Ionic Channels in Cells and Model

Systems

Series of the Centro de Estudios Cientificos de Santiago

Series Editor: Claudio Teitelboim Centro de Estudios Cientfj'icos de Santiago Santiago, Chile and University of Texas at Austin Austin, Texas, USA

IONIC CHANNELS IN CELLS AND MODEL SYSTEMS Edited by Ramon Latorre

Ionic Channels in Cells and Model

Systems

Edited by

Ramon Latorre Centro de Estudios Cientfjicos de Santiago

and Facultad de Ciencias Universidad de Chile

Santiago, Chile

PLENUM PRESS • NEW YORK AND LONDON

Library of Congress Cataloging in Publication Data

Ionic channels in cells and model systems.

(Series of the Centro de Estudios Cientificos de Santiago) Includes bibliographies and index. 1. Ion channels. 2. Cell membranes. 3. Biological models. I. Latorre, Ramon. II.

Series. QH601.I67 1986 574.87/5 86-12265

ISBN-13: 978-1-4684-5079-8 DOl: 10.1007/978-1-4684-5077-4

© 1986 Plenum Press, New York

e-ISBN-13: 978-1-4684-5077-4

Softcover reprint of the hardcover I st edition 1986

A Division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013

All rights reserved

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

Contributors

Osvaldo Alvarez, Departamento de Biologia, Facultad de Ciencias, Uni­versidad de Chile, Santiago, Chile; and Centro de Estudios Cientificos de Santiago, Santiago, Chile

Clay M. Armstrong, Department of Physiology, School of Medicine, Uni­versity of Pennsylvania, Philadelphia, Pennsylvania 19104

Illani Atwater, Laboratory of Cell Biology and Genetics, NIADDK, Na­tional Institutes of Health, Bethesda, Maryland 20205

Juan Bacigalupo, Departamento de Biologia, Facultad de Ciencias, Uni­versidad de Chile, Santiago, Chile

Charles Bare, Agricultural Research Service, Weed Science Laboratory, Agricultural Environmental Quality Institute, Beltsville, Maryland 20705

Francisco J. Barrantes, Instituto de Investigaciones Bioquimicas, Uni­versidad N acional del Sur, 8000 Bahia Blanca, Argentina

Dale J. Benos, Department of Physiology and Biophysics, Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02115; present address: Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Al­abama 35294

v

vi Contributors

Francisco Bezanilla, Department of Physiology, Ahmanson Laboratory of Neurobiology; and Jerry Lewis Neuromuscular Research Center, Uni­versity of California at Los Angeles, Los Angeles, California 90024

Ximena Cecchi, Departamento de Biologfa, Facultad de Ciencias, Uni­versidad de Santiago, Chile; and Centro de Estudios Cientiffcos de San­tiago, Santiago, Chile

John A. Dani, Department of Physiology, University of California Medical School, Los Angeles, California 90024; present address: Section of Mo­lecular Neurobiology, Yale University, New Haven, Connecticut 06510

A. Darszon, Departamento de Bioqufmica, Centro de Investigacion y de Estudios Avanzados deII.P.N., Mexico, D.F. 07000, Mexico

Gerald Ehrenstein, Laboratory of Biophysics, NINCDS, National Insti­tutes of Health, Bethesda, Maryland 20205

George Eisenman, Department of Physiology, University of California Medical School, Los Angeles, California 90024

Richard FitzHugh, Laboratory of Biophysics, NINCDS, National Insti­tutes of Health, Bethesda, Maryland 20205

Robert J. French, University of Maryland School of Medicine, Baltimore, Maryland 21201

Harold Gainer, National Institutes of Health, Bethesda, Maryland 20205

Ana Maria Garcia, Boston Biomedical Research Institute, Department of Muscle Research, Boston, Massachusetts 02114; present address: White­head Institute, Cambridge, Massachusetts 02142

J. Garcia-Soto, Departamento de Bioqufmica, Centro de Investigacion y de Estudios Avanzados de I.P.N., Mexico D.F. 07000, Mexico

Sandra Guggino, Laboratory of Molecular Aging, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Bal­timore, Maryland 21224

Cecilia Hidalgo, Department of Muscle Research, Boston Biomedical Re­search Institute, and the Department of Neurology, Harvard Medical School, Boston, Massachusetts 02114; present address: Departamento de Fisiologfa y Bioffsica, Facultad de Medicina, Universidad de Chile; and Centro de Estudios Cientfficos de Santiago, Santiago, Chile.

