NEUROMETHODS 0 26 Patch-Clamp Applications and Protocols NEUROMETHODS 0 26 Patch-Clamp Applications and Protocols Edited by Alan A. Boulton Uniuersity of Saskatchewan, Saskatoon, Canada Glen B. Baker Unioersity of Alberta, Edmonton, Canada and Wolfgang Walz University of Saskatchewan, Saskatoon, Canada Humana Press * Totowa, New Jersey 0 1995 The Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored m a retrieval system, or transmitted in any form or by any means, electromc, mechanical, photocopying, ml- crofilming, recording, or otherwise without written permisslon from the Pubhsher. All authored papers, comments, opmions, conclusions, or recommendations are those of the author(s) and do not necessarily reflect the views of the publtsher This publication is prmted on acid-free paper. B ANSI 239.48-1984 (American National Standards Institute) Permanence of Paper for Prmted Library Materials. 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Printed in the United States of America, ISBN O-89603-311-2 ISSN 0893-2336 Preface to the Series When the President of Humana Press first suggested that a series on methods in the neurosciences might be useful, one of us (AAB) was quite skeptical; only after discussions with GBB and some searching both of memory and library shelves did it seem that perhaps the publisher was right. Although some excellent methods books had recently ap- peared, notably in neuroanatomy, it is a fact that there was a dearth in this particular field, a fact attested to by the alac- rity and enthusiasm with which most of the contributors to this series accepted our invitations and suggested additional topics and areas. After a somewhat hesitant start, essentially in the neurochemistry section, the series has grown and will encompass neurochemistry, neuropsychiatry, neurology, neuropathology, neurogenetics, neuroethology, molecular neurobiology, animal models of nervous disease, and no doubt many more “neuros.“Although we have tried to in- clude adequate methodological detail and in many cases de- tailed protocols, we have also tried to include wherever possible a short introductory review of the methods and/or related substances, comparisons with other methods, and the relationship of the substances being analyzed to neurologi- cal and psychiatric disorders. Recognizing our own limita- tions, we have invited a guest editor to join with us on most volumes in order to ensure complete coverage of the field. These editors will add their specialized knowledge and com- petencies. We anticipate that this series will fill a gap; we can only hope that it will be filled appropriately and with the right amount of expertise with respect to each method, substance or group of substances, and area treated. Alan A. Boulton Glen B. Baker V Preface E. Neher and B. Sakman were the first to monitor the opening and closing of single ion channels and membranes by conductance measurements. In 1976, they used firepolished micropipets with a tip diameter of 3-5 pm to record currents from a small patch of the membranbe of skel- etal muscles, thereby decreasing background membrane noise. In order to reduce the dominant source of background noise-the leakage shunt under the pipet rim between mem- brane and glass- the muscle membrane had to be treated enzymatically. Despite these early limitations, a new tech- nique was born -the patch-clamp technique. The final break- through came in 1981 when the same authors, in collaboration with 0. P. Hamill, A. Marty, and F. J. Sigworth, developed the gigaohm seal. Not only did this improve the quality of recordings, it was now possible to gently pull the membrane patch with the attached pipet off the cell and study its trapped ion channels in isolation. Another offshoot of the gigaohm seal technique was the whole-cell patch-clamp technique, in which the patch is ruptured without breaking the seal. This technique is really a sophisticated voltage-clamp technique and also allows for the altering of cytoplasmic constituents if the experimenter so wishes. The first part of Patch-Clamp Applications and Protocols presents modern developments associated with the technol- ogy of patch-clamp electrodes, of cell-free ion channel record- ing, and of the whole-cell patch-clamp technique. Chapters on recent offsprings of the patch-clamp method, such as the concentration clamp technique, the pressure clamp method, and the perfusion of patch-clamp electrodes, were written for this Neuromethods volume by authors who were intimately involved in their development. Two increasingly widely used methods are the loose-patch-clamp technique and the perfo- rated patch-clamp technique. An important addition is the vii Preface patch clamp technique in brain slices, developed by A. Konnerth et al., who wrote a dedicated chapter for the present book. Finally, molecular biological aspects of the patch-clamp technique are covered by two additional chapters, one on Xenopus oocyte microinjection and ion channel expression, and one on patch-clamp recording and RT-PCR on single cells. The patch-clamp method is certain to be refined further in the future, as new applications involving the manipula- tion of cellular constituents, molecular biological techniques, and the various imaging techniques emerge. Wolfgang Walz Contents Preface to the Series .................................................................................... V Preface ........................................................................................................... Vii List of Contributors ................................................................................ xvii Technology of Patch-Clamp Electrodes Richard A. Leuis and James L. Rae 1. Introduction .............................................................................................. 1 2. General Properties of Pipet Glass ........................................................ 3 3. Whole-Cell Pipet Properties: Practical Aspects ................................. 5 3.1. Choice of Glass ............................................................................... 5 3.2. Pulling Whole-Cell Electrodes .................................................... 7 3.3. Elastomer Coating Whole-Cell Electrodes ................................ 7 3.4. Firepolishing Whole-Cell Electrodes ......................................... 