Table Of ContentNicotinic Acetylcholine Receptor
Structure and Function
NATO ASI Series
Advanced Science Institutes Series
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H Cell Biology
Series H: Cell Biology Vol. 3
Nicotinic Acetylcholine Receptor
Structure and Function
Edited by
Alfred Maelicke
Max-Planck-Institut fOr Ernahrungsphysiologie
Rheinlanddamm 201, 0-4600 Dortmund
Springer-Verlag
Berlin Heidelberg New York London Paris Tokyo
Published in cooperation with NATO Scientific Affairs Divison
Proceedings of the NATO Advanced Research Workshop on Mechanism of Action of
the Nicotinic Acetylcholine Receptor held on the Island of Santorini, Greece, May 19-
23, 1986
ISBN-13 :978-3-642-71651-5 e-ISBN-13 :978-3-642-71649-2
001: 10.1007/978-3-642-71649-2
Library of Congress Cataloging-in-Publication Data. NATO Advanced Research Workshop on "Mechanism
of Action of the Nicotinic Acetylcholine Receptor" (1986: Thera Island, Greece) Nicotinic acetylcholine
receptor. (NATO ASI series. Series H, Cell biology; vol. 3) "Proceedings of the NATO Advanced Research
Workshop on Mechanism of Action of the Nicotinic Acetylcholine Receptor held on the Island of Santorini,
Greece, May 19-23, 1986"-T.p. verso. "Published in cooperation with NATO Scientific Affairs Division"
Includes indexes. 1. Acetylcholine-Receptors-Congresses. 2. Nicotine-Receptors-Congresses.
I. Maelicke, Alfred. II. Title. III. Series. [DNLM: 1. Receptors, Cholinergic-congresses. 2. Receptors,
Nicotinic-congresses. WL 102.8 N279n 1986] QP364.7.N36 1986 599'.0188 86-28047
ISBN-13:978-3-642-71644-7 (U.S.)
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© Springer-Verlag Berlin Heidelberg 1986
Soft cover reprint of the hardcover 1st edition 1986
2131/3140-543210
Contents
PREFACE IX
WORKSHOP PARTICIPANTS XII
CONFERENCE PHOTOGRAPHS XVI
I. Molecular structure
Three-Dimensional Structure of the Acetylcholine Receptor 1
A. Brisson
Ultrastructural Analysis of the Native Acetylcholine Receptor 7
M. Giersig, W. Kunath, H. Sack-Kongehl, and F. Hucho
Probing Acetylcholine Receptors with Monoclonal Antibodies 19
J. Lindstrom, M. Criado, H. Lam, D. Le Nguyen, M. Luther,
S. Ralston, J. Rivier, L. Swanson, P. Whiting, D. Berg,
M. Jacob, M. Smith, J. Stollberg, P. Sargent, and V. Sarin
Acetylcholine Receptor Conformation Probed by Subunit-Specific 35
Monoclonal Antibodies S.J. Tzartos, and A.A. Kordossi
What can we Learn About the Acetylcholine Receptor from Syn- 49
thetic Peptides? S. Fuchs, D. Neumann, and A.Safran
II. Biophysical and Bioche~ical Studies
of AChR Topography
Topography of the Acetylcholine Receptor as Revealed by Fluo- 61
rescence Energy Transfer P. Taylor, J. Herz, D. Johnson,
and R.D. Brown
Infrared Studies of the Acetylcholine Receptor Structure 75
C.T. O'Konski
Monoclonal Antibodies as Probes of Acetylcholine Receptor 83
Function A. Maelicke, D. Watters, and G. Fels
Antigenic Specificity of Monoclonal Antibodies Against 93
Torpedo 93 and Human Acetylcholine Receptor, and Use
of these Anti-bodies to Investigate Myasthenia gravis
A. Vincent, and H. Wood
VI
Tertiary Structure of the Nicotinic Acetylcholine Receptor 103
Probed by Photolabeling and Protein Chemistry
J. Giraudat, M. Dennis, T. Heidmann, J.P. Changeux,
H. Bisson, C. Montecucco, F. Kotzyba-Hibert, M. Goeldner,
C. Hirth, and J.Y. Chang
A Structural Model of the Ion Channel of the Nicotinic 115
Acetylcholine Receptor F. Hucho, W. Oberthilr,
F. Lottspeich, and B. Wittmann-Liebold
III. Ligand Binding, Ion Flu.x Stu.dies
and Heconstitu.tion
A General Treatment of Ligand Binding to the Acetylcholine 129
Receptor H. Prinz
Lipid-Protein Interactions and Acetylcholine Receptor Func- 147
tion in Reconstituted Membranes M.G. McNamee, T.M. Fong,
O.T. Jones, and J.P. Earnest
Regulation of Intercellular Signal Transmission. New 159
