection of protons was prevented by the inclusion of N-ethylmaleimide to inhibit the phosphate carrier. A higher H+/coupling site ratio would permit a purely electrophoretic uniport of Ca2+to be reconciled with the generally observed influx of two Ca2+ ions per two electrons per site. In summary, there is considerable evidence for an electrophoretic component in the driving force for Ca2+ uptake by mitochondria. Whether or not the carrier catalyzes a purely electrophoretic uniport of Ca2+or a partially charge-compensated exchange with other cations (e.g., H+) is open to question. Unlike the Ca2+ pumps of the sarcoplasmic reticulum and plasma membrane, there is no evidence that the Ca2+transport system is an active pump that transduces the free energy of a chemical reaction into the free energy of a Ca2+gradient. On the contrary, the energy input into the Ca2+influx process appears to be derived from the electrochemical gradient of protons across the inner membrane generated by the primary proton pumps of the respiratory chain. For this reason, experiments designed to elucidate the mechanism of Ca2+ translocation are often complicated by uncertainties involving the primary process of energy transduction.

C. The Capacity of Mitochondria to Accumulate Ca2+ and the Reversibility of the Transport Process in Vivo; the Mechanism of Ca2+ Efflux from Mitochondria

In the absence of added permeant anions respiring mitochondria accumulate about 100 nmoles Ca2+/mg of protein (Lehninger et al.,

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1967; Carafoli and Crompton, 1976; Mela, this volume). It is relevant to mention here that the internal volume of mitochondria is about 1 pl/mg of protein. When phosphate is present, it is accumulated as well. This is understandable, since phosphate is transported by the phosphate carrier by symport with protons (Chappell and Crofts, 1966; Klingenberg et al., 1974), hence its accumulation is driven by the pH gradient, acid outside, generated by the respiration-supported accumulation of Ca2+.For reasons that are still obscure, the presence of ADP or ATP and Mg2+in the external medium promotes the precipitation of calcium phosphate within the mitochondria1 matrix (Brierley and Slautterback, 1964; Greenawalt et al., 1964; Peachey, 1964; Greenawalt and Carafoli, 1966; Weinbach and von Brand, 1965). Under such in vitro conditions as much as 3 pmoles of calcium phosphate are accumulated per milligram of protein. This underlines the remarkable accumulating capacity of mitochondria for Ca2+. Indeed, such a capacity is predicted from the electrophoretic movement of Ca2+ in response to an electric potential difference of 160-200 mV across the inner membrane of magnitude (Mitchell and Moyle, 1969; Nicholls, 1974; but see Padan and Rottenberg, 1973, for lower values). With an electric potential difference of 180 mV, the equilibrium gradient of ionized Ca2+ across the inner membrane is lo6 for passive Ca2+movement (passive uniport). However, if the system responsible for Ca2+transport effectively catalyzed an exchange between one Ca2+ ion and one proton (Reed and Bygrave, 1975), the predicted gradient of Ca2+would be much less than lo6.In this case the equilibrium gradient of Ca2+would be about lo3 if the pH gradient across the inner membrane were ignored. Since the extramitochondrial p H is more acid than the intramitochondrial pH, the p H gradient would decrease the equilibrium gradient of Ca2+ to values less than 103. Unfortunately, the pH gradient in vivo is not known; it seems likely to be small, however, since it is decreased by the accumulation of phosphate. The latter in turn is coupled to the accumulation of dicarboxylic acid cycle intermediates (McGivan and Klingenberg, 1974). One way to gain an idea of the actual in vivo gradient of ionized Ca2+across the inner membrane is to analyze the intramitochondrial and cytoplasmic enzymes that may be subject to Ca2+regulation. To do this it is necessary to assume that Ca-dependent changes in enzyme activity observed in vitro also occur at these concentrations in vivo. In the case of heart and skeletal muscle (Crompton et al., 1976a) this means the free Ca2+ concentration in the cytoplasm and inside the mitochondrion is 0.1-10 pM, This calculation reflects what is known about the myofibrillar ATPase, phosphorylase kinase, and

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glycerol phosphate dehydrogenase of the cytoplasm, and the pyruvate dehydrogenase phosphate phosphatase, pyruvate dehydrogenase kinase, and isocitrate dehydrogenase of the mitochondria (Ebashi and Endo, 1968; Brostrom et al., 1971; Hansford and Chappell, 1967; Zammit and Newsholme, 1976; Denton et al., 1972; Cooper et nl., 1974; Vaughan and Newsholme, 1969).These calculations imply that the gradient of ionized Ca2+across the inner membrane in vivo is less than los and probably less than 101. This excludes the possibility that a Ca2+uniport mechanism approaches equilibrium. More direct estimates of the ionized Ca2+gradient across the inner membrane of liver mitochondria respiring in vitro have been reported by Puskin et al. (1976), who used Mn2+ as an electron paramagnetic analog of Ca2+. These investigators observed that the gradient of what appeared to be ionized Mn2+ approximated lo3, whereas the gradient would have been at least lo5if the Mn2+had attained electrochemical equilibrium (i.e., by passive uniport). Puskin and co-workers interpreted their data to mean that there exist two systems for Mn2+(and Ca2+)transport in liver mitochondria: a system responsible for Ca2+influx (e.g., passive uniport) and an additional system that is partially or completely charge-compensated. This interpretation was supported b y the fact that ruthenium red induces a net efflux of Mn2+ (and Ca2+; see also Sordahl, 1974) but does not decrease the electric potential across the inner membrane (Puskin et al., 1976). This means the efflux process is not sensitive to ruthenium red. The mechanism by which Ca2+efflux is charge-compensated has not been identified. Direct evidence for two separate systems of Ca2+transport has been obtained in the case of heart mitochondria. Following reports that Na+ depresses Ca2+uptake and induces Ca2+release from heart mitochondria (Carafoli et al., 1974; Noack and Greff, 1975; Dhalla et al., 1975), the existence of a distinct Na+-dependent route of Ca2+efflux was reported b y Crompton et al. (1976a), who showed that the addition of Na+ to mitochondria inhibited with ruthenium red induces a rapid efflux of intramitochondrial Ca2+(Fig. 4). The stimulation b y Na+ was interpreted to indicate that the ruthenium red-insensitive system catalyzes an exchange between Na+ and Ca2+,thus permitting Ca2+ efflux against its electrochemical gradient. The Na+-induced efflux of Ca2+from heart mitochondria has a maximum velocity of about 15 nmoles Ca2+/mg of protein per minute at 25"C,and half-maximal velocity is attained with about 8 mM Na+. The rate of Na+-dependent Ca2+efflux is adequate to compete effectively with the rate of Ca2+influx when Ca2+and Na+ are present in physiological concentrations, i.e., 1.5-4.5 p M and 3.0-20 mM, respectively.

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F FIG.4. The release of CaZ+from heart mitochondria by NaC(in the presence of ruihenium red). The preparation of the mitochondria and the use of the Caz+electrode are described in Crompton et al. (1976a). The reaction medium contained 120 mM KCI, 10 mM HEPES (pH 7.2), 2 pg rotenone, and 2 mg mitochondrial protein, in 4.0 ml, at 25°C. K succinate (1.25 mM) was added as indicated to energize the mitochondria. Ruthenium red (0.1p M ) was added at point RR to prevent reuptake of the lost Caz+.Na+ (13 mM) was added at the point indicated. (From Crompton et al., 1976a.)

The curve relating the rate of CaZ+release to the concentration of external Na+ exhibits pronounced sigmoidal qualities, and the kinetic data are consistent with a process in which two or more Na+ ions are involved in each exchange cycle. The sigmoidal properties may be physiologically important, since over a certain range of Na+ (4-10 mM) the rate of Ca2+ efflux is markedly changed by relatively small changes in the Na+ concentration. The intracellular Na+ activity in heart, determined with microelectrodes, is about 6 mM (Lee and Fozzard, 1975). However, the increase in sarcoplasmic Na+ that occurs on depolarization of the sarcolemma, for example, is likely to be very small (i.e., no ,greater than about 0.1 mM, and probably much less than this; Reuter, personal communication; Niedergerke, personal communication). However, the mitochondrial Na+-Ca2+ exchange may be important in the positive ionotropic effect of cardiac glycosides (Crompton et al., 1976a). In this case, glycoside inhibition of Na+ extrusion from the ceIl by the Na+, K+-/ATPase would be predicted to result in increased intracellular Na+ which would induce net Caz+efflux from the mitochondria. A further contribution may also be made by the Na+-Ca2+ exchange in the sarcolemma, although the activity of the sarcolemmal carrier (0.1-1.0 nmole Caz+/gm wet weight per second) appears to b e less than the maximal activity of the mitochondrial

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carrier [about 20 nmoles Ca2+/gm wet weight per second at 25"C, calculated from the data of Crompton et al. (1976a) on the assumption that the mitochondrial protein content of heart is 100 mg/gm wet weight, Scarpa and Graziotti (1973)l. Data obtained by other workers suggest that a Na+-dependent efflux of Ca2+may occur in mitochondria of other tissues. Thorn et al. (1975) reported that Na+ induces a release of Ca2+from mitochondria isolated from the neurohypophysis. Lowe et al. (1976), working with isolated islets of Langerhans, observed that a large influx of Na+ into the cell (induced by veratridine) promoted the release of insulin, which in turn is Ca2+-dependent; since EGTA was included in the external medium, the source of Ca2+ was probably intracellular, and perhaps mitochondrial. Swanson and Stahl (1975) observed that Na+ inhibits the net rate of uptake of Ca2+by brain mitochondria, and this may indicate the existence of a competing Na+-dependent efflux pathway for Ca2+.Such a mechanism is potentially important, since the mitochondria of brain slices sequester most of the Ca2+accumulated (Stahl and Swanson, 1972; Cooke and Robinson, 1971).

IX.

THE TRANSCELLULAR TRANSPORT OF Ca2+

It is clear from what has been discussed in the preceding sections that the intracellular concentration of Ca2+is very precisely regulated at low levels. However, in extracellular fluids, and expecially in blood, Ca2+is also closely controlled. This requires the proper functioning of at least three processes, which are absorption at the intestinal level, reabsorption by the kidney tubules, and reversible deposition in bone. As a consequence, the cells of these three tissues must continuously transport very large amounts of Ca2+ and are therefore continuously exposed to the risk of high levels of Ca2+in the cytosol. The capacity of the outwardly directed pumps in the plasma membrane, and of the sequestering systems in the intracellular organelles, is very large but is not unlimited. Consequently, these systems could easily be swamped by excess Ca2+penetrating the cell. The presence of large insoluble mitochondrial deposits, assumed to b e Ca phosphate, in tissues involved in Ca2+transport has been repeatedly observed in vivo (osteoclasts in healing bone fractures: Gonzales and Kamovski, 1961; condrocytes: Martin and Matthews, 1969). This is consistent with the possibility that mitochondria can serve as calcium buffers. However, accumulation of Ca2+by mitochondria, in addition to constantly dissipating energy, leads eventually to irreversible func-

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tional and structural damage to organelles. As a consequence, in the absence of efficient sequestering systems, mitochondria would probably loose the accumulated Ca2+which would then rise in the cytosol to toxic levels. These problems are circumvented in a model proposed by Terepka et al. (1976) in which Ca2+ is transported across the cells of Ca2+transporting tissues (e.g., small intestine) without mixing with the intracellular Ca2+pool. Ca2+in transit in these cells is in fact still extracellular, and therefore does not impose excessive demands on the outwardly directed pumps or on the sequestering organelles, The model has been developed from studies of Ca2+ transport on the isolated chick chorioallantoic membrane and on isolated preparations of small intestine (Terepka et al., 1976). When incubated in media containing 1.0 mM Ca2+, the Ca2+ content of the tissue increased u p to three times. By mapping the sites of Ca2+accumulation in the tissue with an x-ray microprobe, it was concluded that, during transport, Ca2+ was sequestered within portions of the cytoplasm. Similar conclusions could be reached from electron paramagnetic resonance experiments in which the paramagnetic cation Mn2+ was used as an analog of Ca2+.Then studies revealed discrete, cation-rich loci within the transporting cells. The Ca2+-richloci were localized in areas of the cell in which conventional electron microscopy indicates a scarcity of mitochondria. That mitochondria are not prominently involved in sequestering Ca2+ during its transit in these tissues was also shown by calculations of the total Ca2+content of the tissue, which would require implausibly high levels of mitochondria in the tissue if the mitochondria were the only storage sites. In addition, the amount of Ca2+transported by the tissue was not decreased by uncoupling mitochondria in uiuo with dinitrophenol. Terepka et al. (1976) consider that Ca2+may be enclosed in packets formed b y vesiculation of the plasma membrane. This model (Fig. 5 ) also accounts for two further observations, i.e., the apparent energy dependence of the transport of Ca2+across the chorioallantoic membrane and the dependence of Ca2+ entry into chorioallantoic cells (i.e., into the vesicles formed from the plasma membrane) on the external Na+ concentration (Armstrong et d.,quoted in Terepka et al., 1976). Ca2+movement is thought to occur by exchange with Na+. In addition, the model accounts for the presence of numerous vesicles and numerous fibers which tend to be orientated in a basal-to-apical direction. As depicted in the model, invaginations of the plasma membrane would lead to the formation of endocytotic vesicles, into which

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FIG.5. A model for the transcellular transport of calcium. Step 1 illustrates the invagination of the plasma mebbrane, step 11 the formation of the endocytotic vesicle and the ejection of cytosolic CaZ+into it, step 111 the migration of the interiorized Ca2+loaded vesicle across the cell, and step IV the fusion of the vesicle with the plasma membrane and the extrusion of Ca2+.The crossed circles represent the Na+-dependent ejection of Ca2+from the cytosol. (From Terepka et al., 1976.)

the outwardly directed, presumably Na+-dependent,exchange system would transport the Ca2+that leaked into the cytosol through other portions of the plasma membrane. The vesicles would then move across the cytosol and discharge Ca2+at the other pole of the cell by an exocytotic process. One unclear facet of the model is what controls the vectorial transfer of the vesicles in the cells. It can be speculated that microfilaments play a role in this transfer (Terepka et al., 1976), but proof is lacking.

X.

CONCLUSIONS

In the messenger function of Ca2+,two aspects can be conveniently distinguished. Ca2+may act as a primary messenger, that is, it may directly generate the primary signal at the level of the plasma membrane¶ by carrying the current during depolarization, or modulate the signals generated by the other common current-carrying cations (Na+, K+, Ca2+itself). Or it may act as a secondary messenger, that is, provide a link in transmission of the primary signal from the plasma membrane to the interior of the cell, where the targets are contained (Weber, 1976).

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The primary messenger function has been repeatedly alluded to in the text. An extensive survey of the literature on this point would be beyond the scope of this article, which is primarily concerned with the ways and means of regulation of intracellular Ca2+. As for the secondary messenger function, that is, carrying the signal to the interior of the cell, where the targets mentioned in Table I are contained, it has been discussed extensively by other investigators (e.g., see Rasmussen, 1970). Something could perhaps be added at this point on the biological rationale for the necessity of regulating Ca2+, in the course of its secondary messenger function, at very low levels. For example, one could speculate on whether or not the choice of Ca2+ as a biological messenger, and thus the development of efficient pumping systems to control it, was primarily dictated by the unique fitness of Ca2+,which made it an idea1,evolutionary choice, or whether other factors also played a role in the choice. For example, as recently speculated by Weber (1976) and by Kretsinger (1978), the development of efficient pumping systems for Ca2+could have been dictated by the mandatory high metabolic requirements for inorganic phosphate. To avoid intracellular precipitation of Ca2+ and phosphate, equilibration of the highly concentrated extracellular Ca2+with the intracellular milieu had to be avoided. It seems reasonable that both the biological necessity and the chemical opportunity have been instrumental in shaping the course followed by evolution, entailing the development of both Ca2+transport processes and target processes for Ca2+.As described in this article three types of Ca2+transport processes have been recognized: Ca2+-ATPases(sarcoplasmic reticulum, plasma membrane of erythrocytes), Ca2+-Na+exchanges (many plasma membranes, inner membrane of certain mitochondria), and systems that appear to catalyze a charge-uncompensated movement of Ca2+ not directly coupled to the countertransport of other cations (mitochondrial inner membrane, heart sarcolemma). This report has attempted to bring together the essential information on these different modes of Ca2+transport as they operate in the different membrane systems of the cell. It is hoped that in doing so a vast amount of scattered information has been brought together into a more meaningful picture. ACKNOWLEDGMENTS The authors are indebted to Profs. R. Niedergerke (London), H. Reuter (Bern), L. Venanzi (Zurich), and E. R. Weibel (Bern) for helpful discussions and advice on various aspects of the subject matter of this article. They are also indebted to Miss M. Nadler for her very helpful secretarial assistance. The original work described in this report was aided by financial contributions from the Swiss National Science Foundation (Grants No. 3.592-0.73and 3.592-0.75).

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Tasaki, I. (1968).“Nerve Excitation.” Thomas, Springfield, Illinois. Taylor, A. N., and Wasserman, R. H, (1967). Vitamin Dhinduced calcium-binding protein. Partial purification, electrophoretic visualization, and tissue distribution. Arch. Biochem. Biophys. 119, 536-540. Taylor, A. N., and Wasserman, R. H. (1974). The vitamin D-induced calcium binding protein. Some physiological and physical characteristics. In “Calcium Binding Proteins” (W. Drabikowski, H. Stnelecka-Golaszewska, and E. Carafoli, eds.), pp. 751-769. Elsevier, Amsterdam and Polish Scientific Publ., Warsaw. Terepka, A. R., Coleman, J. R., Armbrecht, H. J., and Gunter, T. E. (1976).Transcellular transport of calcium. Symp. SOC. E x p . Biol. 30, 117-140. Thakar, J. H., Wrogemann, K., and Blanchare, M. C. (1973). Effect of ruthenium red on oxidative phosphorylation and the calcium and magnesium content of skeletal muscle mitochondria of normal and Bio-14.6-dystrophic hamster. Biochim. Bdophys. Acta 314,B-14. The, R., and Hasselbach, W. (1972). Properties of the sarcoplasmic ATPase reconstituted by oleate and lysolecithin after lipid depletion. Eur. J. Biochem. 28,357-363. Thorn, N. A., Russell, J. T., and Robinson, I. C. A. F. (1975). Factors affecting intracellular concentration of free calcium ions in neurosecretory nerve endings. In “Calcium Transport in Contraction and Secretion” (E. Carafoli, F. Clementi, W. Drabikowski, and A. Margreth, eds.), pp. 261-270. North-Holland Publ., Amsterdam. Thorley-Lawson, D. A., and Green, N. M. (1973). Studies on the location and orientation of proteins in the sarcoplasmic reticulum. Eur. J. Biochem. 40,403-413. Tobias, J . M. (1964). A chemically specified molecular mechanism underlying excitation in nerve: A hypothesis. Nature (London) 203, 13-17. Trauble, H., and Eibl, H. (1974). Electrostatic effects on lipid phase transitions: Membrane structure and ionic environment. Proc. Natl. Acad. Sci. U.S.A. 71,214-219. Trautwein, W. (1973). Membrane currents in cardiac muscle fibers. Physiol. Rev. 53, 793-835. Tritthart, H., Volkmann, R., Weiss, R., and Fleckenstein, A. (1975). Inhibition of calcium dependent action potentials in mammalian myocardium by specific inhibitors of the transmembrane calcium conductivity (Verapamil, D600). I n “Recent Advances in Studies on Cardiac Structure and Metabolism” (A. Fleckenstein and N. S. Dhalla, eds.), Vol. 5, pp. 27-33. Univ. Park Press, Baltimore, Maryland. Urry, D. W. (1972). Protein conformation in biomembranes: Optical relation and absorption of membrane suspensions. Biochim. Biophys. Acta 265,115-168. Valberg, L. S., Holt, J. M., Paulson, E., and Szivek, J. (1965).Spectrochemical analysis of sodium, potassium, calcium, magnesium, copper and zinc in normal human erythrocytes.]. C l h Invest. 44,379-389. Vallieres, J., Scarpa, A., and Somlyo, A. P. (1975). Subcellular fractions of smooth muscle. Isolation, substrate utilization and calcium transport by main pulmonary artery and mesenteric vein mitochondria. Arch. Biochem. Biophys. 170, 659-669. Van Breemen, C., Farinas, B. R., Gerba, P., and McNaughton, E. P. (1973). Excitation-contraction coupling in rabbit aorta studied by the lanthanum method for measuring cellular calcium influx. Circ. Res. 30,44-54. Vanderkooi, J . M., and Martonosi, A. (1971). The permeability of phosphatidylcholine vesicles for calcium. Arch. Biochem. Biophys. 147, 632-646. Vaughan, H., and Newsholme, E. A. (1969).The effects of Ca2+and ADP on the activity of NAD-linked isocitrate dehydrogenase in muscle. FEBS Lett. 5, 124-126. Van Rossum, G. D. V. (1970). Net movements of Ca2+and Mg*+in slices of rat liver. J. Gen. Physiol. 55,lB-32.

THE REGULATION OF INTRACELLULAR CALCIUM

21 5

Vasington, F. D., and Murphy, J. V. (1961).Active binding ofcalcium by mitochondria. Fed. Proc., Fed. A m . S O C . E r p . Biol. 20, 146. Vasington, F. D., and Murphy, J. V. (1962).Ca2+uptake by rat kidney mitochondria and its dependence on respiration and phosphorylation.]. Biol. Chem. 237,2670-2672. Vasington, F. D., Gazzotti, P., Tiozzo, R., and Carafoli, E. (1972). The effect of ruthenium red on calcium transport and respiration in rat liver mitochondria. Biochirn. Biophys. Acta 256,43-54. Verkleiy, A. J., Zwaal, R. F. A., Roelofsen, B., Cumfurius, P., Kastelijn, D., and Van Deenen, L. L. M. (1973). The asymmetric distribution of phospholipids in the human red cell membrane. Biochini. Biophys. Acta 323, 178-193. Vinogradov, A,, and Scarpa, A. (1973). The initial velocities of calcium uptake by rat liver mitochondria. ]. Biol. Chem. 248, 5527-5539. Vinogradov, A., Scarpa, A,, and Chance, B. (1972).Calcium and pyridine nucleotide interaction in mitochondria1 membranes. Arch. Biochem. Biophys. 152,646-654. Wacker, W. E. C., and Williams, R. J. P. (1968). Magnesium/calcium balances and steady states of biological systems.]. Theor. Biol. 20, 65-78. Waddell, W. J., and Bates, R. G. (1969). Intracellular pH. Physiol. Reo. 49,285-329. Walser, M. (1961). Ion association. VI. Interaction between calcium, magnesium, inorganic phosphate, citrate and protein in normal human plasma. ]. Clin. Znoest. 40, 723-730. Warren, G . B., Toon, P. A., Birdsall, N. J. M., Lee, A. G . , and Metcalfe, J. C. (1974).Reconstitution of a calcium pump using defined membrane components. Proc. Natl. Acad. Sci. U.S.A. 71, 622-626. Wasserman, R. H., and Taylor, A. N. (1966).Vitamin D,-induced calcium binding protein in chick intestinal mucosa. Science 152, 791-793. Wasserman, R. H., and Taylor, A. N. (1968).Vitamin D-dependent calcium binding protein. Response to some physiological and nutritional variables.]. Biol. Chem. 243, 3978 -3986. Watson, E. L., Vincenzi, F. F., and Davies, P. W. (1971). Ca2+activated membrane ATPase: Selective inhibition by ruthenium red. Biochim. Biophys. Acta 249, 606-610. Weber, A. (1966). Energised calcium transport and relaxing factors. Cum. Top. Bioenerg. 1,203-254. Weber, A. (1971).Regulatory mechanism of the calcium transport system of fragmented rabbit sarcoplasmic reticulum. I. The effect of accumulated calcium on transport and adenosine triphosphate hydrolysis. J. Gen. Physiol. 57, 50-63. Weber, A. (1976). Synopsis of the presentations. Symp. S O C . E x p . Biol. 30,445-455. Weber, A., Herz, R., and Reiss, I. (1966).Study of the kinetics of calcium transport by isolated fragmented sarcoplasmic reticulum. Biochem. Z. 345,329-369. Weed, R. I., LaCelle, P. L., and Merrill, E. W. (1969).The metabolic dependence of red cell deformability. J . Clin.Znuest. 48, 795-809. Weidemann, M. J., Erdelt, H., and Klingenberg, M. (1970). Adenine nucleotide translocation of mitochondria. Identification of carrier sites. Eur. J . Biochern. 16, 3 13-333. Weinbach, E. C., and von Brand, T. (1965). The isolation and composition of dense granules from Ca2+ loaded mitochondria. Biochem. Biophys. Res. Commun. 19, 133-137. Wenner, C. E., and Hackney, J. H. (1967). H+ changes associated with divalent cation uptake by mouse liver mitochondria. ]. B i d . Chem. 242,5053-5058. Wikstrom, M., Ahonen, P., and Luukkainen, T. (1975). The role of mitochondria in uterine contraction. FEES Lett. 56, 120-123.

21 6

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Wilbrandt, W., and Koller, H. (1948). Die Calciumwirkung am Froschherzen als Function des Ionengleichgewichts zwischen Zellmembrane and Umgebung. Helo. Physol. Pharmacol. Acta 6,208-211. Williams, R. J . P. (1975).T h e binding of metal ions to membranes and its consequences. I n “Biological Membranes: Twelve Assays on Their Organization, Properties, and Functions” (D. S. Parsons, ed.), pp. 106-121. Oxford Univ. Press (Clarendon), London and New York. Williams, R. J . P. (1976). Calcium chemistry and its relation to biological function. Symp. Soc. E r p . Biol. 30, 1-17. Winegrad, S. (1970).The intracellular site of calcium activation of contraction in frog skeletal muscle. J . Gen. Physiol. 55,77-88. Winegrad, S., and Shanes, A. M. (1962). Calcium flux and contractility in guinea-pig atria. J . C e n . Physiol. 45, 371-394. Wolf, H. V. (1970). Purification of the Caz+-dependent ATPase of human erythrocyte membranes. Biochim. Biophys. Acta 219,521-524. Wolf, H. V. (1972). Studies on a Caz+-dependent ATPase of human erythrocyte. Biochim. Biophys. Acta 266,361-375. Wollenberger, A., Will, H., and Krause, E. C . (1975). Adenosine 3’,5’-monophosphate, the myocardial cell membrane, and calcium. I n “Recent Advances in Studies on Cardiac Structure and Metabolism” (A. Fleckenstein and N. S. Dhalla, eds.), Vol. 5, pp. 81-93. Univ. Park Press, Balitmore, Maryland. Worsfold, M., and Peter, J . B. (1970). Kinetics of calcium transport by fragmented sarcoplasmic reticulum. J . Biol. Chem. 245,5545-5552. Wray, H. L., Gray, R. R., and Olsson, R. A. (1973). Cyclic adenosine 3‘,5’monophosphate-stimulated protein kinase and a substrate associated with cardiac sarcoplasmic reticulum. J . Biol. Chem. 248, 1496-1498. Yamada, S., and Tonomura, Y. (1972). Reaction mechanism of the Ca2+-dependent ATPase of sarcoplasmic reticulum from skeletal muscle. VII. Recognition and release of Caz+ions.]. Biochern. (Tokyo) 72,417-425. Yamamoto, T., and Tonomura, Y. (1968). Reaction mechanism of the Caz+-dependent ATPase of sarcoplasmic reticulum from skeletal muscle. 11. Intermediate formation of phosphoryl protein. J . Biochem. (Tokyo)64, 137-145. Yamamoto, T., Yoda, A., and Tonomura, Y. (1971). Reaction mechanism of the Ca2+dependent ATPase of sarcoplasmic reticulum from skeletal muscle. IV. Hydroxamate formation from a phosphorylated intermediate and 2-hydroxy-5-nitrobenzyl hydroxylamine. J . Biochem. (Tokyo) 69,807-809. Zammit, V. A., and Newsholme, E. A. (19’76).Effects of calcium ions and adenosine diphosphate on the activities of NAD+-linked isocitrate dehydrogenase from the radular muscle of the whelk and flight muscles of insects. Biochem. J . 154,677-687.

Calcium Transport and the Properties of a Calcium-Activated Potassium Channel in Red Cell Membranes VIRGIL10 L . LEW AND HUGO G . FERRElRA* Physiological Laboratory Cambridge University Cambridge. England

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . A Survey of Ca-Sensitive K Permeability Processes in the Plasma . . . . . . . . . . . . . . . . . . . . . . . Membrane of Cells B . The Sequential Nature of the Process Leading to an Increase in K Permeability . . . . . . . . . . . . . . . . . . . . . . . . . . C . Is the Ca-Sensitive K Channel Present in All Cell Membranes? . . D. Some Considerations on [Ca2+], . . . . . . . . . . . . . . . . . . I1. The Ca-Sensitive K Channel in Red Cells . . . . . . . . . . . . . . . A . Historical Introduction . . . . . . . . . . . . . . . . . . . . . . B . Procedures That Activate the K Channel in Red Cells . . . . . . . C . The Gardos Effect . . . . . . . . . . . . . . . . . . . . . . . . D . Relative Efficacy of ATP-Depleting Procedures . . . . . . . . . . E . Parallelism in ATP Depletion. Ca Entry. and Increased K Flux . . . F. Heterogeneous Behavior of Cells in Relation to the Ca-Induced K Flux 111. Ca Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Ca Permeability of Intact Red Cell Membranes . . . . . . . . . . B. The Problem of the Large-Capacity. Low-Activity Ca Pump . . . . C . Cytoplasmic Ca Buffering . . . . . . . . . . . . . . . . . . . . D . Passive Ca Transport . . . . . . . . : . . . . . . . . . . . . . IV. The Gating Mechanism . . . . . . . . . . . . . . . . . . . . . . . A . Ca Sensitivity and Ca Selectivity . . . . . . . . . . . . . . . . . B . Activation and Inactivation; Time and Voltage Dependence of the KGating Process . . . . . . . . . . . . . . . . . . . . . . . . . V . Pharmacological Effects . . . . . . . . . . . . . . . . . . . . . . . A . Activators . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 . Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . .

218 218 219 222 223 224 224 227 228 229 23 1 232 234 234 237 238 242 247 247 252 256 256 259

* Permanent address: Grupo de Biofisica. Instituto Gulbenkian d e Ciencia. Oeiras. Portugal . 217

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VI. The Movement of K and the Nature of the Permeability Mechanism A. Selectivity ........................... B. KCounterflow . . . . . . . . . . . . . . . . . . . . . . . . . C. Interactions with Internal Na and External K . . . . . . . . . D. The Nature of the Ca-Sensitive K Channel . . . . . . . . . . VII. Conclusions ............................ References

. .

.............................. 1.

. .

..

265 265 266 267 269 270 271

INTRODUCTION

A. A Survey of Ca-Sensitive K Permeability Processes in the Plasma Membrane of Cells

In their various physiological roles, ion-selective channels provide specific dissipative routes for the ionic gradients which ATP-fueled pumps maintain across the cell membrane. (Nothing is known about the molecular nature of these permeability mechanisms, and the term “channel” is used throughout this article in a purely descriptive sense.) In this way, the energy stored in the form of electrochemical potential gradients is converted into electrical and osmotic work. The specific functional pr0pertie.s of each channel are determined by the structures controlling its ion selectivity and the degree and timing of activation and inactivation (gating processes). In recent years it has become apparent that a large variety of excitable and nonexcitable cells possess in their membranes K-selective channels which, unlike the more familiar Na, K, and Ca channels of nerve and muscle, have gating mechanisms controlled mainly by the concentration of ionized Ca in the cytoplasm. In Table I we report briefly various observations made on Ca-sensitive changes in the K permeability of plasma membranes and the various physiological functions attributed to these mechanisms. We have not attempted here a comprehensive survey but rather a representative listing of observations. There are many other less well-documented instances in which Ca-sensitive K channels may be involved (Meech, 1976), sometimes not even noted by the investigators (Inoue et al., 1975). A wider awareness of their existence and mode of operation may lead to a more frequent recognition of their occurrence. The importance of these processes in the regulation of various membrane functions is apparent from Table I, but their full physiological potential has still to be assessed. In this article we concentrate mainly on the Ca-sensitive K perme-

CA-ACTIVATED K CHANNEL IN BIOLOGICAL MEMBRANES

219

ability mechanism of red cells. Although the functional role of this process is completely unknown in these cells, the red cell membrane represents an extremely convenient experimental model for the study of such a mechanism. The extent to which the findings in red cells reflect the properties of this process in other cells remains to be seen. There are many similarities in terms of K selectivity, Ca dependence, and the effects of inhibitors, and there are only a few conflicting observations (see Table I). The present feeling among workers in this field is that we are probably dealing with a closely related family of Casensitive K channels. The identity of the various Ca-dependent K permeability mechanisms described in different cells is questionable. The assumption that they are not identical, however, has no predictive power and, until it is proved wrong, an assumption of identity seems to be the better working hypothesis. 8. The Sequential Nature of the Process leading to an Increase in K Permeability

An increase in the K permeability of the cell membrane usually occurs at the end of a more-or-less long sequence of steps leading to a rise in the concentration of free Ca ions in the vicinity of the cytoplasmic face of a Ca-sensitive K channel. The particular characteris tics of the phenomenon being observed, whether the measurement of a K flux or a Ca-sensitive change in membrane conductance or potential, therefore depend largely on events preceding the interaction between Ca ions and the K-gating mechanism. Because, as we see later, the binding of Ca to, and the dissociation of Ca from, the K gate seem to be the only relevant reactions for the opening and closing of the K channel, and because these reactions are apparently very fast, the onset, evolution and termination of the K permeability change closely follows the local variation in free Ca in the cytoplasm (Ca?'). It is therefore the process causing this variation that is mainly reflected in the magnitude and time course of the K permeability change. This is not totally correct, because the reliability of the K channel as a local Ca2" indicator depends on the constancy of its Ca sensitivity, and this has recently been found to be capable of substantial fluctuations (Lew and Ferreira, 1976). This means that at a constant concentration of ionized Ca in the cytoplasm [Ca2+li,the channel behavior is determined by the factors controlling the apparent Ca2+affinity of its gate. This fact and its physiological significance are considered in more detail in Section IV,A.

REPRESENTATIVE

SURVEY OF CA-CONTROLLED

TABLE K PERMEABILITY

Measurements performed and experimental conditions

Ohsewation interpreted as K penneahility change

Culbred dorsal root ganglion neurones from the spinal cord of 13-day-old mouse emhryos.

Microelectrode recordings of memhrane potential and conductance.

Posttetanic hyperpolarization with reversal potential identical to that or the afterpotential followinga single spike. Simultaneous decrease in input resistance.

Sensory epithelium nt the electroreceptor organ of the skate.

Voltage clamp of the epithelium.

Depolarizing currents evoke delayed outward current.

Cardiac Purkinje fibers (probably from sheep).

I M CaCI,-filled microelectrode for recording of resting and action potentials as well for electrophoretic injection of Ca" to secure recording in regions of elevated Cat+. Also, double-hameled electrode: CaCI, and KCI.

