Contents 7
Preface 11
1 Introduction 13
1.1 Patch Clamping and its Context 13
2 Basic Theoretical Principles 17
2.1 Introduction to Membrane Biology 17
2.1.1 The plasma membrane and its ionic environment 17
2.1.2 Electrochemical gradients and the Nernst equation 19
2.1.3 Maintenance of ion gradients and the membrane potential 20
2.1.4 Ion channels 23
2.2 Electrical Properties of the Cell Membrane 25
2.2.1 Driving force and membrane resistance 25
2.2.2 Membrane capacitance 27
2.2.3 Consequences of membrane capacitance 28
2.2.4 An electronic model of the plasma membrane 29
2.3 Recording Modes and their Equivalent Circuits 30
2.3.1 The basics of equivalent circuits 30
2.3.2 Intracellular recording 35
2.3.3 Voltage clamp and current clamp 40
2.3.4 Introduction to patch clamp configurations 43
2.3.5 The equivalent circuit for the cell-attached patch configuration 49
2.3.6 The equivalent circuit for the whole-cell configuration 51
2.3.7 The equivalent circuit for the excised patch configurations 53
3 Requirements 55
3.1 The Platform 55
3.1.1 Stability: vibrations and drift 55
3.1.2 Where in the building should the set-up be placed? 56
3.1.3 Anti-vibration tables 57
3.2 Mechanics and Optics 59
3.2.1 The microscope 60
3.2.2 Micromanipulators 64
3.2.3 Pipette pressure 68
3.2.4 Baths and superfusion systems 69
3.3 Electrodes and Micropipettes 76
3.3.1 Solid\u2013liquid junction potentials and polarisation 77
3.3.2 The bath electrode 79
3.3.3 Micropipettes 79
3.3.4 Liquid junction potentials 86
3.4 Electronics 87
3.4.1 External noise and Faraday cages 88
3.4.2 Patch clamp amplifiers 93
3.4.3 Noise prevention and signal conditioning 96
3.4.4 Data acquisition and digitisation 102
3.4.5 Computers and software 105
4 The Practice of Patch Clamping 107
4.1 Preparing the Experiment and Making a Seal 107
4.1.1 Setting up 107
4.1.2 Bringing the pipette near the preparation 110
4.1.3 Making the seal 113
4.2 Whole-cell Modes 116
4.2.1 Conventional whole-cell recording 116
4.2.2 Perforated patch recording 120
4.3 Single-channel Modes 122
4.3.1 General notes 122
4.3.2 Cell-attached patch 124
4.3.3 Excised patches 125
5 Whole-cell Protocols and Data Analysis 127
5.1 Standard Cellular Parameters 127
5.2 Voltage-activated Currents 128
5.2.1 Introduction to pulse protocols 128
5.2.2 Signal conditioning and positive/negative subtraction 131
5.2.3 Space clamp artefacts 135
5.2.4 Isolation of a homogeneous population of channels 138
5.2.5 Current\u2013voltage relationships and reversal potential 139
5.2.6 Determination of relative permeabilities 143
5.2.7 Activation and inactivation studies 144
5.3 Non-voltage-activated Currents 149
5.3.1 Introduction to continuous recording 149
5.3.2 Determination of reversal potential using voltage ramps 150
6 Single-channel Protocols and Data Analysis 153
6.1 General Single-channel Practice and Analysis 153
6.1.1 Practical notes 153
6.1.2 Amplitude analysis 155
6.1.3 Event detection 160
6.1.4 Dwell time analysis 164
6.2 Continuous Recording of Single Channels 169
6.2.1 Data acquisition 169
6.2.2 Spontaneous activity 170
6.2.3 Receptor-induced activity 172
6.3 Study of Single-voltage-dependent Channels 172
6.3.1 Step protocols 172
6.3.2 Ramp protocols 174
6.3.3 Correlation with macro-currents 176
Further Reading 179
Index 183