Single-Channel Recording

Single-channel recording has become a widely used tool for the study of ion permeation mechanisms in biological membranes. Whereas the technique might have been considered an "art" after its introduction in 1976, it developed into a relatively simple method after it became possible to obtain high-resistance (several gigaohm) membrane-pipette seals. In the summer of 1982, a course on the technique was held at the Ettore Majorana Center for Scientific Culture in Erice, Sicily. It brought together people from most of the laboratories involved in patch clamping at that time. During the course, it became apparent that the technique had reached a state of maturity. Repeatedly, the opinion was expressed that a detailed description of all the aspects of the technique including representative examples of results should be available. We therefore asked the course instructors, as well as several other colleagues, to provide chapters on selected topics in order to produce this volume. The different variants of patch clamping were described quite extensively in an article by Hamill, Marty, Neher, Sakmann, and Sigworth (Pflugers Archiv 391:85) in 1981. Rather than repeating this survey in an introductory chapter, we chose to reprint that article in the Appendix of this volume (by permission of Springer-Verlag). The methods section will, therefore, go straight into detailed aspects of the technol­ ogy.

I. Methods.- 1 Electronic Design of the Patch Clamp.- 1. Introduction.- 2. Current-Measurement Circuitry.- 2.1. Current-Voltage Converter.- 2.2. Dynamics of the I–V Converter.- 2.3. Correcting the Frequency Response.- 3. Background Noise in the Current-Voltage Converter.- 3.1. Noise in the Feedback Resistor.- 3.2. Noise in the Amplifier.- 3.3. Example of a Low-Noise Amplifier Design.- 3.4. Summary of Noise Sources.- 4. Capacitance Transient Cancellation.- 4.1. Overload Effects in the Patch Clamp.- 4.2. Fast Transient Cancellation.- 4.3. Slow Transient Cancellation.- 5. Series Resistance Compensation.- 5.1. Theory.- 5.2. Effect of Fast Transient Cancellation.- 5.3. Incorporating Slow Transient Cancellation.- 5.4. A High-Speed, “Chopping” Voltage Clamp.- References.- 2 Geometric Parameters of Pipettes and Membrane Patches.- 1. Introduction.- 2. Geometry of Patch Pipettes.- 2.1. Tip Shape of Soft Glass Pipettes.- 2.2. Tip Shape of Hard Glass Pipettes.- 2.3. Tip Shape of Thick-Walled Pipettes.- 3. Geometry of Membrane Patches.- 3.1. Patch Area by Observation in the Light Microscope.- 3.2. Patch Area as Measured by Patch Capacitance.- 4. Conclusions.- References.- 3 Science and Technology of Patch-Recording Electrodes.- 1. Introduction.- 2. Science.- 2.1. Glass Structure.- 2.2. Membrane Structure.- 2.3. Glass-Membrane Interactions.- 3. Technology.- 3.1. Choice of Glass.- 3.2. Pulling.- 3.3. Coating.- 3.4. Polishing.- 3.5. Filling.- References.- 4 Enzymatic Dispersion of Heart and Other Tissues.- 1. Introduction.- 2. Methods.- 2.1. Outline of Dissociation Processes.- 2.2. Access of the Solution to the Tissue.- 2.3. Enzymes.- 2.4. Calcium.- 2.5. Tests of Viability.- 3. Mammalian Heart.- 3.1. Dissociation Techniques.- 3.2. Gigaseals.- References.- 5 A Primer in Cell Culture for Patchologists.- 1. Introduction.- 2. The Spectrum of Cell Cultures.- 2.1. Primary Cultures.- 2.2. Cell Lines.- 3. Some Methodological Considerations.- 3.1. Primary Cell Cultures.- 3.2. Culture Milieu.- References.- 6 Patch-Clamped Liposomes: Recording Reconstituted Ion Channels.- 1. Introduction.- 2. Small Unilamellar Vesicles and Recording Accessibility.- 3. Large Liposomes from Small Unilamellar Vesicles.- 4. Gigaseals and Isolated Patches with Freeze-Thaw Liposomes.