Hyperpolarization and reduced duration of action potential following Ca injection.

Liver slices from male albino rats.

Time course of recovery of tissue levels of Ca, K, and Na on rewarming d t e r incubation at 1°C.

Delayed recovery of K levels in relation to Ca and Na, indicating failure to retain K at high Ca,.

Molluscan neurones (Aplysio, Helix).

Pressure injection of Ca salts during intracellular microelectrode recordings of potential or during voltage clamp.

Immediate reduction in memhrane resistance following Ca injection. Reversal potential was very near K equilibrium potential and sensitive only to K replacement in medium.

Frog motoneurones

Microelectrode recordings from motoneurones in isolated, perfused spinal cord slices. Current injected through recording electrode.

Slow afterhyperpolarization lasting 60-

Frog skeletal muscle fihers.

Microelectrode recordings of membrane potential and resistance in cyanide plus iodoacetate-poisoned fihers.

More than 100-lold rise in K conductance assessed in CI-free media. K conductance relative to CI changed from 2:3 in normal fihers to 5 : I in poisoned fihers.

Cat cortical neurons and spinal motoneurones.

Microelectrode recordings of memhrane potential in cortical neurones and triplebarreled micropipet penetrations of spinal motoneurones for the injection of current and CaCI, and for potential measurement.

DNP increases K conductance and blocks excitability ofcortical neurons reversibly. Hyperpolarization and an increase in K conductance following Ca injections in spinal motoneurones. Reversal potential of Ca effect about 10 mV more negative than resting potential.

Cell, organ. or tissue

220

200 msec following action potentials. Simultaneous increase in K conductance. Also elicited by depolarizing currents when Na current is blocked by tetrodotoxin.

I PROCESSES IN THE PLASMA MEMBRANEOF CELLS Proposed physiological function and mediation of pharmacological effects

Comments and assesment of (Ca"), dependence

One 01 two mechanisms loond to produce relaxing hyperpolarizing responses in CNS neurons. Information processing. Link hetween rapid electrochemical events and lastinz hiochemical phenomena mediated hy Ca. Delayed outward current responsihle for the repolarizing phase of the action potential, Regulation of electrical activity in cell h d i e s . Facilitated onset of repolanzation due to residual Ca:'. Ca:' control 01 K permeability in cardiac muscle.

Strophantidin-insensitive. Ca dependence not tested. Cultured multipolar cells from the same spinal cord show strophantidinsensitive hyperpolarization following prlonged glutamate application.

(a)

Late outward current observed only under conditions where a net Ca influx was expected to have preceded its onset. Ionic basis of outward current not identified yet.

(14

Shortening of action potential might have resulted lrom a reduction in the Ca driving force, decreasing the slow inward current after Ca injection. Tip potential, and coupling artiFacts. may complicate interpretation of the observed hyperpolariza-

(C)

T h e lag in K recovery was ahsent when K was restored to the medium after Ca, had heen reduced. Ca dependence was impossihle to test with Ca-free media because ofgenera1 memhrane leakiness. Injection of Ca-EGTA giving lo-' M Ca" induce resistance changes similar to those induced by CaCI, injections in the millimolar range. TEA prevents hyperpolarization hut not resistance change. Posttetanic hyperpolarization is abolished h y EGTA injection. Quinine appears not to prevent the etlect of Ca injtction. Slow afterhyperpolprization and conductance increase selectively and are reversibly inhihited hy petiiision with low-Ca media or in the presence of Mn or Co and enhanced hy high Ca. TEA-insensitive, while fist afterhyperpolarization in same preparation is TEA-sensitive. External application olcatleine (presumed to release Ca from internal stores) decreased input resistance hy 20% in normal fibers. Direct Ca injection caused irreversible damage. Poisoned fibers had surface and tuhular memhranes intact, as judged from the conservation of memhrane constants. Ca dependence of K Conductance change not established.) Ca sensitivity change not considered, although alteration of gating mechanism suspected in poisoned fiher. Extracellular ionophoretic administration of quinine caused prolonged rise in resting input resistance as well as marked diminution in conductance increase following postspike hyperpolarization. EGTA injections had similar effects.

(d)

C a h u m regulation ineahility

of

membrane per-

In niemliranes where Ca activation is conpled to K activation, stimulation could produce a variety of responses ranging from graded depolarization to long-lasting action POtentials. Posttetanic hyperpolarizabon. A Ca activation-K activation system provides Rexihility for information processing at the memhrane level. Regulation of low-frequency repetitive discharge.

None.

Ca control ofresting memhrane potential and conductance. Postspike afterhyperpolarization. Mechanism of the reversible hyljerpolariaation and lall in excitability oh\erved in cerelxal cortex tollowing hypoxia. Mediation of the central actions of general anesthetics, mainly processes underlying cniisciousiies\.

References

(e)(f )(R)

(h)

(i)

(j)(k)(l)(m)(n)

(continued)

221

222

VIRGIL10 1. LEW AND HUGO G. FERREIRA

TABLE I Cell, organ. or tissue Intestinal smooth muscle of the guinea pig taenia coli.

Measurements performed and experimental conditions

Ohservation interpreted as K permeability change

Tension response, memhrane potential. and resistance measured with double sucrose gap method. Resistance change was estimated Irom amplitude change in electrotonic potential following the application of current pulses.

Ellect of adrenaline and noradrenaline on the memhrane potential follows shifts in K equilibrium potential (hyperpolarization under physiological conditions.) Catecholamines decrease membrane resistance more in high-K media.

Code to references: (a) Ransom et al. (1975). (11) Closin el ol. (1975). (c) Isenberg (1975). (d) van Rossum (1970). (e) Meech and Strumwasser (1970). (f) Meecli and Standeli (1975). (R) Meech (197fi). (h) Barrett and Barrett (1976).

C. Is the Ca-Sensitive K Channel Present in All Cell Membranes?

We know now that red cells from certain species do not have this permeability mechanism in their membranes (Jenkins and Lew, 1973), at least not in a functional state; no systematic search of other cell types has, however, been reported so far in the literature. Most of the original observations concerning this channel have been accidental. In cat cortical neurons, for instance, the original finding was that 2,4-dinitrophenol (DNP) reversibly blocked excitability by inducing a rise in K conductance (Godfraind et aZ., 1970).This led Krnjevic and his collaborators to suspect that an increase in Ca?+, resulting from a reduction in Ca2+ uptake by the DNP-poisoned mitochondria, mediated the effect of DNP on the K conductance of the membrane. This hypothesis was supported by results obtained with direct ionophoretic injections of Ca into the cytoplasm of cat spinal motoneurons (Krnjevic and Lisiewicz, 1972). As early as 1956, Gardos observed a large selective loss of K from human red cells incubated in plasmalike media containing iodoacetic acid (IAA) and adenosine. The great merit of Gardos' original work was that he established, for the first time, that the presence of Ca was an absolute requirement for the K loss to occur (Gardos, 1958a, b, 1959),but the mechanism of action of the peculiar combination of IAA and adenosine has only recently been elucidated (see Section II,D and E). Another interesting chance finding was that of Lassen et al., (1974) who, while measuring the membrane potential of giant red cells ofAmphiuma during brief microelectrode penetrations, found a large hyperpolarizing potential on repuncture of the same cell at short intervals after the first penetration, provided Ca was present in the

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

223

(continued ) Proposed phy*iological tunction and medi;ition ot vli;irmacological eltects

Comments and a5sessment ot (Cay*),dependence

Mediation of the etlect 01 catecliolamines on intestinal smooth niirscle ot guinea pig taenia coli (hyperpolarization and inhiliition nt qpontineous spike activity).

Gradual disappearance o t adrenaline etlecta on removal ot external Ca. Application of' C;t to muscle siipertused with Ca-tree sohrtion produces adrenaline-dependent hyperl,olarization and ii decrease in memlirane reYistance. Side ettectc, hut not specific etlectr olCa removal, prevented b y high external Mg. Catecholamine etlectc insensitive to ouahain.

Reterences (O)(l>)

(i) Fink and Luttgau (1976).(j) Codf'raindet 01. (1970. ( k ) Krnjevic and Lisiewicz (1972).(I) Krnjevic (1974).(m) Pull and Knijewc (1977).(n) Krnjevic et ~ l (1975). . ( 0 ) Hulhring and Tomita (19774. ( p ) Bulbring and Tomita (197711).

medium. Their interpretation was that the hyperpolarization resulted from Ca entry during the first puncture. Failure to detect a Ca-dependent change in K permeability is not necessarily proof of the absence of the Ca-sensitive K channel from a particular cell if it is not certain that the local [Ca2+liat the inner membrane surface has reached a very high level. A channel in a low Ca affinity state (Lew and Ferreira, 1976) or in a strongly Ca-buffered cell may not be easy to reveal. With these considerations in mind and the channel "sightings" reported in Table I, it is clear that one is dealing with a widespread mechanism present in evolutionarily distant cells which seems to fulfill some important physiological functions. It is not, however, found in all cells, and its real distribution has yet to be assessed. D. Some Considerations on [Ca2+ll

Throughout this article we use the symbol [Ca2+lifor the concentration of free, ionized Ca in the cytoplasm. This concentration, however, is not always unambiguously defined, because it may vary in space and time within the cytoplasmic phase. If [Ca2+Iiis to regulate diverse functions within a single cell through several different receptors, either [Ca2+],can vary locally in the uicinity of each receptor type without affecting the local concentration near other receptors, or the different functions must appear to be interdependent. There are examples of fine structural compartmentalization of Ca which tend to optimize the diffusional times for Ca2+to and from the receptor sites (sarcoplasmic reticulum and myofibrils, for instance),

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VIRGIL10 1. LEW AND HUGO G. FERREIRA

but this does not necessarily mean that the variation in Ca2+is locally restricted to the environment of these particular receptors. If [Ca2+Ii were uniform, all Ca2+-controlled functions would be linked. If at least some are independent, [Ca2+Iiwould have only a local meaning. Variations in the Ca sensitivity of individual Ca2+receptors (see Section IV, A), whether induced by intra- or extracellular messengers, would add versatility to the operation of the various functions using Ca:+ as a common mediator. Their interdependence may therefore become apparent only under certain physiological or experimental conditions. The interdependence of apparently unrelated functions known or suspected to be regulated by a single mediator has not yet been investigated systematically. Its study may prove useful for our understanding of the type of functional compartmentalization and sensitivity control operating in the intact cell. The local [Ca2+],has never been assessed directly in relation to the Ca-sensitive K channel. A promising approach is that recently used by Rose and Loewenstein (1976) with aequorin-filled giant cells from the salivary gland of Chironomus thummi. In this system, the effect of a local increase in [Ca2+lifollowing Ca injection was a reduction in membrane resistance accompanied by depolarization of the plasma membrane of the injected cell. This observation is compatible with increased K conductance only if there had been a localized accumulation of K on the external membrane surface due to K leaking out of the cell after the Ca injection. There is, however, no evidence for this, and the ionic basis of this effect has not yet been investigated.

II. THE Ca-SENSITIVE K CHANNEL IN RED CELLS A. Historical Introduction

The earliest observations of a large selective K loss from red cells that have come to our attention date from 1936 and 1937. Henriques and Brskov (1936)found that Pb poisoning, in vivo or in vitro, caused rapid K loss from rabbit red cells. Pb removal induced a faster restoration of the K levels than could be expected from red cell renewal. Wilbrandt (1937) observed a large K loss from human red cells incubated in plasmalike media to which fluoride had been added. That the cation loss was selective for K was correctly inferred by Wilbrandt from the observation of simultaneous cell shrinkage, implying net KC1 loss without an equivalent Na gain. Based on these results and on the

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

225

existence of a correlation between the fluoride-induced inhibition of glycolysis and the K loss, Wilbrandt (1940) advanced a hypothesis containing some of the ideas that were to influence research in this field for the next 30 years. Essentially, Wilbrandt’s point was that the altered glycolytic metabolism resulted in the formation of abnormal metabolic products which in turn directly controlled the K permeability of red cell membranes. Passow and his co-workers (see review by Passow, 1964) embarked on an exhaustive search for such metabolites, but with negative results. This work led, however, to a vast accumulation of data on the various procedures which produce a selective increase in the K permeability of red cell membranes (Table 11). The findings of Gardos (1958a, b, 1959), mentioned in Section I, C, were also interpreted in terms of direct metabolic control of the K permeability with Ca as an essential cofactor. A less specific divalent cation requirement when the K loss was induced by fluoride was described by Lepke and Passow (1960). In 1966, Hoffman demonstrated that the effect of Ca on the K permeability did not necessarily require treatment with IAA and adenosine. Prolonged depletion, incubation with IAA alone, and nucleotide depletion in resealed ghosts, all induced a large K loss on addition of Ca to the medium. Hoffman (1966) concluded that the removal of a protective metabolite under all these conditions was a more likely alternative. Up to the late 196Os, the prevailing belief was that this phenomenon represented a highly artificial condition restricted to red cells. Its main attraction was the hope that it might lead to the understanding of at least some of the mechanisms which regulate the alkali cation permeability of cell membranes, the guiding idea being that of a tight metabolic control of permeability. I n 1968, Whittam (1968) suggested that it was the increase in the internal concentration of ionized Ca which raised the K permeability of the membrane. In 1970, Lew (1970) reported the first measurements of variations in the intracellular Ca content of intact human red cells (in the micromolar range) and showed that they paralleled the changes in K permeability induced by various ATP-depleting procedures. In the same year the observation of Ca-induced K permeability changes in other cells (Godfraind et al., 1970, in cat cortical neurons; Meech and Strumwasser, 1970, in Aplysia neurons) led to the realization of the physiological potential of this mechansim and to an impressive revival of interest in this subject. The particular advantage of red cells as an experimental model for

TABLE I1 PROCEDURES THATINDUCE Incubation of red cells in the presence of Fluoride (40 mM)

Triose reductone Pb (5-60

a)

Propranolol (1 mM) IAA or IAc plus adenosine or other subtrates. Prolonged starvation. Prolonged incubation with IAA or IAc alone or with 2-deoxyglucose

A

SELECTIVE INCREASE

I N THE

K PERMEABILITY

OF

RED CELL MEMBRANES

Conditions for production or prevention of K permeability rise Ca (0.05 mM) and Mg (0.5 mM) are equally effective. Excess of EDTA over divalent cation prevents the effect. Ca contamination in Mg salt is possible, since 1mM Mg in excess of 0.1 mM EGTA had no effect (Lew, unpublished). High-Ca sensitivity? Ca-dependent. Prevented by excess EDTA. NH,NH, or NH$I enhance the effect Prevented by EDTA. Predepletion not required. Direct effect of contaminating traces of Ca made accessible to K gate by Pb impossible to exclude. High-Ca sensitivity? Predepletion not required. Ca-dependent. Prevented by excess EDTA. Large increase in Capermeability. High-Ca sensitivity? Ca-dependent. Prevented by excess EDTA or EGTA

References Wilbrandt (1937) Lepke and Passow (1960) Lepke and Passow (1968) Passow and Vielhauer (1966) Passow (1964) Henriques and Brskov (1936) Passow (1964) Riordan and Passow (1973) Manninen (1970) Szasz et al. (1978) Gardos (1956, 1958a,b) Hoffman (1966) Lew (1970, 1971a)

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

227

the study of this permeability mechanism became more apparent as the sequential nature of the process leading to the increase in K permeability became more fully understood (Lew, 1971a,b, 1974). In 1970, Blum and Hoffman (1970) advanced the challenging and controversial hypothesis, based mainly on a partial inhibitory effect of ouabain, that the K permeability mechanism represents abnormal behavior of the Na pump. I n 1972 the same investigators (Blum and Hoffman, 1972) showed that, in resealed ghosts containing Ca buffers, the free Ca2+concentration required internally to induce the selective increase in K permeability was in the micromolar range, thus confirming Lew’s observations on intact, depleted cells. This was important, since Romero and Whittam (1971), working with aged blood bank cells, described less selective changes in permeability to alkali metal ions induced b y Ca concentrations in the millimolar range. More recent research has been mainly concerned with the action of cofactors and pharmacological agents on the K permeability mechanism, with the basic aim of gaining a better understanding of the molecular nature and mode of operation of this mechanism and of finding ways of testing the identity of similar mechanisms operating in different cells (Manninen, 1970; Szasz and Gardos, 1974; Porzig, 1975; Gardos et al., 1975; Hoffman and Knauf, 1973; Riordan and Passow, 1973; Lassen et al., 1976; Simons, 1976b; Armando-Hardy et al., 1975; Jenkins and Lew, 1973; Knauf et al,, 1975). A recent surprising finding was that the Ca sensitivity of the K channel in intact red cells is not invariant with the experimental conditions (Lew and Ferreira, 1976). If we are to believe that this Ca sensitivity change plays an important physiological role, it has yet to be shown that it can occur under physiological conditions as well. The earlier search for the metabolite presumed to control the K permeability of red cell membranes may eventually have to be reformulated in terms of the search for the physiological effector of the Ca sensitivity change. This, however, is not yet history. B. Procedures That Activate the K Channel in Red Cells

The preceding historical introduction illustrates an important point: There are many procedures that lead to a more-or-less selective increase in the K permeability of red cell membranes. These are listed in Table 11. For many years it had been assumed that all these methods acted on the same basic mechanism. Evidence in favor of this view is relatively recent, and there is no evidence against it. The effects produced b y Pb

228

VlRGlllO 1. LEW AND HUGO G. FERREIRA

and fluoride can be observed only in red cells from species which also exhibit the Ca-sensitive increase in K permeability (Riordan and Passow, 1973; Jenkins and Lew, 1973; Lew, 1974). Quinine (Armando-Hardy et al., 1975) and oligomycin (Riordan and Passow, 1971) selectively inhibit the K loss induced by Ca or Pb but not the increased Na flux observed during Pb poisoning. It is clear that some of these procedures represent abnormal methods of operating this permeability mechanism. Although in this sense they are not physiologically relevant, they may eventually help elucidate the reactions taking place when more is known about the chemistry of this channel.

C. The Gardos Effect

Of all the procedures listed in Table 11, the one originally described b y Gardos (1956, 1958,a,b) has received the most attention and has been the most extensively investigated, possibly because it involves steps which are more likely to occur under physiological conditions. In the following discussion we will consider this particular mode of operating the K channel in detail and center on this scheme the analysis and discussion of all other aspects of the Ca-controlled K permeability mechanism. The mature mammalian red cell has neither nucleus nor Caaccumulating organelles; it is virtually Ca-free (see review by Schatzmann, 1975)and, to a first approximation, can be considered a homogeneous cytoplasm surrounded by a plasma membrane. Although it lacks morphologically recognizable compartments, differences in age and density within a population of cells often simulate compartmentalized behavior. When intact red cells are incubated at 37°C in their plasma, or in a plasmalike medium containing Ca in the presence of IAA and adenosine, a large K loss is observed within the first hour, usually preceded by a short lag period. Addition of a Ca-chelating agent in excess of the Ca concentration in the medium prevents or reverses the increase in K permeability. The increase in K permeability was shown to result from the following sequence of events (Lew, 1971a, 1974; Lew and BeaugC, 1978) (Fig. 1).Incubation of red cells in the presence of IAA and adenosine induces a very rapid and complete depletion of intracellular ATP. This has at least two relevant effects: (1)It increases the Ca sensitivity of the K-gating mechanism (Lew and Ferreira, 1976), and (2) it allows net Ca entry. Ionized Ca, which represents about

229

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

I n c u b a t i o n of human red c e l l s w i t h I A A plus a d e n o s i n e i n the p r e s e n c e of Ca

.1 N e t Ca e n t r y

Unknown step

-1

-1

C y t o p l a s m i c Ca Imftering 1Ca2’1, = alCa1, (0.2 ZZ a ZZ 0.45)

I n c r e a s e d Ca s e n s i t i v i t y of K gate

.1

Ca:+ \

/

G a t i n g mechanism r e a c t s w i t h Ca:+

-1 Activation

.1 Increased K permea\,ility

FIG.1. S e q u e n t i a l model for the m e c h a n i s m of t h e G a r d o s e f f e c t .

20-45% of the total Ca content of the cells (Cai), interacts with the K gate, inducing activation of the permeability mechanism leading to an increased K flux. Ca entry, buffering, and interaction with the K gate are all reversible steps. A proper understanding of this mechanism requires a detailed knowledge of each of the steps involved. In red cells this implies the need to explore a vast diversity of functions, from the metabolism of depleting cells and the cytoplasmic buffering of Ca to the properties of the active and passive Ca transport systems of the plasma membrane. If we are not to lose sight of the Ca-sensitive K channel at the end of it all, it is necessary to establish the link between each step and the final effect on the K permeability of the membrane at every stage during this investigation. I n the following sections w e present a systematic account of our present knowledge of each of the steps outlined in Fig. 1. D. Relative Efficacy of ATP-Depleting Procedures

The peculiar effect of the metabolic “cocktail” used by Gardos (l956,1958a,b) is to reduce the internal ATP concentration from about 1 mM to 1 p M in less than 60-80 minutes (Lew, 1971a). This is extremely fast when compared with the rate of ATP depletion in the

230

VlRGlLlO 1. LEW AND HUGO G. FERREIRA

absence of substrates or even in the presence of IAA or iodoacetamide (IAc) alone (Lew, 1971a). The presence of adenosine, inosine, or glucose in conjunction with the inhibitor accelerates the rate of depletion, because ATP is used up in the early stages of the glycolytic cleavage through phosphorylation of glucose or of fructose 6-phosphate Eormed from the metabolism of adenosine or inosine. The synthesis of ATP is simultaneously blocked by inhibition of the 3-phosphoglyceraldehyde dehydrogenase induced by IAA or IAc. The presence of inorganic phosphate (Pi)inside the cells greatly accelerates the rate of depletion in the presence of inosine; this indicates a rate-limiting effect of Pi , possibly on the phosphorylation of this substrate to ribose 5-phosphate catalyzed by inosine phosphorylase. In the presence of inosine and IAc the ATP content of phosphate-loaded cells falls to about l p M in 20-30 minutes. This is the fastest depleting procedure found, the slowest being the incubation of red cells in substrate-free media containing no inhibitor. In between these two extremes there is a whole gradation of controllable rates. Starvation, IAA or IAc alone, or 2-deoxyglucose acting as a false substrate induces slow depletion and reduces ATP levels to between 200 and 20 pM in 6-24 hours at 37°C. However, the association of a metabolizable substrate with IAA or IAc always produces rapid and more complete depletion, lowering the intracellular ATP concentration to 1 or 2 pM in 20 minutes to 2 hours. The relative depletion rates of the different combinations parallel the ability of the substrate to maintain ATP levels in the absence of inhibitors. Thus, in human red cells, in the presence of IAc, glucose is less effective than inosine, whereas in guinea pig red cells, where inosine entry is rate limiting (Duhm, 1973), inosine is less effective. The fall in ATP concentration is by no means the only substantial metabolic alteration taking place in cells undergoing depletion. During fast depletion, all adenosine nucleotides (ATP, ADP, and AMP) are stoichiometrically converted to IMP which accumulates inside the cells. This conversion is independent of the nature of the substrate and results from the activation of AMP deaminase (Askari and Rao, 1968; Lew, 1971a). During slow depletion, IMP does not accumulate to any measurable extent. We are largely ignorant of other metabolic changes that occur during fast depletion and that may ultimately influence the behavior of the Ca-sensitive K channel. We know, however, that the Mg permeability and the intracellular Mg content, the cytoplasmic buffering of Ca, and the passive Ca permeability of the red cell membrane (Ferreira and Lew, 1975) all remain unchanged.

CA-ACTIVATED K CHANNEL IN BIOLOGICAL MEMBRANES

23 1

E. Parallelism in ATP Depletion, Ca Entry, and Increased K Flux

When Ca entry is measured during ATP depletion (Lew, 1970, 1971a), a strict parallelism is found between the rate at which ATP falls and the rate at which Ca accumulates inside the cells; the faster the rate of depletion, the higher the rate of Ca accumulation. Net Ca entry rates in cells undergoing depletion seldom exceed 10 pmoles/liter of cells per hour and are independent of the external Ca concentration above 2-3 mM (see Fig. 11).When depletion is induced in Pi-loaded cells, it is impossible to measure initial Ca uptake rates because of the formation of minute extracellular precipitates of hydroxyapatite (Passow, personal communication; Lew, 1971a; Schrader, 1973), which centrifuge together with the cells. The levels of Pi found in depleted cells in the presence of inosine or glucose were not high enough for the Pi leaking out into the Pi-free medium to exceed the solubility product of the Ca salt. On addition of the divalent cation ionophore A23187, all the cell-associated Ca of ATPdepleted cells (down to 0.1-0.2 pmolelliter of cells) suspended in a Ca-free medium was released in less than 30 seconds (Ferreira and Lew, 1977), indicating that it had been Ca contained in the cytoplasm. The rate of 42K tracer loss from 42K-loaded cells undergoing depletion in the presence of Ca follows very closely the pattern of the Ca uptake curve (Lew, 1971a). ATP depletion can act by removing the fuel for the Ca pump, as well as by removing a Ca buffer. In intact cells pump inhibition is necessary for net Ca entry, but the effect on the K permeability may be augmented by a decrease in Ca buffering however, this capacity. With a normal content of Mg of about 3.7 d , effect is likely to be negligible. Since Ca entry can be manipulated to some extent not only by varying the rate of depletion but also by varying the monovalent cation composition of the external medium, the parallelism between Ca uptake and rate of 42Kloss can b e studied to some extent independently of the depletion procedure. Thus, in high-Na media containing less than 10 mM K, the uptake of Ca is much faster than in high-K media for equal depletion rates (Lew, 1971a). The rate of net K loss in the high-Na medium exceeds the rate of 42Ktracer loss in the high-K medium, and this occurs in spite of the possible existence of an adverse electric potential in the low-K, high-Na medium (Manninen et al., 1967; Glynn and Warner, 1972). This potential is unlikely to be the cause of the larger Ca uptake observed in high-Na media for two reasons: (1)It is observed even in red cells from species which have no Ca-sensitive K channel and where the membrane potential is presumed not to be significantly disturbed

232

VlRGlLIO 1. LEW AND HUGO G. FERREIRA

during the experiments; and (2) in low-K, high-Li media (Li, like Na, does not permeate through the K channel; Simons, 1976b), Ca uptake is similar to that observed in high-K media (Ferreira and Lew, 1977). The larger 42K loss induced by Ca in low-K, high-Na media may be naively attributed to single-filing if the larger Ca uptake measured under these conditions is overlooked. This illustrates how important it is for the correct interpretation of these effects to assess the relative contribution of each of the steps in the sequential scheme. In guinea pig red cells undergoing depletion in the presence of IAc, the contribution of the Na pump to the overall ATP turnover is much more easily observed than in human red cells (Ellory and Lew, 1970; Lew et al., 1970; Lew, 1971b), probably because the ratio of pumping rate to glycolytic rate (McManus, 1967) is larger in the guinea pig cells. Depending on whether the Na pump is operating a normal Na-K exchange with simultaneous hydrolysis of ATP, or whether it is running in reverse (Garrahan and Glynn, 1967; Glynn and Lew, 1970; Lew et al., 1970), inducing net ATP synthesis, inhibition of the pump can either reduce or enhance the Ca-induced 42Kloss from the guinea pig red cells (Lew, 1971b). Further evidence for the close parallelism in ATP depletion, Ca uptake, and increased K permeability is provided by the observation that in guinea pig red cells Ca entry and K loss are greater when glucose rather than inosine is present during IAc-induced depletion, while the converse is true in human red cells (Lew, 1974; see also Section II,D. The inverse correlation between ATP depletion and IMP accumulation observed during fast depletion of human red cells may suggest a role for IMP in the process leading to the increase in K permeability. This seems unlikely, because incorporation of IMP into ATP-free or ATP-containing resealed ghosts had no effect on the Ca-induced K fluxes compared with those in IMP-free controls (Lew, 1971a). A possible effect of IMP on the Ca sensitivity of the K channel, however, cannot be ruled out at present. F. Heterogeneous Behavior of Cells in Relation to the Ca-Induced K Flux

The Ca sensitivity of the K permeability in ATP-depleted cells, like that in nucleotide-free ghosts, is very high (Lew, 1970; Blum and Hoffman, 1971; Simons, 1976b; Lew and Ferreira, 1976). Lew (1970) in ATP-depleted intact cells, and Blum and Hoffman (1972) in re-

CA-ACTIVATED K CHANNEL IN BIOLOGICAL MEMBRANES

233

sealed ghosts, reported large increments in the K permeability when the total Ca concentration per unit volume of cells ([Cal,) or [Ca2+li was as low as 1 or 2 p M . In a more precise study in resealed ghosts = tetraacetic loaded with Ca-1,2-di(aminoethoxy)ethane-N,N7N’,N’ acid (EGTA) buffers, Simons (1976b) found half-maximum activation of the K permeability at about 0.2 pM [Ca2+Ii,in agreement with earlier observations by Porzig (1975). We consider this point in more detail in Section IV,A, but there is a good reason to mention it here. A familiar observation when working with intact red cells first emphasized by Riordan and Passow (1973), but also observed in earlier work (Gardos, 1958a,b), is that not all the K contained in a given sample of ATP-depleted cells seems to participate in the Ca-induced K flux (Lew, 1974). The heterogeneous behavior of intact cells can be extremely marked and we have performed experiments in which only about 20% of the K in the cells participated in the fast, Ca-induced, tracer equilibration process. Lew (1974) suggested that this might be explained in terms of a heterogeneous Ca content of depleted cells, resulting from differences either in depletion rates or in Ca permeability. The fact that the heterogeneous behavior of the K permeability was also observed when Ca was added ufter all the cells had been depleted of ATP (as estimated from an average ATP content of about 1 p M ) seemed to favor the idea of variable Ca permeability of the cells. Recent experiments by Ferreira and Lkw (1977), however, do not support Ca heterogeneity as the explanation for this effect. The Ca content of red cells depleted of ATP in the presence of Ca was measured after separating groups of cells according to their density (Fig. 2). The results showed that the Ca content of red cells with different densities was the same. Similar results were obtained with cells separated according to their resistance to lysis in hypotonic solutions. This means either that Ca is homogenously distributed or that the same heterogeneity of Ca content is present in cells having different densities and osmotic fragilities. What, then, determines the fact that Ca induces the K permeability change in certain cells and not in others? It cannot be that some cells have no Ca-sensitive K channels, since under certain conditions, by “ghosting” the cells (Simons, 1976a)or by the use ofthe divalent cation ionophore A23187 (Lew and Ferreira, 1976), all the K contained in any red cell sample can be made to participate in the 42Ktracer equilibration process induced by Ca. The observation (Lew and Ferreira, 1976) that the gating mechanism of the Ca-sensitive K channel can be found in different states characterized by a variable Ca sensitivity

234

VIRGIL10 L. LEW AND HUGO G. FERREIRA

I

TUBE No

FIG. 2. Ca content of ATP-depleted human red cells of different densities. ATPdepleted cells were preloaded with 45Cafor about 3 hours at 37°C. They were then centrifuged through mixtures ofethyl phthalate (sp gr 1.117) and di-n-butyl phthalate (sp gr 1.042). A series of 1.5-ml Eppendorf tubes was prepared containing a mixture of both oils in different proportions, each tube containing 0.4 ml of the mixture. One milliliter of a 10% hematocrit suspension of cells was deposited on top of the mixture, and the tubes were centrifuged in an Eppendorf 3200 centrifuge for 1 minute. In the tubes containing up to seven-tenths ethyl, phthalate all the cells remained on top, while in the tubes containing only butyl phthalate all the cells went to the bottom of the tube. This technique was pioneered by Danon and Marikovsky (1964) as a method of separating red cell populations. The incremental changes in the volume of cells that comes through the oil mixture (measured as a percent of the total hemoglobin in the original sample) together with their calcium content are plotted as a function of the tube number. The tubes are numbered in such a way that tube 1 contains 30% and tube 8 contains 100%di-n-butyl phthalate, and consecutive tubes contain 10% increments of this oil compensated by 10% decrements of ethyl phthalate.

could explain the heterogeneous behavior of the cells in terms of a low Ca affinity of the “silent” channels. This possibility, and the factors controlling the reaction between Ca:+ and the K gate are discussed in Section IV, A. 111. Ca TRANSPORT A. Ca Permeability of Intact Red Cell Membranes

The net uptake of Ca b y fresh, fed red cells (which are virtually Ca-free) is immeasurably small (Lew, 1970, 1971a, 1974; Wiley and Gill, 1976; Sarkadi et al:, 1976; Ferreira and Lew, 1977). This may be the result of vigorous extrusion compensating for large inward leaks or the result of a low Ca permeability of the red cell membrane. The first alternative seems more likely, since it is known that red cell mem-

235

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

branes possess a powerful Ca extrusion pump fueled by internal ATP, which is associated with a Mg, Ca-ATPase (for references on the red cell Ca pump, see Schatzmann, 1975). ATP-depleted cells, however, provided they are from fresh blood or from cold-stored blood less than a week old, are extremely impermeable to Ca. Net Ca uptake rates vary between 1 and 10 pmoles/liter cells per hour (Lew, 1970; Szasz and Gardos, 1974; Wiley and Shaller, 1974), and these are extremely low when compared with the maximum Ca pumping rates of 5-40 mmoles/liter of cells per hour proposed for the Ca pump (Schatzman, 1973; Wiley and Gill, 1976; Ferreira and Lew, 1976; Sarkadi et al., 1976). Old blood bank cells (Romero and Whittman, 1971; Lew, 1974) and resealed ghosts (Porzig, 1970, 1972) are muck more permeable to Ca, since the net Ca uptake rates are two or three orders of magnitude larger than those observed in fresh, intact cells after fast ATP depletion (Fig. 3). "Resealed"

20.

15

ghosts

.

CCal,

IU

-

5'

Fed 30

GO

Time (min)

FIG. 3. Representative data from different experiments showing the Ca uptake of human red cells under different conditions. Bank cells were from 3-week-old bank blood; fed cells were incubated in the presence of 10 mM inosine; depleted cells were incubated in the presence of 6 mM IAc and 10 mM inosine; resealed ghosts were isotonically resealed from metabolically depleted red cells.

236

V I R G I L 1 0 1. LEW AND HUGO G. FERREIRA

What, then, is the normal Ca permeability of red cells under physiological conditions? It is difficult to answer this directly, since intact cells (1) do not take up Ca, (2) have no detectable intracellular Ca pool, and (3) must be disturbed in order to measure Ca permeability (ATP depletion, reversible lysis). Ferreira and Lew (1975) provided indirect evidence which suggests that the Ca permeability of fresh cells is very similar to that of ATP-depleted intact cells. The rationale behind their experiments was that, if the intact red cell membrane were largely permeable to Ca, the Ca pump would have to extrude Ca at a substantial rate in order to maintain the physiological, quasi-Cafree steady state. Since the ATP/Ca stoichiometry is about 1 : 1 (Schatzmann, 1973)or 1 :2 (Sarkadi et al., 1977) and the glycolytic rate of human red cells is only about 3 mmoles/liter of cells per hour (McManus, 1967), the operation of the pump could be expected to contribute considerably to the overall turnover of ATP. Ferreira and Lew (1975) could detect no effect of external Ca on the level (Fig. 4) or turnover of ATP in fresh human red cells. With the precision of the techniques employed, this implies that under physiological conditions the Ca pump utilizes ATP at a rate of less than 20 pmoles/liter cells per hour, equivalent to less than about 0.1% of its full capacity. A steady state without net Ca gain can therefore exist only if the Ca permeability of the fresh cells is at least as low as that of the intact, * lodoacetarnide Glucose

1

CATPI,

300 o

Control

+ l O r n M Co

0

30

GO

90

J

120

150

Time (rnin)

FIG.4. Effect of external Ca on the ATP content of human red cells. Medium A (see p. 242) containing 10 mM glucose; IAc was added to the medium to give a final concentration of 6 mM.