A. D. Islas-Trejo, Departamento de Bioqufmica, Centro de Investigacion y de Estudios Avanzados deII.P.N., Mexico, D.F. 07000, Mexico

Kinihiko Iwasa, Laboratory of Biophysics, NINCDS, National Institutes of Health, Bethesda, Maryland 20205

Contributors vii

Enrique Jaimovich, Departamento de Fisiologia y Biofisica, Facultad de Medicina, Universidad de Chile, Santiago, Chile

Bruce K. Krueger, University of Maryland School of Medicine, Baltimore, Maryland 21201

Ramon Latorre, Departmento de Biologia, Facultad de Ciencias, Uni­versidad de Chile, Santiago, Chile; and Centro de Estudios Cientificos de Santiago, Santiago, Chile

Harold Lecar, Laboratory of Biophysics, National Institute of Neurolog­ical and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20205

Irwin B. Levitan, Graduate Department of Biochemistry, Brandeis Uni­versity, Waltham, Massachusetts 02254

A. Lievano, Departamento de Bioquimica, Centro de Investigaci6n y de Estudios Avanzados del I.P.N., Mexico, D.F. 07000, Mexico

Werner R. Loewenstein, Department of Physiology and Biophysics, Uni­versity of Miami School of Medicine, Miami, Florida 33101

M. Luxoro, Laboratorio de Fisiologia Celular, Facultades de Medicina y Ciencias, Universidad de Chile, Santiago, Chile

Christopher Miller, Graduate Department of Biochemistry, Brandeis U ni­versity, Waltham, Massachusetts 02254

Charles Mischke, Agricultural Research Service, Weed Science Labora­tory, Agricultural Environmental Quality Institute, Beltsville, Maryland 20705

Nava Moran, Laboratory of Biophysics, NINCDS, National Institutes of Health, Bethesda, Maryland 20205; present address: Department of Neu­robiology, The Weizmann Institute of Science, Center for Neurosciences and Behavioural Research, Rehovot, Israel 76100

V. Nassar-Gentina, Laboratorio de Fisiologia Celular, Facultades de Med­icina y Ciencias, Universidad de Chile, Santiago, Chile

Ana Lia Obaid, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Harvey B. Pollard, Laboratory of Cell Biology and Genetics, NIADDK, National Institutes of Health, Bethesda, Maryland 20205

Stanley I. Rapoport, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, Maryland 20892

John Rinzel, Mathematical Research Branch, NIADDK, National Insti­tutes of Health, Bethesda, Maryland 20205

viii Contributors

Eduardo Rojas, Laboratory of Cell Biology and Genetics, NIADDK, Na­tional Institutes of Health, Bethesda, Maryland 20205

Frederick Sachs, Department of Biophysics, State University of New York at Buffalo School of Medicine, Buffalo, New York 14214

Brian M. Salzberg, University of Pennsylvania, Philadelphia, Pennsylvania 19104

J. Sanchez, Departamentos de Bioquimica y Farmacologia, Centro de Investigacion y de Estudios Avanzados del I.P.N., Mexico, D.F. 07000, Mexico

Rosa Maria Santos, Laboratory of Cell Biology and Genetics, NIADDK, National Institutes of Health, Bethesda, Maryland 20205

Andres Stu[zin, Laboratory of Cell Biology and Genetics, NIADDK, Na­tional Institutes of Health, Bethesda, Maryland 20205

Benjamin A. Suarez-Isla, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, Maryland 20892; pres­ent address: Centro de Estudios Cientificos de Santiago, Santiago, Chile

