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Patch-Clamp Methods and Protocols METHODS in MOLECULAR BIOLOGYTM Patch-Clamp Methods and Protocols METHODS IN MOLECULAR BIOLOGYTM John M. Walker, SERIES EDITOR 288. Patch-Clamp Methods and Protocols, edited by Peter 265. RNA Interference, Editing, and Modification: Methods Molnar and James J. Hickman, 2007 and Protocols, edited by Jonatha M. Gott, 2004 399. Neuroprotection Methods and Protocols, edited by 264. Protein Arrays: Methods and Protocols, edited by Eric Tiziana Borsello, 2007 Fung, 2004 286. Transgenic Plants: Methods and Protocols, edited by 263. Flow Cytometry, Second Edition, edited by Teresa S. Leandro Peña, 2004 Hawley and Robert G. Hawley, 2004 285. Cell Cycle Control and Dysregulation 262. Genetic Recombination Protocols, edited by Alan S. Protocols: Cyclins, Cyclin-Dependent Kinases, and Waldman, 2004 Other Factors, edited by Antonio Giordano and Gaetano 261. Protein–Protein Interactions: Methods and Romano, 2004 Applications, edited by Haian Fu, 2004 284. Signal Transduction Protocols, Second Edition, edited 260. Mobile Genetic Elements: Protocols and Genomic by Robert C. Dickson and Michael D. Mendenhall, 2004 Applications, edited by Wolfgang J. Miller and Pierre 283. Biconjugation Protocols, edited by Christof M. Capy, 2004 Niemeyer, 2004 259. Receptor Signal Transduction Protocols, Second 282. Apoptosis Methods and Protocols, edited by Hugh J. Edition, edited by Gary B. Willars and R. A. John M. Brady, 2004 Challiss, 2004 281. Checkpoint Controls and Cancer, Volume 2: 258. Gene Expression Profiling: Methods and Protocols, Activation and Regulation Protocols, edited by Axel H. edited by Richard A. Shimkets, 2004 Schönthal, 2004 257. mRNA Processing and Metabolism: Methods and 280. Checkpoint Controls and Cancer, Volume 1: Reviews Protocols, edited by Daniel R. Schoenberg, 2004 and Model Systems, edited by Axel H. Schönthal, 2004 256. Bacterial Artifical Chromosomes, Volume 2: 279. Nitric Oxide Protocols, Second Edition, edited by Aviv Functional Studies, edited by Shaying Zhao and Marvin Hassid, 2004 Stodolsky, 2004 278. Protein NMR Techniques, Second Edition, edited by A. 255. Bacterial Artifical Chromosomes, Volume 1: Library Kristina Downing, 2004 Construction, Physical Mapping, and Sequencing, 277. Trinucleotide Repeat Protocols, edited by Yoshinori edited by Shaying Zhao and Marvin Stodolsky, Kohwi, 2004 2004 276. Capillary Electrophoresis of Proteins and Peptides, 254. Germ Cell Protocols, Volume 2: Molecular Embryo edited by Mark A. Strege and Avinash L. Lagu, 2004 Analysis, Live Imaging, Transgenesis, and Cloning, 275. Chemoinformatics, edited by Jürgen Bajorath, 2004 edited by Heide Schatten, 2004 274. Photosynthesis Research Protocols, edited by Robert 253. Germ Cell Protocols, Volume 1: Sperm and Oocyte Carpentier, 2004 Analysis, edited by Heide Schatten, 2004 273. Platelets and Megakaryocytes, Volume 2: Perspectives 252. Ribozymes and siRNA Protocols, Second Edition, and Techniques, edited by Jonathan M. Gibbins and edited by Mouldy Sioud, 2004 Martyn P. Mahaut- Smith, 2004 251. HPLC of Peptides and Proteins: Methods and 272. Platelets and Megakaryocytes, Volume 1: Functional Protocols, edited by Marie-Isabel Aguilar, 2004 Assays, edited by Jonathan M. Gibbins and Martyn P. 250. MAP Kinase Signaling Protocols, edited by Rony Mahaut-Smith, 2004 Seger, 2004 271. B Cell Protocols, edited by Hua Gu and Klaus 249. Cytokine Protocols, edited by Marc De Ley, 2004 Rajewsky, 2004 248. Antibody Engineering: Methods and Protocols, 270. Parasite Genomics Protocols, edited by Sara E. edited by Benny K. C. Lo, 2004 Melville, 2004 247. Drosophila Cytogenetics Protocols, edited by Daryl S. 269. Vaccina Virus and Poxvirology: Methods and Henderson, 2004 Protocols,edited by Stuart N. Isaacs, 2004 246. Gene Delivery to Mammalian Cells: Volume 2: Viral 268. Public Health Microbiology: Methods and Protocols, Gene Transfer Techniques, edited by William C. Heiser, edited by John F. T. Spencer and Alicia L. Ragout de 2004 Spencer, 2004 245. Gene Delivery to Mammalian Cells: Volume 1: 267. Recombinant Gene Expression: Reviews and Nonviral Gene Transfer Techniques, edited by William Protocols, Second Edition, edited by Paulina Balbas C. Heiser, 2004 and Argelia Johnson, 2004 244. Protein Purification