Table Of ContentThe Receptors
A COMPREHENSIVE TREATISE
Wl/ume 1
General Principles
and Procedures
The Receptors
A COMPREHENSIVE TREATISE
Series Editor for Volume 1:
R. D. 0 'Brien, Cornell University
Series Editor for succeeding volumes:
Palmer Taylor, University of California, San Diego
Volume 1: General Principles and Procedures
Edited by R. D. 0 'Brien
A Continuation Order Plan is available for this series. A continuation order will bring
delivery of each new volume immediately upon publication. Volumes are billed only upon
actual shipment. For further information please contact the publisher.
The Receptors
A COMPREHENSIVE TREATISE
Jtblume 1
General Principles
and Procedures
Edited by
R.D O'Brien
Cornell University
Ithaca, New York
PLENUM PRESS· NEW YORK AND LONDON
Library of Congress Cataloging in Publication Data
Main entry under title:
The Receptors.
Includes bibliographies and index.
CONTENTS: v. 1. General principles and procedures.
1. Cell receptors. I. O'Brien, Richard D.
QH603.C43R43 574.8'75 78-24366
ISBN-13: 978-1-4684-0981-9 e-ISBN-13: 978-1-4684-0979-6
DOl: 10.1007/978-1-4684-0979-6
© 1979 Plenum Press, New York
Softcover reprint of the hardcover 1st edition 1979
A Division of Plenum Publishing Corporation
227 West 17th Street, New York, N.Y. 10011
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
E. J. Ariens, Pharmacological Institute, University of Nijmegen, Nijmegen, The
Netherlands
Eric A. Barnard, Department of Biochemistry, Imperial College, London,
England
A. J. Beld, Pharmacological Institute, University of Nijmegen, Nijmegen, The
Netherlands
Robert Blumenthal, Laboratory of Theoretical Biology, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland
D. Colquhoun, Department of Pharmacology, St. George's Hospital Medical
School, London, England
Pedro Cuatrecasas, The Well come Research Laboratories, Burroughs Wellcome
Company, Research Triangle Park, North Carolina
A. De Lean, Medical Research Council of Canada; Endocrinology and Re
production Research Branch, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, Maryland
Morley D. Hollenberg, Division of Clinical Pharmacology, Departments of
Medicine and of Pharmacology and Experimental Therapeutics, The lohns
Hopkins University School of Medicine, Baltimore, Maryland
Christopher Miller, Graduate Department of Biochemistry, Brandeis University,
Waltham, Massachusetts
R. D. O'Brien, Section of Neurobiology and Behavior, Cornell University,
Ithaca, New York. Present affiliation: Provost, University of Rochester,
Rochester, New York
Efraim Racker, Section of Biochemistry, Molecular and Cell Biology, Cornell
University, Ithaca, New York
D. Rodbard, Endocrinology and Reproduction Research Branch, National In
stitute of Child Health and Human Development, National Institutes of
Health, Bethesda, Maryland
J. F. Rodrigues de Miranda, Pharmacological Institute, University of Nijmegen,
Nijmegen, The Netherlands
Adil E. Shamoo, Department of Radiation Biology and Biophysics, University
of Rochester School of Medicine and Dentistry, Rochester, New York
A. M. Simonis, Pharmacological Institute, University of Nijmegen, Nijmegen,
The Netherlands
v
Preface
The following remarks are intended to serve as an introduction to this particular
volume as well as to the whole series of volumes of which this is the first. The intent
of the series is to provide an authentic and relatively complete statement about
the status of our understanding of the receptors. The models we had in mind
while developing this series are The Enzymes, The Proteins, and comparable
groups of books. The receptors have received a degree of importance and richness
of understanding that makes them deserving of comprehensive and complete
coverage. The study of these molecules, which may well include such diverse
items as the receptors for hormones, neurohumors, pheromones, taste, and many
other chemical signals, have a great deal in common, so that the student of any
one of them will wish to know the status of research about the others. This com
monality is in part substantive, and in part practical and procedural.