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

237

ATP-depleted cells. These depleted cells seem therefore to be the best experimental model for study of the physiological properties of the passive Ca transport in red cells since, unlike resealed ghosts and aged blood bank cells, they possess the extremely low Ca permeability characteristic of the intact membrane. 6. The Problem of the Large-Capacity, low-Activity Ca Pump

The fact that red cell membranes have such a low Ca permeability leaves us puzzled about the function of a large-capacity Ca pump in the same membrane. Under physiological conditions, the magnitude of the unidirectional Ca fluxes at steady state must b e less than about 20 pmoles/liter of cells per hour, and this means that the Ca pump is practically inactive. The active Ca transport mechanism does not seem to represent a relic of “leakier” times since, unlike the Na pump or the Ca-sensitive K channel, it is present in red cells from all species investigated (Schatzmann, 1974; Lassen and Lew, unpublished observations) and there are no known exceptions to the existence of large inward Ca2+ gradients with [Ca2+Iiless than lop6o lop7M . If the presence of interspecies variation and polymorphism in relation to a certain genetic character can be interpreted in terms of coexistent mutational variants with minimum selective pressures (neutral mutations), as suggested by Lew and Beauge (1978), the luck ofvuriution in relation to Ca transport and permeability (Schatzmann, 1974; Jenkins and Lew, 1973) suggests indirectly that these probably represent properties essential for individual survival. A large-capacity pump may constitute a reserve system, but the red cell membrane, at least in some mammals, is not known to have any Ca gradient “dissipators” (Lew and Beauge, 1978) like those present in the membranes of excitable or epithelial cells. Ferreira and Lew (1975), arguing from the observation that aged blood bank red cells become increasingly leaky to Ca after 4-5 weeks’ storage, suggested that the decreased posttransfusional viability of cold-stored cells (Mollison, 1972) may result from a metabolic imbalance following increased ATP consumption b y the Ca pump, when the Ca permeability exceeds a critical value. Lew and Beauge (1978) suggested the possibility of a Ca dissipator which, in a similar way, could mediate the self-destruction of aged red cells, but all these possibilities are purely speculative at present. An alternative and more general interpretation of the function of a pump which normally operates at a smali fraction of its full capacity is

238

VIRGILIO 1. LEW AND HUGO G. FERREIRA

that this might be required to maintain large steady-state gradients of the transported substrate (Lew and Beaugh, 1978) if it were impossible to lower the K , sufficiently. This point can be illustrated in a very simple way. The physiological steady state in relation to Ca fluxes in red cells is described by the equation: leak = pump (the passive Ca efflux is considered negligibly small); this is reasonably approximated by Leak = V,,,

Kca

[Ca2+li + [Ca2+li

where V,,, is the maximum Ca extrusion rate through the Ca pump and Kca is the apparent dissociation constant of Ca at the inner surface of the pump. If the steady-state pump flux is much smaller than V,,,, then [Ca2+li (steady state) must be much smaller than Kca (probably less than lo-’ M ) , and Eq. (1) can be simplified and rearranged to give &a [Ca2+li= leak x -

Vmax

Lew and Beaug6 (1978) argued that at constant external Ca and with a Ca permeability near “ground level” (see Section 111, D) the inward Ca leak in red cells already has a minimum value and so does Kca which at about 1 pM (Schatzmann, 1973; Wolf, 1972; Ferreira and Lew, 1976) is within an order of magnitude of the lowest dissociation constants known for rapid dissociating Ca chelators at physiological pH values. If the product leak X Kca already has a minimum value determined by physiological or chemical constraints, a large inward Ca2+ gradient, which requires a very low [Ca2+Ii,can be sustained only by way of a large -V,,, pump.

C. Cytoplasmic Ca Buffering Measurements of the total Ca content of the cells only provide an upper limit for the level of intracellular free Ca. It is essential, however, to know and to be able to control the level of Ca:+ in any investigation concerning the mechanism of the reaction between this ion and the K-gating mechanism. We review here some recent experiments directly concerned with the assessment of [Ca2+Iiin the intact

cell. Schatzmann (1973) carried out equilibrium dialysis of cells (disrupted by freezing and thawing twice) which had previously been incubated with 10 mM inosine and 5 mM adenine for 60 minutes at

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

239

37°C. The total Ca concentration in the hemolysate was measured by absorption flame photometry, and the Ca2+concentration in the dialysate was assayed with a Ca2+-sensitivedye (murexide). Over a range of Ca concentrations from 0.4 mM to about 4 mM, half of the total Ca was found to be free, but this must have been an overestimate since all the diffusible buffers would have also equilibrated across the dialysis membrane. Ferreira and Lew (1976) measured cytoplasmic Ca buffering in intact red cells. They used the selective divalent cation ionophore A23187 (Reed and Lardy, 1972) to render the cell membrane highly permeable to Ca. The concentration of all other permeant ions in the medium (OH-, C1-, Mg2+,K+) was kept at electrochemical equilibrium with the corresponding internal ions, thus effectively voltageclamping the membrane at about - 10 mV. Under these conditions, the membrane of the cell itself acts like a dialysis bag for Ca and, by exploring the equilibrium or steady-state isotopic distribution of 45Ca at various external Ca concentrations, a buffer curve can be constructed. Figures 5 and 6 illustrate this procedure. Figure 5 shows the change in external and internal Ca concentration when A23187 was added three times in succession to a well-stirred 10%hematocrit suspension of ATP-depleted red cells followed by the addition of excess EGTA (Ferreira and Lew, 1977). After addition of the ionophore, Ca uptake is extremely fast, reaching a plateau which corresponds to the equilibrium distribution of Ca and is independent of the ionophore concentration. This indicates that the ionophore per se does not partition Ca into the cells to a measurable extent. Figure 6 shows a similar type of experiment carried out with inosink-fed cells using a single ionophore addition at different external Ca concentrations. In these

FIG.5. Equilibration of ionized Ca across the membrane of ATP-depleted red cells after successive additions of ionophore (A23187)and EGTA. Open circles, [Ca2+lo;solid circles, [Ca],. (From Ferreira and Lew, 1977.)

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V I R G I L I O 1. LEW AND HUGO G. FERREIRA

575 _.

307

_.

. . . . . . ..

0

10

195.

20

Time (min)

FIG.6. Time course of Ca uptake by inosine-fed human red cells after the addition of ionophore (A23187, 6 p M ) at different external Ca concentrations. Cells were from 8 day-old bank blood. Ionophore and 42Kwere added at the indicated times.

cells, the activity of the Ca pump can maintain steady-state levels of Ca inside the cells far below equilibrium only at relatively low external Ca concentrations. When the Ca pump becomes saturated, however, the steady-state Ca content of the cell increases in a manner depending on the membrane potential and the buffering behavior of the cell. The curious initial kinetic behavior (the Ca “peaks”) is analyzed in Section IV,A. Figure 7 (Ferreira and Lew, 1977) shows the steady-state distribution of internal total Ca versus external free Ca concentration obtained

24 1

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

1

i 0.1

1

10

102

I

103

FIG.7. Intracellular Ca bufferin n inosine-fed cells and in ATP-depleted cells. Pooled data from five different experiments. Each point corresponds to an equilibrium or steady-state distribution of Ca such as the ones described in Figs. 5 and 6. The ionophore concentration was >25 pLM in the experiments with inosine-fed cells.

from experiments such as those illustrated in Figs. 5 and 6 in ATPdepleted and inosine-fed red cells. The results indicate that, over a wide range of internal Ca concentrations, from about 0.5 pM to nearly 10 mM, fed and depleted cells behave as if they have a single largecapacity, low-affinity Ca buffer. [Ca2+Iitherefore represents a constant fraction a of the total Ca content of the cell (Ferreira and Lew, 1976). The value of (Y varied between 0.2 and 0.45 in cells from different donors, indicating that between 20 and 45% of the total Ca content of the cells is ionized (Ferreira and Lew, 1976). ATP depletion made no significant difference in the buffering behavior of fresh cells after saturation of the Ca pump, indicating that the metabolic changes occurring during depletion are either immaterial or self-compensating in relation to Ca buffering. The technique applied by Ferreira and Lew (1976) in these studies has proved very convenient, since it allows the levels of Ca?+ to b e controlled not only in depleted cells, but also in fed cells where the Ca pump is fully operational, simply by varying the external Ca concentration. The K fluxes through the Ca-sensitive K channel can thus be investigated at different constant levels of C a t + representing equilibrium or pump leak steady states, and the results of such studies are considered in Section IV.

242

V l R G l L l O 1. L E W AND HUGO G. FERREIRA

D. Passive Ca Transport

The experiments to be described in this section have been reported by Ferreira and Lew (1977) and are presented here only because of their relevance to the Ca entry step of the sequential model (Fig. 1).The reader interested primarily in the K channel may omit this section without breaking up the flow of the review. Fast ATP depletion was induced by the preincubation of fresh cells or blood bank cells less than a week old in media containing 6 mM IAc and 10 mM inosine for about 2-3 hours at 37°C. All the experiments described here were performed in a medium containing 75 mM KCl, 75 mM NaCl, 10 mM Tris-HC1 (pH 7.7 at 37"C), and either 0.1 mM Tris-EGTA (Ca-free) or the various concentrations of CaC12indicated in the text. (In the figure legends the Ca-free medium is referred to as medium A,) The uptake of Ca by fresh, ATP-depleted cells is shown in Fig. 3, together with the uptake of Ca in the same cells before depletion, the uptake in 3-week-old blood bank cells, and the uptake in ATP-free resealed ghosts. When Ca uptake is measured during depletion, the intracellular accumulation of Ca proceeds at rates which increase in inverse proportion to the rate at which ATP falls (Lew, 1971a, Fig. 1).In cells depleted of ATP, however, there is always an initial faster rate of uptake (up to 30 pmoles/liter of cells per hour) which decreases gradually during the first 20-60 minutes and tends to a constant value (up to 10 pmoles/liter of cells per hour; see Figs. 3,8, and 10).The nature of this faster initial uptake component is uncertain. Its magnitude varies considerably with the monovalent cation composition of the

Time (hours1

FIG.8. Time course of Ca uptake and release in ATP-depleted human red cells. Cells were from 7-day-old bank blood. At 1,2, and 3 hours cells contained in aliquots of the cell suspension used to measure net Ca uptake were washed and Ca release measured in the absence and presence of Ca. In a parallel group of cells treated in the same way as the cells in which net uptake was being measured except for the omission of '"a, "Ca was added at 1,2, and 3 hours, and the 'Wa uptake followed for 30 minutes (as ap. Ca,; see text).

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

243

medium and is, at least in part, an artifact, since it can also be elicited simply by cooling and rewarming a cell suspension during the constant-uptake phase. In order to characterize the behavior of the mechanisms responsible for passive Ca translocation, Ca uptake and release were studied as functions of the internal and external Ca concentrations. Before analyzing the results it is necessary to consider a few points of method. The Ca content of the cells was measured according to Lew (1971a), but with the 45Ca-loadingor 45Caefflux reaction terminated by addition to the cell suspension of about 2.5 volumes of ice-cold medium. Determination of the 45Cauptake into nominally Ca-free cells or the release of 45Cainto a Ca-free media is straightforward and involves no change in the specific activity of 45Cathroughout the experiment, But when the entry of 45Cais measured in cells preloaded with tracer-free calcium, as well as when the release of 45Cais determined in media containing tracer-free calcium, the specific activity of 45Caconstantly changes inside the cells but remains practically constant or negligibly small outside. The curve representing the time course of the variation in 45Cacontent of the cells under these conditions is a measure of the rate at which tracer equilibration proceeds plus the rate of change in Ca content of the cells. Although this representation is useful for the calculation of rate constants, the comparison of actual Ca movements is not possible unless specific activities are also given. I n order to bypass this difficulty, an apparent Ca content of the cells was calculated (ap. Ca,) using the original specific activity of 45Cain the external medium. It can be shown that the initial rate of change in ap. Cai provides a direct measure of the influx or efflux. This convention therefore permits the comparison of tracer curves at variable specific activities inside cells, with the curves indicating the true Ca content of cells (see Fig. 8). 45Caloading was performed either by incubating the cells at a constant external Ca concentration for different periods of time (time loading) or by incubating at various external Ca concentrations for a fixed time (concentration loading). Figure 8 shows the results of an experiment in which Ca uptake and release were measured under a variety of conditions at various times during a time-loading experiment, and Fig. 9 shows the release of 45Ca in the presence and absence of external Ca from concentration-loaded cells. Figures 10-12 show the dependence of net uptake and release on external and internal Ca concentrations as obtained from experiments such as those illustrated in Figs. 8 and 9. These results indicate that: (1)the component of the net Ca uptake

244

VlRGlLlO 1. LEW AND HUGO G. FERREIRA

that is linear with time saturated with the external Ca concentration with a K , of about 0.4-0.7 mM and a V,,, which varied between 1and 20 pmoles/liter of cells per hour in different experiments (Figs. 10 and 11); (2) external Ca accelerates 45Carelease from 45Ca-loadedcells (Figs. 8 and 9); this is accompanied by a Ca influx (Fig. 8, ap. Ca influx) which accounts for a large Ca-Ca exchange flux during net uptake; (3)the initial 45Carelease, whether in the absence or presence of external Ca, can be described by a single exponential (Fig. 9). The plot of these fluxes as a function of the initial internal Ca concentration (Fig. 12) reveals apparent saturation kinetics. The interpretation of these results is not simple. It is clear that passive Ca translocation in intact red cell membranes is largely carriermediated and has a large exchange diffusion component. Even with 10 mM Ca in the medium, no linear flux component which could represent diffusional Ca movement was detected. If we set the measurable limit of such a flux at, say, 0.2 pmoles/liter of cells per hour, the diffusional Ca permeability of intact cell membranes must be less than 3 x cm per second.

I

TIME lrninl 15 25 35 -7

0 5

J

2

FIG.9. &Carelease from concentration-loadedhuman red cells as a function of time. Fresh cells were depleted of ATP in the presence of 0.5, 1 , 2 , 4 , and 8 mM radioactive Ca. The cells were then suspended in medium A containing either no Ca (open circles) or 2.1 mM tracer-free Ca (solid circles). (From Ferreira and Lew, 1977.)

245

CA-ACTIVATED K CHANNEL IN BIOLOGICAL MEMBRANES

r Cg.W 13

5

2

0 1-5

0

0

I

a0

1

160

TIME Iminl

FIG. 10. The dependence on external Ca of the Ca uptake and release in ATPdepleted human red cells. Cells from 4-day-old bank blood were loaded with radioactive Ca during depletion. The cells were then divided into eight lots, all suspended in medium A but containing, respectively, 0, 1 , 2 , 5 , and 10 mM radioactive calcium (open circles) and 1, 2, and 5 mM tracer-free calcium (solid circles). Cat and ap. Ca, were measured at the indicated times. (From Ferreira and Lew, 1977.)

A puzzling feature of these results is that they contain apparent self-contradictory observations. Inspection of Figs. 8,9, and 12 shows that, within individual experiments and in the pattern which emerges from pooled data, Ca efflux saturates with internal Ca. However, in experiments such as those shown in Figs. 9 and 10 it was seen that (1) when 45Caefflux is measured in the presence of external tracer-free Ca, there is never any effect on the efflux rate attributable to dilution

FIG. 11. The dependence on external Ca of the net Ca uptake by ATP-depleted human red cells. Same experiment as in Fig. 10. The linear regression slopes of the four upper curves of Fig. 10 (between 60 and 160 minutes) are plotted as a function ofthe external Ca. (From Ferreira and Lew, 1977.)

246

VlRGlLlO 1. LEW AND HUGO G. FERREIRA

- 70

01

"

GO-

50-

.40_I

E

-3

30-

20

"0

40

60

Initial Co content of cells

80

( p mole /

L cells)

FIG. 12. The dependence on internal Ca of the Ca efflux from ATP-depleted human red cells. Pooled data, Open circles, Efflux into solution A containing at least 2 mM Ca; solid circles, efflux into Ca-free solution. (From Ferreira and Lew, 1977.)

of the intracellular specific activity (see Fig. lo), and (2) in every single measurement of the efflux rate constant, whether in the presence or absence of external Ca, even when there was a very substantial reduction in Cat during the efflux period, as in the experiment in Fig. 13, no increase in the rate constant of efflux was observed, as would have been expected if the flux path were to become progressively unsaturated. If anything, a slower efflux rate became apparent, but only after several hours. These considerations suggest very strongly that for the net and ex-

-

1 0

I

I

1

2

3

4

I

I

5

6

Time (hours)

FIG. 13. Time course of Ca efflux from ATP-depleted human red cells preloaded with Ca. Cells from 5-day-old bank blood containing 56 pmoles Ca/liter of cells resuspended in Ca-free solution A. (From Ferreira and Lew, 1977.)

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

247

change components of the Ca efflux the Ca exit route is always far below saturation. What, then, is the explanation for the saturation pattern observed in Figs 8 , 9 , and 12? From experiments like that in Fig. 2 a heterogeneous Ca distribution seems very unlikely, and so do explanations based on precise compensatory effects giving false single exponentials. Perhaps a more plausible explanation is that, within the range of Ca, explored in these experiments, the eMux path is indeed far below saturation but that the number of pathways available for Ca efflux is irreversibly reduced b y increasing the initial Ca concentration inside the cell. According to this hypothesis, the inner membrane surface seems to have a “memory” of the highest Ca concentration it has “seen” in the form of some change imprinted on it, which reveals itself b y a reduced Ca efflux. The effect of Ca could not have been reversible; if it had been, the efflux rate would have increased with time. Since apparent saturation is observed both after time or concentration loading, with very different cell histories preceding the efflux period, the only common factor that can be responsible for this effect seems to be Cai. On a completely different scale, Porzig (1972) had also observed self-limiting effects of internal Ca on the Ca permeability of resealed ghosts. If a common process mediates this Ca effect in intact cells and resealed ghosts, where the permeabilities are so different, it probably involves a more general alteration in the membrane, perhaps a physical or chemical change in its lipid components.

IV.

THE GATING MECHANISM

A. Ca Sensitivity and Ca Selectivity

The K permeability of intact red cells containing different [Ca2+l, can b e investigated b y adding 42K to the medium and measuring the 42K tracer equilibration rate during any of the equilibrium or steadystate conditions shown in Figs. 5 and 6 in the presence of the ionophore A23187 (Lew and Ferreira, 1976). The results of this type of experiment are shown in Figs. 14-16. It can be seen that there are large variations not only in Ca sensitivity but also in the general kinetic pattern. Intact, inosine-fed cells exposed to a low ionophore concentration, a condition we described (Lew and Ferreira, 1976) as causing only a “minimal disturbance” to the cell, showed the lowest Ca sensitivity and the highest K permeability rise. Higher ionophore concentrations in the same cells increased the Ca sensitivity by two to

248

VlRGlLlO 1. LEW AND HUGO G. FERREIRA

8: -.. 10

RIY

fLC; 0 8

.'I

Z 6 6 c

/

1

3

0 'i" 2

*Fed cells

i$ . v -

do-o\o

I=100

-

.b

three orders of magnitude, and in ATP-depleted cells the Ca-induced rise in K permeability was already saturated at the lowest [Ca2+],investigated (0.5 and 1pM).We cannot tell at present whether there is a whole gradation of Ca sensitivities rather than two or three discernible states, and the kinetic pattern is bewilderingly complex. These results are, however, very reproducible and consistent. The ionophoric effect of A23187 can be easily reversed by washing the cells or simply by diluting the cell suspension (Lew and Ferreira, 1976; Sarkadi et al., 1976). Unfortunately, the only way of maintaining a steady Cat+ once the ionophore has been removed is to inhibit the Ca pump. When this is done by removal of internal Mg, an essential cofactor for Ca extrusion (Lew and Ferreira, 1976), the cells exhibit a high-Ca afY N

qI z, o r

-

1

9-

.

0 -

-Fed cells - 1=10 /O

/*

[Ca2*], (mM)

FIG. 15. The rate constant of "K tracer equilibration as a function of the internal Ca2+concentration in intact red cells in the presence of a low ionophore concentration. The proportion of ionophore to cells was about 10 pmoles/liter of cells. (From Lew and Ferreira, 1976.)

CA-ACTIVATED K CHANNEL IN BIOLOGICAL MEMBRANES

$

249

1

0 ‘

0

100 [Co2’],

200

(JJM)

FIG. 16. The rate constant of ‘*K tracer equilibration as a function of the internal Ca2+concentration in intact cells under various conditions. Fed cells were from fresh blood incubated in the presence of 10 mM inosine; depleted cells were from the same blood sample depleted of ATP by preincubation for 3 hours with 6 mM IAc and 10 mM inosine. Ionophore concentrations are given in micromoles per liter of cells, assuming complete partition of the ionophore into the cells (as from Lew and Ferreira, 1976).

finity pattern similar to that of the high-ionophore cells. High and low Ca affinities are observed at normal internal ATP concentrations, and in ATP-depleted cells where the Ca sensitivity is highest. This suggests that ATP has some effect on the affinity change, but it cannot be the only factor. There is some evidence indicating that the change from a low to a high Ca affinity is reversible. Whenever a small concentration of ionophore is added to a suspension of intact fed cells in a medium containing relatively low Ca concentrations, a transient increase in [Cali is observed (Ferreira and Lew, 1976). These Ca peaks can be clearly seen in Fig. 6. If 42Kis also present in this medium, a large 42K influx is observed during the peak (Lew and Ferreira, unpublished observations). At least some cells must have gone through a transient high-Ca sensitivity phase during the Ca peak, since the magnitude of the highest [Ca2+livalue during the peak is not high enough to produce such a large *K flux if the Ca sensitivity were that observed during the subsequent steady state. A high Ca affinity can therefore revert to a low Ca affinity under certain conditions, but we do not know yet whether or not low Ca sensitivities can be recovered after a more prolonged exposure to factors inducing the high-affinity state, such as a high ionophore concentration (Lew and Ferreira, 1976). Ferreira and

250

VlRGlLIO 1. LEW AND HUGO G. FERREIRA

Lew (1976) suggested that the peaks result from an initial heterogeneous distribution of ionophore, but since nearly all the K in the cells can become equilibrated with tracer 42Kduring the peak, it is more likely that all the cells experience a transient higher concentration of ionophore. This could occur if Ca-translocating forms of the various metal ion-ionophore complexes (Pfeiffer and Lardy, 1976) happen to be transiently more concentrated in the membrane before equilibrium is established. The basic conclusion of these experiments is that the Ca-activated K channel can be found in states with different Ca sensitivities. It is unlikely that these results are simply an artifact due to local heterogeneities in [Ca2+Ii,since the variable Ca sensitivity of the K channels was originally measured in precisely those experiments in which a constant Ca affinity for the Ca pump of about 1 p M , independent of the ionophore concentration, had been determined (Ferreira and Lew, 1976; Lew and Ferreira, 1976). With hindsight, earlier observations point to the possibility of variations in Ca sensitivity. To elicit the K permeability change fluoridepoisoned ghosts need external Ca concentrations two orders of magnitude lower than controls without fluoride (Riordan and Passow, 1973). Tetracaine increases the apparent Ca sensitivity of the K channel in resealed ghosts (Porzig, 1975). Yet the possibility of genuine changes in Ca sensitivity had never been suggested before, probably because it conflicted with strong expectations of a constant Ca affinity within the physiological Ca:+ concentration range. With similar expectations, the physiological [Ca2+],had often been estimated using the Ca2+sensitivity of a particular Caz+receptor determined under certain experimental conditions as a calibrated indicator (Lew, 1970; Baker, 1972; Schatzmann, 1973; Porzig, 1975; Simons, 1976b). Changes in Ca2+sensitivity like those reported here restrict the validity of such estimates. As mentioned before, a variable Ca sensitivity only adds versatility to the possible physiological roles of this channel. Even with the mechanism in a low Ca affinity state, variations in the physiological levels of Cat+ could induce substantial changes in the K permeability of the membrane if the maximum effect of CaF+ is large enough (Lew and Ferreira, 1976). The factors determining the affinity change need not be strictly localized to the channel. Because the low-affinity response was observed only at lower ionophore concentrations, a treatment interpreted as producing minimal cell injury, we suggested that it reflects the native state of the red cell K channel under physiological conditions (Lew and Ferreira, 1976). Conversion to a high-affinity state can

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

25 1

be induced by very diverse treatments (depletion, high ionophore concentration, Mg depletion, reverse hemolysis), suggesting the response of a labile mechanism to a more general membrane disturbance. At present, research on different aspects of the Ca sensitivity change is being actively pursued in various laboratories. A detailed analysis of this work is therefore premature, and the following discussion is intended only to be an account of some preliminary results and of the basic ideas guiding this research. Brown and Lew (unpublished results) examined the inhibitory effect of Ca,2+ on the ouabainsensitive Na efflux from inosine-fed cells at high and low ionophore concentrations and found apparent K , values of about 5-10 p M and 400 pM, respectively, under the same experimental conditions as those in which the affinity shift of the K channel was observed. This means either that the Na pump and the K channel are different aspects of the same mechanism, as suggested by Blum and Hoffman (1970), or that a diffuse membrane change affects the Ca sensitivity of different Ca-reactive transport systems in a similar way. Beaug6 and Glynn (personal communication) measured a low inhibitory affinity for Ca of about 400 p M for the Na, K-dependent ATPase activity of a purified preparation of Na, K-ATPase from the pig kidney inner medulla (Jorgensen, 1975).Although this was measured in the absence of an ionophore, it supports the view that the low Ca sensitivity configuration represents more the native state of the isolated enzyme, and that in order to induce the high Ca sensitivity state other membrane components may be required. One possibility is that internal Ca activates membrane phospholipases and that, at high ionophore concentrations, this activation occurs at a very low Ca;+. Allan and Michell (1975) found a Ca?+-dependent accumulation of 1,2-diacylglycerol (DAG) in the red cell membrane of intact human red cells in the presence of the ionophore A23187 under experimental conditions similar to those in which we observed the Ca affinity change (Lew and Ferreira, 1976). According to Allan and Michell (1975), the steady-state level of DAG results from Cat+-dependent formation from an unknown precursor and ATP-dependent breakdown to an “inactive” phosphatide. A relattively high content of DAG or a similar intermediate may cause a high-Ca sensitivity of the K gate, or this may be determined by reactions parallel to those involved in regulation of the DAG level. I n any case Ca;+ would have effects other than direct activation or inhibition of the transport systems. T h e implicit assumption here is that Ca?+ activates internal enzymes which produce a change in the lipid components or in the lipid environment of transport mechanisms, af-

252

VlRGlLIO 1. LEW AND HUGO G. FERREIRA

fecting their reactivity toward Ca. A similar process may also provide the irreversible imprinting mechanism responsible for the “memory” effect of the passive Ca transport system discussed in Section II1,D. Diffuse membrane alterations, whether mediated through lipid changes or not, may be the cause of the Ca sensitivity change induced by the various experimental procedures investigated. However, if there is a physiological regulator of the Ca sensitivity, it is likely to be, at least in certain cells, a more specific agent acting directly on the Ca-reactive part of the K channel. Perhaps in systems such as the metabolically exhausted frog skeletal muscle fiber (Fink and Luttgau, 1976), where the K channel seems to function as a reserve mechanism to reduce excitability, specific control does not apply. It was not possible to demonstrate a rise in [Ca:+l in these cells. If the K channel responsible for the K conductance change is at all Ca-sensitive, increased Ca affinity, probably induced by the metabolic condition of the cell, seems to be the most likely explanation of the observed effects. Other divalent cations can react with the K channel but none as efficiently as Ca. In hypotonically resealed ghosts from human red cells, containing ethylenediamine-N,N,N’,N‘-tetraacetic acid (EDTA) or N-(2’-hydroxyethyl)ethylenediamine-N,N,N’,N’-triaceticacid (HEDTA) as divalent cation buffers, Simons (1976b) found the following activating sequence: Ca > Sr >> Ba. Internal Mg acts like a weak Ca antagonist (Porzig, 1975; Simons, 1976b). The effects of, and even the requirement for, divalent cations can be drastically modified if fluoride or traces of Pb are present in the medium (Wilbrandt, 1940; Lepke and Passow, 1960). For a descriptive review of this subject, the reader is referred to Riordan and Passow (1973). B. Activation and Inactivation; Time and Voltage Dependence of the K-Gating Process

Gardos (1958a,b) observed that the addition of a chelating agent in excess of the Ca concentration in the medium reversed the Ca effect on the 42Kloss from cells being depleted of ATP. Lew (1974) reported similar results, but only for cells which had not been loaded with too much Ca. As indicated in Section 111, D and Fig. 13, net Ca efflux from ATP-depleted cells into a Ca-free medium is a relatively slow process, and it seems that the K impermeability recovers earlier than would be expected from the fall in Cai. If, as is usually the case, the cells exhibit

CA-ACTIVATED K CHANNEL IN BIOLOGICAL MEMBRANES

253

substantial heterogeneity in relation to the K permeability change (see Section 11, F), one can interpret the apparent restoration of impermeability after chelation of external Ca as resulting from K emptying from the leaky cells with no participation of the “tight” cells. But whatever the explanation of the “premature” recovery in metabolically depleted cells, it is impossible to rule out modifying effects of external Ca on the K permeability mechanism. In cells being depleted, residual ATP may aid Ca extrusion through the Ca pump, and faster reversal is not surprising (Gardos, 1958a,b; Lew, 1974). The parallelism between Ca uptake and increased K permeability (Lew, 1971a) implies a rapid response to changes in [Ca2+liby the K-gating mechanism. But the time resolution of these experiments is very poor. This, together with heterogeneity and the sluggishness of the Ca movements in ATP-depleted cells, makes human red cells rather unsuitable for study of the on-off reactions of the K gate. Lassen et al. (1974, 1976) measured the hyperpolarizing effect of Ca-containing media when giant red cells from the salamander Amphiuma means were prepunctured or exposed to a sudden increase in external Ca. Unfortunately, it is technically impossible to apply the hyperpolarizing condition while recording the membrane potential of a single cell. The distribution of potentials recorded from individual cells after treatment confirmed heterogeneity at a cellular level and changed with time in a manner expected from possible changes in [Ca2+Ii.More recently, Lassen et al. (1977) measured 42K and 45Ca fluxes during a steep rise in external Ca (Lassen et al., 1976)and found a large increase in 42Kinflux which was not accompanied by a measurable entry of Ca (Ca content < lOpmoles/liter of cells). The time course of the increased K flux paralleled the previously observed transient hyperpolarization (Lassen et al., 1976). This result again suggests a rapid opening of the channel following Ca addition, but the interpretation of the subsequent time course of the effect is obscure. If an increased level of external Ca induces a new pump-leak steady state with a slightly higher [Ca2+Ii,why is the effect on the K permeability transient? There seem to be four possibilities: (1)a transient increase in [Ca2+],, which can result only from changes in Ca permeability or pumping, (2) spontaneous inactivation of the K permeability mechanism, (3) a transient change in Ca sensitivity, and (4) unknown effects of external Ca on the K channel. We cannot decide among these various possibilities, except that in human red cells there is evidence that spontaneous inactivation does not occur. This follows from the observation that, when the steady-state Ca level is controlled by use of the ionophore A23187, as in the experiments in Figs. 5 and 6,

254

V l R G l L l O 1. L E W AND HUGO G. FERREIRA

the K permeability estimated from the tracer flux remains virtually constant for periods of at least 1 hour (Brown and Lew, unpublished observations). A maximum value for the duration of the onset and inactivation reactions of the K permeability mechanism following a steep change in internal Ca can be estimated from an experiment reported by Reed (1976, Fig. 4).H e measured the effect of EGTA addition on the release of K from rat red cells incubated in a medium containing lo-‘ M A23187 and contaminating concentrations of Ca, probably in the micromolar range. Before the addition of EGTA the K concentration in the medium was increasing at a rate of 100 pM per minute. In the 30 seconds following EGTA addition (2 p M ) the external K concentration increased by less than 5 pM (so far as we can tell from the figure). This suggests that it must have taken less than 3 seconds to inactivate the K permeability mechanism. On subsequent addition of CaC12 to give a final concentration of 20 pM in the medium, the K permeability increased more than before, since the new rate of external K accumulation was about 130 pmole per minute. The lag between the “zero” rate, during the excess EGTA period, and the new rate was about 10 seconds. We need much better time resolution, probably in the millisecond range, if we are to estimate the true activation and inactivation rates of the K-gating mechanism. The basic difficulty in obtaining the right experimental conditions for these measurements is precisely controlling rapid changes in Ca:+ at fixed (and known) Ca sensitivity and, at the same time, measuring with sufficient time resolution some parameter reflecting the K permeability of the membrane. A promising approach may be the sudden change in [Ca2+liwhich can be induced in resealed ghosts containing Ca-EGTA buffers when pulsed with a pH change in the medium while measuring the membrane potential with carbocyanine dyes (Simons, 1976a,c; Hladky and Rink, 1976).In excitable cells, where conductance changes can be recorded with sufficient time resolution, the control of [Ca2+l,and the assessment of the Ca sensitivity of the K gate present awkward problems. One system worth analyzing is the sensory epithelium of the electroreceptor (ampulla of Lorenzini) of the skate (Clusin et al., 1975), although there is no evidence so far to indicate that the Ca-sensitive K conductance found in this organ is identical to the Ca-sensitive K channel of red cells or other cells. The epithelium can generate allor-none action potentials of about 60 mV. Voltage-clamp experiments showed that these action potentials resulted from an initial inward depolarizing Ca current, followed by an outward K current responsible

CA-ACTIVATED K CHANNEL IN BIOLOGICAL MEMBRANES

255

for the repolarizing phase. Voltage-clamp pulses below or above the reversal potential of the initial Ca current at different external Ca concentrations showed that the late K current was not activated unless there had been a net influx of Ca into the cytoplasm during the early phase of the pulse. The rising phase of the outward K current during suitable voltage steps lasted about 150 msec and was followed by a plateau due to a lack of inactivation of the early Ca current for the duration of the voltage-clamp pulse. On repolarization, outward tail currents lasting about 600 msec were observed. Their size increased, but their duration was not affected by repolarizing the epithelium to more lumen-positive voltages. This indicates that the inactivation process of the K conductance was independent of the membrane potential. It is impossible to say at present whether the 150-msec rising phase of the late outward current, or the slow time course of the tail currents, represent the true activation and inactivation times of the K gate after the binding or dissociation of Cayor whether they reflect the local variation in [Ca2+],,in which case the activation and inactivation reactions must be much faster. The second alternative seems more plausible, since it is difficult to imagine a process which gives a sufficiently fast change in Ca?+. Clusin et al. (1975) also observed that, if two voltage-clamp pulses were applied 2 seconds apart, while the size of the inward current remained the same and the membrane conductance returned to normal in between the pulses, the onset of the outward K current occurred considerably earlier. A facilitated onset was observed u p to 10 seconds after a conditioning stimulus, Unfortunately, no information was provided about the effect of a conditioning pulse on the duration of the tail currents. If residual Caiz+ is responsible for facilitation, as suggested by these investigators, and if also the tail currents reflect [Ca2+Ii,their duration might be expected to be prolonged and their amplitude increased. If tail current duration is not affected, Caindependent inactivation is a possibility. That this might be the case is supported b y the fact that the epithelium conductance returned to normal between pulses. If the lack of inactivation of the K channel observed in red cells at constant Cai2+and constant Ca sensitivity is to be compatible with inactivation of the late K current at increased Ca?+ in the sensory epithelium of the skate electroreceptor, either the facilitating level of Ca:+ is considerably lower than that required for threshold activation of the K channel, or inactivation is mediated by a reduction in Ca sensitivity. More experiments are needed to decide between these alternatives. The possibility that an affinity change may play a role in the on-off reaction sequence of the K gate is margin-

256

VIRGlLlO 1. LEW AND HUGO G. FERREIRA

ally supported by observations on red cells which can be more easily explained as resulting from high Ca sensitivity transients. These are the ionophore A23187-induced Ca peak (Ferreira and Lew, 1976), the effect of a sudden rise in external Ca on the membrane potential of Amphiuma red cells (Lassen et al., 1976), and a propranolol-induced Ca peak recently described by Szasz et al. (1978) in human red cells, which is considered in Section V,A. In conclusion, what the analysis of the available experimental information suggests (assuming a single mechanism is involved in all cases), is that the activation and inactivation rates of the K permeability are very fast. The on-off reactions are completed in less than 1second, perhaps in much less; more experiments specifically designed to measure these times are necessary. The gating mechanism of the K channel does not seem to inactivate with time and shows no voltage dependence, but the possibility that transient changes in Ca2+sensitivity take part in the on-off reactions cannot be ruled out. It is worth pointing out that there are some kinetic similarities between this channel and the acetylcholine sensitive channel. In both instances the permeability change is triggered by the association of a mediator with a receptor molecule close to or even part of the permeability mechanism itself, and inactivation follows dissociation and removal of the intermediate, although we do not know how fast this followup is in case of the Ca-sensitive K channel. Below saturation both responses are graded and follow the variation in the concentration of mediator in the vicinity of the receptor sites. V.

