Patch Clamping: An Introductory Guide to Patch Clamp Electrophysiology

ISBN-13: 9780471486855 / Angielski / Miękka / 2002 / 186 str.

Areles Molleman

Opis

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

Spis treści

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
2.3.3 Voltage clamp and current clamp 28
2.3.4 Introduction to patch clamp configurations
2.3.5 The equivalent circuit for the cell–attached patch configuration
2.3.6 The equivalent circuit for the whole–cell configuration 39
2.3.7 The equivalent circuit for the excised patch configurations

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
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
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

Blurb

Patch Clamping: An Introductory Guide to Patch Clamp Electrophysiology is a concise introduction to 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 fundamentals of the subject, platforms, equipment and environmental control. This self–contained guide provides everything a practising patch clamp electrophysiologist needs to know to master this important technique, including an overview of membrane biophysics, standard experimental design, data analysis and technical concerns.

Aimed at novice and intermediate researchers within neurobiology laboratories, this text will also be of interest to people new to this technique, in universities and research labs as well as research technicians. A useful reference for undergraduates specialising in neurophysiology, neuropharmacology and applied physiology.

Features:

• A self–contained guide introducing basic principles and practical applications
• A practical approach to the subject and written in an accessible non–technical style
• Extensive coverage of recording modes, analysis of results and numerous handy hints on how to avoid common pitfalls.