Robert E. Taylor, Laboratory of Biophysics, NINCDS, National Institutes of Health, Bethesda, Maryland 20205

Cecilia Vergara, Departmento de Biologia, Facultad de Ciencias, Univ­ersidad de Chile, Santiago, Chile; and Centro de Estudios Cientificos de Santiagos, Santiago, Chile

Daniel Wolff, Departmento de Biologia, Facultad de Ciencias, Universidad de Chile, Santiago, Chile; and Centro de Estudios Cientificos de Santiago, Santiago, Chile

Jennings F. Worley, III, University of Maryland School of Medicine, Bal­timore, Maryland 21201

Preface

This book is based on a series of lectures for a course on ionic channels held in Santiago, Chile, on November 17-20, 1984. It is intended as a tutorial guide on the properties, function, modulation, and reconstitution of ionic channels, and it should be accessible to graduate students taking their first steps in this field.

In the presentation there has been a deliberate emphasis on the spe­cific methodologies used toward the understanding of the workings and function of channels. Thus, in the first section, we learn to "read" single­channel records: how to interpret them in the theoretical frame of kinetic models, which information can be extracted from gating currents in re­lation to the closing and opening processes, and how ion transport through an open channel can be explained in terms of fluctuating energy barriers. The importance of assessing unequivocally the origin and purity of mem­brane preparations and the use of membrane vesicles and optical tech­niques in the stUGY of ionic channels are also discussed in this section.

The patch-clamp technique has made it possible to study ion channels in a variety of different cells and tissues not amenable to more conven­tional electrophysiological methods. The second section, therefore, deals with the use of this technique in the characterization of ionic channels in different types of cells, ranging from plant protoplasts to photoreceptors. Several chapters on ionic channel reconstitution form part of the third section. Here we learn how this methodology has made it possible to

ix

x Preface

study the channels contained in membrane systems inaccessible to patch electrodes such as sarcoplasmic reticulum or muscle transverse tubules. Furthermore, single-channel recording in cells, in cell-free membrane patches, and in planar lipid bilayers allows us to reveal how the metabolic machinery of the cell and the cell-to-cell interaction regulates the behavior ofthese proteins. These subjects are covered in the fourth section. Finally, chapters on models, channel structure, and function form part of the last section.

I would like to thank Osvaldo Alvarez, Cecilia Hidalgo, Enrique Jaimovich, and Maria Luisa Valdovinos for their invaluable help in the organization of the course. The enormous enthusiasm put forward by the assisting students and professors provided a very pleasant and exciting environment that resulted in an excellent meeting. I would also like to thank the Tinker Foundation and the PNUD, UNESCO program for funding.

Ramon Latorre Santiago, Chile

Foreword

Robert E. Taylor

In the United States it is often asked why there are so many Chileans working on channels and on active transport. There are probably a number of reasons for this, but surely prominent among them is the existence of a small laboratory in Montemar, near Vifia del Mar, Chile. How did it start, what happened, and who did it?

It started because Mario Luxoro got his Ph.D. with Francis O. Schmitt at MIT in Cambridge, Massachusetts in 1957. Luxoro and Schmitt were interested in the axoplasm of the giant axon of the squid then available in Massachusetts. In 1955, Schmitt and friends had caught specimens of the large squid, Dosidicus gigas, off Iquique, in northern Chile. With the cooperation of Dr. P. Yanez, a unit was set up in the Estaci6n de Biologia Marina in Montemar for the procuring, processing, and shipping of chilled as well as frozen and dried axoplasm to Cambridge. On his return to Chile, Luxoro was involved in this arrangement. That year Eduardo Ro­jas, as a medical student, spent some time in Montemar, and Pancho Hunneus-Cox went to MIT for 2 years.