PHARMACOLOGICAL EFFECTS

A proper classification of activators and inhibitors of the Casensitive K channel according to their mechanism of action is not yet possible simply because we do not know enough. Ideally, such a classification should be based on the effects of each drug on the various steps of the sequential model. In this section we consider in some detail only agents about which enough information is available to suggest possible mechanisms of action. A. Activators

1. PROPFMNOLOL

Manninen et al. (1967), Ekman et al. (1969),and Manninen (1970) found that propranolol, a well known P-adrenergic blocking agent, in-

CA-ACTIVATED K CHANNEL IN BIOLOGICAL MEMBRANES

25 7

duces a sharp Ca-dependent increase in the K permeability of fresh human red cells. Manninen (1970, Fig. 6) observed that, when Ca was present in a medium containing about 0.4 mM propranolol, the accumulation ratio of ['4Clpropranolol b y the cells increased from 8 to about 20. This must reflect an increased propranolol content of the cells, since isolated membranes did not appreciably accumulate this drug under any of the conditions tested. The Ca-induced propranolol accumulation probably results from a reduction in the efflux rate of propranolol in Ca-containing cells (Manninen, 1970, Fig. 7), but the mechanism of this effect is obscure. The uptake of propranolol was considerably slower at pH 7.0 than at pH 7.5 and 8.0. Manninen (1970) suggested that for this pH effect charged membrane groups are probably more important than the fraction of uncharged propranolol molecules since, with a pK, of 9.45, this would be very small in the pH range 7.0-7.5. The Ca-dependent effect of propranolol on the K permeability is superimposed on a general leakiness of the cell membrane, and considerable haemolysis is observed even after short incubation times in isotonic media. It is also interesting to note that, while the dextro (+) isomer of propranolol was more effective than the lev0 (-) isomer in inducing the Ca-dependent K loss, the converse is true for the P-adrenergic blocking action of this agent (Kaumann and Blinks, 1967; Vaugham Williams, 1970). Porzig (1975) investigated further the effect of propranolol on resealed human red cell ghosts. In these experiments the drug was added during hemolysis, so that it was possible to explore drug action independently of the rate of penetration across the membrane. Porzig found that, in the absence of added Ca, 1 mM propranolol induced a large K loss from the ghosts, which was prevented when the cells also contained EGTA. The K loss from ghosts containing neither propran0101 nor EGTA was unfortunately not reported. Higher propranolol concentrations (7 mM) inhibited the K loss. Porzig (1975) assumed that, under these conditions, the only Ca source was membranebound Ca. Propranolol must have therefore displayed Ca from internal binding sites and increased [Ca2+],.Using the curve of K outflow in 1hour as a function of [Ca2+],,measured separately in the same ghosts, as an indicator of [Ca2+],,he estimated the Ca:+ released by propranolol to be between 0.7 and 3 pM, a figure amounting to between 5 and 20%of the membrane-bound Ca reported by Harrison and Long (1968). The argument that propranolol acts by releasing Ca from binding sites on the internal surface of the membrane is considerably weakened, however, by the following considerations: (1)About 90% of the membrane-bound Ca measured by Harrison and Long (1968) could be washed out by using strong external chelators, indi-

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V I R C i l L I O 1. L E W AND HUGO G. FERREIRA

cating that it was mainly externally bound Ca. (2) The chemicals used b y Porzig (1975) “were of the highest purity which was commercially available,” but there is no suggestion that they were spectroscopically pure. Commercially available Analar-quality chloride salts of Na and K (BDH Analar salts, for instance) specify on their labels maximum limits of impurities for the “calcium group and magnesium (Ca)” of 0.004% (w/w). For isotonic solutions of these salts this is equivalent to a maximum of about 10 pM Ca. Ca contamination in the absence of added Ca can therefore provide more than enough Ca in the medium to induce the observed effects, as was also noted by Reed (1976) in experiments on rat red cells. (3)Possible effects of propranolol(1 mM) on the Ca sensitivity curve used as a “calibrated” estimate of [Ca2+li were not investigated, This is a rather important point, since in the same series of experiments Porzig (1975) showed that ghosts containing 2 mM tetracaine exhibited about twice the Ca sensitivity of the controls. The lack of controls containing neither propranolol nor EGTA in Fig. 2 of Porzig’s paper does not allow even a rough assessment of this possibility and raises the question of whether or not propranolol had any effect at all under these conditions. I n a more recent study, Szasz et al. (1978) measured directly the effect of propranolol on the binding of Ca by isolated human red cell membranes in a low-ionic-strength medium buffered with Tris-HC1 and containing 40 pM Ca2+.They found that in the presence of 0.5 mM propranolol membrane-bound Ca is reduced at all p H values between 6 and 9. Although these experiments indicate that propran0101 can indeed interfere with Ca binding, they are not proof that it does so in intact cells or that this effect is involved in the mechanism leading to increased K permeability. Szasz et al. (1978)also examined the releasing effect of propranolol on residual 45Caremaining in fresh intact cells (1)after exposure to the tracer for 3 hours at 37°C and (2) during the tailing off of the active Ca extrusion process after ionophore-induced Ca loading (Sarkadi et al., 1976). The results showed enhanced release of Ca at all propranolol concentrations tested (0.25-1 mM), but similarity with the Ca-releasing action of A23187 (Ferreira and Lew, 1977, Fig. 6) suggests that this effect was probably due to increased membrane permeability to Ca. In intact red cells propranolol produces a sharp, partially transient increase in Ca permeability, resembling the A23 187-induced Ca peak (Szasz et al., 1978, Fig. 1; Ferreira and Lew, 1976) discussed above. This does not seem to be the only effect of propranolol. Labilizing effects on the membrane have also been postulated based on the “nonspecific” action of propranolol (Szasz et al., 1978; Manninen 1970).

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A more-or-less transient increase in the Ca sensitivity of the K channel, together with the Ca ionophoric effect, could explain all the experimental observations concerning the effect of low propranolol concentrations on the Ca-dependent K channel. A recent observation by Gardos et al. (1978) supports this view. These workers found that the internal Ca concentration required to induce an equivalent loss of K from ionophore-pretreated fresh cells was reduced from 0.5-1 mM to 10-20 p M by the presence of 0.5-1 mM propranolol in the medium. It remains to be seen whether or not further Ca sensitivity shifts are induced b y propranolol under high Ca affinity conditions like those investigated by Porzig (1975). The extent to which this effect of propranolol is responsible for any of its multiple pharmacological actions is not clear. 2. OTHERACTIVATORS Szasz and Gardos (1974) reported a variety of drugs other than propranolol which activated K permeability: pronethalol, tetracaine, histamine, and theophylline. The level of ATP was little affected by these drugs, but the size of the K permeability change roughly paralleled the effect on the Ca permeability of the membrane. This seems to differ from the effect of triose reductone plus Ca (Passow and Vielhauer, 1966), which required preincubation of the cells in the absence of substrate. Energy-depleted resealed ghosts containing 2 mM tetracaine displayed increased Ca sensitivity (Porzig, 1975), but in general the precise mechanism of action of this group of drugs is poorly characterized. 6. Inhibitors

1. QUININEAND QUINIDINE Among a variety of organic cations and anesthetic agents tested [guanidine, strychnine, procaine, benzocaine, chloroquine, mepacrine, quinine, quinidine, 4-aminopyridine, tetraethylamine (TEA), phloridzin, butanol, ethanol, and ether] only the chloride salts of quinine and quinidine were found to have a clear inhibitory effect on the Ca-induced K loss from ATP-depleted human red blood cells (Armando-Hardy et al., 1975). The inhibitory effect of quinine is fast and reversible. Figure 17 shows the dose-response curves of quinine and its opitical isomer qoinidine in human red cells. Half-maximum

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V I R G I L 1 0 1. L E W AND HUGO G. FERREIRA

Y

c W

xL [QUININd OR CQUlNlOlNE] (mM)

FIG. 17. Inhibition of the Ca-induced K loss by quinine and by quinidine. Inhibitory effect tested on the loss of 42K at 15 minutes from 4xK-loadedcells predepleted of ATP for 3 hours in the presence of Ca.

inhibition is observed at concentrations between 0.1 and 0.2 mM in the medium, and there is a marked sigmoidicity at the onset of the inhibitory effect. Quinidine is more effective than quinine at low concentrations, and the reverse is true at higher concentrations. The inhibitory effect of quinine is not mediated through actions on ATP or on Ca permeability, since (1)inhibition is equally effective in fed and depleted cells whether or not a Ca ionophore (A23187) is present (Lew and Ferreira, 1976), (2) quinine has virtually no effect on the Ca fluxes in depleted cells, and (3)it is equally effective in cells preloaded with Ca. Figure 18 shows the dose-response curve of quinine in ATPdepleted cells loaded with different concentrations of Ca. The fact that the KI of quinine was not increased when the internal Ca was raised indicates that there is no competitive displacement of Ca by quinine at the Ca receptor site of the K-gating process. These observations suggest two possible mechanisms for the action of quinine: (1)a noncompetitive interaction with the Ca receptor which would either inhibit the reaction with Ca:+ or the subsequent activation of the gate, or (2) a direct blocking effect on K movement through the K channel. Fleminger, Ferreira, and Lew (unpublished results), following a suggestion by R. A. Klein, tested the inhibitory effect of cinchonine and cinchonidine, a stereoisomeric pair closely related to quinine and quinidine, differing only in the presence of a 6-methoxy group. Cinchonine, at a concentration of 1mM, inhibited the Ca-induced 42Kloss

261

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005

0.1

OUlNlNE

0.5

1

CONCENTRATION (mM)

FIG.18. Inhibition of the Ca-induced K loss by quinine at two different internal Ca concentrations. Cells from 5-day-old bank blood were depleted of ATP in the presence of 0.75 and 6 mM Ca in the medium to give the indicated Ca contents at the beginning of the final incubation. The cells were loaded with 42Kduring the depletion period.

from ATP-depleted cells by only 16-22%, while cinchonidine had no effect. This result, together with the lack of effect of aminoquinoline derivatives (chloroquine, primaquine, mepacrine) illustrates the marked specificity with which the Ca-sensitive K channel is inhibited by quinine and quinidine. It should be emphasized, howeuer, that although the K permeability mechanism in red cells is specijically inhibited by quinine and quinidine these agents are, unfortunately, not specijic inhibitors of this mechanism. They have a variety of other well-known pharmacological actions as local anesthetics, antiarrhythmics, antimalarial agents, and so on, at concentrations below those which block the K channel. In spite of this shortcoming they have proved useful in testing the similarity among the Ca-dependent mechanisms of different cells. Thus Puil and Krnjevic (1977) found that extracellular ionophoretic administrations of quinine to cat lumbosacral motoneurons produced a prolonged rise in input resistance and a marked reduction in or disappearance of the conductance increase associated with postspike hyperpolarization. Quinidine had similar effects. These actions of quinine and quinidine are fully consistent with a primary block of the K conductance, and this was interpreted by Puil and Kmjevic (1977) as further proof that the Caactivated channels contribute significantly to the resting K conductance of neuronal bodies (Krnjevic et al., 1975).

262

VlRGlLIO 1. LEW AND HUGO G. FERREIRA Ca - LOADED CELLS

TIME (rntn)

Pb- TREATED CELLS

TIME (min)

FIG.19. Effect of quinine on the Na and K loss from Ca-loaded or Pb-treated red cells. Cells from Sday-old bank blood were loaded with %*Na and "K while being depleted of ATP in the presence of either Ca,(2m M )or Pb (Pb acetate, 120 ELM).Final incubation under the same conditions but without IAc plus inosine.

In the barnacle photoreceptor, Hanani and Shaw (1977) found that the hyperpolarized component of the response to light was inhibited by quinine and quinidine with half-saturation concentrations in the 0.15-0.2 mM range, a value similar to that observed in human red cells. A similar inhibitory constant also seems to hold for the Cainduced hyperpolarization observed in Amphiuma red cells following a sudden increase in external Ca (Lassen, personal communication). In molluscan neurons, however, Meech (1976) reported briefly that quinine does not prevent the K permeability change induced by intracellular Ca injection. In the absence of more detailed information this negative result is difficult to interpret. Armando-Hardy et al. (1975) also reported that quinine and quinidine inhibited the Pbinduced increase in K permeability but not the p-chloromercuribenzene sulfonate (PCMBS)-induced K loss which supposedly results from reversible, nonselective membrane leakiness to small ions (Garrahan and Rega, 1967). Neither quinine nor quinidine had any effect on the Pb-induced increase in Na flux. This suggests that different sites mediate the action of Pb on the increase in Na permeability (Fig. 19). 2. OUABAIN Blum and Hoffman (1970, 1971) reported that ouabain, a wellknown specific inhibitor of the N a pump (Schatzmann, 1953),exerts a partial inhibitory effect on the Ca-dependent K loss from energy-

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depleted red cells. The K loss from cells exposed to M ouabain during the final hour of their energy depletion was reduced between 11 and 46% in relation to that from ouabain-free controls. Failure to detect the inhibitory effect of ouabain under a variety of conditions was reported by Manninen (1970), Riordan and Passow (1971), Romero and Whittam (1971), Gardos et d.(1975), Lassen et d.(1974, 1976), Reed (1976), and Simons (1976b). Lew (1974), however, confirmed Blum and Hoffman’s (1971) observations in intact human red cells but found that the inhibitory effect of ouabain becomes vanishingly small as the cell become progressively depleted of ATP. Lew (1971b) had previously shown that in guinea pig red cells, where the contribution of the Na pump to the overall turnover of ATP is considerably larger than in human red cells (see Section 11, E), the presence of ouabain can considerably alter the internal ATP levels of IActreated cells. Thus in the presence of external K, when the Na-pump runs forward, ouabain should reduce the rate of ATP hydrolysis, whereas in the K-free, high-Na medium, when the pump runs backward synthesizing ATP (Lew et d.,1970), ouabain should reduce the rate of ATP formation. If Ca entry parallels the rate of ATP depletion, and if the action of ouabain is mediated through the ATP level of the cells, ouabain ought to inhibit or stimulate the Ca-induced K loss depending on whether it inhibits an ATP-consuming or an ATP-sparing pump. The results obtained confirmed these predictions. More recently, Isern and Romero (1977), working with resealed human red cell ghosts, found that small internal ADP and ATP concentrations were necessary for ouabain to elicit a’net Ca efflux under conditions in which the Na pump was reversed, thus providing further support for an ATP-mediated effect of ouabain, as proposed by Lew (1971b). The effect of ouabain was originally interpreted as being indicative of an identity between the Na pump and the Ca-sensitive K permeability mechanism (Blum and Hoffman, 1970). The results of Lew (1971b, 1974), however, suggest that the ouabain effects are ATPmediated and need not involve a direct interaction between ouabain and the K channel. Such an interaction may seem superfluous for the interpretion of the available data but is difficult to rule out. Blum and Hoffman (1971) argued against ATP- mediation by saying that the same degree of inhibition was obtained when ouabain was added only during the final incubation medium, without prior ouabain treatment. This was presumed to minimize cumulative differences in the ATP levels of control and ouabain-treated cells and to make the ATPmediated effect negligible. In the experiments of Lew (1974), inhibition of the K flux was also observed even when ouabain-induced dif-

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VIRGIL10 1. LEW AND HUGO G. FERREIRA

ferences in ATP levels were barely detectable. If a direct interaction between ouabain and the K permeability mechanism is to be entirely ruled out, one has to postulate that minute differences in ATP concentration markedly affect the K permeability. According to the sequential model this could work if the ATP differences are “amplified” by multiplying their effect on the Ca entry and Ca sensitivity steps. Whatever the explanation, small variations in the internal concentrations of ATP, whether induced by ouabain or by other means (the various ATP-depleting procedures discussed above), have in the end large effects on the K fluxes (Lew, 1970, 1971a). 3. CARBOCYANINE DYES

Carbocyanine dyes are widely used as fluorescent probes of membrane potential in isolated cell systems (Hladky and Rink, 1976). Simons (1976~)recently showed that these dyes had a direct inhibitory effect on the Ca-induced hyperpolarization or K flux observed in suspensions of Ca-buffered resealed ghosts, provided the external K concentration was low. In these experiments, a concentrated solution of 3,3‘-dipropyl- or 3,3’-diethylithiodicarbocyanineiodide in ethanol was added to a suspension of ghosts resealed in a medium containing 2 mM Ca, 3 mM HEDTA, 2 mM phosphate, and about 100 mM KCl and suspended in a K-free choline solution. This led to a large initial hyperpolarization followed by rapid depolarization to the zeropotential level (estimated from the variation in the fluorescence signal of the ghost suspension). The addition of 1 pM valinomycin at that point caused a large hyperpolarization. This means that the dissipation of the K gradient had not caused the depolarization. Simons (19764 also measured the inhibitory effect of these dyes on the net K efflux. He obtained a rough dose-response curve giving 50% inhibition at about 20-50 pM in the presence of 0.1 mM external K. The dyes apparently had no inhibitory effect when the external K concentration exceeded 2 mM. The ouabain-sensitive K influx into intact human red cells in the presence of 0.16 mM 42K was not affected by the dye. However, it inhibited the net K loss induced by 0.1 mM Pb acetate or by incubation with inosine plus IAc plus Ca. The protective action of external K suggests that the inhibitory reaction occurs at the external surface of the membrane, but the nature of the inhibitory effect is unclear. It is possible that the interaction of the dye with an external membrane site reduces the Ca sensitivity of the K gate. A more likely explanation is that it directly blocks the K movements through the channel. More experiments will be needed to de-

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cide between these possibilities. This inhibitor is of interest mainly because it has a very high inhibitory affinity and, unlike oligomycin, seems to b e fairly specific. Because of these properties it may be useful as a channel marker during membrane purification (Simons, 1976~).

4. OTHER INHIBITORS A large variety of agents has been reported as having inhibitory effects on the Ca, Pb, or fluoride plus Ca-induced increase in K permeability observed in red cells. Their site of action on the sequential scheme is, however, not well understood in most cases. When TEA, a well-known blocking agent of the K currents in nerve, was added to the outside medium, it partially inhibited the undirectional K influx but had very little effect on the net K loss from ATP-depleted cells (Gardos et al., 1975). Simons (1976b) also described partial inhibitory effects of TEA and tetramethylamine (TMA). A list of other inhibitors includes furosemide (Blum and Hoffman, 1971), oligomycin (Blum and Hoffman, 1971; Riordan and Passow, 1971; Gardos et al., 1975), ethacrynate, mersalyl, and p-chloromercuribenzoate (PCMB) at low concentrations (Gardos et al., 1975), chlorpromazine, promethazine, and high concentrations of histamine (Szasz and Gardos, 1974; Gardos et al., 1976). Oligomycin has been widely used in concentrations up to 20 pg/ml (Blum and Hoffman, 1971, 1972; Riordan and Passow, 1971, 1973; Gardos et al., 1975) and was shown to inhibit the increased K permeability induced by adenosine plus IAA plus Ca, by fluoride plus Ca, and by Pb (Riordan and Passow, 1971).

VI.

THE MOVEMENT OF K AND THE NATURE OF THE PERMEABILITY MECHANISM

Large net and unidirectional K fluxes can take place through the Ca-sensitive K channel. In this section we consider the main properties of the K movement. A. Selectivity

K, Rb, and to some extent Cs ions move through the Ca-sensitive channel; choline, Na, and Li are not measurably transported (Man-

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VlRGlLlO 1. LEW AND HUGO G. FERREIRA

ninen et al., 1967; Simons, 1976b). In resealed ghosts the relative rates for the penetrating alkali metal cations K, Rb, and Cs are 1: 1.5:> 0.05 (Simons, 1976b), but in such ghosts the membrane is generally more leaky than in the intact cell and selectivity ratios better than 50 or 100 cannot be determined. In intact cells, the ground permeability of the membrane to K and Na is about cm per second, equivalent to a rate constant of 0.007 per hour (Lew and Beaug6,1978). This corresponds to the linear components of the unidirectional Na and K fluxes, after allowing for all other saturable components including the fluxes through the Na pump. The increase in K permeability induced by Ca must be compared with the ground permeability of the membrane in order to assess the true permeability increase and the order of selectivity in relation to Na. Lew and Ferreira (1976) reported a maximum increase in K permeability equivalent to rate constants of about 10 per hour. This corresponds to more than a 1000-fold rise in the K permeability of the intact red cell membrane. At the same time, the Na flux in the presence of ouabain was unchanged or, if anything, slightly reduced by internal Ca (Lew and Ferreira, 1976; Brown and Lew, unpublished observations). When the minimum detectable increase in the rate constant for Na efflux is set at about 0.001-0.005 per hour, the size of two standard errors of the mean of our best estimates, the lack of measurable effects on the Na flux implies a K/Na selectivity ratio better than lo3 and probably even better than 104. In red cells at least, the Ca-sensitive K channel is therefore extremely K-selective in relation to Na. B.

K

Counterflow

Activation of the K channel when intact red cells are incubated in low-K or low-Rb media containing 42Kor ssRb leads to a transient accumulation of tracer against its concentration gradient. This was observed in Pb-poisoned red cells from rabbits (Joyce et al., 1954) and humans (Grigarzik and Passow, 1958), in propranolol plus Ca-treated human red cells (Ekman et al., 1969; Manninen, 1970), and in energy-depleted red cells in the presence of Ca (Blum and Hoffman, 1971; Gardos et al., 1975). Manninen et al. (1967) suggested that the diffusion potential arising from the increase in K permeability could provide the energy for tracer accumulation, but Manninen (1970), believing the red cell membrane to be los times more permeable to

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

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small anions than to small cations, rejected the possibility of potential-coupled counterflow. After the demonstration by Hunter (1971)that the diffusional chloride permeability is about four orders of magnitude smaller than the exchange permeability in human red cells, Glynn and Warner (1972) indicated that the rejection of potential-coupled counterflow may no longer be justified. Evidence in support of this view was provided by actual measurements of Cainduced hyperpolarized potentials in prepunctured Amphiuma red cells (Lassen et al., 1974),and more recently in intact human red cells (Lassen et al., 1977)and in resealed ghosts (Simons, 1976~)~ when the cells were incubated in low-K media. Further support was provided by the observed reduction in the net loss of K from energy-depleted cells in Ca-containing media when either dipyridamole or 4acetamido-4’-isothiocyanostilbene-2,2’-disulfonic acid (SITS), two well-known inhibitors of anion permeability, were added to the cell suspension (Hoffman and Knauf, 1973).Inhibition of the Ca-induced K outflow by dipyridamole and SITS was much reduced (but surprisingly not entirely abolished) at high external K concentrations, as expected from a primary limiting effect on the net anion movement. All these results are consistent with the view that the net movement of K is able to generate large diffusion potentials across the membrane, which are directly responsible for the counterflow effects described above. C. Interactions with Internal Na and External K

In many of the experiments in which K counterflow was investigated (see Section VI,B), external Ca was used to induce the increase in K permeability (Manninen, 1970;Blum and Hoffman, 1970, 1971).The internal Ca concentration must have therefore increased during the experiments, and the effect of the various external conditions on the Ca permeability of the membrane (Ferreira and Lew, 1977;see also Section III,E), which might have finally affected the K flux measurements, was overlooked (Kregenow and Hoffman, 1972). This leads to interpretations which bypass possible interactions at the early steps of the sequential process and attribute nonlinearities in the K flux or the action of inhibitors directly to reactions at the level of the translocating mechanism (Hoffman and Knauf, 1973).The decrease in Ca uptake when the external K concentration is increased (see Section III,E), for instance, appears to be an inhibitory effect of external K on

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VlRGlLlO 1. LEW AND HUGO G. FERREIRA

the K permeability (Kregenow and Hoffman, 1972; Riordan and Passow, 1973; Lew, 1974) if the K flux is measured during net Ca uptake. When the K flux is measured at constant internal Ca in resealed ghosts (Simons, 1976b), the inhibitory effect of high external K concentrations is no longer observed. The findings in red cells are also in agreement with the hyperpolarizing effects of Ca observed in other cells (Meech, 1976), in which the limiting permeabilities of other co- or counterions are different from those in red cell membranes. Knauf et aZ. (1975) reported that low concentrations of external K (half-maximum about 0.7 mM) were required to stimulate the net K loss induced by Ca entering human red cell ghosts. The stimulatory effect of external K was also observed when the rate constant for 42K tracer exchange was measured in the absence of K gradients (Knauf et al., 1975). Since below 5-10 mM the external K concentration has no effect on the uptake of Ca in intact cells (Lew, 1974), differences in Ca content cannot explain these results. In Amphiuma red cells, Lassen et al. (1976) reported that the hyperpolarizing effect of 15 mM Ca is also observed in K-free media (KO> 0.1 p M ) . If the results in Amphiuma and human red cells are to be compatible, the effect of low concentrations of external K would be to increase the C1 permeability, thus allowing the net outflow of K. However, the K exchange rate is stimulated by K even under equilibrium conditions. This strongly supports a direct effect of external K on or near the transport mechanism in the human red cell. The absence of such an interaction in the Amphiuma red cell suggests that the external K site is perhaps not a constant component of the Ca-sensitive K channel. In resealed human red cell ghosts, whether during net Ca uptake (Knauf et al., 1975) or with incorporated internal Ca buffers (Simons, 1976b), internal Na inhibits the Ca-induced K flux in the presence or absence of an outward K gradient, but only at low external K concentrations. The curve showing the efflux rate constant as a function of the K concentration becomes sigmoid in Na-containing ghosts. This indicates complex competitive interactions between Na and K at more than one internal site (Simons, 1976b). An interesting observation made by Simons (1976b) is that under equilibrium exchange conditions the K effEux expressed as a function of the K concentration did not saturate up to 200 mM K. We do not know yet whether these effects of internal Na and external K on the K flux are the result of interactions affecting the Cabinding site or the K-translocating path.

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D. The Nature of the Ca-Sensitive K Channel

The molecular apparatus responsible for the effect of Ca on the K permeability in a variety of biological membranes (see Table I) has not yet been identified. The finding by Jenkins and Lew (1973) that this permeability mechanism is functionally absent from red cells of certain species suggested that a comparative exploration of the protein composition of their membranes might reveal significant differences. A visual inspection of disk gel electrophoresis patterns of hemoglobin-free red cell membranes (J. C. Ellory and B. Hinton, personal communication) revealed distinct species patterns but no correlation with Ca-mediated effects on K permeability. Two more familiar transport systems have also been considered as possible candidates or mediators of the Ca-induced effects: the TEAsensitive K channel found in the excitable membranes of nerve and muscle cells (see review by Meech, 1976; Szasz and Gardos, 1974) and the Na pump (Blum and Hoffman, 1971; Lew, 1974). The distribution, Ca sensitivity, and apparent lack of time and voltage dependence of the Ca-sensitive channel argue against identity with the K channel of nerve membranes, although the molecular structures responsible for K translocation and selectivity may well be similar. The Ca-sensitive K channel reacts with many of the cofactors of the Na pump. It was, however, mainly the action of ouabain and other less specific inhibitors of the Na pump (furosemide, oligomycin) that led Blum and Hoffman (1970, 1971) to suggest that the mechanism which mediates K translocation is a Ca-modified form of the Na pump. Although there is no conclusive evidence against this hypothesis, the facts used in its support admit alternative interpretations which, on the balance of the available evidence, seem more plausible. The cases for and against the identity between the Na pump and the Casensitive K channel have been argued before (Kregenow and Hoffman, 1972; Hoffman and Knauf, 1973; Lew, 1974; Lew and Beauge, 1978) and deserve only brief consideration here. The suggestion of Blum and Hoffman (1971) was based on inhibitor studies and on the assumption that the &K counterflow discussed above was the result of carrier-mediated coupling, the carrier complex being in a charged form (Hoffman and Knauf, 1973). Conversion of the Na pump machinery so that it operated as a K channel was, they supposed, triggered by an interaction with internal Ca perhaps aided by additional alterations in cell metabolism. The identity of the Na pump and the Ca-sensitive K channel also provides the simplest explanation for the parallel variations in Ca sensitivity .of pump inhibition and

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V I R G I L 1 0 L. L E W AND HUGO G. FERREIRA

channel activation (Lew and Ferreira, 1976; Brown and Lew, unpublished observations). The alternative interpretations of these effects have all been considered above. Two additional observations are relevant to this discussion: (1) A specific antipump antibody which inhibits all pump-mediated transport and ATPase activities (Glynn et al., 1974) has no effect on the Ca-induced K permeability system (Karlish, Glynn, and Lew, unpublished experiment); it may be important to observe, however, that the K-dependent phosphatase activity associated with the Na pump (Bader et al., 1968) is actually slightly stimulated by a similar antibody (Askari, 1974). (2) The Ca-induced K permeability mechanism is absent or inactive in red cells from several species which contain high-K or low-K-type Na pumps (cow, sheep, and goat) or no Na pumps at all (dog) (Jenkins and Lew, 1973). Cells lacking the Na pump but having the Ca-dependent K channel, however, have yet to be found.

VII.

CONCLUSIONS

This article has been concerned with a Ca-sensitive K channel, the physiological importance of which is only now beginning to emerge. From the initially isolated and mostly casual observations to the present flood of experimental data concerning this mechanism (see Table I) we have learned a great deal, but much of the information available is confusing, Perhaps this is because, at least in red cells, the Ca-induced K flux is so easy to measure and so richly responsive to all sorts of treatments that it has stimulated more extensive than analytical research. As an experimental model, the red cell has clear advantages in that CaF+ can be assessed and controlled better than in any other cell, and in that even minute selective changes in K permeability can be unambiguously determined by relatively simple methods. Red cells provide therefore an ideal system in which to investigate the interaction between Cat+ and the K channel, the properties of the K flux, and the site and mechanism of action of various pharmacological agents. Excitable cells, however, remain the model of choice for the study of the rates of activation and inactivation. Great experimental ingenuity will be required to be able to discriminate between intrinsic kinetic rates, changes in Ca sensitivity, and Ca:+-induced variations in K permeability. Future research in this field will have to cover two main areas: (1) the physiological functions of the Ca-dependent K channel and (2) the dynamic and structural properties of this molecular mechanism.

CA-ACTIVATED K CHANNEL I N BIOLOGICAL MEMBRANES

27 1

We know that this channel exists in the membrane of central nervous system (CNS) neurones, and we also know how it operates under certain experimental conditions, but we are still largely ignorant of the way in which it functions under physiological conditions. Similar considerations apply to other cells, such as striated muscle cells (Fink and Liittgau, 1976), for instance, where it remains to be seen whether the increased K conductance induced by metabolic exhaustion also occurs during “physiological” fatigue. The physiological potential of this mechanism in the control of excitability, particularly in the CNS, is immense. If the resting membrane potential is regulated by the operation of this channel, the factors that control the Ca sensitivity or the intracellular level of ionized Ca become immediate candidates for the mediation of many integrated responses of the CNS. The channel is present in neuronal bodies but seems to be absent from the axonal membrane, since injection of Ca into giant squid axons failed to elicit any clear changes in K conductance (Begenisch and Lynch, 1974). This tends to support the idea of a role for the channel in the generation of the nervous signal by the cell body. Future research in this field looks arduous but promising. Investigation of the molecular nature of the Ca-sensitive K channel is still at a very early stage. A few attempts (Armando-Hardy et al., 1975; Simons, 1976c) have been made to search for specific highaffinity inhibitors with which to “label” the channels, measure their surface density, and attempt isolation and, eventually, functional reconstitution, but the results so far are not very promising. ACKNOWLEDGMENTS We thank the Wellcome Trust and the Medical Research Council of Great Britain for funds, and I. M. Glynn, A. M. Brown, and A. J. Kaumann for helpful discussions and for reading the manuscript for this article. REFERENCES Allan, D., and Michell, R. H. (1975). Accumulation of 1,2-diacylglycerol in the plasma membrane may lead to echinocyte transformation of erythrocytes. Nature (London) 258,348-349. Armando-Hardy, M., Ellory, J. C., Ferreira, H. G., Fleminger, S., and Lew, V. L. (1975). Inhibition of the calcium-induced increase in the potassium permeability of human red blood cells by quii1ine.J. Physiol. (London) 250,32P-33P. Askari, A. (1974). The effects of antibodies to Na+, Kf-ATPase on the reactions catalyzed by the enzyme. Ann. N.Y. Acad. Sci. 242,372-388. Askari, A., and Rao, S . N. (1968). Regulation of AMP deaminase by 2,3diphosphoglyceric acid: A possible mechanism for the control of adenine nucleotide metabolism in human erythrocytes. Biochitn. B i o p h y s . Acta 151, 198-203.