8 4. Patch Electrode Fabrication for Single-Channel Recording.. ........ 10 4.1. Choice of Glass ............................................................................. 10 4.2. Pulling Single-Channel Electrodes ........................................... 11 4.3. Coating Single-Channel Pipets with Elastomers ................... 11 4.4. Firepolishing Single-Channel Pipets ........................................ 13 4.5. Fabrication Methods Specific to Quartz .................................. 13 4.6. Low-Noise Recording ................................................................. 15 5. Noise Properties of Patch Pipets ........................................................ 17 5.1. Noise Contribution of the Pipet ................................................ 17 5.2. Thin-Film Noise ........................................................................... 19 5.3. Distributed RC Noise .................................................................. 21 5.4. Dielectric Noise ............................................................................ 24 5.5. R -C Noise .................................................................................... 28 5.6. Sialhoise ...................................................................................... 29 5.7. Summary of Plpet Noise Sources ............................................. 30 5.8. Noise Sources for Whole-Cell Voltage Clamping ................ .32 References ............................................................................................... 36 Whole-Cell Patch-Clamp Recordings Harald Son theimer 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . .. .*..*..*.....**..*.*.*.........* 37 2. Principles (Why Voltage Clamp?) .,..,.,.,,..,,.................,......*............... 38 3. Procedure and Techniques ,,......,,............*.... ..*.*......,..............*.*.......... 39 3.1. Pipets . .. .**.................*......*....... .,...,....,.................,.,..,,..................... 39 3.2, Electronic Components of a Setup ,.*..........*..............*....*....*.... 40 ix Con tents 3.3. Recording Configuration ........................................................... 42 3.4. Experimental Procedure ............................................................. 44 4. Data Evaluation and Analysis ............................................................ 46 4.1. Data Filtering/Conditioning, Acquisition, and Storage .... ..4 6 4.2. Leak Subtraction ....................................................................... ...4 9 4.3. Determination of Cell Capacitance ......................................... .52 4.4. Dissecting Current Components .............................................. 52 4.5. I-V Curves ..................................................................................... 57 4.6. Fitting of Time-Constants ......................................................... .63 4.7. Data Presentation.. ...................................................................... .64 5. Limitations, Pitfalls, and Errors .......................................................... 65 5.1. Series Resistance and Its Consequences .................................. 65 5.2. Voltage Clamp Errors ................................................................. 68 6. Special Applications ............................................................................. 70 7. Conclusions ............................................................................................ 72 Acknowledgments ................................................................................ 72 Recommended Readings ..................................................................... 72 References ............................................................................................... 73 Pressure/Patch-Clamp Methods Owen P. Hamill and Don W. McBride, Jr. 1. Introduction ............................................................................................ 75 2. General Cell-Attached Patch Recording Procedures.. ................... .76 3. Methods of Applying Suction ............................................................. 77 3.1. Steady-State Methods ................................................................. 77 3.2. Perturbation Methods ................................................................. 78 4. Properties of the Pressure Clamp ....................................................... 79 4.1. Stimulation Protocols ................................................................. .79 4.2. Speed of the Pressure Clamp.. .................................................. .81 4.3. Sensitivity and Noise of the Pressure Clamp ........................ .82 4.4. Range of the Pressure Clamp .................................................... 82 5. Applications of Pressure/Patch-Clamp Methods .......................... .83 5.1. Sealing Protocols and Determination of Functional Membrane-Cytoskeleton Interactions .................................... .83 5.2. Membrane Viscoelastic and Mechanical Properties ............. 84 5.3. Characterization of Mechano-Gated Channels ..................... .84 6. Conclusion .............................................................................................. 85 Acknowledgments ................................................................................ 85 References ............................................................................................... 85 Cell-Free Ion-Channel Recording C. G. Nichols, M. B. Cannell, and A. N. Lopatin 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..~............................. 89 2. Making an Inside-Out Membrane Patch: The Problem of Vesicle Formation .. .*..*........*.....*..*.........*...*....*... 90 Con tents xi 2.1. How to Tell When You Have a Vesicle ................................... 90 2.2. How to Deal with the Problem ................................................ 94 3. Analysis of Ion Channels in Cell-Free Patches: Dealing with the Problem of Channel “Rundown” ........................................ 95 3.1. Mechanisms of Rundown .......................................................... 97 3.2. Accounting for Rundown: Statistical Approaches fo I Analyzing Current Records ................................................. 98 4. Methods for Rapid Change of the Solution Bathing Cell-Free Membrane Patches .............................................................................. 