Approaches to Chemical Kinetic Measurements on ·Cell
Surfaces G.P. Hess
Hysteresis and Channel Properties of the Acetylcholine 177
Receptor from Torpedo Californica E. Neumann
IV. Electrophysiological Stu.dies
States of the Nicotinic Acetylcholine Receptor: Enumeration, 197
Characteristics and Structure D. Colquhoun and
D.C.Ogden
Function of Mammalian Nicotinic Acetylcholine Receptors 219
J.H. Steinbach, M.Covarrubias, S.M. Sine, and J. Steele
Single Channel Studies of Acetylcholine Receptors Covalent- 233
ly Alkylated with Acetylcholine D. Siemen, S. Hellmann,
and A. Mael i cke
Effects of Allosteric Ligands on the Gating of Single Chan- 243
nel Currents in BC3H-l Cells H.L. Papke, and H.E. Oswald
On the Role of Trophic Factors vs. Muscle Activity in the 259
Development of Acetylcholine-Receptor Ion Channels in Rat
Endplates H. Brenner
Molecular Electrophysiology of Cloned AChR Channels Expressed 263
in Xenopus Oocytes C. Methfessel
VII
V. AChH Associated Proteins
The Transglutaminase of Acetylcholine Receptor Rich Membranes 275
F. Hucho and G. Bandini
Association of the Postsynaptic 43K Protein with Sites of 281
Clustered and Diffusely-Distributed Acetylcholine Receptor
S.C. Froehner, W.J. LaRochelle, and A.A. Murnane
cAMP-Dependent Phosphorylation of the Nicotinic Acetylcholine 291
Receptor: Characterization of the Protein Kinase in Torpedo
Electric Organ, Lack of Correlation in Myotubes Between
Increased Intracellular Levels of cAMP and Influx of Mono
valent Ions, Block of Influx by Forskolin Independently of
cAMP Levels E. Heilbronn, H. Eriksson, and J. Haggblad
Common Antigenic Determinants Between Acetylcholine Receptor 305
and Protozoan Membranes K. Soteriadou, A. Tzinia, and
S. Tzartos
VI. Ganglionic and Brain AChH
Identification and Reconstitution of a Neuronal Acetylcholine 319
Receptor from Insects H. Breer, G. Hinz, U. Madler, and
W. Hanke
Studies on the Nicotinic Cholinergic Receptor of Sympathetic 333
Neurones P. Leprince
Radioligand Labelling qf Nicotinic Receptors in Mammalian 345
Brain P.B.S. Clarke
VII. Molecular Genetics
Molecular Biology of Muscle and Neural Acetylcholine Recep- 359
tors S. Heinemann, J. Boulter, J. Connolly, D. Goldman,
K. Evans, D. Treco, M. Ballivet, and J. Patrick
Molecular Biology of Nicotinic Acetylcholine Receptors from 389
Chicken Muscle and Brain E.A. Barnard, D.M.W. Beeson,
V.B. Cockcroft, M.G. Darlison, A.A. Hicks, S.G. Moss,
and M.D. Squire
Acetylcholine Receptor Related Genes Expressed in the Nervous 417
System P. Nef, C. Oneyser, T. Barkas, and M. Ballivet
Expression Cloning and Fusion Proteins as Tools to Study 423
Receptor Structure T. Barkas, A. Mauron, B. Roth,
C. Alliod, S.J. Tzartos, and M. Ballivet
V"I
Establishing a System for the Stable Expression of Torpedo 431
Acetylcholine Receptors T. Claudio
Characterization of the mRNA and the Gene of a Putative Neu- 437
ronal Nicotinic Acetylcholine Receptor Protein from
Drosophila E.D. Gundel finger, I. Hermans-Borgmeyer,
D. Zopf, E. Sawruk, and H. Betz
VIII. Structural Models
Review and Revision of Structural Models for the Trans- 447
membrane Portion of the Acetylcholine Receptor Channel
H.R. Guy
A Structural and Dynamic Model for the Nicotinic Acetyl- 465
choline Receptor E.M. Kosower
AUTHORS INDEX 485
SUBJECT INDEX 487
Preface
The most critical event in chemical cell to cell communications
is the conversion of the chemical signal into the primary
response of the receiving cell. In the case of cholinergic cell
to cell communication, i.e. in cholinergic nerve-nerve and nerve
muscle systems, a single molecule, namely the nicotinic acetyl
choline receptor is responsible for both signal reception and
primary response: Through binding of the neurotransmitter acetyl
choline released from the associated nerve ending, the receptor