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Bader, H., Post, R. L., and Bond, G. H. (1968). Comparison of sources of a phosphorylated intermediate in transport ATPase. Biochim. Biophys. Acta 150,41-46. Baker, P. F. (1972). Transport and metabolism of calcium ions in nerve. Prog. Biophys. Mol. Biol. 24, 177-223. Barrett, E. F., and Barrett, J. N. (1976). Separation of two voltage-sensitive potassium currents, and demonstration of a tetrodotoxin resistant calcium current in frog motoneurones. J . Physiol. (London)255, 737-774. Begenisch, T., and Lynch, C. (1974). Effects of internal divalent cations on voltageclamped squid ax0ns.J. Gen. Physiol. 63,675-689. Blum, R. M., and Hoffman, J. F. (1970). Carrier mediation of Ca-induced K transport and its inhibition in red blood cells. Fed. Proc., Fed. Am. S O C . E x p . Biol. 29, 663a. Blum, R. M., and Hoffman, J. F. (1971). The membrane locus of Ca-stimulated K transport in energy depleted human red blood cells. ]. Membr. Biol. 6,315-328. Blum, R. M., and Hoffman, J. F. (1972). Ca-induced K transport in human red cells: Location of the Ca-sensitive site to the inside of the membrane. Biochem. Biophys. Res. Commun. 46, 1146-1151. Bulbring, E., and Tomita, T. (1977a). The K-action of catecholamines on the guinea-pig taenia coli in K-free and Na-free solution and in the presence of ouabain. Proc. R. SOC., Ser. B 197,255-269. Biilbring, E., and Tomita, T. (1977b). Calcium requirement for the a-action of catecholamines on guinea-pig taenia coli. Proc. R. SOC., Ser. B 197,271-284. Clusin, W., Spray, D. C., and Bennett, M. V. L. (1975). Activation of a voltageinsensitive conductance by inward calcium current. Nature (London) 256, 425-427. Danon, D., and Marikovsky, Y. (1964). Determination of density distribution of red cell populations. J . Lab. Clin. Med. 64,668-674. Duhm, J. (1973). 2,3-Diphosphoglycerate metabolism of erythrocytes and oxygen transport function of blood. In “Erythrocytes, Thrombocytes, Leucocytes” (E. Gerlach, K. Moser, E. Deutsch, and W. Wilmanns, eds.), pp. 149-157. Thieme, Stuttgart. Ekman, A., Manninen, V., and Salminen, S. (1969). Ion movements in red cells treated with propranolol. Acta Physiol. Scand. 75,333-344. Ellory, J. C., and Lew, V. L. (1970). Sodium pump reversal in the erythrocytes ofvarious species. J. Physiol. (London)206,36P-37P. Ferreira, H. G., and Lew, V. L. (1975). Ca transport and Ca pump reversal in human red blood cells. J. Physiol. (London)252,86P-87P. Ferreira, H. G., and Lew, V. L. (1976). Use of ionophore A23187 to measure cytoplasmic Ca buffering and activation of the Ca pump by internal Ca. Nature (London) 259, 47-49. Ferreira, H. G., and Lew, V. L. (1977). Passive Ca transport and cytoplasmic Ca buffering in intact red cells. In “Membrane, Transport in Red Cells” (J. C. Ellory and V. L. Lew, eds.), Academic Press, New York. pp. 53-91. Fink, R., and Luttgau, H. C. (1976). An evaluation of the membrane constants and the potassium conductance in metabolically exhausted muscle fibres. J. Physiol. (London) 263,215-238. Gardos, G. (1956). The permeability of human erythrocytes to potassium. Acta Physiol. 10,Acad. Sci. Hung. 185-189. (Budapest). Gardos, G. (1958a). Effect of the ethylendiaminetetraacetate on the permeability of human erythrocytes. Acta Physiol. Acad. Sci. Hung. 14, 1-5. Gardos, G. (195%). The function of calcium in the potassium permeability of human erythrocytes. Biochim. Biophys. Acta 30,653-654.

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Gardos, G. (1959).The role of calcium in the potassium permeability of human erythrocytes. Acta Physiol. Acad. Sci. Hung. 15, 121-125. Gardos, G., Szasz, I., and Sarkadi, B. (1975). Mechanism of Ca-dependent K-transport in human red cells. FEBS (Fed. Eur. Biochem. Soc.) Proc. Meet. 35, 167-180. Gardos, G., Lassen, U. V., and Pape, L. (1976).Effect of antihistamines and chlorpromazine on the calcium-induced hyperpolarization of the Amphiuma red cell membrane. Biochim. Biophys. Acta 448,599-606. Gardos, G., Szasz, I., and Sarkadi, B. (1978). Effect of intracellular calcium on the cation transport processes in human red cells. Acta Biol. Med. Germ. (in press). Garrahan, P. J., and Glynn, I. M. (1967). The incorporation of inorganic phosphate into adenosine triphosphate by reversal of the sodium pump.]. Physiol. (London) 192, 237-256. Garrahan, P. J., and Rega, A. F. (1967). Cation loading of red blood cells. J. Physiol. (London) 193,459-466. Glynn, I. M., and Lew, V. L. (1970). Synthesis of adenosine triphosphate at the expense of downhill cation movements in intact human red cells./. Physiol. (London) 207, 393-402. Glynn, I. M., and Warner, A. E. (1972). Nature of the calcium dependent potassium leak induced by ( +)-propranolol, and its possible relevance to the drug’s antiarrhythmic effect. Br. J. Pharmacol. 44,271-278. Glynn, I. M., Karlish, S. J. D., Cavieres, J. D., Ellory, J. C., Lew, V. L., and Jbrgensen, P. L. (1974). The effects of an antiserum to Na+, K+-ATPase on the ion transporting and hydrolytic activities of the enzyme. Ann. N.Y. Acad. Sci. 242,357-371. Godfraind, J. M., Kmjevic, K., and Pumain, R. (1970).Unexpected features of the action of dinitrophenol on cortical neurones. Nature (London) 228,562-564. Grigarzik, H., and Passow, H. (1958). Versuche zum Mechanismus der Bleiwirkung auf die Kalium-permeabilitat roter Blutkorperchen. P’uegers Arch. Gesamte Physiol. Menschen Tiere 267,73-92. Hanani, M., and Shaw, C. (1977).A potassium contribution to the response of the barnacle photoreceptor. J . Physiol. (London) 270, 151-163. Harrison, D. G . , and Long, C. (1968). The calcium content of human erythr0cytes.J. Physiol. (London) 199,367-381. Henriques, V., and 0rskov, S. L. (1936). Untersuchungen iiber die Schwankungen des Kationengehalts der roten Blutkorperchen. 11. Anderung des Kaliumgehaltz der Blutkorperchen bei Bleivergiftung. Skand. Arch. Physiol. 74, 78-85. Hladky, S. B., and Rink, T. J. (1976). Potential difference and the distribution of ions across the human red blood cell membrane: A study ofthe mechanism by which the fluorescent cation, diS-C,-(5) reports membrane potentia1.J. Physiol. (London) 263, 287-319. Hoffman, J . F. (1966). The red cell membrane and the transport of sodium and potassium. Am. J. Med. 41,666-680. Hoffman, J. F., and Knauf, P. A. (1973).The mechanism of the increased K transport induced by Ca in human red blood cells. In “Erythrocytes, Thrombocytes, Leucocytes” (E. Gerlach, K. Moser, E. Deutsch, and W. Wilmanns, eds.), pp. 66-70. Thieme, Stuttgart. Hunter, M. J. (1971). A quantitative estimate of the non-exchange-restricted chloride permeability of the human red cel1.J. Physiol. (London) 218,49P-50P. Inoue, M., Utsumi, K., and Seno, S. (1975). Effect on concanavalin A and its derivative on the potassium compartmentation of Ehrlich ascites tumour cells. Nature (London) 255,556-557.

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Proton-Dependent Solute Transport in Microorganisms A . A . EDDY Department of Biochemistry University of Munchester Institute of Science and Technology Manchester. England

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Principles of Gradient Coupling . . . . . . . . . . . . . . . . . . A . Proton Cycling at Equilibrium . . . . . . . . . . . . . . . . . B . Charge Neutralization during Net Substrate Absorption with Protons C . Leak Pathways . . . . . . . . . . . . . . . . . . . . . . . . . D . Carrier Models . . . . . . . . . . . . . . . . . . . . . . . . . E . Sodium Ion Cycling Dependent on Proton Circulation . . . . . . F. Potassium Ion Antiport . . . . . . . . . . . . . . . . . . . . . 111. Cation Transport . . . . . . . . . . . . . . . . . . . . . . . . . . A . Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Neurospora crassa . . . . . . . . . . . . . . . . . . . . . . . . C . Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . D . Streptococcus faecalis . . . . . . . . . . . . . . . . . . . . . E . Halobacterium halobium . . . . . . . . . . . . . . . . . . . . IV . Carbohydrate Transport . . . . . . . . . . . . . . . . . . . . . . . A . Neurospora crassa . . . . . . . . . . . . . . . . . . . . . . . B . Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Chlorella vulgaris . . . . . . . . . . . . . . . . . . . . . . . D . Escherichia coli and Other Bacteria . . . . . . . . . . . . . . . V . Amino Acid Absorption in Fungi . . . . . . . . . . . . . . . . . . . A . Proton-Dependent Concentration of Glycine by Energy-Depleted . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yeast B . The Role of K+ Ions . . . . . . . . . . . . . . . . . . . . . . C . Multiple Systems for Proton-Dependent Amino Acid Absorption . D . The Magnitude of the Proton Gradient during Energy Metabolism VI . Amino Acid Transport in Bacteria . . . . . . . . . . . . . . . . . . A . Binding Protein Systems . . . . . . . . . . . . . . . . . . . . B . Amino Acid Transport Linked to the Energized Membrane State . C . Streptococcus faecalis .......... . . . . . . . . . . . D . Staphylococcus aureus . . . . . . . . . . . . . . . . . . . . . VII . Sodium-Dependent Systems . . . . . . . . . . . . . . . . . . . . . A . The Melibiose Permease (TMG 11) of Salmonella typhimuriuni . . B. Amino Acid Uptake in a Marine Pseudomonad . . . . . . . . . .

280 282 282 284 285 286 288 288 290 290 296 298 302 303 304 304 306 310 311 320 322 322 325 326 327 327 328 332 333 335 335 336 279

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Light-Driven Leucine and Glutamate Transport in Holobacterium

halobium . . . . . D. Mycobacterium phlei

VIII.

IX.

. . . . . . . . . . . . . . . . .. Miscellaneous Compounds . . . . . . . . A. Succinate . . . . . . . . . . . . .. B. Citrate . . . . . . . . . . . . . . . . C. Lactate . . . . . . . . . . . . . . . D . Gluconate . . . . . . . . . . . . . . E. Glucose 6-Phosphate . . . . . . . . . F. Sulfate . . . . . . . . . . . . . . . . G. Phosphate . . . . . . . . . . . . . .

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. . General Conclusions . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . 1.

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340 341 343 346

INTRODUCTION

It was Mitchell (1963) who first suggested that the accumulation of nutrients, such as lactose or specific amino acids, by bacteria might be energized b y coupling the influx of the compound in question, by means of an appropriate carrier system, to the spontaneous influx of protons across the plasmalemma. The resultant influx of positive charge would be neutralized, elsewhere in the plane of the membrane, by the ejection of protons from the cell interior back into the extracellular solution. The latter process would involve an exergonic chemical reaction, the carrier-mediated solute influx itself being a purely physical process, only indirectly dependent on ATP or similar compounds, at least in the simplest version of the scheme. Mitchell (1963) recognized the fundamental similarity between the above proposal and the now well established (Eddy, 1977) notion of sodium and potassium ion cycling that Christensen (Christensen and Riggs, 1952; Riggs et al., 1958) and Crane et al. (1961) had put forward to explain the ability of various vertebrate tissues to concentrate amino acids or carbohydrates. As Riggs et al. (1958) pointed. out, the proposed coupling between the flows of the primary substrate and the ionic cosubstrate (H+,Na+, or K+) represents an extension of the principle of counterflow observed in facilitated diffusion systems, whereby a concentration gradient of one solute across a cell membrane is used to form a gradient of a second solute interacting with the same carrier system. These hypotheses focused attention, on the one hand, on the possible roles of ionic gradients as the immediate source of energy driving solute accumulation and, on the other hand, on the processes whereby both the prevailing electric potential difference across the plasmalemma and the ionic gradients themselves are maintained. The

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

28 1

K+

FIG. 1. Model of a proton symport coupled to a proton pump driven by the hydrolysis of ATP. The solute S is absorbed with n equivalents of protons. The proton pump ejects rn equivalents of protons during the hydrolysis of 1 g-mole of ATP. The dashed line represents a channel through which potassium ions might enter or leave the cell under the influence of the electric field acting across the membrane boundary.

main purpose of this article is to review some of the more important evidence currently available on several microbial systems in which ionic gradients appear to be implicated in the transport of amino acids, carbohydrates, and other nutrients such as phosphate, sulfate, and succinate. The availability of an explicit hypothesis about the mechanism of energy coupling in microbial transport stimulated attempts to establish both the molecular and genetic bases of the processes concerned. This has led to an appreciation of the multiplicity of genetically distinct systems that may participate in the uptake of a given compound by any one strain of organism grown under a particular set of physiological conditions (Slayman, 1973). A proper understanding of the functions of these different systems and of the factors governing their use has not yet been achieved.

1. Meanwhile, compelling evidence in favor of the proton cycling mechanism has been obtained, but only as one of several mechanisms that microorganisms may utilize to concentrate nutrients (Harold, 1972, 1977; Mitchell, 1973; Hamilton, 1977). 2. A second mechanism, seemingly acting in parallel with a proton pump, appears to involve Na+ ions as cosubstrate. 3. A third type of mechanism, involving the so-called binding proteins of gram-negative bacteria, may be driven by ATP or a similar compound. 4. The fourth category, to which no further reference is made in this article, involves the chemical conversion of the substrate as it penetrates the plasmalemma. The sugar phosphotransferase systems discovered by Roseman are examples of this kind of vectorial metabolism (Roseman, 1977).

282

A. A. EDDY

While future work may demonstrate novel aspects of these four mechanisms that are common to some or all of them, it seems more useful at present to emphasize the differences between them, if only as an aid in the search for further categories. In this connection a brief exposition of the principles underlying gradient coupling is followed by a discussion of the characteristic properties of microbial systems belonging to each of the first three categories.

II. PRINCIPLES OF GRADIENT COUPLING

A. Proton Cycling at Equilibrium

1. SUBSTRATE

WITH N O

NET

CHARGE

In the simplest model based on a cosubstrate function for protons, 1 equivalent of the uncharged solute S enters a membrane-bounded vesicle along with n equivalents of protons (Fig. 1).These are then expelled through the proton pump. If the latter is actuated by ATP, the whole system will work as an ATPase circulating protons in the indicated manner. In the absence both of leaks, through which protons, other anions and cations, or S itself may separately traverse the membrane, and of side reactions involving the chemical reactants, the following equilibria are set up, where the subscript indicates the location of each ligand:

+ nHZ,, * S,, + nHZ ATP + mHL = ADP + Pi + n H A t So,,

(1) (2)

The supposition is that m equivalents of protons are expelled per equivalent of ATP hydrolyzed. Clearly, raising the ratio n / m increases the magnitude of the ratio [Slin/[S]o,t reached at equilibrium. Calculations based on estimates of the free energy of hydrolysis of ATP, under conditions resembling those in the bacterial or fungal cytosol, show that a concentration ratio for S of at least lo8can be achieved in this way when n / m is unity. Reference to Eq. (1)shows that the chemical potentials of the participating species are related b y pi"n

-

poit =

n(

-

pYJ

(3)

The factor (pt& - p 3 = A p H + ,expressed in millivolt equivalents,

283

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

is sometimes referred to as the proton motive force in the current literature (Harold, 1972; Hamilton, 1977). It is convenient to assume activity coefficients of unity for S and to express Eq. (3) as the approximation

~Sli,/[Sl,,l = 10"IPHexp(- nVF/RT)

(4)

Here V is the membrane potential, F , R, and T have their usual significance, and ApH = pHi, - pHout. In general, since the value of m in Eq. (2) is unknown, one could suppose that a p H gradient of much more than 2 units and a negative membrane potential exceeding about 0.2 V are unlikely to be established across the plasmalemma of fungi and bacteria. The accumulation ratio [Sli,/[S],,l is therefore unlikely to exceed about lo5 when n = 1, and to exceed 10'O when n = 2. Experience has shown that the accumulation ratios for various carbohydrates and amino acids lie in the range 101-105in bacteria and fungi, so that the above model appears feasible on energetic grounds, provided some explanation is found for the diversity of ratios encountered in practice. The central importance of the parameter n in the energetics of the coupling is at once apparent. These very rough estimates justify moreover the common assumption that n is expected to be unity if the proton cycling model applies (Fig. 2). However, experimental evidence (see Section V,C) has shown that this assumption is not always correct.

2. CHARGEDSUBSTRATES Equation ( 3 ) also applies when the ligand S is charged (S+, S-, or S z - ) , the appropriate forms of Eq. (4) then being

[S+],,/[S+],,,

=

lonAPH exp[- ( n + 1)VF/RT]

[S-I~,/[S-l,,, = lonAPH exp[- ( n

(5)

1)VF/RTl

(6)

[S2-l~,/[S2-],,, = 10"'pH exp[ - (n - 2)VF/RTl

(7)

-

The overall distribution of an anionic or cationic substrate may also be affected by its ionization at the p H values prevailing in each of the two phases, a circumstance that complicates the application of these equations. Perusal of Eq. (6)shows that, when n > 1, the absorption of anion into the intracellular compartment is assisted by a large negative membrane potential and is not merely dependent on ApH being large as in the conventional model of electroneutral anion absorption (Fig. 2).

A. A . EDDY

cationic S

basic model

S+

*a

-4

neutral S

,"H"+

H + and Na+

H + and M I 2 +

::I%: 7t : :2

x

Na:

=

FIG.2. Different ways of coupling the flows of cosubstrate ions to the influx of solute molecules, with corresponding variations in the energy input. The basic model follows the proposals of Mitchell (1973). The other possibilities are derived from observations made with yeast (Seaston et al., 1973; Cockbum et ul., 1975) and various other systems discussed in the text. B. Charge Neutralization during Net Substrate Absorption with Protons

The scheme envisaged in Fig. 1 is simplified in Fig. 3 to show how the net uptake of S might require the simultaneous ejection of H+ through the proton pump. Thus, in the absence of ATP, S would fail to penetrate the vesicle, However, an alternative mode of charge neutralization may be available if the vesicle were permeable to K+ ions, when S would be absorbed with H+ and an equivalent number of K+ ions expelled. Similar schemes apply when the ligand S carries a net charge. Some simple possibilities are outlined in Fig. 3. For instance, uptake of S+ might then couple with the proton pump to expel 1 equivalent of protons. Another interesting situation concerns the behavior of S- when n = 2. Only one of the protons would be ejected through the pump, unless another ion such as K+ could be absorbed

285

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

MI:

["+I K t out.

n H * I"]

[n-l

Kt out,

n

in]

n+

- -

[n K + n H+

out. In]

IKt

H+ fosl. K+slow net:

(n +I)Ht

[in+aut]

nH+

[I K

In]

-nH+ [no n*]

FIG. 3. Charge neutralization during absorption of the solute. The primary symport may he neutralized (a) by proton cycling through the proton pump during energy metabolism, (I)) by the efflux of potassium when energy metabolism has stopped, or (c) by a combination of these processes. It seems possible that proton ejection is potentially faster than either the influx or efflux of K+, or the primary process in which the solute is absorbed. The net movements of ions are those that would be recorded in conventional assays (see, e.g., Figs. 19 and 20).

and the second proton would be expelled. It is seen later that the intrinsic carrier stoichiometry is only indirectly related to the net flow of ions that occurs when the proton pump is functioning. C. leak Pathways

The system may permit the passage of S without the proton cosubstrate through a second carrier system (Fig. 4).This type of behavior is envisaged in the classical pump leak model of active transport, one obvious function of which is to restrict solute accumulation to osmotically tolerable levels (Johnstone and Scholefield, 1965). I n contrast to Eq. (4), the ratio [S],,/[S],,, at a given value of the proton gradient A p H + is not in these circumstances independent of the magnitude of [SI,,, (Morville et al., 1973). At a given value of [S] the precise degree of coupling to the proton gradient depends on the relative rates of passage of S through the two competing pathways. Analogous arguments apply when the passage of H+ without S occurs through a leak pathway. The arrangement illustrated in Fig. 4 requires that ATP be consumed continuously in order. to maintain [S]in/[S]out at a given

286

A. A. EDDY

initial

steady state

sn so +sH:

- -Ht

H+

s-

H+

(I-dos

(I- d)H+

FIG.4. A leak pathway for a substrate operating in parallel with its proton symport. The initial state represents the uptake of S. I n the steady state S is shown as penetrating largely through the proton symport with 1 equivalent of protons. A substantial fraction (1 - a)of the efflux of S occurs through the leak pathway, the remainder (a)occurring through the proton symport operating in reverse. Note that a is a measure of the flux, not of the stoichiometry (a). Under these conditions a continuous ejection of protons through the pump is required to keep the cellular pH constant.

value. Gradient coupling has accordingly been treated by the methods of irreversible thermodynamics (Lagarde, 1976). D. Carrier Models

The stoichiometry n of the coupling, in the carrier, between the flows of the substrate S and the cosubstrate protons is reflected in the way the rate of uptake of S varies with the pH and with [SlOut.This complicated problem has been considered by several workers, but a full treatment, taking into account the role of the membrane potential, is outside the scope of this article (Schultz and Curran, 1970; Heinz et al., 1972; Jacquez, 1972; Geck and Heinz, 1976). The carrier system may function in different modes (Fig. 5a), so that n is either 0 or 1, depending on the concentrations of S and of H+ (Schultz and Curran, 1970). However, it is important to note that, provided ApH’ and n are constant, the ratio [S]i,/[S]o,t is independent of the magnitude of [Slout, even when n is nonintegral (Eddy, 1968). In general the carrier may bear either no net charge, or a small net positive or negative charge which would either hinder or assist its translocation in an electric field. The term “translocation” is used here in the broad sense of a conformational change, or a movement by rotational diffusion, that results in the carrier alternating between its inward- and outward-facing aspects (Baker and Widdas, 1973; Edwards, 1973). Rottenberg (1976) has assumed explicitly that the complex formed by the protonated carrier and the substrate S is neutral. However, this need not be so. Seaston et al. (1976) postulated that a positively

287

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

-a OUT

-b IN -

I.

eur

H+I

7

sjT

I

zi“

EH

LK

S

T--%

E - E

FIG.5. Steps in the translocation of the substrate and cosubstrate. (a) (I) Translocation involves the formation of the ternary complex with the carrier E and the return of the latter to the outer membrane face. Movement of the binary complexes EH and ES would be forbidden if tight coupling were to be achieved between the flows of S and H+. (11) A special mode of uncoupling postulated for sugar transport in Chlorella (Section IV,C) in which S causes the translocation of H+ without itself being translocated. (I)) Schemes illustrating the possible role of the formal charge borne by the various carrier complexes. Imposition of an electric field across the membrane would change the relative magnitudes of the rate constants of the translocation steps. (I) The neutral species E is presumed to traverse rapidly, and the species ESH carrying a positive charge might only move inward rapidly when the membrane potential is large and negative. The efflux of S, either in the presence or absence of extracellular S, may be very slow in these circumstances unless the membrane potential becomes large and positive (Seaston et al., 1976).(11) In this case the electric field accelerates the movement of the unloaded camer, and exchange between the cellular and extracellular compartments will be fast even when the membrane potential is small. However, net efflux might be slow. (111)In this case the translocation of both the loadedand unloaded carrier may be assisted by an electric field. This behavior requires that two protons serve as cosubstrate. This type of system resembles that in (I) in several respects. All these models lead to specific predictions about the way in which K, and V,,, vary with the electric field and with the ligand concentrations on either side of the membrane (see, e.g., Seaston et al., 1976).

288

A. A. EDDY

charged complex was involved in the absorption of glycine by a certain yeast, in order to explain why the influx of the amino acid occurred very much faster than its efflux. Figure 5b illustrates the issues involved. These concern both the way in which (1) the pH on each side of the plasmalemma and (2) the magnitude of the electric field across it affect the kinetic parameters K, and V,,, expressing the relative rates of influx and efflux of both S and H+. E. Sodium Ion Cycling Dependent on Proton Circulation

A circulation of Na+ ions appears to b e maintained in certain vertebrate systems by the coupling of a sodium pump (Na+,K+-ATPase)to a Na+ symport (Eddy, 1977).Various microorganisms expel Na+ ions, in a process linked to energy metabolism, either with an accompanying anion or in exchange for another cation such as H+, K+, or M$+. There are grounds for thinking that these processes depend on a functioning proton pump and that the expulsion of Na+ ions is not itself directly linked to the hydrolysis of ATP. Some possible mechanisms are illustrated in Fig. 6, where the accumulation of the neutral substrate S is maintained by the circulation of Na+. Schemes similar to those outlined in Fig. 2 would apply if S were an anion or a cation. Likewise Eq. (3) would take the form pf,, - piut= n( poNu"; - pp:+ ), from which the appropriate forms of Eqs. 5-7 are readily derived. The function of Na+, in transducing the flow of H+ set up by the proton pump, into a flow of S across the plasmalemma can be compared with the role phosphate ions play in the accumulation of certain dicarboxylate anions in mammalian mitochondria (Chappell, 1968). F. Potassium Ion Antiport

This type of mechanism was first suggested by Riggs et al. (1958) and would function in parallel with a mechanism for reabsorbing K+. The sodium pump might serve this purpose in vertebrate systems. The ejection of K+ during absorption of S (Fig. 7) would tend to make the membrane potential more negative, whereas the symport with protons (Fig. 2) wouId make it more positive. Another possibility to be considered is that of solute absorption linked, in an electrically neutral process, to the absorption of H + and the simultaneous efflux of K+. In contrast to the model shown in Fig. 2 a compulsory turnover of both K+ and H+ occurs when the proton pump is functioning. Crane (1977) has repeated his earlier suggestion (Crane, 1965)that

289

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORl

-b

a -

(n64 : Notin. S In I

m.

Ht

(rial: S In. H + h )

___)

K+

( n r l . Na+in. K+oull

-

H+

(1181. Na+out, H + in)

FIG.6. Models illustrating coupling between the flows of sodium ions, protons, and the solute S. ( I ) (a) The primary process ofproton absorption with S becomes converted to a net influx of Na+ as a result of the operation of a Na+ symport-H+ antiport. The means of neutralizing the net inflow of charge is not represented. (b)The corresponding situation in which the primary process of Na+ uptake leads to an absorption of protons (West and Mitchell, 1972).(11) Sodium ejection dependent on a proton pump driven by metabolism. (a) Coupling between K+ absorption and Na+ ejection, the latter by electroneutral exchange for protons. The dashed line represents a pathway for absorbing Na+; yeasts, for instance, appear to do this when K+ is in short supply. (b) Sodium ion cycling leading to the absorption of the solute S coupled to the functioning ofthe proton pump. (111) (a) A net uptake of Na+ may occur in the presence of a proton conductor that accelerated the efflux of K+. (b) An electrogenic expulsion of Na+ that couples with the electrogenic proton pump (Lanyi and MacDonald, 1976).

I

II

FIG. 7 . Solute absorption linked to the simultaneous ejection of potassium ions either with (I) or without (11) the absorption of protons.

290

A. A. EDDY

coupling to the energy source represented by the electrochemical gradient of K+ across the plasmalemma might occur without the actual passage of K+ down this gradient in the manner suggested by Riggs et a2. (1958). This suggestion appears to contravene the fundamental principle of thermodynamics that removal of energy from a given source necessarily depletes the source. It is nevertheless quite possible that the presence simultaneously of a large concentration of K+ in the cytosol and a low extracellular concentration of K+ may increase the extent to which solute influx is coupled to the flow of the cosubstrate proper (H+ or Na+). Such an effect of K+, as a modulator of the carrier system, might be equivalent to raising the value of n, the stoichiometric ratio for the symport ion (Fig. 1). If this happened, the presence of a gradient of K+ might be required, but not its expenditure as an energy source. Thus the substrate would enhance the flow of Na+ or H+ rather than the flow of K+. It is only in this sense that Crane’s proposal (Crane, 1965, 1977) seems acceptable in thermodynamic terms, 111.

CATION TRANSPORT

A. Yeasts

Washed cell preparations of Saccharomyces cereuisiae, partially depleted of K+ ions by preliminary starvation, have long been known to absorb K+ ions in exchange for protons when provided with a substrate such as glucose or ethanol (Conway and O’Malley, 1946; Rothstein and Enns, 1946). The cellular pH meanwhile increases by about 0.5 units (Ryan and Ryan, 1972). The omission of K+ ions from the system results in a somewhat lower rate of proton ejection and an equivalent flow of anions, principally succinate and bicarbonate, out of the yeast (Conway and Brady, 1950). However, the anions C1- and S042- penetrate very slowly, the former probably being excluded from the yeast. These processes, which occur both in the presence of air and in its absence, result in the formation of both a substantial pH gradient across the plasmalemma and a large concentration gradient with respect to K+ ions. For instance, when pH,,, = 4.5, pHt, is probably 6.5 and [K+]i,/[K+l,,t is about 2 x lo3 (Conway and O’Malley, 1946; Rothstein, 1960a,b).The cellular content of K+ appears to be regulated by a mechanism that lowers the initially rapid influx of K+ until a balance between its influx and efflux is obtained.

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

291

Na+ ions compete with K+ ions for entry into the yeast in a manner that varies in a complicated way with the extracellular pH, the concentration of other alkali cations and, seemingly, with the intracellular pH (Rothstein, 1974; Ryan and Ryan, 1972). For instance, in the presence of low extracellular concentrations of K+ near pH 6.5, yeast cells concentrate Na+ relative to the extracellular phase. In other words, the cells appear to be equipped with an inwardly directed Na+ pump (Fig. 8a). However, yeast cells in which Na+ replaced a large fraction of the normal complement of K+ absorbed K+ ions from solution, losing roughly an equivalent amount of Na+ (Fig. 8b). This happened even with solutions containing a larger concentration of Na+ than the cytosol contained. Conway et al. (1954) attributed this process to the operation of an outwardly directed sodium pump. I n the absence of extracellular K+, Na+ was excreted with anions, or by exchange with H+ (Rothstein, 1974). The interpretation of all these effects necessitates measurements of the various ionic fluxes. Such measurements are complicated by the presence of a cell wall compartment that may delay the escape of K+ from the yeast sufficiently for it to be reabsorbed. Unfortunately, no agreement has yet been reached in the literature about the carrier basis of cation exchanges. A single carrier system may normally function so as to excrete H+ and Na+ in preference to K+ and absorb K+ in preference to Na+ or H+ (Foulkes, 1956; Rothstein, 1974). Alternatively, two carrier systems might serve, respectively, to excrete H+ in exchange for K+ and to excrete Na+ in exchange for extracellular K+ (Ryan and Ryan, 1972).I n the absence of extracellular K+, Na+ would be absorbed by the first system and excreted by the second (Conway et al., 1954).

RELATION

TO

CHEMIOSMOTIC CONCEPTS

The question arises whether the above findings can be reconciled with the concept of an electrogenic proton pump (Mitchell, 1963). This concept differs in several important ways from the view expressed much earlier by Conway and Downey (1950)that proton ejection from yeast is a redox process involving cytochromes. Indeed, the yeast plasmalemma probably lacks respiratory carriers. The functions of a proton pump can therefore more plausibly be attributed to the membrane-bound oligomycin-insensitive ATPase (Matile, 1970). However, in contrast with the bacteria discussed in Section 111, no selective inhibitor of proton ejection during glycolysis has been described.

A. A. EDDY

292 750

P +Y

' 500 b

+

z

0

120

60

180

minutes (0)

0.5

0.4

-:

c D O

\

v

0.3

I.

c 0 I.

U

I.

+

0

z

-

.)

0.2

0

E L

0.

c

I

I

I

I

I

8

16

24

32

40

minuter

(b)

FIG.8 . Reciprocal movements of sodium ions and potassium ions in the yeast Saccharomyces uvamm (strain NCYC 74). (a) Sodium uptake and postassium efflux at 30°C in the presence of 5% (w/v) glucose and 0.2 M sodium citrate (Eddy et al., 1970a). The ionic composition of the cells was determined by flame photometry and is expressed in units of yeast dry weight. The large amount of Na+ hound during the first few minutes

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

293

1. The net ejection of protons is nevertheless inhibited by various proton conductors (Conway and Brady, 1950; Rothstein, 1960a,b; Peiia, 1975). 2. At pH 4.5 and 30°C, in the absence of Na+, various proton conductors accelerated the efflux of K+ from yeast preparations depleted of ATP in the presence of antimycin and 2-deoxyglucose (Seaston et al., 1976). The maximum rate of efflux of K+ was similar to the rates of uptake of K+ and efflux of H+ observed in the presence of glucose and K+ (Fig. 9). The efflux of K+ was coupled to the absorption of H+ through the proton conductors (Fig. 10). When 1 mM azide was used, about 30% of the cellular K+ content was lost in 4 minutes from the preparations depleted of ATP. Further loss of K+ occurred much more slowly (Borst-Pauwels et al., 1971; Peiia, 1975), possibly because the cytosol became acidified, the cellular pH falling from about 6.5 to 5.7 (Seaston et al., 1976). A similar efflux of K+ occurring in the presence of high concentrations of uncoupling agents during energy metabolism was probably not simply due to the depletion of the cellular ATP content (Borst-Pauwels et al., 1971; Peiia, 1975). Eventually, acidification of the cellular interior (Ryan and Ryan, 1972), in the presence of glucose and low concentrations of dinitrophenol may cause protons to b e ejected through the proton pump and some of the K+ might be recaptured. Thus the efflux of dinitrophenolate ions appeared to be speeded up under these conditions, possibly because the membrane potential increased (Borst-Pauwels and Huygen, 1972). 3. Peiia (1975) showed that at p H 8 both proton efflux from the yeast cells and the uptake of Rb+ are simultaneously accelerated by the proton conductor tetrachlorosalicylanilide. Both ions apparently moved passively under the influence of their respective ionic gradients. 4. Studies with lipophilic anions and cations have also been inter-

may be associated with the cell wall. (b) Sodium efflux and potassium uptake at 30°C in the absence of glucose. The yeast was loaded with Na+ as illustrated in Fig. 8a. The cells were washed with water, resuspended in 5 mM 2-(N-morpholino)ethanesulfonic acid at pH 6, and shaken with access to air. No K+ was added to the control suspension (open circles), whereas 2 mM KCI was added to the test suspensions (triangles, closed circles). Potassium ion uptake (triangles) was assayed by determining the amount of K+ remaining in the extracellular solution at various times. No K+ leaked from the cells in the control preparation. The observations show that absorption of the K+ led to roughly an equivalent amount of Na+ leaving the cells. However, the coupling between the two processes was evidently an indirect one, as the efflux of Na+ was initially unaffected by the uptake of K+ (Eddy and Seaston, unpublished observations).