- 5. Reconstituted AChR and Chloride Channels from Torpedo Electroplax.- 6. Conclusions.- References.- 7 Tight-Seal Whole-Cell Recording.- 1. Introduction.- 2. Procedures and Techniques.- 2.1. General Description of the Method.- 2.2. Pipettes.- 2.3. Electronics.- 2.4. Cell Capacitive Current.- 2.5. Solutions for Filling Whole-Cell Pipettes.- 3. Evaluation of the Whole-Cell Clamp.- 3.1. Ease of Penetration.- 3.2. The Equivalent Circuit.- 3.3. Cell Size and Quality of Clamp.- 3.4. Exchange of Cell Content with Pipette Solution.- 3.5. Junction Potential Drift Caused by the Loss of Cell Constituents...- 3.6. Modification of Channels following the Loss of Cell Constituents..- 3.7. Background Noise of a Whole-Cell Voltage-Clamp Measurement.. 117 4. Conclusion.- References.- 8 The Loose Patch Clamp.- 1. Introduction.- 2. Setup.- 2.1. Pipettes.- 2.2. Principle of Method and Recording Circuitry.- 2.3. Main Amplifier.- 2.4. Series Resistance Correction.- 2.5. Digital Hardware.- 3. Some Examples and Applications.- 3.1. Potential Control.- 3.2. Some Further Applications.- 3.3. Limitations of the Method.- References.- II. Concepts and Analysis.- 9 The Principles of the Stochastic Interpretation of Ion-Channel Mechanisms.- 1. The Nature of the Problem.- 1.1. Reaction Mechanisms and Rates.- 1.2. Rate Constant and Probabilities.- 2. Probabilities and Conditional Probabilities.- 3. The Distribution of Random Time Intervals.- 3.1. Another Approach to the Exponential Distribution.- 3.2. Generalization.- 3.3. Relationship between Single-Channel Events and Whole-Cell Currents.- 3.4. The Length of Time Spent in a Set of States.- 4. A Mechanism with More Than One Shut State: The Simple Open Ion Channel-Block Mechanism.- 4.1. A Simple Ion Channel-Block Mechanism.- 4.2. Relaxation and Noise.- 4.3. Open Lifetimes of Single Channels.- 4.4. Shut Lifetimes of Single Channels.- 4.5. Bursts of Openings.- 4.6. Lifetime of Various States and Compound States.- 4.7. Derivation of Burst Length Distribution for Channel-Block Mechanism.- 5. A Simple Agonist Mechanism.- 6. Some Fallacies and Paradoxes.- 6.1. The Waiting Time Paradox.- 6.2. The Unblocked Channel Fallacy.- 6.3. The Last Opening of a Burst Fallacy.- 6.4. The Total Open Time per Burst Paradox.- 7. Reversible and Irreversible Mechanisms.- 7.1. A Simple Example.- 7.2. Distribution of the Lifetime of an Opening.- 7.3. Probabilities of Particular Sequences of Transitions When the Open States are Distinguishable.- 8. The Problem of the Number of Channels.- 8.1. Estimation of the Number of Channels.- 8.2. Evidence for the Presence of Only One Channel.- 8.3. Use of Shut Periods within Bursts.- 9. Distribution of the Sum of Two Random Intervals.- 10. A More General Approach to the Analysis of Single-Channel Behavior...- 10.1. Specification of Transition Rates.- 10.2. Derivation of Probabilities.- 10.3. The Open Time and Other Distributions.- 10.4. A General Approach to Bursts of Ion-Channel Openings.- 10.5. Some Conclusions from the General Treatment.- References.- 10 Conformational Transitions of Ionic Channels.- 1. Introduction.- 2. Two-State Channel with a Single Binding Site.- 2.1. Concentration Dependence of Conductance.- 2.2. Carrierlike Behavior of Channels.- 2.3. Single-Channel Currents with Rectifying Behavior.- 3. Nonequilibrium Distribution of Long-Lived Channel States.- 4. Current Noise in Open Channels.- 5. Conclusion.- References.- 11 Fitting and Statistical Analysis of Single-Channel Records.- 1. Introduction.- 2. Acquiring Data.- 2.1. Transient Recorders.- 2.2. Computer On Line or from Magnetic Tape.- 2.3. Filtering the Data.- 2.4. Digitizing the Data.- 3. Finding Channel Events.- 3.1. Description of the Problem.- 3.2. Choosing the Filter Characteristics.