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