294

A. A. EDDY conditions:

GLUC

ANT

ANT

DOG

DOG

+

DNP

conditions:

.: c

E

CONTROL

ANT DOG

At1 DOG DNP

D

c

-10

o

FIG.9. Sodium ion, proton, and potassium ion exchanges across the yeast plasma membrane as a function of energy depletion. Proton ejection was energized by glucose, the potassium ions being absorbed from a 1 mM solution of KCI at pH 4.5 and 30°C. The yeast was alternatively depleted of ATP in the presence of antimycin and 2deoxyglucose, and the rate of efflux of K+ and the influx of H+ assayed. Similar assays were performed in the presence of 2,4-dinitrophenol as well. Efflux is represented as a negative rate, and absorption as a positive one. The mean value obtained in from three to nine assays of the net flow of the ion, observed during the first minute after exposure to the relevant conditions, is represented by the total height of the histogram, the corresponding shaded area donating the standard error of the mean. The net efflux of sodium was assayed under similar conditions, except that glucose was omitted from the control. The procedure used corresponded to that illustrated in Fig. 8 b for the preparation lacking added potassium ions. The mean rate of sodium efflux was determined in the interval up to 6 minutes (adapted from Seaston et al., 1976, and unpublished observations).

preted to mean that proton translocation is the primary process that causes such ions to traverse the plasmalemma (Riemersma and Alsbach, 1974). Taken together, the above evidence and certain other arguments presented by Pefia (1975)indicate that a major route for the influx and efflux of K+ ions involves their passive diffusion across the plasmalemma, possibly in association with a carrier. All the observations are consistent with, but do not prove, that the flow of K+ in these yeasts is electrically coupled to the electrogenic ejection of H+ from the yeasts.

295

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

I

10

IS

20

2s

Ht uptake (naquiv/mln permg)

FIG.10. The efflux of potassium ions from energy-depleted yeast as a function of the proton influx observed in the presence of various proton conductors. The observations were made at 30°C with cell preparations depleted of ATP in the presence of antimycin and deoxyglucose. Solid squares, control; solid circles, 50 or 100 p M azide; open squares, 50 or 100 pA4 dinitrophenol; triangle, 100 pM carbonyl cyanide m chlorophenylhydrazone; open circle, 100 p M tetrachlorosalicylanilide (adapted from Seaston et a!., 1976).

The availability of a method for preparing vesicles derived from the yeast plasmalemma may help to answer these questions (Fuhrmann et al., 1976; Christensen and Cirillo, 1972).

5. The efflux of Na+ was partially inhibited by cyanide but not by dinitrophenol (Conway et al., 1954). Other work shows that Na+ efflux is in fact partially stimulated b y dinitrophenol or azide, but not to the same extent as the efflux of K+ (Foulkes, 1956). Moreover, Na+ efflux continues under conditions in which energy metabolism is probably not involved (Fig. 9). These passive movements of Na+ out of the yeast occur at a rate comparable to the rate of uptake of Na+ in the presence of glucose. Three tentative conclusions can be drawn about the influx and efflux of Na+. (a) It seems possible that the K+ carrier referred to above also facilitates the uptake of Na+, but only when the membrane potential has reached sufficiently large negative values and the cellular p H (Ryan and Ryan, 1972) and extracellular pH (Rothstein, 1974) are optimal. This hypothesis may also account for the relatively small increase that dinitrophenol caused in the rate of efflux of Na+ from yeast depleted of ATP (Fig. 9). (b)The Na+ exit pump (Conway et al., 1954) and the Na+ excretion with anions may both depend on the coupled exchange of Na+ for H+ (Fig. 6), which might function in parallel with an electroneutral symport of anions with H+ (Rothstein, 1974). One specific prediction based on the above hypothesis is that the absorption of Na+ at p H 6.5, by yeast depleted of ATP, would be accelerated

2 96

A. A. EDDY

by proton conductors. The supposition is that the exit mechanism would then function in reverse and, in cooperation with the K+ carrier, effectively exchange extracellular Na+ for cellular K+ (Fig. 6). (c) The existence of relatively fast “passive” diffusion pathways for Na+ and K+ across the yeast plasmalemma, at least in certain circumstances, seems undeniable in light of the above evidence. This point has been neglected in the recent literature (see, e.g., Rothstein, 1972).

B. Neurospora crassa

The concept of an electrogenic proton pump located in the plasmalemma and driven by ATP has received important support from work with preparations of Neurospora in which the hyphal cells are large enough.to give stable recordings from micropipet electrodes (Slayman and Slayman, 1975). The resting membrane potential in the mature hyphae of these obligate aerobes is -0.18 to - 0.25 V, values that are much too large to represent diffusion potentials due to the prevailing gradients of ions such as Na+, H+, or K+. However, there are reasons for thinking that the membrane potentials of the small cells present in shaken cultures are in a lower range of values than the above limits (Slayman et al., 1973). Whereas partial depolarization of the hyphal plasmalemma occurred in the presence of extracellular K+ or Na+, the presence of cyanide dramatically lowered the membrane potential to about - 0.05 V within 20 seconds (Slayman, 1965a,b). Chemical assays on individual hyphae are not feasible, but they can b e made with smaller cells that are not suitable for microelectrode work. Comparison of the two systems indicated that the cellular ATP content and the membrane potential underwent a strictly parallel decrease with time in the presence of cyanide. I n other assays the net proton efflux was correlated with the membrane potential. These important observations are consistent with the presence of an ATPase system expelling protons across the fungal plasmalemma. Work with isolated plasma membranes of Neurospora has shown that they contain an ATPase activity which drives the accumulation of thiocyanate ions into the plasma membrane vesicles, This biochemical evidence supports the contention that the Neurospora plasma membrane ATPase is an electrogenic pump (Scarborough, 1976; Bowman and Slayman, 1977). In work with the hyphae, the putative proton conductor sodium azide lowered the membrane potential more rapidly than the cellular ATP content, without causing an immediate change in resistance.

ENERGY COUPLING I N MICROBIAL SOLUTE TRANSPORT

297

This behavior is not necessarily inconsistent with the proton conduction hypothesis, however, because (1) the changing resistance of the proton pump itself has to be taken into account, (2) azide may act directly on the ATPase (Slayman et al., 1973), and (3) other studies (Gradman and Slayman, 1975) showed that depolarization from 0.2 V to 0.1 V caused the membrane conductance to fall by 40%. Interestingly, dinitrophenol and another uncoupling agent, a chlorophenylhydrazone, lowered both the membrane potential and the membrane resistance. However, these parameters subsequently both increased (Slayman and Slayman, 1975). Further work is evidently needed to explain the time course of the actions of these reagents on this system which obviously exhibits more complicated properties than the simple proton conductor hypothesis envisages (Felle and Bentrup, 1977). Slayman et al. (1973) intimated that the free energy equivalent available to the proton pump from the hydrolysis of ATP was about 0.5 V/mole of ATP hydrolyzed. As the voltage recorded was near 0.23 V at pH 5.8 when the cellular p H was about 6.8, these workers suggested that the proton pump ejected two protons per ATP hydrolyzed. The implicit assumption is that the electrochemical gradient of protons across the plasmalemma was at equilibrium with the ATP and the products of its hydrolysis. This assumption may b e wrong if the processes of ATP hydrolysis and the attendant excretion of anions and absorption of K+ were still continuing. The possibility that the proton stoichiometry is 1 has not therefore been excluded altogether. The effect of raising the extracellular concentration of K+ was to lower the resting potential in a complex fashion. In the range up to about 10 mM K+ a fall of about 45 mV occurred with a 10-fold increase in [K+],,,, whereas in the next range up to 100 mM Kf a fall of about 85 mV occurred. Thus values smaller and larger than the 58 mV predicted by the Nernst relationship were observed. The addition of sodium also partially depolarized the system. The effects of Na+ and of K+ were smaller in the presence of relatively small amounts of Ca2+ (0.1 mM), possibly because CaZ+altered the relative permeability to the other two ions. T h e membrane potential fell by about 0.08 V from about -0.25 V when the pH was lowered from near pH 8 to pH 4 in the presence of Ca2+ (Slayman, 1965a,b). This effect may arise from the tendency for protons to flow back through the proton pump as the pH falls (Spanswick, 1974). While the notion that an electrogenic proton pump functions in the plasmalemma of Neurospora thus explains in a qualitative fashion the magnitude of the observed potentials, further work is needed to

298

A. A. EDDY

understand the way extracellular ions affect the system. The absorption of K+ during respiration, in exchange for Na+ and H+, superficially resembles these processes in yeast (Slayman and Slayman, 1968, 1970). However, in contrast to the situation in yeast the maximum rate of proton ejection was reported to be about twice as large as the maximum rate of K+ absorption, possibly because anion excretion was involved. Another difference concerns the effect of external p H on Na+ efflux in the presence of K+, which was much smaller for Neurospora at p H 7 than at pH 5 (Slayman and Slayman, 1970). Such behavior is compatible with an Na'-H+ exchange process being involved. However, the depolarization effected by external Na+ or K+, when Ca2+is lacking, points to the conclusion that the absorption of these monovalent ions involves the uptake of positive charges. It is interesting therefore that both azide and dinitrophenol caused only a slow efflux of 42K+from Neurospora, substantially slower than the rate of absorption of the isotope (Slayman and Tatum, 1964a,b). This behavior is in striking contrast to the rapid efflux of K+ from yeast that these compounds caused. However, as noted above, the actions of azide and dinitrophenol on Neurospora are complex, and further work is needed to show whether or not there are fundamental differences in the mechanism of K+ transport in the two systems. In particular the efflux of K+ has not been studied under conditions strictly analogous to those applying when K+ was absorbed. For instance, if K+ uptake occurs only in response to a large negative membrane potential, the rapid efflux of K+ may require actual reversal of the sign of the membrane potential. In principle a proton conductor might reverse the sign of the membrane potential at low extracellular pH values. C. Escherichia coli

The net absorption of K+ by Escherichia coli is a complex process in which four genetically distinct systems participate and which is subject to osmotic regulation (Rhoads et al., 1976; Rhoads and Epstein, 1977; Kepes et al., 1977). One of the transport systems, Kdp, can be repressed by K+,exhibits a high affinity for K+ corresponding to a K, value of 2 has a V,,, of 150 pmoles per minute per gram dry weight and, alone among the four'systems, may involve a periplasmic binding protein. A second system, TrkA, is constitutive, exhibits a K, of 1.5 mM and aV,,, of 550 pmoles per minute per gram dry weight. The other two systems, TrkD and TrkF, are constitutive and relatively slow. Electroneutrality during K+ uptake is maintained either by the extrusion of protons or the extrusion of Na+ (Schultz et al., 1963), and

a,

ENERGY CQUPLING IN MICROBIAL SOLUTE TRANSPORT

299

all four systems appear to couple K+ uptake with Na+ extrusion (Weiden et al., 1967; Rhoads et al., 1976). Early work indicated that E . coli absorbs chloride, the further study of which in relation to these systems seems to be required. Other mutant classes (TrkB, TrkC)have been detected with a defective ability to retain cellular K+. 1. PROTONPUMPING There are now clear indications that these bacteria eject protons by two distinct mechanisms: 1. Everted membrane vesicles, prepared by sonication of rightside-out vesicles, absorbed protons in the presence of ATP and valinomycin in a reaction that was prevented by either azide or dicyclohexylcarbodiimide (see, however, Gutowski and Rosenberg, 1976b, for a cautionary comment), two inhibitors of Ca2+,M$+-ATPase (West and Mitchell, 1974b; West, 1974). Such a system thus ejects protons from the bacterial cell during ATP hydrolysis. However, the H+ stoichiometry of the reaction, expressed as the value of the coefficient m in Eq. (2),has not been established for E . coli. A value of 2 may apply in rat liver mitochondria (Moyle and Mitchell, 1973). Confirmation has come from demonstration of the reverse reaction, namely, the synthesis of ATP, in circumstances where protons tended to flow into the bacteria, or vesicle preparations, under the influence of a pH gradient or electric field created by the valinomycin-catalyzed efflux of K+ (Grinius et al., 1975; Tsuchiya and Rosen, 1976; Wilson et al., 1976) ATP synthesis was inhibited by a proton conductor, by dicyclohexylcarbodiimide, or by mutations leading to defects in Ca2+, M$+ATPase. 2. Oxidative reactions are also associated with the stoichiometric extrusion of protons from the E . coli cell, provided a conducting pathway is available to neutralize the movement of charge (Jones et al., 1975; Lawford and Haddock, 1973). For instance, the addition of small amounts of oxygen to anaerobic suspensions of E . coli treated with valinomycin caused the ejection of protons and the absorption of an equivalent number of K+ ions. However, everted vesicles absorbed protons during respiration, whereas right-side-out vesicles ejected them in a fashion similar to that observed in intact cells (Tsuchiya, 1976). These and other observations strongly support the idea that E . coli is equipped with two distinct mechanisms specifically oriented to eject protons from the cells, one associated with electron transport (Mitchell, 1976) and the other with Ca2+,M$+-ATPase. It is of course the possibility of coupling occurring between these two types of pro-

300

A. A. EDDY

ton pump that provides the basis of the chemiosmotic hypothesis of oxidative phosphorylation. The adequacy for this purpose of the observed values of ApH+,the proton electrochemical gradient, has recently been discussed (Collins and Hamilton, 1976). There is growing evidence that, in the absence of oxygen, anaerobic electron transport, to nitrate or fumarate, for instance, may involve the vectorial ejection of protons which can be coupled to the transfer of sugar and amino acids (Konings, 1977).

2. GENERATIONOF

A

MEMBRANEPOTENTIAL

Studies with lipophilic cations and anions, with various fluorescent dyes both in everted vesicles, right-side-out vesicles, and intact E . coli, all indicate that the respiratory mode of proton ejection, as well as the system driven by ATP, both generate a vectorial charge movement that tends to hyperpolarize the plasmalemma. The generation of a membrane potential is inhibited by proton conductors (Singh and Bragg, 1976a; Grinius and Braienaitk, 1976; Griniuvienk et al., 1975a). This interpretation has been strengthened by studies with strains of E . coli with defects in the membrane ATPase complex (Rosen and Adler, 1975; Tsuchiya and Rosen, 1975). It is known that, when the lipophilic triphenylmethylphosphonium ion is absorbed, a cation that probably penetrates the membrane phase without the assistance of a carrier, protons are ejected (Griniuvienk et al., 197513). The interpretation of the fluorescence studies in terms of membrane potential changes is equivocal, and it is the combination of all these observations, rather than any one of them, that justifies the above conclusion (Hoeberichts and Borst-Pauwels, 1975).

3 . RELATIONSHIP

TO

Kf TRANSPORT

Rhoads and Epstein (1977)studied the net absorption of K+ through the K d p , TrkA, and TrkF transport systems in circumstances where the oxidative mode of proton ejection, which is characteristically inhibited by low concentrations of dinitrophenol, was not coupled to ATP formation owing to a genetic defect (occurring in unc mutants) in the Mg2+,Ca2+-ATPasesystem. Alternatively, the system was provided with glucose anaerobically to make ATP available. The ATP content was further varied by the addition of arsenate. The application of these and other tests showed that the TrkF system was probably actuated by proton ejection, whereas the K d p system required ATP as such. However, the TrkA system appeared to require both sources of

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

301

energy, possibly because it was fueled by ATP and regulated by the membrane potential. Some of the observations made by Klein and Boyer (1972) on Rb+ transport in intact E . coli might b e interpreted similarly to the behavior of the TrkA system, in that Rb+ uptake seemed to require ATP specifically and yet was blocked by uncoupling agents. Rhoads and Epstein (1977) also suggested that the failure to demonstrate rapid K+ absorption in certain E . coli vesicle preparations, unless valinomycin was present, might be due to the fact that they lacked both a binding protein associated with the K d p system and a means of generating the ATP needed by the TrkA system. It is to be emphasized that the K d p system functioned anaerobically, in the presence of glucose, in unc mutant strains despite the lack of functional Mg2+,Ca2+-ATPase. If protons were expelled from the bacteria under these conditions, as these workers imply, it is necessary to postulate yet a third type of proton pump associated with the K d p system. A function for anaerobic electron transport under these specific conditions seems unlikely in view of (1)the behavior shown toward dinitrophenol and (2) the way proline and glutamine transport were affected in parallel tests (see Section VI). It is interesting that all the mutant bacteria lost K+ rapidly in the presence of dinitrophenol, especially when the cell suspension contained Na+. West and Mitchell (19744 observed that raising the extracellular “a+] caused protons to b e expelled from anaerobic suspensions of E . coli, apparently by an electroneutral process. A sodium ion-proton antiport may couple with a potassium ion antiport, in the presence of dinitrophenol, to exchange Na+ for K+ effectively (Fig. 6). A respirationdependent efflux of Na+ from E . coli vesicles in the presence of valinomycin and Rb+ was reported by Kaback (1972) and deserves further study. These complex observations give some indication that the movement of K+ into and out of the bacteria is in certain circumstances governed by its diffusion in the electric field. However, in general cellular K+ is clearly not in equilibrium with the electric field. Thus E . coli grown in 1 p M K+ concentrated the ion in excess of 105-fold, equivalent to a membrane potential of at least - 0.3 V, whereas bacteria grown with 1 mM K+ or 10 mM K+ concentrated it about 300-fold (0.15 V) or 40-fold (0.09 V), respectively. Reported values of the membrane potential, assayed in the presence of valinomycin, range from about -0.07 to -0.14 V (Griniuvieng et al., 1975b; Collins and Hamilton, 1976; Padan et al., 1976). Thus at low K+ concentrations (
302

A. A. EDDY

lation of K+, whereas at higher concentrations (1-10 mM K+) accumulation governed by the electric field seems more feasible, even if the natural permeability to K+ is probably relatively small. Moreover, evidence that in the absence of valinomycin the membrane potential falls as the extracellular [K+] rises is at present lacking. D. Streptococcus faecalis

In contrast toE. coli, S.faecalis, except possibly under certain growth conditions, lacks cytochromes and the systems effecting oxidative phosphorylation, so that the fermentation of glucose or arginine is required to drive the transport of solutes. Pioneering work by Harold and his associates led to a penetrating series of studies showing that the absorption of K+, which could be replaced by other ions such as dibenzyldimethylammonium or triphenylmethylphosphonium, was accompanied by the vectorial extrusion of an equivalent of protons or Na+ in varying proportions. Membrane-bound Ca2+,Mg2+-ATPasewas inhibited by concentrations of dicyclohexylcarbodiimide that stopped all the above transport. processes. Moreover, both the ATPase and the ionic exchanges became insensitive to the reagent as a result of a mutational change in the enzyme. It was inferred that proton extrusion was electrogenic and involved the ATPase system (Harold et al., 1970a,b; Harold and Papineau, 1972a,b). The efflux of Na+, which was defective in certain mutants, appeared to occur by direct exchange with an equivalent number of protons rather than by exchange with K+ itself. Indeed, the exchange with H+ occurred in the absence of added K+, was not inhibited by the organic imide, and seemed therefore not to involve the proton pump. However, the presence of glucose was required. The explanation is uncertain. Harold (1977) has proposed that ATP either regulates the exchange of Na+ for H+ or actually drives the process. The efflux of Na+ is known to occur both against its own concentration gradient and the opposing electric field (Harold and Papineau, 1972a,b). The existence of a vectorial proton pump driven by ATP has been confirmed in elegant work with a related organism, Streptococcus lactis (Maloney and Wilson, 1975).ATP synthesis occurred as a result of the spontaneous influx of protons into cells under the combined influence of a pH gradient across the plasmalemma and an electric potential difference, established in the presence of valinomycin by the efflux of K+. Proton conductors or dicyclohexylcarbodiimide stopped the reaction. A A p H +of at least -0.215 V equivalent was required to start net ATP synthesis.

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

3 03

The above findings are, at first sight, qualitatively consistent with the notion that the absorption of K+ occurred in response to the electric field generated by the proton pump. However, Harold and Papineau (197213) have emphasized certain difficulties with this view. For instance, either the removal of glucose or the presence of proton conductors stopped the uptake of K+ without causing its efflux, unless valinomycin was present, Further, K+ displaced dibenzyldimethylammonium cations from the bacteria, but the reverse process required valinomycin. Both points concern the reversibility of the influx pathway for K+. One could postulate either that the pathway showed voltage-gating characteristics, or that a carrier with properties like the one illustrated in Fig. 5b was involved. Harold (1977) emphasizes the different view, namely, that the absorption of K+ may be driven directly by ATP through a distinct type of mechanism analogous perhaps to the binding protein systems discussed in Section VI. Since the absorption of K+ can displace lipophilic cations, which themselves appear to move electrophoretically, it is necessary to assume that K+ uptake by the proposed mechanism depolarizes the cells. The same K+ pathway is also required to couple with the proton pump. These specific difficulties have to be weighed against the lack of clear evidence that the prevailing membrane potential is in fact large enough to maintain the large gradients of K+ concentration (2 x lo3-fold) these bacteria produce in certain circumstances. E. Halobacterium halobium

When this organism is grown under low oxygen tension in the light,

the plasmalemma contains distinct patches that can be isolated as a purple, highly ordered (Henderson, 1975) membrane fraction containing one major protein, bacteriorhodopsin (Lozier et al., 1975). The purified protein was incorporated into everted lipid vesicles and then acted as a proton pump, absorbing protons in the presence of light (Racker and Stoeckenius, 1974). The further incorporation of mitochondrial ATPase produced a system that synthesized ATP in the light. These important observations provide a direct demonstration of the feasibility of the principle underlying the chemiosmotic hypothesis of oxidative phosphorylation. Light absorption by the intact purple bacteria caused a complex response due to the ejection of protons initially being modulated by their inflow through the ATPase. During this period ATP synthesis occurred, apparently b y the chemiosmotic mechanism (Bogomolni et al., 1976).Right-side-out vesicles were prepared b y sonication of the

304

A. A. EDDY

bacteria and retained various ion transport functions (Kanner and Racker, 1975; Renthal and Lanyi, 1976). These included the ability to generate a p H gradient and a membrane potential of up to 0.14 V, to accumulate Rb+ in the absence of valinomycin, and to accumulate triphenylmethylphosphonium cation. Similar functions were recognized in the intact bacteria in which the value of Ap”+ reached the equivalent of 0.14 V (Bakker et al., 1976) or up to 0.28 V (Michel and Oesterhelt, 1976). The relation between proton pumping and K+ accumulation in this organism has not been studied in detail, but the behavior of the membrane vesicles suggests that K+ moves in the electric field generated by the proton pump (Renthal and Lanyi, 1976; Lanyi and Hilliker, 1976).These bacteria typically grow in the presence of 30 mM K+ and 4 iM Na+, and the cells may contain as little as 10 mM Na+ and 3-5 M K+ under these conditions. The absorption of large amounts of Na+ by the membrane vesicles was slow, whereas the ejection of most of the Na+, in a 0.1 M solution, from the vesicle lumen took only about 5 minutes provided K+ was present in the surrounding medium. Characteristic changes in the extracellular pH and in the membrane potential across the vesicle boundary accompanied the ejection of Na+. Thus loading the vesicles with Na+ both lowered the number of protons immediately ejected when the light was switched on and led to a progressive rise in pH that subsequently reversed. The more Na+ the vesicles initially contained, the later the reversal occurred. The explanation of all these effects is uncertain, but Lanyi and MacDonald (1976) plausibly argue that the efflux of Na+ depended on an electrogenic proton symport-Na+ antiport leading to a net flow of positive charge into the vesicles (Fig. 6). In the presence of extracellular K+ the operation of such a system, in parallel with the electrogenic proton pump, would lead to a large gradient of Na+ and K+ developing across the vesicle boundary. The natural permeability of the vesicles to K+ nevertheless appeared to be relatively small (Lanyi and Hilliker, 1976) but may have been restricted by the impermeability of the membranes to the other ions.

IV.

CARBOHYDRATE TRANSPORT

A. Neurospora crassa

Wild-type cells that had been starved without a carbon source for

1-2 hours absorbed various hexoses, including glucose, 3-0-

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

3 05

methylglucose, 2-deoxyglucose, and L-sorbose, by an inducible mechanism (system 11) that depended on energy metabolism (Schneider and Wiley, 1971b; Neville et d., 1971; Scarborough, 1970a,b; Klingmuller and Huh, 1972). A second carrier system for carbohydrates was found in other cell preparations. System I1 concentrated 3-0-methylglucose, as the unphosphorylated carbohydrate, u p to about 6 x lo3-fold with respect to the extracellular solution (Slayman and Slayman, 1975). Other workers found smaller ratios, about 3.5 X 10' (Schneider and Wiley, 1971a) and 102 (Neville et al., 1971). The corresponding hyphal preparations exhibited striking changes in membrane potential in the presence of each of the above carbohydrates. The study of these changes and the accompanying proton movements has provided powerful support for the cosubstrate hypothesis (C. L. Slayman and Slayman, 1974; C . W. Slayman and Slayman, 1975). T h e main findings are: 1. Provided system I1 had been induced, the addition of any one of its substrates caused a rapid depolarization of the hyphal plasmalemma of up to 0.2 V per second and to the extent of about 0.1 V for glucose. The depolarization was not due to depletion of the cellular ATP content. 2. Partial repolarization ensued within 1 minute, behavior that was attributed to the acceleration of the proton pump. 3. The magnitude of the initial depolarization of the hyphae varied systematically with the concentration of glucose or 3-O-methylglucose applied. The K , values deduced, 40 and 80 pM, respectively, were similar to those observed when the rate of uptake of the sugars was assayed in small cells from shaken cultures. 4. The membrane resistance fell slightly during glucose depolarization. 5. Approximately 1 equivalent of H+ was absorbed along with 2deoxyglucose or 3-0-methylglucose. The way in which electroneutrality was preserved was not established. 6. The presence of cyanide abolished the absorption of protons with 3-0-methylglucose. This may mean that the proton symport functioned rapidly only when the membrane potential was sufficiently large. This behavior merits further investigation, because it implies that the system required an input of metabolic energy to function. In all other respects the observations provide an impressive body of evidence for the existence of the two components of the model, namely, an electrogenic proton symport functioning in parallel with an electrogenic proton pump.

306

A. A. EDDY

The absorption of 3-0-methylglucose and L-sorbose by germinated or ungerminated conidia is reversible (Scarborough, 1970b; Neville et al., 1971). For instance, the addition of either glucose or azide caused a fast efflux of 3-0-methylglucose. Working with germinated conidia, Schneider and Wiley (1971a) found that, when a steady state was achieved, the ratio of the cellular to the extracellular concentration of 3-0-methylglucose was about 350, whether the cells were given a 10 or a 100 pM solution of the compound. It is interesting that the substrate accumulation ratio varied with the nature of the carbohydrate accumulated and with its concentration in preparations of another fungus, Aspergillus nidulans (Mark and Romano, 1971). How this behavior can be reconciled with Eq. (3) is not clear, but one factor that might complicate the application of these simple thermodynamic concepts is the possibility that sugar uptake is limited by feedback regulation (Romano and Kornberg, 1969). 0. Yeasts

Strains of Saccharomyces related to brewer’s or baker’s yeast absorb hexoses such as glucose and deoxyglucose by facilitated diffusion (Cirillo, 1961; Heredia et al., 1968; Wilkins and Cirillo, 1965; Kou et al., 1970; Fuhrmann et al., 1976). The evidence for the claim (Jaspers and van Steveninck, 1975)that, additionally, certain of these monosaccharides are phosphorylated by a separate mechanism during their entry into the yeast is equivocal (Kotyk and Michaljanieovi, 1974). Glucose also appears to be a substrate of a-methylglucoside permease (Brocklehurst et al., 1977). Kotyk and Hofer (1965) observed that a strain of Rhodotorula gracilis concentrated various monosaccharides, including D-XylOSe, by a route that D-glucose also appears to use. Relatively small concentrations of D-xylose were concentrated up to about 103-fold.The addition of either sugar to the yeast suspension at pH 4.5 caused a transient increase in pH (Misra and Hofer, 1975). D-Ribose, however, neither changed the pH nor was it concentrated. Treatment of the yeast with dicyclohexylcarbodiimide, which may have acted at the mitochondria1 level, retarded proton ejection from the yeast and diminished the uptake of D-xylose. These observations establish a preliminary case for supposing that the absorption of xylose and glucose involved proton cycling. An interesting property of this Rhodotorula is that both the influx and efflux of the carbohydrates were markedly inhibited by azide or dinitrophenol. A slow exchange of extracellular carbohydrate for intracellular carbohydrate persisted under

307

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

I min

FIG. 11. The absorption of protons and the ejection of potassium ions caused by the addition of 4 mM TEG, 20 mM maltose, or 40 mM a-methylglucoside to preparations of strain NCYC 240 of S. cereuisiae (adapted from Brocklehurst et al., 1977).

these conditions (Hofer and Kotyk, 1968; Hofer, 1970, 1971a,b). It is not easy to interpret this behavior in terms of the models shown in Fig. 5b. Various a-glucosides (Fig. 11 and 12) and lactose (Seaston et al., 1973) are absorbed along with protons by certain strains of Saccharomyces. The statement applies to a-thioethylglucoside (TEG) a substrate of a-methylglucoside permease, to trehalose, to maltose, and to sucrose (Seaston et al., 1973; Brocklehurst et al., 1977; Eddy et al., 1977). Proton uptake with TEG occurred in yeast preparations thoroughly depleted of ATP (Fig. 13).Thus energy metabolism was evidently not involved. T h e assay of proton absorption caused by 0.5 mln

'

1405-7A

I

1403-17A

12. Proton uptake following the addition of 10 mM sucrose to a strain of Sacal., 1973)and the absence of this effect in a related strain lacking the permease 1403-17a) (unpublished observations of P. Earnshaw and A. A. Eddy). FIG.

churoozyces (1403-7a) carrying sucrose permease (Khan et

308

A. A. EDDY

..

I

0

I

1.0

I

2 .o

( m M carbohydrate I-'

FIG. 13. Proton uptake by energy-depleted yeast in the presence of amethylglucoside (open circles) or TEG (closed circles). The reciprocal of the induced rate of proton uptake is shown as a function of the reciprocal of the carbohydrate concentration (redrawn from Brocklehurst et al., 1977).

these carbohydrates depends on the simultaneous efflux of K+ ions (Fig. 11). Studies with a fluorescent carbocyanine dye were taken to imply that the carbohydrate first caused depolarization and then repolarization when the proton pump was actuated (Eddy et al., 1977). It seems likely therefore that the efflux of K+ in the presence of the carbohydrates is due to the same cause as the efflux of K+ in the presence of proton conductors (Fig. lo), namely, a fall in the membrane potential. In other strains of Saccharomyces sucrose is hydrolyzed by extracellular invertase, the glucose and fructose so produced subsequently entering the yeast by facilitated diffusion (Jennings, 1974). Yeasts from other genera utilize maltose and trehalose by a similar mechanism (Janda and von Hedenstrom, 1974). The intimate connection between the flow of protons stimulated b y lactose, maltose, or trehalose and the uptake of the carbohydrate itself is shown by their numerical equivalence under various conditions (Fig. 14). Further, the physiological conditions that lead to formation of the sugar carrier system are those giving rise to the p H response. The view that a-methylglucoside is absorbed intact and then metabolized (Okada and Halvorson, 1964) was disputed by van Steveninck (1970) who claimed that the sugar was phosphorylated during entry. His findings have not been confirmed by subsequent work (Kotyk and MichaljaniCovL, 1974; Brocklehurst et al., 1977) which instead seems

309

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

0

I 0

.

I

20

40

maltose

( n mole / mp

60

1

FIG. 14. Comparison of the uptake of protons in the presence of maltose with the amount of maltose absorbed during the same time interval (Eddy et d., 1977).

fully consistent with the earlier observations of Okada and Halvorson (1964). The behavior of TEG, also a substrate of a-methylglucoside permease, is interesting from several standpoints : 1. T h e system functions optimally near pH 4.5, concentrating T E G by a factor of about 3 from a 5 mM solution and up to 100 times at concentrations of T E G below 0.5 mM (Okada and Halvorson, 1964; De Kroon and Koningsberger, 1970; Brocklehurst et al., 1977).T h e K , values for the variation in (a) the rate of uptake of protons with T E G concentration and (b) the rate of uptake of T E G itself were both about 1-2 mM (Okada and Halvorson, 1964; Brocklehurst et al., 1977). Hence it seems from these preliminary studies that the concentrating mechanism is relatively inefficient in the range of T E G concentration in which the proton uptake is largest. If this behavior is not due simply to a deficiency of energy metabolism, it raises the question of how tightly the flow of protons is coupled to the flow of TEG. 2. Okada and Halvorson (1963, 1964) observed that, whereas either maltose or a-methylglucoside caused the efflux of TEG, azide which prevented the concentration of T E G caused only a slow efflux of the compound. Moreover, the first-order rate constant for T E G exit had roughly the same value whether the yeast was grown with glucose, and TEG penetrated the cells by facilitated diffusion ( K , for TEG = 50 mM), or whether it was grown with a-methylglucoside, and 2 mM). Nevertheless, efflux the yeast concentrated T E G ( K , through the concentrative mechanism was evidently not simply a passive process, because azide inhibited the efflux observed in the presence of maltose. Further study of this system is evidently required.