101 4.1. M’ethods for Rapid Bulk Application of Solution ................ 102 4.2. Laminar Flow Methods of Separating Parallel Solutions . .103 4.3. Separation of Solutions Using an “Oil-Gate” ...................... .103 4.4. Separation of Solutions Using an “Air Gate” ..................... ..lO 6 5. Analysis of Responses to Rapid Concentration Changes.. ......... .107 5.1. Modeling the Pipet Geometry ................................................. 107 5.2. Time Course of Solution Change: The Effects of Pipet Geometry ...................................................................... 108 5.3. Experimental Measurement of Diffusion Delays ............... .109 5.4. Correcting for Diffusion Delays in Analysis of Concentration Jump Experiments ...................................... 112 5.5. Advantages and Disadvantages of the Analysis ................ -113 6. Twenty Eight Hints and Tips for Successful Cell-Free Ion-Channel Recording’ ...................................................................... 114 7. Concluding Remarks .......................................................................... 118 References ............................................................................................. 119 Perfusion of Patch Pipets John M. Tang, F. N. Quandt, and R. S. Eisenberg 1. Introduction .......................................................................................... 123 2. Methods ................................................................................................. 124 2.1. Patch-Clamp of Neuroblastoma Cells ................................... 124 2.2. In ternal Perfusion Technology ................................................ 125 3. Results ................................................................................................... 129 3.1. Time Course of Exchange of Internal Solution ................... .129 3.2. Efficiency of Exchange of Internal Solution ........................ .129 3.3. Selectivity of K Channels Measured by Reversal Potentials ............................................................... 131 3.4. Pharmacology of 4-Aminopyridine ....................................... 133 3.5. Parameters Controlling the Rate of Exchange of Solution ................................................................................... 134 4. Discussion ............................................................................................. 135 4.1. Applicability of the Technique.. ............................................. .135 4.2. Possible Problems ...................................................................... 137 4.3. Improvements ............................................................................ 138 References ............................................................................................. 139 xii Contents Concentration Clamp Techniques Norio A kaike 1. Introduction .......................................................................................... 141 2. Setup of Rapid Solution Change ...................................................... 142 3. Preparations ......................................................................................... 144 3.1. Whole-Cell Recording Mode ................................................... 144 3.2. Excised Cell Membranes .......................................................... 146 4. Kinetic Studies Using Concentration Clamp Technique ............ .146 4.1. Receptor-Mediated Ionic Currents ......................................... 146 4.2. Voltage-Dependent Ionic Currents ........................................ 147 4.3. Rapid Change of Physical Conditions ................................... 148 4.4. G-Protein Mediated Response ................................................. 150 4.5. Measurement of Ca2+ Release from Intracellular Ca2+ Store Sites ............................................................................ 151 5. Limitations ............................................................................................ 151 References ............................................................................................. 151 Perforated Patch-Clamp Techniques Wolfgang Walz 1. Introduction .......................................................................................... 155 2. Dialysis of Cytoplasm by the Patch Micropipet Filling Solution ................................................................................................. 155 3. Strategies Used to Prevent Dialysis ................................................. 158 3.1. Increase of Pipet Resistance ..................................................... 158 3.2. Addition of a Cytosolic Extract to the Pipet Solution ......... 159 3.3. Use of ATP .................................................................................. 160 3.4. Use of Polyene Antibiotics ....................................................... 160 4. Use of Nystatin .................................................................................... 160 4.1. Properties of Nystatin Pores .................................................... 160 4.2. Perforating the Patch Membrane with Nystatin ................. .162 4.3. Intrapipet Dialysis of Nystatin ............................................... .163 4.4. Composition of Pipet Solutions .............................................. 164 4.5. Detailed Protocol for Use of Nystatin .................................... 165 4.6. Use of a Nystatin-Fluoroscein Mixture ................................. 166 5. Use of Amphotericin B ....................................................................... 167 6. Special Application: The Perforated Vesicle .................................. 168 7. Conclusions .......................................................................................... 169 Acknowledgment ................................................................................ 170 References ............................................................................................. 170 The Loose Patch Voltage Clamp Technique J. H. Calduel and R. L. Milton 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 2. Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 2.1. Amplifier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174