The idea of the facilities of the Estacion in Montemar being used as merely a source of axoplasm for someone at MIT was not too attractive to the Chilean scientists. Luxoro worked with Eduardo Rojas in the sum-

ROBERT E. TAYLOR. Laboratory of Biophysics, NINCDS, National Institutes of Health, Bethesda, Maryland 20205.

xi

xii Robert E. Taylor

mer of 1959-60 on the microinjection of trypsin into squid axons, Hun­neus-Cox returned in 1962 and worked in Montemar on squid, studying S-S bonds, and in 1963 Mitzy Canessa returned from postdoctoral work in the United States to become involved in studies of the biochemistry of the axon membrane with Sigmund Fischer. Fernando Vargas was there, and Ichichi Tasaki went to Montemar in 1964 and introduced internal perfusion with Luxoro.

In November of 1963, a group consisting of Clay Armstrong and Daniel Gilbert, from the Laboratory of Biophysics at the NIH in Bethesda, along with Clara Franzini-Armstrong, Rita Guttman, and Werner Loe­wensteinjoined Luxoro in Montemar for a few months. This arrangement grew out of discussions that Luxoro had had with Dr. Kenneth S. Cole, who wanted to take advantage of the availability of the large squid in Chile. Cole was not able to go, and the administration devolved on me. I went to Chile in February of 1966 and continued to spend part of each Chilean summer there until 1972. That is basically the reason why I am the one who is writing this.

By the summer of 1964-65, Eduardo Rojas had completed his Ph.D. in Chicago and was working at the NIH biophysics laboratory, and with Gerald Ehrenstein of that laboratory, he went to Montemar to perfuse the axon of the giant squid. About this time various problems arose be­tween the workers and the administration of the Estacion, and the Chan­cellor of the University of Chile provided some money to buy an old house across the street, and the Laboratorio de Fisiologia Celular was born; it was in operation when I first went there in 1966.

The many students who appeared in the lab in Montemar from time to time make an impressive list., The ones I got to know well include Ramon Latorre, Cecilia Hidalgo, Eugenia Yanez, Francisco Bezanilla, Julio Vergara, and Veronica Nassar. There were others, like F. Zambrano, Cristian Bennett, and many more after 1972.

It is important to remember that the axon of the giant squid, available only in Chile during the period of about 1957 to 1971, was of great im­portance to our understanding of channels. Not only was it a superb preparation, mainly because of the absence of branches, but it served to gather people together with common interests. Some people worked on the voltage-dependent ion movements, some on the biochemistry of the membrane, and some were, and still are, interested in the role of the axoplasm. We might recall that in the 1960s it was not known for sure that there were individual ionic channels and that they were composed of protein (or lipid-protein-carbohydrate complexes). Many people thought so, but the pioneering work of Luxoro and Hunneus-Cox and then Mitzy Canessa with Sigmund Fischer and later the work of Rojas, Armstrong,

Foreword xiii

and Atwater with internally perfused pronase was all important in focusing attention on the proteins.

These are only a few comments about this small but important lab­oratory. Perhaps someone will write a proper history that would include the work of Mario Luxoro, Veronica Nassar, Francisco Bezanilla, Julio Vergara, Juan Bacigalupo, Cecilia Vergara, Elizabeth Bosch, Rafael Torres, and Victor Corvalan since there have been no giant squid. Some of these people are still associated with this laboratory, and many are spread about the world, but the work goes on. Most of the great ideas are probably wrong, but by training students and fighting about the ideas, progress occurs.

Contents

Introduction.............................................................. 1

I. Methodologies

Chapter 1

Kinetic Models and Channel Fluctuations

Osvaldo Alvarez

1. Introduction ......................................................... 5 2. Two-State Channel .................................................. 5 3. Two Two-State Channels ........................................... 11 4. Three-State Channel with Three Conductances .................. 13 5. Three-State Channel with Only Two Conductances.............. 13

References ........................................................... 15

Chapter 2

Single-Channel Currents and Postsynaptic Drug Actions

HaroLd Lecar

1. Introduction ......................................................... 17 2. Channel Gating as a Stochastic Process ........................... 18

xv

xvi Contents

3. Postsynaptic Channels in the Presence of Drugs ................. 24 4. Reconstructing the Postsynaptic Current .......................... 30 5. Macroscopic and Molecular Consequences ....................... 33