310

A. A. EDDY

C. Chlorella vulgaris

This organism concentrates 6-deoxyglucose in the presence of air in the dark, by a mechanism that is inhibited by azide and other proton conductors. A concentration gradient of about 1.5 x lo3 was formed at pH 6.5 from solutions containing 1-10 pM deoxyglucose. Anaerobic cell suspensions also concentrate 6-deoxyglucose in the light, as well as such analogs as l-deoxyglucose, 3-O-methylglucose, and glucose, according to Komor et al. (1972,1973),Decker and Tanner (1972), and Komor and Tanner (1974~). These workers made a penetrating analysis of the aerobic system in terms of the proton cycling model. At pH 6.5 sugar uptake and proton uptake (Komor, 1973) were similarly affected by varying the carbohydrate concentration, by anaerobiosis, by light, and by varying the Na+ or K+ concentration in the suspension medium (Komor and Tanner, 1974a,b). Raising the pH value had the following effects:

1. It lowered the rate of uptake of 6-deoxyglucose from dilute solutions and revealed the presence of an alternative, deprotonated form of the carrier (pK = 6.8) exhibiting a much lower affinity for the carbohydrate. 2. The ratio H+ absorbed/sugar absorbed fell from 1 at pH 6.5 to 0.2 at pH 8. One reservation about this interpretation concerns the fact that inhibitors were not used to stop proton recycling. 3. Raising the pH caused accumulated carbohydrate to be ejected from the cells, a new steady-state distribution being achieved. 4. The net eHux of carbohydrate, which was very slow at pH 6, was much faster at pH 8. These findings are consistent with the general model shown in Fig. 5a. The substrate would enter the cells, either with or without a proton, depending on the prevailing pH. The key question which arises is whether exit occurs by the protonated carrier or by its unprotonated form. Komor and Tanner (1974b) originally suggested that sugar efflux exhibited the entry characteristics of the unprotonated carrier. If this were so, the steady state with respect to the sugar distribution would correspond to a continuous absorption of protons balanced by the proton exit pump (Fig. 4). Indeed, the addition of nonmetabolized analogs of glucose stimulated respiration of the algae by an amount that remained constant from the start of the experiment to the period in which the carbohydrate distribution reached a steady state (Decker and Tanner, 1972). Initially, about 1 equivalent of ATP appeared to be required per equivalent of 6deoxyglucose absorbed, and the accelerated respiration was accompanied by proton ejection. This type of steady state corresponds to a lower accumulation ratio than the equilibrium between the sugar gra-

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

31 1

dient and A p H + defined by Eq. (3).However, the maximal sugar accumulation ratio was observed with <10 pM substrate, whereas about 200 p M substrate gave half-maximal stimulation of respiration. It is therefore possible, though unproven, that neither the acceleration of the respiration nor net proton absorption occurred in the steady state with very low external substrate concentrations, in which case true equilibrium with A p H + may have been achieved. In this connection, Komor and Tanner (1976) estimated the membrane potential in this system by studying the rate of uptake of triphenylmethylphosphonium cation. At p H 6, in the presence of low concentrations of K+, the membrane potential was estimated to e about -0.13 V and ApH was about 1.1 units. Accordingly, A p H +was large enough to maintain the maximum accumulation ratio of 1.6 x lo3. The latter ratio appeared to be independent of the concentration of 6-deoxyglucose in the low range up to 10 pM (Komor et al., 1973), as required by Eq. ( 3 ) . If it is assumed that the indirect method of estimating the membrane potential is valid, it is interesting that the value obtained in the presence of 10 mM substrate was -0.11 V. The fall in the accumulation ratio with rising substrate concentration is therefore not d u e to a substantial decrease in the membrane potential. One problem concerns the ability of proton conductors to inhibit both the influx and efflux of 6-deoxyglucose in circumstances where the membrane potential appeared to be at least - 0.07 V. Whether this type of behavior is consistent with the schemes shown in Fig. 5 is not apparent. An unexpected property of the system concerns the fact that 6deoxyglucose was absorbed with 1 equivalent of protons at p H 6, whereas 2 proton equivalents were absorbed with l-deoxyglucose, apparently through the same carrier system. On the basis of a study of sugar exchange fluxes, Gruneberg and Komor (1976) suggested that, during the uptake of l-deoxyglucose, statistically only each second carrier molecule appearing on the inner side of the plasmalemma carried sugar. This idea represents a novel means of “uncoupling” the flow of the cosubstrate from that of the primary substrate. It envisages a partial dissipation of the proton gradient caused by the substrate, but without the performance of useful work (Fig. 5a). D. Escherichia coli and Other Bacteria

1. LACTOSETRANSPORT Genetic and biochemical studies have shown that E. coli possesses three transport systems for P-galactoside (Rotman et al., 1968) and at

312

A. A. EDDY

least seven systems for transporting galactose (Kornberg, 1976). The methylgalactoside-specific (MglP) system appears not to be a proton symport, whereas the galactose-specific (GalP) system (Henderson et al., 1977) and the widely studied lactose permease (TMG I) (West and Mitchell, 1973) are proton symports. Melibiose permease (TMG 11) of E . coli and Salmonella typhimurium, for which P-thiomethylgalactoside (TMG) is a better substrate than lactose, requires Na+ ions (Section VII). A characteristic property of the lactose transport system is that the initial rate of uptake of its substrates, such as the lactose analog TMG, is maintained in the steady state when the influx and efflux of substrate are equal. The reversibility of the system is shown by the fact that inhibitors of energy metabolism, or simply dilution of the cell suspension, causes a fast efflux of TMG. Exhaustive treatment with appropriate metabolic inhibitors is necessary to lower the accumulation ratio ([TMGli,/[TMGl,,t), or the corresponding ratio for lactose, from a maximum value of about 2 x lo3 in certain strains (Lancaster et al., 1975) and 300 or less in others (Maloney and Wilson, 1975; Kepes and Cohen, 1962) to a ratio near 1 (Winkler and Wilson, 1966). In discussing the role of proton gradients in the transport of lactose and its analogs there has been tendency to overlook the striking variations in the accumulation ratio that are observed (1) in different strains of E . coli, (2) with different analogs of lactose, and (3) at different stages of the physiological induction of the carrier system. For instance, Kepes and Cohen (1962) reported that a strain that concentrated lactose 2 x 103-fold concentrated TMG no more than 65-fold. Another interesting property concerns the so-called capacity to absorb a given substrate. For instance, a particular washed cell preparation accumulated about 100 pmoles of p-galactosyl-p-thiogalactosideper gram dry weight in the steady state whether 0.2 or 2 mM substrate was initially present (Kepes, 1970). It seems unlilely, though the point needs to b e demonstrated experimentally, that the electrochemical gradient of protons would have been lowered very much by raising the substrate concentration in these circumstances. From another standpoint, the fall in the ratio [Sl,,/[Sl,,t as [SI,,, is increased has been described in terms of a pump leak model (Fig. 4).Influx through the permease has been represented as a saturation process, and solute efi3ux has been represented as a first-order process, the rate of which varies appreciably from one galactoside analog to another (Kepes, 1960,1970).The change in accumulation ratio with [SI,,, is in some cases not very large. For instance, [TMG]i,/[TMG],,t varied by no more than a factor of 2 when [TMGI,,,

ENERGY COUPLING I N MICROBIAL SOLUTE TRANSPORT

31 3

varied from 0.05 to 0.5 mM. The notion that efflux of the galactosides involves at least two components has received experimental support. The first component of efflux is not saturated at high intracellular concentrations of TMG or lactose. There have been different assessments of the extent to which efflux occurs b y this route (Maloney and Wilson, 1973; Kepes, 1970; Lancaster et al., 1975). This particular concept of the leak as a first-order process, which is in principle reversible, is incompatible with the claim that the bacteria have a strictly limited capacity to absorb the galactoside. However, the notion of a finite capacity may be erroneous, being based on the extrapolation of data obtained with small range of substrate concentrations of the same order as the K , for influx. T h e first-order component of TMG efflux is said to be inhibited by azide and to be insensitive to thiol reagents (Kepes, 1970). It may be independent of lactose permease, being found in uninduced cell preparations. The second component is saturable ( K , = 16-60 mM), but m l y at much larger lactose concentrations than are required to saturate the influx mechanism ( K , = 0.3-0.6 mM), with which it appears to couple in counterflow experiments. This second efflux component is inhibited b y thiol reagents and becomes prominent after energy depletion. It may represent the inward-facing form of the carrier, determination of the properties of which is apparently complicated by the existence of a diffusion barrier facilitating recapture of the escaping galactoside. T h e implication of all these findings appears to be that, especially at elevated substrate concentrations, the steady state with respect to TMG or lactose accumulation does not in general represent an equilibrium catalyzed by the permease. In the view expressed by Kepes (1970) the need for a continuous expenditure of energy explains why the absorption of TMG, or its analogs, stimulates endogenous respiration both initially and during the period in which the sugar distribution reaches a steady state. There may of course be other explanations of the enhanced oxygen absorption, which in any case has been demonstrated only with relatively large substrate concentrations. a. Proton Involvement. Energy-depleted preparations of E . coli absorbed protons with lactose and TMG, expelling an equivalent of K+ ions, provided the bacteria had been rendered permeable to that ion by treatment with EDTA and valinomycin (West and Mitchell, 1972, 1973). The uptake of thiocyanate ions apparently provided an alternative means of neutralizing the charge displaceent associated with the uptake of protons. This behavior is consistent with the postulated electrogenicity of the process of lactose uptake. When a 3-mM lactose

314

A. A. EDDY

solution was used, the ratio of the number of protons absorbed to the number of lactose molecules absorbed fell from 0.8 at 15 seconds to 0.5 at 70 seconds. A stoichiometric ratio of 1 was inferred by extrapolation to zero time. A mutation in, or near, the lac y gene lowered the proton/lactose stoichiometric ratio. The defect was associated with (1) an increased sensitivity to thiol reagents, (2) a faster influx of the galactoside, and (3)its considerably faster efflux. Consequently, the net accumulation of TMG by these strains was impaired. This behavior is in keeping with the idea that a molecular defect in the permease protein loosened the coupling with the proton gradient (Wilson and Kusch, 1972; West and Wilson, 1973). b . Role of Metabolic Energy: The Energized Membrane State. According to the symport hypothesis the accumulation of lactose or its analogs with protons is coupled either to proton ejection through the respiratory chain or to proton ejection through the Mg2+,Ca2+-ATPase system (Section 111, C). Both phenomena appear to occur, though some workers prefer to define such coupling in terms of a hypothetical high-energy state of the cell membrane. It is of course true that the postulated turnover of protons (Fig. 1) is a cryptic process, the existence of which can at present only be inferred on indirect evidence. 1. Coupling of the energized membrane state to TMG accumulation is inhibited by dinitrophenol without the cellular ATP content necessarily being depleted (Pavlasova and Harold, 1969). 2. Anaerobic coupling occurs in two ways. The first uses ATP, is blocked by arsenate (Klein and Boyer, 1972) because of ATP depletion, and requires the Mg2+,Ca2+-ATPasesystem lacking in unc mutants (Schairer and Haddock, 1972). The second anaerobic mode involves a coupled electron flow to nitrate or fumarate without intervention of the ATPase system (Konings and Kaback, 1973). 3. Under aerobic conditions TMG accumulation occurred in whole cells depleted of ATP, in the presence of arsenate, provided respiration was not inhibited (Klein and Boyer, 1972). A strain carrying an unc ATPase mutation likewise accumulated TMG aerobically in the presence of maltose, whereas the wild-type strain accumulated it both aerobically and anaerobically (Schairer and Haddock, 1972). Furthermore, membrane vesicles that were unable to make ATP by oxidative phosphorylation nevertheless accumulated TMG during respiration in the'presence of arsenate (Klein and Boyer, 1972). It is especially significant that this mode of transport can be energized by artificial electron donors such as ascorbate-phenazine methosulfate (Konings et al., 1971).

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

315

The crucial role of proton gradients in TMG accumulation is further illustrated by evidence that unc mutations leading to enhanced permeability of the plasmalemma to protons lowered the accumulation of TMG below the levels achieved by wild-type preparations. Proton impermeability was restored and TMG uptake was increased in the presence of dicyclohexylcarbodiimide (Rosen and Adler, 1975; Altendorf et al., 1974; Rosen, 1973a,b). Another observation indicating that optimal accumulation of lactose b y the vesicles required an elevated membrane potential was that valinomycin lowered the accumulation in the presence of K+ ions (Kaback, 1972). The above examples provide a set of experimental criteria defining the role of the energized membrane state in the transport of TMG. Now electrogenic proton ejection was activated or inhibited by the very same genetic factors and by the same proton conductors and metabolic inhibitors that modulated the energized membrane state. TMG and lactose each caused an influx of protons, moreover, which the proton pumps might be expected to recycle. The energized membrane state can thus plausibly be equated with the generation of a proton gradient that the absorption of sugar tends to dissipate. This view leads to the prediction that TMG accumulation would take place in the absence of metabolic energy if a proton gradient were artificially imposed across the plasmalemma. Supporting evidence is outlined in c below. c. Galactoside Accumulation Driven b y Proton Diffusion in the Absence of Metabolic Energy. Escherichiu coli membrane vesicles containing K+ absorbed TMG transiently in the presence of valinomycin to an extent that varied systematically with the concentration of K+ outside the vesicles, owing apparently to a graded variation in the membrane potential (Hirata et ul., 1974). Accumulation ratios of up to about sixfold appear to have been achieved, although the total amount of TMG absorbed was in fact of the same order as the total amount of lactose carrier said to be present (Section IV, D, 1, e). Flagg and Wilson (1976)made similar observations with intact unc mutant E . coli preparations. When the extracellular pH was lowered from pH 8 to p H 6 a transient accumulation of TMG occurred, leading to a fivefold gradient between the cells and medium. T h e absence of an effective Ca2+,M$+-ATPase ensured that ATP was not an intermediate in the system. Somewhat similar observations, leading to a 20-fold gradient of sugar concentration, were made with a strain of S. lactis (Kashket and Wilson, 1972a). I n this case the ATPase system was blocked by dicyclohexylcarbodiimide without affecting TMG ac-

31 6

A. A. EDDY

cumulation, and the membrane potential was raised by the addition of valinomycin. Kasket and Wilson (1973) observed that the TMG accumulation ratio varied linearly with the estimated value of A p H t , in the range from 0.02 to 0.13 V (Fig. 15).The results are in fair though not exact agreement with Eq. (3). d . ApH' and Galactoside Accumulation during Proton Pumping. A simple expectation based on the proton cycling hypothesis is that the same relationship between A pHt and the TMG accumulation ratio would hold during energy metabolism as in preparations in which a proton gradient had been established b y artificial means. Kasket and Wilson (1974)showed that this was very roughly true for S . Zactis (Fig. 16). In these further assays the proton gradient was established at a steady level determined by the hydrolysis of ATP, rather than transiently at a selected maximum value by the diffusion of K+ out of the bacteria. The phosphorylation of TMG did not occur to a significant extent (Kasket and Wilson, 1972b). There are two implications. First, it appears that the proton cycling model applies to this system. Second, the steady state of TMG accumulation seems to be near equilibrium with the proton gradient, provided it is correct to assume that 1 equivalent of protons was absorbed with each equivalent of TMG. It I

0

50 TMG r a t i o

(mV)

FIG. 15. Comparison of the accumulation ratio for TMG observed in preparations of S. Zactis and the magnitude of the proton gradient (Ap"') imposed across the plasma membrane in the absence of energy metabolism. The galactoside accumulation ratio and the proton gradient are both expressed in millivolt equivalents (redrawn from Kashket and Wilson, 1973).

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

100

-

I

> E

-Iz

50-

0

c

c 0

2n

0

317

/ 50

TMG rotio

(mV)

FIG.16. Comparison of the accumulation ratio for TMG observed in preparations of S . lactis and the magnitude of the proton gradient (A@"+)established across the plasma membrane during energy metabolism. The galactoside accumulation ratio and the proton gradient are both expressed in millivolt equivalents (redrawn from Kashket and Wilson, 1974).

'

would be interesting, as a further test of these relationships, both to study the effect of varying the TMG concentration and to show directly that the proton stoichiometric ratio is 1. Again, with regard to the behavior of E . coli, Collins and Hamilton (1976)estimated that, during respiration in the presence of 0.3 mM K+ and valinomycin, ApH was about 1.6 units and the membrane potential was about -0.13 V. Thus ApH' was about 0.23 V. This is in the range required to permit ATP synthesis b y the chemiosmotic mechanism. Padan et al. (1976) used different methods with another strain of E . coli. They concluded that A p H +was about 0.13 V equivalents at p H 6-7 in the presence of valinomycin and low concentrations of K+ and fell to 0.07 V at high K+ concentrations. At p H 6, ApH was about 2 units. Ramos et al. (1976) studied vesicles prepared from a related strain of E . coli. During the oxidation of ascorbate the p H of the vesicle lumen was near 7.5 when the extravesicular value varied from 5.5 to 7.5. The membrane potential was roughly constant near 0.07 V. The net effect was that A p H + fell from about 0.18 to about 0.07 V as the pH was raised. Comparison of the lactose accumulation ratio in this system with the value of the membrane potential deduced from the distribution of triphenylmethylphosphonium cation has led to conflicting conclusions. In one study at p H 6.5, when ApH was apparently small, the membrane potential and the equivalent lactose gradient were almost linearly related (Fig. 17). The general trend of the results is similar to the

31 8

A. A. EDDY 100

-

D .c W c

50

0

100

50 lactose ratio

(mV)

FIG. 17. Comparison of the accumulation ratio for lactose observed in membrane vesicles from E . coli and the membrane potential estimated from the distribution of triphenylmethylphosphonium cation. The lactose accumulation ratio is expressed in millivolt equivalents (adapted from Schuldiner and Kaback, 1975).

observations made with S . lactis. However, subsequent work has shown that the lactose accumulation ratio can exceed the estimated equivalent value of ApH+b y about 30 mV (Kaback, 1977). Taken at face value these observations are not consistent with Eq. ( 3 ) unless n > 1. The presence of 10 m M lactose, a larger concentration than was used for the work shown in Fig. 17, partially inhibited the uptake of the phosphonium cation by the bacterial vesicles, as though lactose partially depolarized the system (Schuldiner and Kaback, 1975). The possibility that some of the vesicles burst through swelling at these high lactose concentrations needs to be borne in mind, nor is it certain that the system was in a steady state. Leaving aside these difficulties, the apparent reduction in membrane potential implies that a net flow of charge into the vesicles was maintained in the steady state. If this was so the lactose gradient produced at these high lactose concentrations was probably not in equilibrium with the proton gradient. e. Galactoside Binding to the Lactose Carrier. The M protein specified b y the lac y gene carries a cysteine residue that is protected against reaction with thiol reagents when certain sugars are bound to the protein. Neither lactose nor TMG exerted much protection, but galactosyl-thio-P-galactoside(thiodigalactoside) did, as did certain m-galactosides such as melibiose and p-nitrophenyl-a-galactoside (Jones and Kennedy, 1969; Kennedy et al., 1974). Kennedy et al. (1974) referred to the relevant site on the M protein as site I1 to distin-

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

319

guish it from the lactose- and TMG-binding site. Nitrophenyl-agalactoside is in fact a potent competitive inhibitor of lactose transport that is not accumulated to a significant extent in right-side-out vesicles (Rudnick et ul., 1976). A recent genetic analysis of the lac y gene has cast some doubt on the existence of two separate binding sites for lactose and melibiose (Hobson et al., 1977). The amount of thiodigalactoside or nitrophenylgalactoside bound to vesicles prepared by sonication, some of which may therefore have been everted, was about 0.11 nmole/mg of membrane protein derived from a particular M L strain of E . coli (Kennedy e t al., 1974). Belaich et al. (1976) studied the binding of thiodigalactoside both to sonicated and to right-side-out vesicles by equilibrium dialysis in the presence of 10 mM azide. They detected 0.6-0.9 nmole of binding sites per milligram of protein and assumed that the substrate penetrated all of the vesicular water without being concentrated there. The dissociation constant of the binding process roughly equaled the K , for thiodigalactoside absorption. Study of the heat changes associated with binding of the galactoside allowed the entropy and enthalpy of binding to lie determined. All these observations seem consistent with earlier work showing that in the presence of azide the lactose carrier functions so as to equilibrate its substrates between the inside and the outside of the bacteria. After energy depletion in the absence of azide the functioning of the carrier appears to b e restricted by the absence of a pathway that allows the backflow of protons across the plasmalemma (Cecchini and Koch, 1975; Simoni and Postma, 1975). A series of studies by Kaback and his associates led to very different conclusions. These workers claimed that the binding of various, seemingly impermeant, dansylated galactosides to the lactose carrier, whether assayed directly or in terms of fluorescence changes, was greatly enhanced in three sets of circumstances: (1)during lactate oxidation, (2) during hyperpolarization of the vesicle membrane, or ( 3 ) when lactose was made to flow out of the vesicles and membrane hyperpolarization was apparently not involved in the response (Schuldiner et ul., 1975; Rudnick et ul., 1976). Similar behavior was observed in binding studies with nitrophenyl-a-galactoside. The total number of binding sites was estimated to b e about 2 nmoles/mg of membrane protein. It was inferred that the lactose carrier existed in a cryptic form, with a low affinity for the galactosides, that required energization before the galactoside could be bound (Rudnick et d., 1976; Kaback, 1977). It was supposed that the cryptic form of the carrier underwent a conformational change, bringing it to the outer membrane surface under each of the three conditions referred to above.

320

A. A. EDDY

The latter inference may be correct if the assays used simply detected the amount of carrier available at the outer membrane surface during a relatively short time interval. The number of such sites may increase with time. Binding studies made over a longer time interval, with a substrate that reaches the inner surface, might detect the presence of binding sites there, as well as other cryptic forms of the carrier. The observations made by Belaich et al. (1976) appear to be in this category, but the number of binding sites detected by Kennedy et al. (1974) requires some subsidiary explanation. It would be interesting to determine how everted vesicles bind fluorescent galactosides, as this might show whether the postulated low-affinity cryptic form of the carrier is simply the inward-facing carrier, and whether indeed it exhibits a low affinity for its substrates, a conclusion that is inconsistent with the argument presented by Belaich et aZ. (1976).

2. GALACTOSE TRANSPORT Henderson et al. (1977) showed that galactose absorption by anaerobic preparations of E . coli bearing the GalP transport system (K, = M ) was associated with proton absorption in a strain lacking the MglP transport system ( K , = M ) . Proton conductors abolished the induced proton uptake. Membrane vesicles, lacking detectable binding protein, concentrated galactose during respiration of D-lactate or ascorbate, in a process that was inhibited by proton conductors, by valinomycin in the presence of K+, but not by arsenate (Kewar et aZ., 1972). Galactose caused no extra proton uptake in anaerobic preparations of bacteria carrying the MglP system (a binding protein system) but lacking the GalP system (Henderson et d., 1977). The simplest interpretation of these observations is that protons are cosubstrates in the latter but not in the former system. One of the arabinose transport systems also functions with protons (Henderson, 1974). Scylloinositol absorption by anaerobic preparations of Klebsiella aerogenes was accompanied by the absorption of somewhat less than 1 equivalent of protons (Reber et al., 1977). Studies with selective inhibitors showed that both ATP and respiration energized this system and that it was inhibited by proton conductors.

V.

AMINO ACID ABSORPTION IN FUNGI

Genetic and kinetic studies have shown that fungi possess various permeases that are selective for a particular small group of amino

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

32 1

acids, as well as certain less selective systems, the so-called general permeases handling many of the common amino acids. The induction and repression of these systems, in strains of Saccharomyces, Neurospora, Penicillium, and Aspergillus, has been studied in relation to the . physiological needs of the parent organism (Slayman, 1973; Whitaker, 1976). Amino acid absorption by fungi is known in several cases to be inhibited by dinitrophenol and azide, which has been taken to mean that proton gradients may be involved (Slayman and Slayman, 1975; Hunter and Segel, 197313). Direct evidence for the participation of proton cycling, however, has so far been obtained only with strains of Saccharomyces. The kinetics of the absorption of amino acids, such as glycine and histidine, by strains of Saccharomyces differ strikingly from the corresponding behavior ofthe lactose permease ofE. coli described in Section IV, D.

1. The process is almost irreversible. The I4C-amino acid was released only at a very low rate when the yeast was transferred either to a fresh solution lacking the amino acid or to a similar solution containing %-amino acid. Moreover, neither the presence of dinitrophenol, nor the presence of dinitrophenol plus the metabolic inhibitors antimycin and 2-deoxyglucose which depleted the cells of ATP, caused the efflux or more than a small fraction of the I4C-aminoacid in the cellular pool (Crabeel and Grenson, 1970; Kotyk et al., 1971; Saiyid and Kotyk, 1972; Seaston et al., 1976). 2 . The amount of amino acid absorbed by preparations of washed yeast cells is governed, among other factors, by the phenomenon of transinhibition, one aspect of which is that amino acid influx falls off progressively as the amino acid, or possible some compound derived from it, accumulates in the cells (Crabeel and Grenson, 1970; Morrison and Lichstein, 1976; Indge et al., 1977). When glucose was present, for instance, a steady state was eventually reached in which the slow influx of glycine just balanced the combined rates of glycine efflux from the yeast and conversion of the amino acid to other compounds. The size of the glycine pool in the steady state also depended on whether or not glycine penetrated the main vacuole of the yeast. The presence of physically discrete pools of amino acids in fungal cells (Wiemken and Nurse, 1973; Weiss, 1976) complicates the study of the energetics of the accumulation process. Indge et al. (1977)observed that the inhibition of further glycine absorption by glycine already present in the yeast was greatly reduced by first treating the yeast with glucose for 2 hours. The cells then absorbed so much gly-

322

A. A. EDDY

cine that many of them burst. This behavior indicates that the regulatory inhibition, the mechanism of which is largely unknown, is probably an accessory to the main pumping machinery (see also Hunter and Segal, 1973a). The progressive fall in the rate of entry of glycine with loading is evidently not due to an approach to equilibrium in a system which is virtually irreversible. A. Proton-Dependent Concentration of Glycine by Energy-Depleted Yeast

Preparations of washed cells depleted of ATP in the presence of antimycin and deoxyglucose absorbed glycine from a 200-pM solution, at the optimum p H 4.5, at about 30% of the rate observed when glucose was present to maintain energy metabolism (Eddy et al., 1970a,b; Seaston et al., 1976).The depleted yeast absorbed only about 30 nmoles of glycine rapidly, apparently because further absorption was hindered b y the lowering of the cellular p H from about p H 6.5 to pH 6.0. This effect may be due to the change in the cellular pH as such, rather than to the small drop in the pH gradient across the plasmalemma. The absorption of glycine under these conditions, through the general amino acid permease, caused 2 equivalents of protons to enter the yeast cells and 2 equivalents of K+ ions to leave the cells (Fig. 18).The glycine taken in was not simply exchanged with other cellular amino acids, nor was it chemically changed in the process. Absorbed glycine appeared to be located outside the vacuole in the cytosol (Indge et aZ., 1977).The maximum gradient of amino acid concentration ([glyli,/[gly],,t) set up under these conditions, apparently without the intervention of metabolic energy, varied from 1.8 X lo4to 6.8 x lo4in the presence of 0.1 pM glycine (Seaston et al., 1976).The choice of such low amino acid concentrations was dictated by the need to establish a low rate of glycine uptake that balanced the low natural rate of efflux. Concentration of glycine by the depleted yeast was greatly inhibited by 0.2 mM dinitrophenol, by the presence of extracellular K+, and by working at pH 7. All these circumstances point to involvement of the H+ and K+ gradients as the energy source for concentrating the glycine in yeast preparations in which the proton pump was not functioning. B. The Role of K+ Ions

Because the absorption of glycine during energy metabolism, in the presence of glucose, failed to stimulate the turnover of K+, it seemed

323

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

40.

30 B

glycine

( n mole / mg

1

proline

(

n mole /mg 1

FIG. 18. Proton uptake as a function of the amount of glycine or proline absorbed by preparations of S. cereoisine depleted of ATP (redrawn from Seaston et al.,1973, 1976).

unlikely (Eddy and Nowacki, 1971; Seaston et al., 1976) that K+ served an indispensable antiport function on the glycine carrier, but rather that the efflux of K+ from energy-depleted yeast represented the facultative major route in these yeast preparations for neutralizing the flow of positive charges into the cells during the electrogenic uptake of glycine with protons (Fig. 3). The action of dinitrophenol can be interpreted in a similar way but is complicated by the speed with which the cellular p H falls to inhibitory levels in the presence of this compound, owing to the loss of cellular K+. Thus a short treatment with dinitrophenol greatly lowered the subsequent rate of uptake of glycine in the absence of the phenol (Seaston et al., 1976). Indeed, one function of K+ appears to be to maintain the cellular pH in the optimal range for amino acid absorption. In order to explore the function of K+ further, Eddy and Nowacki (1971) studied the effect of replacing most of the cellular K+ by Na+. Under these conditions the number of equivalents of protons absorbed with each of several amino acids, through the general amino acid permease, fell from about 2 equivalents per mole of amino acid to 1 equivalent per mole. The induced efflux of K+ stopped. Also, amino acid uptake into cells containing mainly Na+ rather than K+ depended on metabolic energy. Thus both proton absorption and absorption of the amino acid ceased in the presence of deoxyglucose and antimycin. An explanation of this behavior has not been given. It raises the ques-

324

A. A. EDDY

tion whether Na+ replaced one but not both of the cosubstrate protons normally involved. The effects of extracellular K+ on glycine absorption are complicated. Yeast preparations depleted of ATP absorbed glycine at p H 7 at about one-seventh of the rate at pH 4.5. The presence of Na+ or K+ considerably inhibited glycine uptake, especially at pH 7. However, the effects of changing the concentrations of K+, Na+, and H + were smaller in the presence of glucose. It seems possible that extracellular ions affect the membrane potential, hence the rate of glycine uptake. The plasmalemma may become hyperpolarized when the proton pump is activated. C. Multiple Systems for Proton-Dependent Amino Acid Absorption

Yeast preparations depleted of ATP concentrated various amino acids with the simultaneous absorption of protons and release of K+ ions. Studies with genetically defined strains (Seaston et al., 1973) TABLE I

THE NUMBEROF EQUIVALENTS OF PROTONS (n) ABSORBED WITH EACH EQUIVALENTOF A GIVEN AMINO ACID BY DIFFERENTROUTES Amino acid and source

Saccharomyces (Eddy and Nowacki, 1971; Seaston et al., 1973; Cockbum et al., 1975) General amino acid permease G1ycine Methionine Lysine Citrulline Phenylalanine Leucine Specific permeases Lysine Methionine Proline Glutamate Candida (Eddy et al., 1977) Glycine Lysine Arginine Glutamate

Number of equivalents

2 2 2 2 2 2 1 1 1 2 or 3 1 1

1 2

325

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

showed that the ratio of the number of protons absorbed to the number of amino acid molecules absorbed varied from one system to another (Table I). For instance, L-methionine was absorbed with 2 equivalents of protons through the general permease and with 1 equivalent of protons through one of the methionine-specific permeases. Coupling between these two systems, leading to net proton absorption without net uptake of methionine, is unlikely to occur because both systems appear to be almost irreversible under physiological conditions. Proline absorption through its specific permease also exhibited a stoichiometry of 1 (Fig. 18). The absorption of glutamic acid is an interesting example in that this compound is absorbed in effect as a positively charged entity both in S. cerevisiae and in Candida utilis (Fig. 19).A further comparison of proton and K+ ion movements during the absorption of glutamate, lysine, and glycine is shown in Fig. 19. During lysine absorption protons were ejected from the yeast in preference to K+ (Eddy and Nowacki, 1971), a process illustrated in Figs. 3 and 20.

I min

JJ-1 I'

GLT

=

k7

0

\"

FIG. 19. Displacements of protons and potassium ions during the absorption of 0.5 pmole of glutamate, glycine, or lysine by preparations of C. utilis (50 mg dry weight), at about pH 5, depleted of metabolic energy (Eddy et nl., 1977, and unpublished observations).

326

A. A. EDDY

H 0.5 rnin

FIG. 20. Proton and potassium ion displacements following the addition of 0.5 pmole of glycine or lysine to respiring preparations (50 mg dry weight) of C . utilis (strain NCYC 193) at pH 5 (unpublished observations of P. Earnshaw and A. A. Eddy).

D. The Magnitude of the Proton Gradient during Energy Metabolism

Only very rough estimates can b e made of the magnitude of A p H +in yeast, owing to the lack of a reliable means of determining the membrane potential. However, one might assume that the distribution of K+ is an indicator of the membrane potential (cf. Fig. 10).Washed cell preparations containing glucose at pH 4.5 appear to maintain a pH gradient of about 2 units across the plasmalemma, in association with a K+ gradient equivalent to about -0.2 V. This A p H + may b e as large as 0.3 V equivalent at low concentrations of K+ (Seaston et al., 1976). Whether or not such a mechanism is compatible with an ATPase with a stoichiometry of two protons per molecule of ATP hydrolyzed is hard to say. A stoichiometry of one proton per molecule of ATP seems more plausible. In any case, the issue is purely speculative because there is no evidence in yeast, as opposed to Neurospora, that the plasmalemma ATPase (Matile, 1970) is involved in proton transport. Given that ApH+at pH 4.5 is 0.3 V equivalent, a glycine accumulation ratio of 5 x lo4 (0.28 V equivalent) could b e maintained even if the proton stoichiometry were one proton rather than the two protons observed with the general amino acid permease. However, at pH 7 the larger stoichiometry would be required, especially when extracellular [K+] was large. These speculative arguments indicate that the largest amino acid concentration gradients that have been observed in this system are probably compatible with the gradient hypothesis. VI.

AMINO ACID TRANSPORT I N BACTERIA

Genetic and kinetic studies have defined a large number of systems involved in the transport of amino acids in bacteria, some absorbing only one and others absorbing several of the common amino acids. For

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example, E. coli K12 possesses at least five transport systems for the aromatic amino acids, including separate specific systems for tyrosine, phenylalanine, and tryptophan (Slayman, 1973). Following Berger and Heppel (1974) two major categories of behavior have been recognized: 1. A periplasmic binding protein is involved, the release of which during controlled osmotic shock of the bacteria results in diminished transport activity. Such systems are not found in membrane vesicles, presumably because the binding protein has been lost. Genetic and physiological evidence has shown that at least one other component, presumably a protein, is required in addition to the binding protein (Ames and Spudich, 1976)for the functioning of the high affinity histidine permease of S. typhirnuriurn. The P-methylgalactoside permease ofE. coli (Robbins et d ,1976) likewise contains two uncharacterized components, the mgl A and mgl C products, in addition to the mgl B product, the galactose binding protein. 2. Transport in whole cells is insensitive to osmotic shock and can be demonstrated in membrane vesicles. The binding of specific amino acids to detergent-solubilized membrane fractions has been demonstrated in a few instances (Gordon et al., 1972). A. Binding Protein Systems

There is now compelling evidence that the above two types of transport systems differ in the way in which they use metabolic energy to concentrate amino acids, carbohydrates, and certain other solutes. Glutamine and methionine transport in E . coli are taken as examples of the first type. Glutamine uptake is known to depend on a binding protein, and methionine uptake may do so (Kreishman et al., 1973; Kadner and Winkler, 1975). Methionine or glutamine absorption failed to occur in bacterial vesicles (Lombardi and Kaback, 1972) and only utilized oxidative energy derived from, for instance, lactate or phenazine methosulfate-ascorbate when the bacteria contained functional Caz+, Mg2+-ATPase (Berger and Heppel, 1974; Kadner and Winkler, 1975). However, an unc mutant with a defective ATPase system maintained glutamine transport during glycolysis by a mechanism in which anaerobic electron transport was not involved (Gutowski and Rosenberg, 1976a). Further, the absorption of these amino acids, especially in mutants with a defective ATPase system, was relatively, though not completely, insensitive to the presence of dinitrophenol, whereas it was inhibited b y arsenate which lowered the cellular ATP content.

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A. A. EDDY

This behavior is broadly consistent with the idea that glutamine and methionine uptake in E . coli are driven by ATP rather than by the energized membrane state (see, however, Singh and Bragg, 197613, for a dissenting view). The partial sensitivity to dinitrophenol of the binding protein systems could be attributed to nonspecific effects of this compound, to partial depletion of the cellular ATP pool, or to depletion of cellular K+ and acidification of the cytosol when the bacteria have first been starved in the presence of the proton conductor. Because of the possibility of the metabolic conversion of the absorbed amino acids, there seems to b e little information available about the maximum extent to which amino acids are concentrated by the binding protein mechanisms, but ratios of at least lo3 seem to be indicated. Methionine uptake appears to be regulated by the cellular content of methionine (Kadner, 1975) in a manner reminiscent of the fungal systems described in Section V, A. In contrast with E . coli glutamine transport in both Bacillus subtilis and in Staphylococcus aureus, like methionine transport in B. subtilis, has been shown to occur in membrane vesicles respiring appropriate substrates (Konings, 1977).