Substantively, the receptors are all macromolecules whose function is to re
ceive some form of chemical signal and transduce it to a form which is usable by
the receiving cell. In this way, a chemical signal may lead to a neural response,
to the turning-on of a cell's chromosomes, or to the activation of some enzymic
apparatus to produce or release a substance. Because most of these processes
are noncatalytic, special techniques not previously commonplace in biochemistry
have been developed in order to study the receptors. Procedurally, then, there
are also a number of common themes. The special technique that is most widely
used is that of the binding of the signaling agent itself (hormone, neurohumor,
or pheromone) or some analog which may be agonistic or antagonistic, employed
as a probe to reveal the existence of the binding macromolecule. Another common
characteristic which sets such studies apart from classical biochemistry is the
concern to reconstitute some essential features of the intact system which were
necessarily lost when the cells were initially disrupted in order to release the
receptor.
The function of the first volume in the series is to focus on some of the
principles which have applicability to a great variety of receptor systems. In
every case the author has some particular interest in a relatively limited number
of receptor systems, but in every case he has been asked to discuss the subject
matter in a way which is perfectly general. It is hoped that the resultant volume
will be of interest to anyone working with any of the receptors and who is anxious
to get a broader view of the status of understanding developed elsewhere in the
vii
viii Preface
field. In addition, anyone contemplating the exploration of a new receptor should
find this an excellent place to begin the search, with the accumulated understanding
that has developed as many other investigators have faced the same fundamental
problems.
Each subsequent volume in the series will deal with a particular receptor
and will examine it from both the biochemical and physiological points of view.
The intent is that each volume will provide a definitive and authoritative descrip
tion of the status of our knowledge with respect to that particular receptor,
and thus provide a point of departure for those continuing exploration in this
area. For instance, the second volume will deal with the nicotinic receptor, and
one would like to think that anyone starting anew in the study of that receptor
would find that volume the obvious starting place to read. Taken as a whole, the
series of volumes should describe the totality of our understanding of the major
receptors.
It is simultaneously a joy and a concern that these volumes are being written
at the very time in which new understanding is growing daily. The problem which
is posed is the need to prepare volumes which are not only useful today but which
will be valuable references for many years to come. Thus we have sought to make
each volume not in any way like an "annual review" for the area, but rather
(in addition to being the most recent work in the area) a comprehensive account
of all that has been done up to the present time. The pleasurable feature of the
tremendous activity in the field is of course that one enjoys participating in a
search which is moving so fast and with such a high sense of excitement, and in
which the rate of publication is so large that a series of the present kind may well
be the only one that can save a researcher from being aware only of the research
within his own particular system. It is a hallmark of current research that, when
the time for exploration of a particular system falls due, the progress of under
standing is astonishingly fast. Consequently, we run the risk of laying down, as
in the fossil record, a series of outdated statements about each receptor which
will serve only as a reminder in the future of how partial our information was.
We hope to minimize this risk by selecting the timing of each volume (which we
anticipate will appear approximately annually) in such a way as to coincide with
the time of maximal elucidation of the particular receptor, knowing that the
frenzied activity in any particular system cannot last eternally. The success of this
endeavor cannot be measured until at least another decade has gone by. It is
our ambition that this series will then constitute a benchmark describing the new
understanding of macromolecules whose existence has been accepted for many
years. It was the early decades of the twentieth century in which the existence of
receptors for these chemical signals was postulated, but it is the closing years
of the century in which the actual composition and mechanism of their action
will be understood.
After completing this first volume, I accepted the position of Provost at the
University of Rochester, simultaneously ending my research career. The function
of the editor of this series is, above all, to be aware of areas of timeliness for the
preparation of volumes in this series and of the key individuals to be consulted
about the contents of each volume. Obviously, this is no task for an ex-researcher.
Fortunately, Dr. Palmer Taylor of the Division of Pharmacology of the School
Preface ix
of Medicine at the University of California, San Diego, has undertaken the re
sponsibility for continuing this series. His outstanding excellence as a researcher,
his experience as an editor, his breadth of knowledge, and his taste for excellence
surely guarantee the success of future volumes.
