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Patch Clamping: an Introductory Guide to Patch Clamp Patch Clamping An Introductory Guide to Patch Clamp Electrophysiology Areles Molleman University of Hertfordshire, UK Patch Clamping Patch Clamping An Introductory Guide to Patch Clamp Electrophysiology Areles Molleman University of Hertfordshire, UK Copyright© 2003JohnWiley&SonsLtd,TheAtrium,SouthernGate,Chichester,WestSussex PO19 8SQ, England Telephone (+44) 1243 779777 Email (for orders and customer service enquiries): [email protected] Visit our Home Page on www.wileyeurope.com or www.wiley.com All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except under the terms of the Copyright, Designs and Patents Act 1988 or under the terms of a licence issued by the Copyright Licensing Agency Ltd, 90 Tottenham Court Road, London W1T 4LP, UK, without the permission in writing of the Publisher. Requests to the Publisher should be addressed to the Permissions Department, John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England, or emailed or [email protected], or faxed to (+44) 1243 770571. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the Publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. OtherWileyEditorialOffices John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA Jossey-Bass, 989 Market Street, San Francisco, CA 94103-1741, USA Wiley-VCH Verlag GmbH, Boschstr. 12, D-69469 Weinheim, Germany John Wiley & Sons Australia Ltd, 33 Park Road, Milton, Queensland 4064, Australia John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop #02-01, Jin Xing Distripark, Singapore 129809 John Wiley & Sons Canada Ltd, 22 Worcester Road, Etobicoke, Ontario, Canada M9W 1L1 Library of Congress Cataloging-in-Publication Data Molleman, Areles. Patch clamping : an introductory guide to patch clamp electrophysiology / Areles Molleman. p. cm. Includes bibliographical references and index. ISBN 0 471 48685 X 1. Patch-clamp techniques (Electrophysiology) I. Title. QP341.M684 2002 572’.437 — dc21 2002027413 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN 0 471 48685 X Paperback Typesetin11/13 ½ SabonbyKeytecTypesettingLtd Printed and bound in Great Britain by TJ International, Padstow, Cornwall This book is printed on acid-free paper responsibly manufactured from sustainable forestry, in which at least two trees are planted for each one used for paper production. Contents Preface ix 1 Introduction 1 1.1 Patch Clamping and its Context 1 2 Basic Theoretical Principles 5 2.1 Introduction to Membrane Biology 5 2.1.1 The plasma membrane and its ionic environment 5 2.1.2 Electrochemical gradients and the Nernst equation 7 2.1.3 Maintenance of ion gradients and the membrane potential 8 2.1.4 Ion channels 11 2.2 Electrical Properties of the Cell Membrane 13 2.2.1 Driving force and membrane resistance 13 2.2.2 Membrane capacitance 15 2.2.3 Consequences of membrane capacitance 16 2.2.4 An electronic model of the plasma membrane 17 2.3 Recording Modes and their Equivalent Circuits 18 2.3.1 The basics of equivalent circuits 18 2.3.2 Intracellular recording 23 2.3.3 Voltage clamp and current clamp 28 2.3.4 Introduction to patch clamp configurations 31 2.3.5 The equivalent circuit for the cell-attached patch configuration 37 2.3.6 The equivalent circuit for the whole-cell configuration 39 2.3.7 The equivalent circuit for the excised patch configurations 41 3 Requirements 43 3.1 The Platform 43 3.1.1 Stability: vibrations and drift 43 3.1.2 Where in the building should the set-up be placed? 44 3.1.3 Anti-vibration tables 45 3.2 Mechanics and Optics 47 3.2.1 The microscope 48 vi CONTENTS 3.2.2 Micromanipulators 52 3.2.3 Pipette pressure 56 3.2.4 Baths and superfusion systems 57 3.3 Electrodes and Micropipettes 64 3.3.1 Solid–liquid junction potentials and polarisation 65 3.3.2 The bath electrode 67 3.3.3 Micropipettes 67 3.3.4 Liquid junction potentials 74 3.4 Electronics 75 3.4.1 External noise and Faraday cages 76 3.4.2 Patch clamp amplifiers 81 3.4.3 Noise prevention and signal conditioning 84 3.4.4 Data acquisition and digitisation 90 3.4.5 Computers and software 93 4 The Practice of Patch Clamping 95 4.1 Preparing the Experiment and Making a Seal 95 4.1.1 Setting up 95 4.1.2 Bringing the pipette near the preparation 98 4.1.3 Making the seal 101 4.2 Whole-cell Modes 104 4.2.1 Conventional whole-cell recording 104 4.2.2 Perforated patch recording 108 4.3 Single-channel Modes 110 4.3.1 General notes 110 4.3.2 Cell-attached patch 112 4.3.3 Excised patches 113 5 Whole-cell Protocols and Data Analysis 115 5.1 Standard Cellular Parameters 115 5.2 Voltage-activated Currents 116 5.2.1 Introduction to pulse protocols 116 5.2.2 Signal conditioning