References ........................................................... 34

Chapter 3

Voltage-Dependent Gating: Gating Current Measurement and Interpretation

Francisco Bezanilla

1. Introduction ......................................................... 37 2. Voltage Gating ...................................................... 37 3. Gating CurrentIs a Capacitive Current ........................... 38 4. Measurement of Gating Currents .................................. 39 5. Gating of the Sodium Channel ..................................... 45

References ........................................................... 51

Chapter 4

Characterizing the Electrical Behavior of an Open Channel via the Energy Profile for Ion Permeation: A Prototype Using a Fluctuating Barrier Model for the Acetylcholine Receptor Channel

George Eisenman and John A. Dani

1. Introduction ......................................................... 53 2. Theory ............................................................... 54 3. Confrontation with Experimental Data for the AChR Channel.. 63 4. Discussion ........................................................... 81

Appendix ............................................................ 83 References ........................................................... 85

Chapter 5

The Use of Specific Ligands to Study Sodium Channels in Muscle

Enrique Jaimovich

1. Introduction ......................................................... 89 2. Molecular Pharmacology of the Sodium Channel in Muscle ..... 90 3. Sodium Channel in Cardiac Muscle: Are All Sodium Channels

Alike? ................................................................ 93 4. Surface and Tubular Sodium Channels in Skeletal Muscle ...... 94

Contents

5. Models for Sodium Channels in Muscle Membranes References .......................................................... .

Chapter 6

Isolation of Muscle Membranes Containing Functional Ionic Channels

Cecilia Hidalgo

1. Introduction 2. 3. 4. 5.

Excitation-Contraction Coupling ................................. . Ionic Channels and E-C Coupling ................................ . Isolation of Muscle Membranes ................................... . Concluding Remarks ............................................... . References .......................................................... .

Chapter 7

Methodologies to Study Channel-Mediated Ion Fluxes in Membrane Vesicles

Ana Maria Garcia

xvii

96 98

101 101 102 105 120 120

1. Introduction ......................................................... 127 2. Channel-Mediated TI + Flux Measured by Fluorescence

Quenching ........................................................... 129 3. Channel-Mediated Ion Fluxes Measured by Light Scattering ... 136

References........................................................... 139

Chapter 8

Optical Studies on Ionic Channels in Intact Vertebrate Nerve Terminals

Brian M. Salzberg, Ana Lia Obaid, and Harold Gainer

1. Introduction ......................................................... 141 2. Equivalence of Optical and Electrical Measurements of

Membrane Potential ................................................ 142 3. Optical Recording of Action Potentials from Nerve Terminals

of the Frog Xenopus ................................................ 147 4. Properties of the Action Potential in the Nerve Terminals ...... 151 5. Ionic Basis of the Depolarizing Phase of the Action Potential .. 154 6. Concluding Remarks ................................................ 159

References ........................................................... 160

xviii

Chapter 9

Optical Detection of ATP Release from Stimulated Endocrine Cells: A Universal Marker of Exocytotic Secretion of Hormones

Eduardo Rojas, Rosa Maria Santos, Andres Stutzin, and Harvey B. Pollard

Contents

1. Introduction ......................................................... 163 2. Methodological Considerations .................................... 164 3. Acetylcholine-Induced ATP Release from Chromaffin Cells:

Calcium Dependence ............................................... 168 4. Nicotinic Receptor Desensitization ................................ 169 5. Granular Nature of the Secreted A TP ............................. 170 6. ATP Release Evoked by Membrane Depolarization Is Mediated

by Activation of Voltage-Gated Calcium Channels ............... 173 7. ATP Release from Collagenase-Isolated Islets of Langerhans ... 174 8. Conclusion ........................................................... 175 9. Summary ............................................................ 176