R. D. O'Brien
Ithaca
Contents
Chapter J
Reconstitution of Membrane Transport Functions
Christopher Miller and Efraim Racker
1. Introduction ..................... . . . . . . . . . . . . . . . . . . . . . . . 1
2. Reconstitution of Active and Passive Transport Systems .......... 2
3. General Techniques of Reconstitution ........................ 5
3.1. Liposomes: Test Tubes with a Difference. . . . . . . . . . . . . . . . . . . 5
3.1.1. Multilamellar Liposomes ......................... 5
3.1.2. Unilamellar Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.2. Methods for Inserting Proteins into Liposomes . . . . . . . . . . . . . . 6
3.2.1. Cholate Dialysis ................................ 6
3.2.2. Sonication..................................... 7
3.2.3. Incorporation .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.2.4. The Use of Superstable Membrane Proteins. . . . . . . . . . . 10
4. What We Can Learn from Reconstitution. . . . . . . . . . . . . . . . . . . . . . 10
4.1. Oxidative Phosphorylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.2. Ca2+ -ATPase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
4.3. (Na+ + K+)-ATPase . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . 13
4.4. Acetylcholine Receptor .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4.5. The Problem of Orientation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
5. Reconstitution in Planar Bilayer Membranes . . . . . . . . . . . . . . . . . . . 18
5.1. Sucrase-Isomaltase Complex. . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5.2. Acetylcholine Receptor in Planar Bilayers . . . . . . . . . . . . . . . . . . 20
5.3. Insertion of Whole Membrane Vesicles. . . . . . . . . . . . . . . . . . . . 21
5.4. Proton Pumps .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
References ..............................._ . . . . . . . . . . . . . . . . . . 23
Chapter 2
The Pharmacon-Receptor-Effector Concept: A Basisfor Understanding
the Transmission of Information in Biological Systems
E. J. Ariens, A. J. Beld, J. F. Rodrigues de Miranda, and A. M. Simonis
1. Introduction .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2. Biological Action ........................................ 34
3. Receptors and Receptor Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
xi
xii Contents
4. Pharmacon-Receptor Interaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
5. Spare Receptors ......................................... 40
6. Structure and Action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
7. Accessory Receptor Sites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 47
8. Steric Structure and Action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
9. Selectivity in Action ...................................... 57
10. Differentiation in Closely Related Receptor Types . . . . . . . . . . . . . . . 59
11. Receptor Binding and Receptor Isolation . . . . . . . . . . . . . . . . . . . . . . 64
12. Dualism in Receptors for Agonists and Their Competitive
Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
13. The Aggregation-Segregation Concept. . . . . . . . . . . . . . . . . . . . . . . . 67
14. Dual Receptor Model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
15. Combination of Pharmaca ................................. 71
16. The Slope of the Concentration-Effect Curves. . . . . . . . . . . . . . . . . . 77
17. The Allosteric Receptor Model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
18. Binding and Displacement on Two or More Independent Classes of
Receptor Sites .......................................... 83
19. Two-Site Model ......................................... 84
20. Reflection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
References ................................................. 86
Chapter 3
The Link between Drug Binding and Response: Theories and Observations
D. Colquhoun
1. The Response to Acetylcholine-Like Drugs. . . . . . . . . . . . . . . . . . . . . 93
1.1. Methods of Investigation of the Response . . . . . . . . . . . . . . . . . . 93
1.2. The Nature of the Response to Acetylcholine ............... 95
1.3. The Response-Concentration Curve at Equilibrium ....... . . . 95
1.4. The Kinetics of the Response. . . . . . . . . . . . . . . . . . . . . . . . . . .. 99
1.4.1. Relationship between Methods of Studying Kinetics. . . . . 99
1.4.2. Concentration-Jump Studies. . . . . . . . . . . . . . . . . . . . . .. 100
1.4.3. Fluctuation Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 102
1.4.4. Voltage-Jump Relaxation Studies. . . . . . . . . . . . . . . . . .. 106
1.5. Anesthetics, Local Anesthetics, and Channel Blocking ........ 107
2. The Binding of Drugs to Acetylcholine Receptors. . . . . . . . . . . . . . .. 110
2.1. Methods for Investigation of Binding ..................... 110
2.2. Binding at Equilibrium ................................ 111
2.2.1. Cooperativity in Binding. . . . . . . . . . . . . . . . . . . . . . . . .. 115
2.2.2. Is There a Single Sort of Binding Site? ............... 115
2.2.3. Binding to Junctional and Extrajunctional Receptors in
Muscle ....................................... 116
2.3. The Kinetics of Acetylcholine Binding. . . . . . . . . . . . . . . . . . . .. 117
3. The Link between Drug Binding and Response. . . . . . . . . . . . . . . . .. 118
3.1. What Should a Mechanism Explain? . . . . . . . . . . . . . . . . . . . . .. 118
3.2. Some Mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 119