Protocols, Second Edition, edited 266. Genomics, Proteomics, and Clinical Bacteriology: by Paul Cutler, 2004 Methods and Reviews, edited by 243. Chiral Separations: Methods and Protocols, edited by Neil Woodford and Alan Johnson, 2004 Gerald Gübitz and Martin G. Schmid, 2004 METHODS IN MOLECULAR BIOLOGYTM Patch-Clamp Methods and Protocols Edited by Peter Molnar and James J. Hickman ©2007 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com 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. Methods in Molecular BiologyTM is a trademark of The Humana Press Inc. All papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials Cover illustration: Production Editor: Jennifer Hackworth Cover design by For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected]; or visit our Website: www.humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $30 copy is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [1-58829-698-9/07 $30]. Printed in the United States of America. 10987654321 eISBN 978-1-59745-529-9 Library of Congress Control Number: 2007931074 Preface Patch-clamp electrophysiology became commonly used since its development by Bert Sakmann and Erwin Neher in 1981. The past two decades brought major advances not only in the patch-clamp technique but in the method’s extensive applications to solve diverse scientific problems. In addition to an increase in the number of people utilizing this technique, there has been a tremendous improvement in the range of sophisticated equipment, elevating this method from the level of “wizardry” to routine usage. Recently, with the emergence of automated/parallel patch clamp, a major reorganization has taken place in the pharmacological applications of this methodology. The goal in this volume was to summarize the typical patch-clamp appli- cations and to assist scientists in identifying problems, which could be best addressed by this technique. In addition, this volume will provide the step-by-step procedures to perform the experiments that will answer those questions. The experiments described in this book will require a basic level of electrophysiological training. Our intention was not to teach the reader patch- clamp instrumentation and technique, as several basic and advanced level books have already been published, but to help an experimenter safely and effec- tively venture into new areas of electrophysiology and to routinely use the patch-clamp technique to answer scientific questions. To enhance the enthusiasm of non-electrophysiologists toward this method, we have introduced several scientific problems as examples where combined techniques (e.g., electrophysiology + imaging or molecular biology) are most effective. Thus, encouraging scientists with diverse backgrounds to attempt to utilize patch-clamp electrophysiology. This book was organized by the major patch-clamp application areas, namely pharmacology, physiology, and biophysics. As there are strong overlaps in the techniques utilized in these application areas, we have discussed individual techniques where they are most commonly used. Thus, avoiding any unnec- essary repetition. The first chapter provides examples and step-by-step instructions on how to use whole-cell and single-channel patch-clamp methods for testing drugs in v vi Preface industrial settings. The reader will learn how to perform the electrophysiological method as well as gain insight into the drug-screening process and the standard procedures in using it for this application. A reader will also learn about how electrophysiology can be best utilized in drug research and development. The second part of this chapter provides an opportunity to compare the current automated patch-clamp systems for typical applications and thus filling a gap in this literature. The second chapter provides a wide selection of patch-clamp applica- tions in physiological studies. Emphasis was given to techniques where patch clamp was combined with other methodologies such as photostimu- lation, force measurement, polymerase chain reaction (PCR), cell patterning, or computer modeling. Also, in this chapter, two typical applications of the relatively new dynamic-clamp method are introduced. In addition, emphasis was given to the high diversity of cell types as
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