References ........................................................... 177

II. Channels in Biological Membranes

Chapter 10

Mechanotransducing Ion Channels

Frederick Sachs

1. Introduction ......................................................... 181 2. Recording SA Channels ............................................ 183 3. General Characteristics ............................................. 184 4. Conductance Properties ............................................ 185 5. Kinetic Properties................................................... 185 6. The Model ........................................................... 188 7. Comparing the Model to the Data ................................. 189 8. Future Prospects .................................................... 192

References........................................................... 192

Chapter 11

Ionic Channels in Plant Protoplasts

Nava Moran, Gerald Ehrenstein, Kunihiko Iwasa, Charles Bare, and Charles Mischke

1. Introduction ......................................................... 195 2. Some Methodological Considerations ............................. 197 3. Voltage-Dependent Channels Opened by Hyperpolarization .... 199

Contents xix

4. Channels Affected by TEA ......................................... 201 5. Conclusions ......................................................... 203

References ........................................................... 204

Chapter 12

Channels in Kidney Epithelial Cells

Sandra Guggino

1. Introduction ......................................................... 207 2. Cell Culture ......................................................... 209 3. Patch-Clamp Methodology ......................................... 209 4. Potassium Channel Characteristics ................................ 211 5. Channel Modulation ................................................ 215 6. Conclusions ......................................................... 215

References ........................................................... 218

Chapter 13

Channels in Photoreceptors

Juan Bacigalupo

1. Introduction ......................................................... 221 2. Vertebrate Photoreceptors ......................................... 223 3. Invertebrate Photoreceptors ........................................ 225

References ........................................................... 232

Chapter 14

Inactivation of Calcium Currents in Muscle Fibers from Balanus

M. Luxoro and V. Nassar-Gentina

1. Introduction ......................................................... 235 2. Methodological Considerations .................................... 236 3. Characteristics of Inward Currents ................................ 237 4. Mechanism of Inactivation ......................................... 239

References ........................................................... 241

Chapter 15

Electrophysiological Studies in Endocrine Cells

Clay M. Armstrong

1. Introduction ......................................................... 243

xx Contents

2. Whole-Cell Patch-Clamp Methodology ............................ 245 3. Cell Culture ......................................................... 247 4. Ionic Currents in GH3 Cells ....................................... 247 5. Characteristics of Calcium Channels .............................. 249 6. Conclusions .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 252

References ........................................................... 254

ITI. Ionic Channel Reconstitution

Chapter 16

Ion Channel Reconstitution: Why Bother?

Christopher Miller

1. Introduction and Background ...................................... 257 2. Unexpected Surprises .............................................. 263 3. Unconstrained Variables ........................................... 264 4. Unrealized Hopes ................................................... 267

References ........................................................... 269

Chapter 17

From Brain to Bilayer: Sodium Channels from Rat Neurons Incorporated into Planar Lipid Membranes

Robert J. French, Bruce K. Krueger, and Jennings F. Worley III

1. Perspectives and Background ...................................... 273 2. Electrophysiology without Cells ................................... 277 3. A Closer Look at Batrachotoxin-Activated Sodium Channels in

Bilayer Membranes ................................................. 280 4. Looking Ahead ...................................................... 287

References ........................................................... 288

Chapter 18

Ionic Channels in the Plasma Membrane of Sea Urchin Sperm

A. Darszon, J. Garda-Soto, A. Lievano, J. A. S{mchez, and A. D. Islas-Trejo

1. Introduction ......................................................... 291 2. Are There Channels in Sea Urchin Sperm? ....................... 293 3. Reconstitution Studies with Isolated Sea Urchin Sperm Plasma

Membrane ........................................................... 294

Contents xxi

4. Channels in the Plasma Membrane of Sea Urchin Sperm: Implications for the Acrosome Reaction .......................... 300

5. Are There Receptors to the Egg Jelly in the Sea Urchin Sperm Plasma Membranes? ................................................ 300

6. Perspectives ......................................................... 302 References ........................................................... 302

Chapter 19

Characterization of Large-Unitary-Conductance Calcium-Activated Potassium Channels in Planar Lipid Bilayers

