This volume describes a range of standard and novel methodological approaches used to probe ion channel function across different modalities. Chapters guide readers through methods and protocols from an introduction to the decades old patch clamp method for the ion channel neophyte to more complex, recent protocol advances, such as optogenetics. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Patch Clamp Electrophysiology: Methods and Protocols aims to be a reference guide for current and future ion channel physiologists.
Channels and transporters are multi-functional proteins that mediate substrate transport and signal transmission and simultaneously act as regulators for other proteins and biosensors for environmental materials. Patch clamping is an epoch-making technique that allows researchers to perform real-time measurements of electrogenic channel/transporter functions at the single/multiple molecular level. This book describes not only the conventional patch clamp techniques but also their newly developed variations or applications, such as perforated patch, slice patch, blind patch, in vivo patch, imaging patch, smart patch, and automated patch clamping. These patch clamp techniques are now essential and are extensively used across the life sciences and in related industries. With plain and practical descriptions of patch clamping and how to carry it out, especially for beginners, the book also shows how widely and exquisitely the patch clamp techniques can be applied by expert electrophysiologists. This work serves as a useful guide for young researchers and students in training and laboratory courses as well as for senior researchers who wish to extend their repertoire of techniques.
Since its inception, patch-clamp has continued to be widely considered the gold standard method to record ion channel activity. Patch-Clamp Methods and Protocols, Second Edition, provides a comprehensive collection of new techniques for the development of automated, high-throughput screening systems for pharmacological evaluation, the use of various patch-clamp configurations together with novel molecular biological and imaging methodologies and enhanced stimulation protocols and perfusion systems. Divided into sections on pharmacology, physiology and biophysics, the chapters cover methods to generate more physiologically relevant conditions for drug application and screening technologies, recently developed applications such as optogenetic stimulation, advances in whole-cell recordings in freely-moving animals and novel technologies to create custom microelectrodes designed for reducing the access resistance and improving the rate of molecular diffusion. Patch-clamp is an indispensable technique for conducting pharmacological, physiological and biophysical research aimed at understanding crucial aspects of cellular and network function. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Patch-Cla mp Methods and Protocols, Second Edition will provide a useful technical and methodological guide to diverse audiences of electrophysiologists, from students to experienced investigators.
Patch clamping is a widely applied electrophysiological technique for the study of ion channels; membrane proteins that regulate the flow of ions across cellular membranes and therefore influence the physiology of all cells. Patch Clamping aims to cover the basic principles and practical applications of this important technique. Starting with a review of the history of patch clamping, the text then goes on to cover the basic principles, platforms, equipment and environmental control, and will also include coverage of preparation types, recording modes and analysis of results. This book will explain the basic principles and practical application of patch clamp electrophysiology Written in a non-technical style to ensure its broad appeal to novice users Takes a practical approach This self-contained guide provides everything a practising patch clamp electrophysiologist needs to know to master this technique, including an overview of membrane biophysics, standard experimental design, data analysis, and technical concerns
Patch-Clamp Analysis: Advanced Techniques describes in reproducible detail all applications that involve patch pipet. Beginning with updated basic patch-clamp techniques, the book presents the newest developments, ranging from fast external solution switching and the switching of the pipet solution during recording, to the loose patch, the perforated patch, and the patch cram detection technique. The advanced techniques covered combine molecular biology and imaging to produce the patch pipet with RT-PCR and fluorometric techniques.
Patch Clamp Methods and Protocols surveys the typical patch clamp applications and advises scientists on identifying problems and selecting the best technique in each instance. The experiments described require a basic level of electrophysiological training and aid the researcher in pursuing new areas of electrophysiology and using the patch clamp technique effectively. Patch Clamp Methods and Protocols is divided into three sections that cover the major areas of patch clamp application: Pharmacology, Physiology, and Biophysics. The first section provides examples and step by step instructions on how to use whole-cell and single-channel patch clamp methods for testing drugs in industrial settings. The second section provides a wide selection of patch clamp applications in physiological studies. The last part focuses on the biophysical applications of the patch clamp method using single channel recordings or statistical analysis of whole-cell currents in order to obtain parameters that describe ion channel properties or transmitter release. Individual techniques are explored within the area that they are applied most often. Researchers will find Patch Clamp Methods and Protocols to be an invaluable aid in the design and execution of a wide variety of patch clamp experiments, both on their own and in conjunction with other state-of-the-art methodologies.
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 sk- 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 m- the muscle membrane had to be treated brane and gla- enzymatically. Despite these early limitations, a new te- nique was born -the patch-clamp technique. The final bre- 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 techn- ogy of patch-clamp electrodes, of cell-free ion channel reco- ing, and of the whole-cell patch-clamp technique.
This Methods in Molecular Biology book offers strategies and protocols for studying a large group of proteins that form ionic channels in the plasma membrane and intracellular membranes of cells. Includes step-by-step protocols, materials lists, tips and more."
Calcium Entry Channels in Non-Excitable Cells focuses on methods of investigating the structure and function of non-voltage gated calcium channels. Each chapter presents important discoveries in calcium entry pathways, specifically dealing with the molecular identification of store-operated calcium channels which were reviewed by earlier volumes in the Methods in Signal Transduction series. Crystallographic and pharmacological approaches to the study of calcium channels of epithelial cells are also discussed. Calcium ion is a messenger in most cell types. Whereas voltage gated calcium channels have been studied extensively, the non-voltage gated calcium entry channel genes have only been identified relatively recently. The book will fill this important niche.
Filling the gap created over the past five years, during which many new techniques have entered the market, this book is directed at both the new and the experienced ion channel researcher wishing to learn more about the considerations and methods for studying recombinant ion channels. These latest developments are covered here for the first time, contributed by editors and authors working for major pharmaceutical companies and who routinely apply these techniques in their daily work. The first three chapters cover the use of the Xenopus oocyte expression system for structure-function studies, from basic approaches for manipulating ion channel cDNAs to more specialized but powerful techniques. This is followed by reviews of strategies and methodologies available for expressing channels in mammalian cells and for their analysis by patch-clamp electrophysiology. Chapters 6 to 8 review the latest methodologies for ion channel drug discovery, including high throughput screening using fluorescence and luminescence, as well as automated planar array electrophysiology. The remaining two chapters focus on approaches for determining ion channel crystal structures and on computational approaches to understanding channel mechanisms at atomic resolution. Rather than provide detailed protocols, indicated by references in each chapter, the authors provide a comprehensive and easily accessible overview of the techniques involved, reviewing underlying principles and providing working guidelines as well as an understanding of the key theoretical and practical considerations associated with each topic. In each case, this practical advice is illustrated by real life examples, taken either from the author's own experience or from key examples in the literature, providing valuable practical hints not found elsewhere. The result is a compendium of practical ion channel information that will prove a valuable resource to academic and industrial workers alike.