- 3.3. Setting the Threshold.- 3.4. Practical Event Detection.- 4. Characterizing Single-Channel Events.- 4.1. Direct Fitting of the Current Time Course.- 4.2. Half-Amplitude Threshold Analysis.- 4.3. Event Characterization Using a Computer.- 5. The Display of Distributions.- 5.1. Histograms and Probability Density Functions.- 5.2. Missed Brief Events: Imposition of a Consistent Time Relationship.- 5.3. The Amplitude Distribution.- 5.4. The Open and Shut Lifetime Distributions.- 5.5. Burst Distributions.- 5.6. Cluster Distributions.- 6. The Fitting of Distributions.- 6.1. The Nature of the Problem.- 6.2. Criteria for the Best Fit.- 6.3. Optimizing Methods.- 6.4. The Minimum ?2 Method.- 6.5. The Method of Maximum Likelihood: Background.- 6.6. Maximum Likelihood for a Simple Exponential Distribution.- 6.7. Errors of Estimates: The Simple Exponential Case.- 6.8. Maximum Likelihood Estimates: The General Case.- 6.9. Errors of Estimation in the General Case.- 6.10. Numerical Example of Fitting.- 6.11. Effects of Limited Time Resolution.- Appendix: Some Numerical Techniques for Single-Channel Analysis.- References.- 12 Automated Analysis of Single-Channel Records.- 1. Introduction.- 2. Levels of Analysis.- 2.1. Global Methods of Analysis.- 2.2. Empirical Methods of Analysis.- 3. A Heuristic Approach to Channel Detection.- 3.1. Base-Line Restoration.- 3.2. Finding Background Noise Variance.- 3.3. Frequency Response.- 3.4. Detection Schemes.- 3.5. Validation of Events.- 3.6. Outputs.- 3.7. Two-Pass Processing.- 3.8. Convenience Features.- References.- 13 Analysis of Nonstationary Channel Kinetics.- 1. Introduction.- 2. A Nonstationary Process Has Occupancy Probabilities That Change with Time.- 3. Relaxation of Current after a Voltage-Clamp Step Is a Nonstationary Process.- 4. An Ensemble Is a Set of Identical Experiments.- 5. Ensemble Averaging Gives the Time-Dependent Probability of a Channel Being Open.- 6. Why Use Single-Channel Records?.- 6.1. Single-Channel Recording Avoids Some Artifacts of Macroscopic Current Recording.- 6.2. Single-Channel Statistics Provide Further Bases for Testing Channel Models.- 6.3. Multiple Channels in a Patch Reduce the Amount of Information Available.- 6.4. Conditional Averaging Correlates Channel Behavior with Past or Future Channel Behavior.- 7. Conclusion.- References.- 14 An Example of Analysis.- 1. Introduction.- 2. The Computer Programs.- 2.1. CATCH: An Event-Catching Program.- 2.2. THAC: Threshold Analysis of Continuous Records.- 2.3. LHI: Histogram and Statistical Analysis Program.- 3. Description of the Data.- 4. Statistical Analysis.- 4.1. Event Characterization.- 4.2. Amplitude Distribution.- 4.3. Open-Time Distributions.- 4.4. Closed-Time Distribution.- 4.5. Burst Kinetics.- References.- 15 Membrane Current and Membrane Potential from Single-Channel Kinetics.- 1. Introduction.- 2. Probabilistic Interpretation of Hodgkin-Huxley Kinetics.- 3. Gate Kinetics.- 4. Channel Kinetics.- 4.1. First Method.- 4.2. Second Method.- 4.3. Flickering.- 5. Results.- 5.1. Step Voltage Clamp.- 5.2. Arbitrary Voltage Clamp.- 5.3. Undamped Membrane.- 6. Summary.- References.- III. Patch Clamp Data.- 16 Bursts of Openings in Transmitter-Activated Ion Channels.- 1. Introduction.- 1.1. Background.- 1.2. Interpretations of Fluctuation and Relaxation Experiments.- 2. Observation of Bursts of Single-Channel Openings.- 2.1. Are the Shut Times Exponentially Distributed?.- 2.2. Bursts on a Slow Time Scale.- 2.3. Bursts on a Fast Time Scale.- 3. Properties of Nachschlag Bursts.- 3.1. Fitting of Apparently Incomplete Channel Closures.- 3.2. Complete or Partial Channel Closure?.- 3.3. Effect of Agonist Concentration and Membrane Potential on Nachschlag Bursts.