B. Amino Acid Transport linked to the Energized Membrane State

Membrane vesicles from an M L strain of E . coli concentrated each of 16 amino acids during respiration with ascorbate-phenazine methosulfate (Lombardi and Kaback, 1972). Proline transport is typical of this type of system. Glutamine, methionine (see above), and arginine were not concentrated. This and subsequent work with mutants with defective ATPase systems, lacking either the enzyme activity or exhibiting defective proton permeability, showed very clearly that ATP was not directly involved in this mode of transport in the vesicles (Altendorfet al., 1974; Hirata et al., 1974). Absorption was inhibited by proton conductors and by other conditions that lowered the membrane potential. Accumulation of the amino acids could also be linked to various modes of anaerobic electron transport (Boonstra et al., 1975). Thus amino acid uptake appeared to be coupled to the energized membrane state. Amino acid absorption into the vesicles, like that into the intact bacteria, was in general found to be a reversible process. Either anaerobiosis or proton conductors caused efflux of the absorbed amino acid. Oxamate, however, inhibited uptake without causing efflux. The explanation is not known.

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329

In intact E . coli cells the aerobic uptake of proline, which was inhibited by proton conductors, was maintained even when the cellular ATP content was depleted b y arsenate (Klein and Boyer, 1972). Further, anaerobic uptake of proline, which was also inhibited by proton conductors, required either the presence of a coupled Ca2+, Mg2+ATPase system or anaerobic electron transport (Gutowski and Rosenberg, 1976a). More direct evidence for a role for an electric potential in the absorption of proline and glycine was provided by Hirata et ul. (1973) who showed that, in the presence of valinomycin, the efflux of K+ from vesicles lacking respiratory substrate caused a transient accumulation of the amino acids representing up to a 60-fold concentration gradient relative to the medium. While the amount of proline absorbed was small, the fact that the amino acid was concentrated to some extent focused attention on the likely role of the proton gradient in this system.

1. PROTON SYMPORT

WITH

AMINO ACIDS

Collins et al. (1976) demonstrated that an inflow of protons occured during the uptake of alanine, serine, and glycine by intact E . coli cells. Each of these amino acids is absorbed by membrane vesicles. Isoleucine and valine failed to cause proton absorption into the bacteria. The system was calibrated b y comparison with the p H changes caused by the addition of lactate or TMG, which were each assumed to absorb 1 equivalent of protons and to enter the cells until their internal and external concentrations were equal. Whether these assumptions are strictly true is not known. I n any case serine and glycine gave rise to an uptake of protons of roughly the same magnitude as alanine. A proton stoichiometric ratio of 0.76 +- 0.06 equivalent of protons per alanine equivalent was determined in six such assays. Some interesting spontaneous mutants of the bacteria collected in chemostat cultures maintained with alanine as the sole source of carbon. I n certain instances these variant strains absorbed 2, and in other instances 4,equivalents of protons per alanine equivalent absorbed. Serine and glycine behaved like alanine in this respect. It was suggested that the mutant cultures had been selected on the basis of their enhanced ability to absorb alanine from very dilute solutions. The enhanced growth rate of the organisms and their performance in the chemostat supported this interpretation. This work shows that the quantitative aspects of the coupling with protons are physiologically significant and provides the only direct evidence

330

A. A. EDDY

available for the coupling between amino acid absorption and proton uptake in this organism. Attempts to demonstrate proton absorption in bacterial vesicles have given negative results, 2. THE RELATIONBETWEEN THE CONCENTRATION GRADIENT OF AMINO ACIDS AND A@*+ Kaback (1977)made a start on the problem of testing Eq. (3)in respiring vesicles. Proline accumulated about 2 x 103-fold, equivalent to about 0.2 V, at p H 5.5 from a 8 pM solution of the amino acid. At pH 7.5 it accumulated to an extent corresponding to at least 0.15 V. ApHf was about 0.19 V in the former circumstances and and about 0.075 V in the latter. To explain this behavior, Kaback (1977) suggests that the proton stoichiometry of the system varied from 1 at pH 5.5 to 2 at p H 7.5. I t would be interesting to examine the accumulation of proline in the intact bacteria from this standpoint, since much larger gradients of proline concentration might be expected to form at pH 7.5 when ApH+ appears to exceed 0.13 V equivalent in these preparations (see Section IV, D). Similar observations were made with lysine, another system exhibiting a high affinity ( K , = 1 pM) for its substrate (Lombardi and Kaback, 1972).

3. GLUTAMATETRANSPORT IN Escherichia coli MEMBRANE VESICLES Both glutamate and aspartate were concentrated by strain K12 of E . coli and b y bacterial mutants selected on the basis of their ability to use glutamate as the sole source of carbon for growth (Kahane et al., 1976a,b). The intracellular concentration of glutamate in the steady state was about 7 x lo2 times higher than the concentration in the medium in succinate-grown cells (Halpern and Even-Shoshan, 1967). A value of 4 x lo4 was reported for strain B (Frank and Hopkins, 1969). There is some evidence (Halpern et al., 1973; Halpern and Ennis, 1975) that entry of glutamate and at least a portion of the exit process are mediated by distinct carrier systems (cf. Section IV, D). Genetic and kinetic analysis, combined with a comparison of the behavior of membrane vesicles and the bacteria from which they were derived, has revealed a complex picture involving both a glutamatebinding protein and an unresolved role for Na+ ions. (1) Na+ in the range u p to 20 mM lowered the K , for glutamate from 60 to 10 p M (Kahane et al., 1975)without increasing the V,,,. This happened both in

ENERGY COUPLING I N MICROBIAL SOLUTE TRANSPORT

33 1

assays with vesicles and in those with intact cells. (2) Na+ inhibited glutamate efflux. ( 3 )The Na+ ion apparently was not involved in the binding of glutamate to the glutamate-binding protein. No binding protein was detected in the vesicle preparations. (4)No influx of Na+ with glutamate was detected. (5)A need for cellular K+ was demonstrated, which may represent an indirect requirement for maintaining the cellular pH. These observations and others made with strain W of E . coli (Willis and Furlong, 1975a,b) neither prove nor disprove the notion that Na+ ions s e n e as a cosubstrate in the transport of glutamate. It seems possible that the glutamate-binding protein may interact with the system demonstrated in the vesicular preparations, which appears to be driven by respiration and the high-energy membrane state (Willis and Furlong, 1975b; Kahane et ul., 1975). Nevertheless there were some conditions under which glutamate uptake by the intact bacteria was not inhibited markedly b y azide (Halpern and Even-Shoshan, 1967). Thus there seem to be conditions under which the energized membrane state was not involved. Willis and Furlong (1975b) have suggested that a complex made up of the binding protein and the amino acid may compete with the unbound substrate for the Na+-stimulated translocation process. However, it has not been shown in this system how energy coupling is affected under these conditions. If the criteria discussed in Section VI, A are valid, a crucial question seems to be whether or not a given membrane carrier can participate in both types of coupling mechanisms. Whether a binding protein is involved or not, the absorption of glutamate anions at physiological p H values necessarily involves the displacement of charges, a process that might be expected to interact with the cellular ionic pumps. Recent work with membrane vesicles derived from E . coli B indicates that the uptake of glutamate is both stimulated by Na+ and is driven by a mechanism based on cotransport of glutamate with Na+. Tsuchiya et ul. (197711) deduced that both the sodium concentration gradient and the membrane potential were components of the driving forces. Thus the transport system for glutamic acid appeared to be electrogenic. MacDonald et al. (1977) inferred that glutamate transport was driven by the chemical gradient of sodium and did not respond to changes in the membrane potential. These observations and the work outlined above suggest that E . coli contains several different transport systems for glutamate, and it is not yet clear whether one or more of these is H+ coupled or whether only Naf serves as the cosubstrate ion. The role of protons in aspartate absorption was studied by Gutowski

332

A. A. EDDY

and Rosenberg (1975) who used relatively high concentrations of the amino acid with preparations of E . coli K12. The bacteria were grown either with succinate or with glucose. In the former case aspartate was absorbed with about 1.3 equivalents protons per amino acid equivalent, b y a mechanism shared with succinate, malate, and fumarate (Section VIII, A). I n the latter case aspartate was absorbed without a net uptake of protons. Proton uptake was studied in a system lacking extracellular Na+. These workers suggested that the true proton stoichiometry for aspartate was the same as that for succinate, namely, 2 equivalents of protons per mole of substrate absorbed. Thus aspartate uptake may be electrogenic in this system (Fig. 2).

C. Streptococcus faecalis

Ashgar et al. (1973) studied a strain of this organism which concentrated glycine, alanine, serine, and threonine up to about 400-fold from 0.1 mM solutions of these amino acids in the presence of glucose. The process was inhibited by proton conductors, by the ATPase inhibitor dicyclohexlcarbodiimide, and by valinomycin in the presence of high concentrations of K+. This and other circumstances implicated the proton gradient as the driving force. The argument was strengthened by the demonstration that threonine accumulated in the bacteria in the absence of glucose when a p H gradient or potassium diffusion potential was established across the cell membrane. Because the concentration of threonine under these conditions was not blocked by the ATPase inhibitor, neither the proton pump nor the synthesis of ATP appeared to be involved. Another interesting property is that the starving bacteria exchanged extracellular amino acids for their intracellular counterparts, even though little net movement of amino acid appeared to occur in the absence of glucose. Nevertheless, the amino acid pool accumulated under the influence of an artificially imposed proton gradient leaked out in the presence of a proton conductor, showing that a net flux of amino acid persisted to some extent when the proton gradient was relatively small. The above arguments clearly implicate the proton gradient as the driving force in these neutral amino acid systems. Attempts to demonstrate the influx of protons with the amino acids, however, were unsuccessful. The evidence outlined in Section 111, D indicates that, on the basis of available estimates of the magnitude of AkH' in this organism, an amino acid gradient of at least lo2 could be maintained if

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333

equilibrium were achieved and the symport operated with a proton/amino acid ratio of 1. Glutamate and aspartate differed from the above amino acids in being absorbed, in the presence of glucose or arginine, by a mechanism that was neither inhibited by various proton conductors nor by dicyclohexylcarbodiimide, nor was it activated by an artificially imposed proton gradient. The extracellular amino acids seemed to be absorbed as such. They were concentrated about 200-fold and rapidly degraded. Exchange between the cellular glutamate or aspartate and the extracellular amino acid was not detected. Furthermore, valinomycin inhibited aspartate transport in the absence of extracellular K+ but not in its presence, an effect that appears to involve maintenance of the content of cellular K+ rather than a requirement for an elevated membrane potential. As Harold and Spitz (1975) emphasize, the above properties are quite inconsistent with a mechanism of absorption based on proton cycling. Instead, and by analogy with the binding protein systems (Section VI, A), it appears that ATP or a similar compound directly drives the aspartate pump, though without a periplasmic protein seemingly being involved. There is as yet no evidence for a specific involvement of Na+ in glutamate absorption, but further investigation seems desirable in view of the need to maintain electroneutrality during anion absorption. Gale and Llewellin (1972) found that neither aspartate or glutamate accelerated the absorption of protons into a related strain of these bacteria. Indeed, their study of the absorption of aspartate, glutamate, lysine, and alanine by this organism showed that, whereas 1 mM dinitrophenol inhibited aspartate and glutamate uptake 50-60%, it failed to inhibit lysine and alanine uptake. Moreover, the absorption of none of the amino acids was inhibited significantly by 100 mM K+ in the presence of valinomycin. Hence some mechanism other than proton cycling appeared to be involved in the uptake of alanine by this bacterial strain. It may b e relevant that another strain of Streptococcus appeared to absorb glycine, serine, and threonine by exchanging these compounds with alanine synthesized endogenously (Brock and Moo-Penn, 1962). D. Staphylococcus aureus

Short et al. (1972a,b) and Short and Kaback (1974) observed a marked accumulation of 16 amino acids by the bacterial vesicles in the presence of various electron donors. Accumulation clearly depended on electron transport rather than ATP formation, and either proton

334

A . A. EDDY

conductors or anaerobiosis caused an efflux of the accumulated amino acids. An amino acid distribution ratio of about 500 was observed for serine. Other neutral amino acids, lysine, glutamate, and aspartate were extensively concentrated by several distinct systems. These observations clearly implicate the proton gradient as the likely driving force in the vesicle preparations. The behavior of intact cell preparations has been interpreted in similar terms: (1) Gale and Llewellin (1972) found that aspartate was concentrated at least 2 x 103-fold at p H 7 by a mechanism that was inhibited by (a) oligomycin, which was presumed to act as an ATPase inhibitor, (b) valinomycin in the presence of K+, or (c), especially effectively, by the two antibiotics acting together. The accumulation observed at p H 8.5 corresponded to a ratio of about 5 x 10'. The process was largely inhibited by valinomycin, whereas the accumulation at p H 5.5, which corresponded to a ratio of lo4,was relatively insensitive to valinomycin. (2) In nine assays aspartate uptake in energydepleted cells at p H 5.5 displaced 0.62 ? 0.11 SEM equivalents ofprotons into the bacteria. The corresponding value for glutamate in three assays was 0.91 & 0.15 equivalents. The presence of lysine caused no increase in proton absorption. (3)Niven and Hamilton (1974)found that the energy-depleted cells of another strain of S . aureus absorbed both acidic amino acids in response to a pH gradient imposed artificially in the presence of valinomycin and a proton conductor. The cellular K+ content was assayed in some of their experiments, and the results appeared to support the idea that acidic amino acids were absorbed as electroneutral species with a proton. Similar studies with lysine were taken to mean that it accumulated without H+ as cosubstrate, in response to the membrane potential. Glycine uptake, however, because it responded both to a p H gradient and to the membrane potential, was presumed to be absorbed with 1 equivalent of protons (Niven et al., 1973; Niven and Hamilton, 1973). (4) At p H 6.3 the respiring organisms maintained an intracellular pH about 1.3 units more alkaline. The membrane potential was estimated to b e 0.13 V, so that the ApHf was equivalent to about 0.21 V (Collins and Hamilton, 1976). As Cockburn et al. (1975) pointed out, the findings of Niven and Hamilton (1974) with the energy-depleted cells are not consistent with the behavior of glutamate or aspartate during energy metabolism that Gale and Llewellin (1972) reported. There is of course the question of metabolic degradation of the amino acid. Leaving that issue aside, it appears that glycine, which is concentrated only about 10'fold, may use the mechanism proposed by Niven and Hamilton (1974). However, lysine was concentrated about 103-fold at pH 7, and a significant energy deficiency appears to be involved unless a

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

335

cosubstrate ion such as H+ is implicated (Fig. 2). The discrepancy is even larger for acidic amino acids. These were in fact concentrated about 5 x 104-foldat p H 6.5 and would be expected to be concentrated only about 20-fold if they were absorbed as electroneutral species. This system provides an important test of the proton cycling model. If future work shows that the proton stoichiometry is indeed unity and no other cation such as Na+ is involved as cosubstrate, the hypothesis must be rejected. Critical evidence about the number of cosubstrate ions involved may be difficult to obtain. First, proton cycling may occur as a result of imperfect inhibition of the proton pump. Second, it may b e necessary to assay the absorption of the cosubstrate protons under conditions in which A p H + is poised in the physiological range. One relevant point is that cellular [K+] appeared to be small in the assays that Gale and Llewellin (1972) made with S . aureus. VII.

SODIUM-DEPENDENT SYSTEMS

Reference was made above to (1)the inhibitory effects of Na+ or Li+ on proline uptake in E. coli (Kawasaki and Kayama, 1973) and (2) the inhibition of glycine uptake by Na+ in yeast. (3) Na+ also inhibited amino acid uptake in Streptomyces hydrogenans (Ring et al., 1976). Further, Na+ stimulated glutamate uptake in E . coli (Section VI, B); (4) 2-aminoisobutyrate uptake in an alkalophilic Bacillus (Koyama et al., 1976); (5)proline uptake in Mycobacterium phlei (Section VII, D); (6) threonine uptake and that of four other amino acids in Brevibacterium jlavium (Araki et al., 1973); as well as (7) the uptake of succinate, glucose, and L-valine by Micrococcus lysodekticus (Ariel and Grossowicz, 1974); and (8) various transport functions in a pseudomonad (Kodama and Taniguchi, 1977). (9) Sodium ions stimulated citrate uptake in membrane vesicles of K . aerogenes (Johnson et al., 1975). It exhibited inhibitory effects on other bacterial strains (Wilkerson and Eagon, 1974). Further work is required to show whether examples 4 to 9 are d u e to Na+ acting as a cosubstrate in the transport of the ligand, or whether the ions act in some other way, for example, as a cofactor that is not itself absorbed with the primary ligands. More information is available about the following three examples. A. The Melibiore Permeare (TMG It) of Salmonella typhimurium

Stock and Roseman (1971) observed that the absorption of TMG by this organism was markedly stimulated by concentrations of Na+ in

336

A. A . EDDY

the range up to 5 mM. Na+ lowered the K, for the galactoside without changing the V,,,. Because TMG stimulated Na" uptake these workers suggested that the Na+ ion served as cosubstrate in concentrating the sugar. Within 10 seconds the organism ejected the absorbed Na+ to a significant extent, and only the earliest time samples obtained indicated that the Na+ flux and the TMG flux were of similar magnitude. The preliminary findings are consistent with the role of Na+ illustrated in Fig. 6. West and Mitchell (1972) suggested that the marked effect of Na+ in this system might be due to an effect of Na+ on the metabolism of this organism. This possibility needs to be borne in mind, as do the modes of indirect coupling illustrated in Fig. 6 (West and Mitchell, 1972). Recent work greatly strengthens the idea that the TMG uptake by the melibiose permease in membrane vesicles derived from Salmonella typhinzurium is driven b y the sodium electrochemical gradient (Tokuda and gaback, 1977). Melibiose permease of E . coli appears to function in a similar manner (Tsuchiya et al., 1977a).

8. Amino Acid Uptake in a Marine Pseudomonad

Extracellular Na+ ions were required for the transport of L-alanine and 2-aminoisobutyrate both into the cells of a certain Pseudomonad and into membrane vesicles oxidizing ethanol or ascorbate (Sprott and MacLeod, 1972; Sprott et al., 1975). The amount of amino acid absorbed both during a short and a long time interval was proportional to the cellular K+ content (Thompson and MacLeod, 1974). When K+ was in short supply, there were conditions under which the amino acid merely equilibrated with the intracellular space. The addition of K+ resulted in the concentration of both K+ and the amino acid in the bacteria and the efflux of Na+. Thompson and MacLeod (1973) drew attention to the fact that the K, and V,,, for 2-aminoisobutyrate influx were unchanged when the standard suspension medium was replaced by one containing the concentrations of Na+ and K+ present in the respiring cells. They accordingly suggested that gradients of these ions across the plasmalemma were not required for amino acid accumulation to take place. However, this interpretation ignores the possibility that an elevated membrane potential may be required with either Na+ or H+ acting as cosubstrates. Studies with ionophores might throw further light on the behavior of this interesting system. The K+ requirement may be analogous to that shown in Halobacterium hdobium (see Section VI1,C). The apparent dependency of the absorption of K+ on the presence of NaC is a finding (Hassan and

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

337

MacLeod, 1975) not easily reconciled with the schemes discussed in earlier sections. C. Light-Driven Leucine and Glutamate Transport in Halobacterium halobium

This system extrudes protons, generating an electric potential, a pH gradient, and a Na+ gradient as outlined in Section 111, E. The uptake of leucine by the envelope vesicle showed an absolute requirement for Na+, as well as a requirement for K+ in the vesicular lumen, the nature of which has not been established. A p H gradient across the vesicular membrane seemed not to be required, whereas an elevated membrane potential appeared to be obligatory. The influx of Na+ with leucine was not demonstrated as such, but clear evidence was obtained for the accumulation of leucine in the dark in response to a K+ diffusion potential established in the presence of valinomycin (MacDonald and Lanyi, 1975). The accumulation of glutamate, which also required extraluminal Na+ and intraluminal K+ appeared to involve an electrically neutral species, on the basis of studies with a lipophilic cation (Lanyi et al., 1976a,b). A p H gradient was not required, but a Na+ gradient was needed. Lanyi et al. (1976a) estimated that the maximum glutamate gradient generated was 4 x lo4. Their interpretation requires that a Na+ concentration gradient of at least this magnitude be formed. This point merits further investigation. T h e results do not seem to rule out completely the possibility that the cosubstrate stoichiometry for glutamate was larger than 1. Indeed, the leucine gradient driven by the Na+ electrochemical gradient was said not to exceed 200-fold (MacDonald and Lanyi, 1975), whereas the much larger glutamate gradient was presumed to be driven only by the Na+ concentration gradient. Some assessment of the importance of leak pathways (Fig. 4) is required in order to interpret these findings in terms of the possible cosubstrate stoichiometry (Fig. 2). D. Mycobacterium phlei

A Na+ or Li+ ion-dependent absorption of proline was observed both in intact cells and in vesicles derived from this organism. Conflicting reports are available both about the effect of uncoupling agents on this system and the role of the proton gradient (Hirata et al., 1974; Prasad et al., 1976). Proline uptake in E . coli vesicles was inhibited by Na+ (Lombardi and Kaback, 1972).

338

A. A. EDDY

VIII.

MISCELLANEOUS COMPOUNDS

A. Succinate

Membrane vesicles prepared from E . coli grown in the presence of succinate concentrated succinate during the respiration of appropriate electron donors. The mechanism was inhibited by azide or dinitrophenol. Preparations lacking both succinate dehydrogenase and fumarate reductase concentrated succinate about 50- to 150-fold (Lo et al., 1972; Rayman et al., 1972; Martin and Konings, 1973). Gutowski and Rosenberg (1975) studied the absorption of protons with succinate, malate, and fumarate in whole cells of this organism. Succinate uptake was markedly stimulated by the addition of glucose and, under the conditions of the assays, required the presence of oxygen. The procedure used involved the addition first of a small amount of glucose which led to rapid acid production for about 1 minute. When succinate was subsequently added, some of it was absorbed along with protons, 2 equivalents with each equivalent of succinate. The same ratio of 2 was found at various succinate concentrations, at p H 6.6 or pH 6.9, and with malate or fumarate as substrates. It is not clear whether the apparent requirement for metabolic energy in this system is merely to set up a small p H gradient across the plasmalemma that facilitates the uptake of succinate, or whether it serves some other purpose. The choice of experimental conditions does not rule out the possibility that some proton recycling occurred. If succinate were absorbed with three protons, that is, as a cation, one of the cosubstrate protons, as opposed to the other two protons, might be rapidly cycled. An apparent stoichiometry of two protons may arise in this way (Fig. 3 ) . Against this view, one could argue that the reported gradients of 10' are relatively small and could be supported by a p H gradient of only 1 unit. This is essentially the view taken by Gutowski and Rosenberg (1975). However, Postma and van Dam (1971) emphasized that the transport of di-and tricarboxylic acids in Axotobacter vinelandii required metabolic energy, and they suggested that binding proteins might be involved. The yeast S . cerevisiae is known to excrete succinate from the cytosol near p H 6.5 into solutions near pH 4 when the acid is formed as a minor product of the metabolism of glucose. It seems unlikely that this process represents the excretion of succinate2-, with two protons, as an electroneutral species. In other circumstances similar yeasts also absorb succinate as their main source of carbon for growth.

ENERGY COUPLING IN MICROBIAL SOLUTE TRANSPORT

B.

339

Citrate

The requirement for extracellular Na+ in citrate uptake in certain bacteria was referred to in Section VII. The need for cellular K+ in a similar system has also been defined (Eagon and Wilkerson, 1972). The mechanism of these interesting effects has not, however, been determined. Citrate uptake in B. subtilis was stimulated 10-fold by the presence of Mg2+and certain other divalent cations. Stoichiometrical amounts of Mg+ were absorbed with citrate, b y a route that appeared to be independent of other pathways for Mg2+absorption (Willecke et d., 1973; Oehr and Willecke, 1974). Absorption was inhibited by proton conductors. Whereas K+ was not a cosubstrate, a requirement for cellular K+ was demonstrated for optimal citrate absorption. The mechanism available for neutralizing the negative charge borne by the Mg2+-citrate complex is unknown. It seems possible that at least one proton is involved (compare Fig. 2). C. lactate

Lactate absorption by S. faecalis appears to be an electroneutral process governed by the pH gradient across the plasmalemma (Harold and Levin, 1974). Whether or not a similar mechanism governs the uptake of lactate by membrane vesicles of Paracoccus denitrificans (Nichols and Hamilton, 1976) has not been established. Collins et d . (1976) have proposed that lactate uptake by intact E. coli is an electroneutral process. D. Gluconate

Robin and Kepes (1973) showed that gluconate was concentrated at least 200-fold by aerobic preparations ofE. coli in the virtual absence of penetrating cations other that H+. Gluconate was absorbed from a solution containing K+ with about 1 equivalent of protons. Since no special attempt was made to inhibit energy metabolism when the proton uptake was assayed, the stoichiometry of 1 must be regarded as a minimum value. The observed accumulation appears to b e too large to be accounted for simply in terms of the pH gradient across the plasmalemma. Booth and Morris (1975), however, obtained evidence that gluconate was absorbed by Clostridium pasteurianium simply in response to a pH gradient.

340

A. A. EDDY

E. Glucose 6-Phosphate

Membrane vesicles from E . coli concentrated this compound during respiration of lactate or glycerophosphate (Dietz, 1972). An 80-fold concentration gradient was set up in the intact bacteria (Winkler, 1973). Proton conductors were powerful inhibitors of the system both in the presence and absence of oxygen. Essenberg and Kornberg (1975) found that, under anaerobic conditions that restricted but may not have stopped proton recyling, 1 equivalent of protons was absorbed along with the doubly charged carbohydrate anion. While the presence of K+, Na+, Mg2+,or Ca2+increased the rate of absorption, no absolute requirement for these ions was demonstrated. Nevertheless, these workers suggested that K+ (or Na+) might function as cosubstrates. The function of K+ may alternatively be the indirect one illustrated in Fig. 3. Preliminary work with membrane vesicles at pH 7.5, when the pH gradient across the vesicle boundary appeared to be small, indicated that during respiration a glucose-6-phosphate gradient on the order of 50-fold was found. To reconcile this behavior with a gradient hypothesis based on H+ ions as the cosubstrate seems to require that the substrate anion be absorbed with at least 2 equivalents of protons (Kaback, 1977). F. Sulfate

In an elegant study, Burnell et al. (1975a)showed that right-side-out membrane vesicles prepared from P . denitrificans concentrated sulfate during respiration, to the extent of about 100-fold. The mechanism was inhibited by a proton conductor, inhibition resulting in an efflux of the accumulated sulfate. In contrast, everted vesicles failed to accumulate sulfate during respiration, thereby demonstrating the vectorial character of the process and reflecting the orientation, presumably, of the redox-driven proton pump. Sulfate was also absorbed, in small amounts, when the vesicle lumen was made alkaline relative to the suspension medium by the addition of K+ and nigericin. The sulfate was not simply bound to its carrier. Because the response to an artificiaIly induced pH gradient was observed with both types of vesicles, the sulfate carrier itself evidently responded to an inwardly as well as to an outwardly directed pH gradient. Cuppoletti and Segal(l974,1975) found that sulfate transport in Penicillium notatum was stimulated by protons and required metal ions as well. Mg2+,Ca2+,and other divalent ions were effective at low coccentrations and could be replaced by larger concentrations of Na+.

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These properties are reminiscent of the behavior of E . coli toward glucose-6-phosphate. A detailed study of the respective roles of Ca2+ and of H+ suggested that these became bound to the carrier before sulfate and that the role of H+ was indispensable. Because only about 0.2 equivalent of Ca2+was absorbed with each equivalent of sulfate, it is not certain that the ion is a cosubstrate rather than a cofactor in the system. Furthermore, Ca2+ could b e replaced by Na+. Whether protons were absorbed was not examined. T h e facultative role of the metal ions is consistent with various hypotheses. G. Phosphate

1. Yeast Early work b y Rothstein and his associates led to the idea that bakers yeast absorbed phosphate as H2PO; in exchange for OH(Rothstein, 1960b). A high cellular K+ content assisted the process, possibly because an alkaline cytosol was required. The inhibition of phosphate uptake by dinitrophenol or azide appeared not to be due to depletion of the cellular ATP content (Borst-Pauwels and Huygen, 1972). Phosphate absorption is usually regarded as irreversible, but efflux can occur in specific circumstances (Button et al., 1973).Three transport systems have been detected in baker’s yeast, exhibiting K , values with respect to [H2PO;I of about 0.5 mM, 10-30 p M , and 1pM (Borst-Pauwels and Jager, 1969: Borst-Pauwels et al., 1975; Roomans et al., 1977). T h e two last-named systems have been studied in cell preparations starved of phosphate so as to induce a subsequent fast rate of absorption of the anion. Cockburn et al. (1975) observed that a rapid absorption of protons accompanied the uptake of phosphate or arsenate at p H 5 into yeast preparations depleted of ATP in the presence of antimycin and deoxyglucose. The system involved appears to be the one exhibiting a K , of about 10 p M . Under these conditions phosphate uptake appeared to be driven by the flow of H+ into the yeast coupled to the efflux of Kf. The ratio of H+ to H2PO; absorbed varied from 3 to about 2 as the amount of phosphate absorbed was raised from about 2 to 10 nmoles/mg of yeast dry weight. Similar observations made with a strain of C. utilis are illustrated in Fig. 21. The work shows that phosphate was absorbed, in effect, as a positively charged complex. Rough estimates of the driving forces involved [Eq. (6)l suggested that the coupling to the proton gradient might in that way lead to the large

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FIG. 21. The uptake of protons and the displacement of potassium ions following successive additions of arsenate (0.1 pmole) to a suspension of C. utilis (NCYC 193) at pH 5. The assay was performed essentially as described by Cockburn et al. (1975) in their work with preparations of Saccharomyces.

phosphate gradients, on the order of lo5- to 10s-fold, that have been observed in some yeast strains (Cockburn et al., 1975).Phosphate absorption in yeast mitochondria, like that in rat liver mitochondria, appears to involve an electroneutral symport with protons (Chateaubodeau et al., 1974). Roomans et al. (1977) showed that phosphate uptake at p H 7, by the route exhibiting a K , of about 1 p M , was greatly increased in the presence of Na+ or Li+. Two affinity constants and therefore two binding sites for each of these ions appeared to be involved. Moreover, phosphate uptake immediately accelerated Na+ uptake and, for reasons that are not understood but may involve lowering of the membrane potential, inhibited Rb+ uptake. These investigators suggested that the two Na+ ions may serve as cosubstrates in this system. The cycling of Na+ may occur by the mechanism discussed in Section 111. Phosphate uptake in two marine fungi was found to require Na+ specifically (Belsky et al., 1970).

2. BACTERIA Phosphate transport in E . coli appears to proceed by at least two parallel mechanisms, one of which utilizes a binding protein and one of

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which may be coupled to the high-energy membrane state (Rae et al., 1976; Rae and Strickland, 1976). There are some conditions under which phosphate uptake by bacterial preparations is closely linked to the absorption of K+ ions (Weiden et al., 1967). Burnell et al. (1975b) showed that membrane vesicles prepared from P . denitrificuns accumulated phosphate during respiration, while everted vesicles failed to do so. However, they accumulated phosphate in response to an artificial pH gradient, as did right-sideout vesicles, when the vesicular lumen was made alkaline b y the addition of K+ and nigericin. Thus phosphate absorption in this instance appeared to be an electroneutral process driven by the pH gradient. Very different behavior was observed by Harold and Spitz (1975) in a study of the accumulation of phosphate and arsenate b y S . faecalis. These workers took the view that the neutral symport mechanism, driven b y the pH gradient (Fig. 2), was implausible in view of the extent to which arsenate was concentrated, namely, up to 103-fold.Conditions were found under which arsenate uptake was not greatly affected by selected concentrations of proton conductors, or of dicyclohexylcarbodiimide, that virtually stopped the absorption of TMG, apparently b y lowering the proton gradient across the plasmalemma. Hence arsenate absorption appeared not to depend on this gradient. Nevertheless, other evidence showed that factors which lowered the cellular p H hindered arsenate uptake. These and other observations led to the suggestion that phosphate and arsenate absorption occurred b y electroneutral exchange with hydroxyl or by the equivalent absorption of H+. Assay of the p H changes in the medium due to phosphate absorption was not feasible, and a role for Na+, for instance, seems not to have been excluded. The system would be energized by ATP or some similar compound, rather than by the membrane proton gradient acting across the plasmalemma (Harold and Spitz, 1975). Phosphate exchange and uptake across the plasma membrane of S. aureus, in the range of pH 5.5-8.5, exhibited a relatively large K , of about 0.8 mM with respect to [H,PO,]-. When the bacteria respired, a net uptake of phosphate occurred, at the same rate at which phosphate exchanged with intracellular phosphate or arsenate in the resting bacteria (Mitchell, 1954). Mitchell (1970) has drawn an analogy between this system and the neutral symport mechanism of the mitochondria1 phosphate carrier. IX.

GENERAL CONCLUSIONS

The above discussion shows that there is a large amount of evidence available about numerous transport systems found in wide range of

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microorganisms, which has a general bearing on the problem of the validity of the proton cycling hypothesis. However, only in a few instances is the evidence sufficiently direct to provide a strong presumption in favor of the hypothesis. The main predictions of the model (Figs. 1 and 2) relate to the following points.

1. The plasma membrane in any given case contains an appropriate carrier, which may function electrogenically, as well as a range of ionic pumps driven by appropriate chemical reactions. The direct demonstration of electric current generation by mitochondria1 proton-translocating ATPase, by cytochrome oxidase, and by bacteriorhodopsin (Drachev et al., 1974) strongly supports this concept. Likewise, the partial purification of an alanine carrier from membranes of a thermophilic bacterium and its reconstitution into functional vesicles with natural phospholipids produced a system that appeared to concentrate alanine when a K+ diffusion potential was imposed (Hirata et al., 1977).Provided the carrier in this system was not simply binding, as opposed to translocating, the absorbed alanine, this work provides important confirmation of the feasibility of the gradient coupling mechanism. 2. Demonstration of the postulated proton symports, of defined stoichiometry, has proved elusive in that an accelerated influx of protons with specific substrates has been shown to occur in relatively few instances. The possible roles of other ions such as Na+, Ca+, and Mg2+ require further examination, as do the compensating processes of proton ejection and K+ absorption which, even in circumstances of partial energy depletion, may obscure the primary movements of ions with the main substrate (Fig. 3 ) .It may be that the detection of a significant influx of cosubstrate ions requires the imposition of a relatively large proton gradient across the plasma membrane simply to cause sufficiently fast absorption of the substrate. 3 . Depending on the proton stoichiometry and the charge carried on the substrate, depolarization of the plasm