Daniel Wolff, Cecilia Vergara, Ximena Cecchi, and Ramon Latorre

1. Introduction ........................ '................................. 307 2. Channel Gating ...................................................... 307 3. Channel Conductance and Selectivity ............................. 311 4. Conductance of the Calcium-Activated K + Channels ............ 312 5. Selectivity of the Ca-K Channels .................................. 313 6. Blockade of the Ca-K Channels ................................... 314 7. Conclusions ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 320

References ........................................................... 321

IV. Ionic Channel Modulation

Chapter 20

Metabolic Regulation of Ion Channels

Irwin B. Levitan

1. Introduction ......................................................... 325 2. Second Messengers ................................. .... . . . . . . . . . . . .. 327 3. Protein Phosphorylation ............................................ 329 4. Summary ............................................................ 331

References ........................................................... 332

Chapter 21

The Cell-to-Cell Membrane Channel: Its Regulation by Cellular Phosphorylation

Werner R. Loewenstein

1. Introduction ......................................................... 335

xxii Contents

2. The Cell-to-Cell Channels Are Up-Regulated by cAMP-Dependent Phosphorylation ................................ 338

3. The Cell-to-Cell Channels are Down-Regulated by Tyrosine Phosphorylation ..................................................... 344 References ........................................................... 349

Chapter 22

The fJ-Celi Bursting Pattern and Intracellular Calcium

IUani Atwater and John Rinzel

1. Introduction ......................................................... 353 2. Role of [Ca2+]i: Dependence on Glucose ......................... 354 3. A Biophysical/Mathematical Model.. ................ .............. 356 4. Burst Frequency Depends on the Ratio

[free Ca2+]/[total Cali .............................................. 359 5. Summary ............................................................ 361

References ........................................................... 361

Chapter 23

Neurotrophic Effects of in Vitro Innervation of Cultured Muscle Cells. Modulation of Ca2+ -Activated K+ Conductances

Benjamin A. Suarez-Isla and Stanley I. Rapoport

1. Introduction ......................................................... 363 2. Methodological Considerations .................................... 364 3. Innervation and Muscle Cell Electrical Activity .................. 367 4. Conclusions ......................................................... 380

References ........................................................... 381

V. Ionic Channel Structure, Functions, and Models

Chapter 24

Correlation of the Molecular Structure with Functional Properties of the Acetylcholine Receptor Protein

Francisco J. Barrantes

1. Introduction ......................................................... 385 2. The AChR Macromolecule ...................... .... .. .. .. .. .. .. ... 386 3. Arrangement of Subunits in the AChR Macromolecule .......... 387 4. The AChR Primary Structure, cDNA Recombinant Techniques,

and Modeling Receptor Structure .................................. 389

Contents xxiii

5. Immunochemistry of AChR and the Testing of Models .......... 392 6. Voltage-Gated and Agonist-Gated Channels: A Comparison .... 392 7. Dynamics of AChR and Lipids in the Membrane ................ 393 8. Acetylcholine-Receptor-Controlled Channel Properties .......... 395

References ........................................................... 397

Chapter 25

Amiloride-Sensitive Epithelial Sodium Channels Dale 1. Benos

1. Introduction ......................................................... 401 2. Amiloride-Sensitive N a + Transport Processes ................... 403 3. Characterization of Amiloride-Sensitive Na + Channels in

Intact Epithelia ...................................................... 405 4. Incorporation of Amiloride-Sensitive N a + Channels into

Planar Bilayers ...................................................... 412 5. Concluding Remarks ................................................ 416

References ........................................................... 417

Chapter 26

A Channel Model for Development of the Fertilization Membrane in Sea Urchin Eggs

Gerald Ehrenstein and Richard FitzHugh

1. Introduction ......................................................... 421 2. Processes Following Fertilization .................................. 421 3. Experimental Basis for Model ..................................... 422 4. Description of Model ............................................... 423 5. Equations of Model ................................................. 425 6. Solutions of Model Equations ...................................... 426

References ........................................................... 429

Index ..................................................................... 431