- 3.4. Dependence of the Nachschlag Phenomenon on the Nature of the Agonist.- 3.5. The Burst-Length Distribution.- 4. Definition and Interpretation of Bursts.- 4.1. What Do We Mean by a Burst?.- 4.2. Some Interpretations of the Experimental Observations.- 4.3. Criteria for an Efficient Fast Transmitter.- References.- 17 Is the Acetylcholine Receptor a Unit-Conductance Channel?.- 1. Introduction.- 2. What Is an Acetylcholine Receptor?.- 3. Multiple Conducting States in Rat Muscle Tissue Culture.- 4. Multiple Conducting States in Chick Muscle Culture.- 5. Multiple Conducting States of the AChR in Other Cell Types.- 6. Other Open States of the AChR.- 7. Subconductance States of other Biological Channels.- 8. Multiple Conducting States of Model Systems.- 9. Implications of Subconductance States for Channel Modeling.- 10. Summary.- References.- 18 Analysis of Single-Channel Data from Glutamate Receptor-Channel Complexes on Locust Muscle.- 1. Introduction.- 2. Gating Properties of Single-Channel Currents.- 3. Nonrandom Activation.- 4. Agonist Dependence.- 5. A Kinetic Model.- References.- 19 Experimental Approaches Used to Examine Single Glutamate-Receptor Ion Channels in Locust Muscle Fibers.- 1. Introduction.- 2. Single Glutamate-Activated Channels.- 3. Distribution of Lifetimes.- 4. Recording of Miniature Currents with Patch Clamp.- 5. Internal Perfusion of Patch Electrodes.- 6. Burst Kinetics of Glutamate-Activated Channels.- 7. Agonist-Activated Channels.- References.- 20 Cholinergic Chloride Channels in Snail Neurons.- 1. Introduction.- 2. Methods.- 3. Results.- 3.1. Whole-Cell Recording.- 3.2. Outside-Out Patches.- 3.3. Cell-Attached Recording.- 4. Discussion.- References.- 21 Single-Channel Analysis in Aplysia Neurons: A Specific K + Channel is Modulated by Serotonin and Cyclic AMP.- 1. Introduction.- 2. Serotonin Produces a Slow epsp in Sensory Neurons of Aplysia.- 3. Single-Channel Recording: Insight into the Molecular Mechanism of Transmitter Action.- 4. Patch-Clamp Technique Applied to Sensory Neurons.- 5. Serotonin Closes Single K+ Channels.- 6. Voltage-Dependent Properties of the Serotonin-Sensitive Channel.- 7. Single-Channel Opening is Independent of Calcium.- 8. Cyclic AMP Also Closes the Serotonin-Sensitive Channel.- 9. Kinetics of Serotonin Action.- 10. Conclusion.- References.- 22 Cholecystokinin and Acetylcholine Activation of Single-Channel Currents via Second Messenger in Pancreatic Acinar Cells.- 1. Introduction.- 2. Methods.- 3. Cation Channels in the Excised Inside-Out Patch.- 4. Indirect Activation of Unitary Inward Currents by Cholecystokinin and Acetylcholine in the Cell-Attached Recording Configuration.- 5. Is the Cation Channel Permeable to Calcium?.- 6. Relationship between Macroscopic Current and Unitary Currents: Numbers of Channels per Cell.- 7. Conclusion and Perspective.- References.- 23 Observations on Single Calcium Channels: An Overview.- 1. Introduction.- 2. Separation of Calcium Currents.- 3. Gating Properties of Calcium Channels.- 3.1. Distribution of Open Times, Closed Times and Burst Duration.- 3.2. Noise Spectra from Patch Calcium Currents.- 3.3. Patch Calcium Tail Currents.- 3.4. Latencies to First Openings.- 4. Conductance of Single Calcium Channels.- 5. Conclusions.- References.- 24 Potassium and Chloride Channels in Red Blood Cells.- 1. Introduction.- 2. Results.- 2.1. Sealing on Red Blood Cells.- 2.2. Cell-Attached Patch Recording.- 2.3. Recordings from Cell-Free Inside-Out Membrane Patches.- 2.4. Whole-RBC Recording.- 3. Discussion.- References.- 25 The Influence of Membrane Isolation on Single Acetylcholine-Channel Current in Rat Myotubes.- 1. Introduction.- 2. Results.- 3. Discussion.- References.