and positive/negative subtraction 119 5.2.3 Space clamp artefacts 123 5.2.4 Isolation of a homogeneous population of channels 126 5.2.5 Current–voltage relationships and reversal potential 127 5.2.6 Determination of relative permeabilities 131 5.2.7 Activation and inactivation studies 132 5.3 Non-voltage-activated Currents 137 5.3.1 Introduction to continuous recording 137 5.3.2 Determination of reversal potential using voltage ramps 138 6 Single-channel Protocols and Data Analysis 141 6.1 General Single-channel Practice and Analysis 141 6.1.1 Practical notes 141 6.1.2 Amplitude analysis 143 6.1.3 Event detection 148 vii 6.1.4 Dwell time analysis 152 6.2 Continuous Recording of Single Channels 157 6.2.1 Data acquisition 157 6.2.2 Spontaneous activity 158 6.2.3 Receptor-induced activity 160 6.3 Study of Single-voltage-dependent Channels 160 6.3.1 Step protocols 160 6.3.2 Ramp protocols 162 6.3.3 Correlation with macro-currents 164 Further Reading 167 Index 171 Preface Since the advent of patch clamp electrophysiology at the end of the 1970s, there has been an ever-increasing interest in the application of its power to investigate ion-channel-related bioscientific questions in unprecedented detail. Whereas patch clamping was pioneered in specialist biophysical laboratories,latertherewasanexpansiontomanyotherfieldsinbiology, as well as to basic research in medicine and related areas. This has not occurred without problems: patch clamping was developed by biologists who were strongly orientated towards physics and who were collaborating closely with engineers and physicists. In the expansion to traditionally less- biophysical fi elds it is very apparent that concepts and methods in patch clamping are fundamentally different from the mostly chemistry-based work that is performed there. Many undergraduate biosciences pro- grammes have been slow to react to this situation, sometimes owing to the lack of experienced lecturers. Even if expert teachers are available, the absence of an appropriate textbook makes it hard for the teachers to communicatethebasicconcepts.Consequently,itisoftendifficultfor researchers to recruit workers with the appropriate training to perform patch clamping. At the same time researchers who are considering using patchclampingintheirworkcanfindithardtoevaluatetheimplications if they lack training and experience themselves. Textbooks on patch clamping are too general for practical laboratory use or are written for experienced biophysicists. Most of them also have a format in which each chapter is written by different authors who write about their respective specialist areas, which inevitably means that the books lack continuity if not coherence. This book is intended to provide, in a single coherent volume, the basic knowledge and an overview of the materials, skills and procedures required to start patch clamping success- fully. The book is written for those who are novices at patch clamping but have some background in Biology or Medical Sciences, be they final-year x PREFACE undergraduate or MSc/PhD students, post-doctoral researchers or estab- lished workers from other fields who may require help in the concepts of electronics, for example. A portion of the book is therefore spent on this subject with the emphasis on relevance to membrane biology. The book also should be useful as a basic method reference, particularly in relation to data acquisition protocols and analysis. One of the developments since the expansion of patch clamping into a wider field has been that equipment and software have become much more commercially available and ‘user-friendly’. The unfortunate side-effect of this has been that it has become easier to work with the equipment, obtain data and have them analysed automatically without the experimenter evaluating the validity of the results or the analysis. Although this is a problem with all automated systems, from statistics software to nuclear magnetic resonance spectral analysis, in patch clamping there are many stages in which input (judgement) from the experimenter is required for proper application of the equipment and analysis techniques. The right input comes with experience and appreciation of the experimental situa- tion. This book is meant to provide the start of that appreciation. The book is not a dictionary of patch clamping. The main fundamentals are touched upon, but the level of discussion is measured to convey the concepts in enough depth to apply in practice. There should then be enough understanding to provide access to specialist literature if required. A selection of this literature is listed in a ‘Further Reading’ section.
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