receives the chemical message. Short-lived openings of the recep
tor-integral ion channel constitute the initial reaction to this
stimulation, i.e. the primary response. This integration of the
receiving and the responsive unit into a single protein structure
seems to be typical for transmi tter-acti vated ion channel pro
teins of many excitable cells including those of the central ner
vous system. Elucidation of the mechansim of action of the
acetylcholine receptor is of interest, therefore, not only for
cholinergic cell to cell communications but, even more impor
tantly, as a general model for the less accessible neurotransmit
ter receptors of the central nervous system.
While the basic reactions in the, course of cholinergic cell to
cell communication and the molecular components involved are
rather well understood, a molecular mechanism for cholinergic ex
ci tation has not yet been established. In view of the rapid
progress in the structural and functional elucidation of the re
ceptor, it therefore appeared timely to bring together exponents
of the different disciplines engaged in such studies and to
assess their results with the mechanism of function of the re
ceptor in mind. This aim was largely achieved with the Santorini
workshop. With a few notable exeptions, it was a representative
forum of the leading laboratories working on the AChR. The work
shop attempted - and I believe it was also successful - to pro
vide the desired assessment of the present state of ultrastruc
tural research, molecular biology, biochemistry, immunology and
electrophysiology of the AChR. Since the emphasis during the
workshop was placed on discussion of the material presented
x
these proceedings may serve as a workbook for the workshop par
t icipants. For those of the field who could not part icipate, it
may be a useful source of informat ion and reference. To secure
the timeliness of the proceedings, the contributors and the pub
lisher have made every effort to assemble the book without delay.
Meeting reports on the Workshop have appeared in the August
issues of Trends in the Pharmacological Sciences (by David
Colquhoun) and in Nachrichten aus Chemie und Technik (by Alfred
Maelicke).
In putting together this workshop, I received excellent advice
from many colleagues. The final result was a program of 35 spea
kers and a total attendance of 61 participants. Some of the
thoroughly pleasant atmosphere of the workshop is reflected in
the photographs printed on the following pages. A prerequisite
for the scientific depth of the meeting was this relaxed mood of
the participants all of whom thought that the Santorini workshop
may well be remembered as a cornerstone for future developments
in the field.
The workshop was made possible by an award from the NATO Special
Programme Panel on Cell-to-Cell Signals. This enabled us to cover
the meeting and travel costs of our invited speakers and to pro
vide needed support for other part icipants. Dr. Mario di Lullo
from the Scientific Affairs Division was instrumental in solving
the technical aspects of the award, and I wish to express my par
ticular esteem for this able science administrator whose sudden
death on June 24 has left the European scientific community in
mourning.
I selected the village of Oia as the location for the workshop
because it represents the credo of the is land' s dramat ic land
scape and, at the same time, offers the seclusion required for
undisturbed scientific discussions. The development of the neces
sary infra-structure for the workshop required many months of
active engagement on the part of my codirector Prof. Socrates
Tzartos from the Hellenic Pasteur Institute in Athens, Mrs. Irene
Sigala and Mrs. Evdoxia Nicolaidou from the Oia office of the
Greek Tourist Organisation (EDT), my good friend Stelios
