Textbook of Pulmonary Vascular Disease

The current chapter provides a description of the patch-clamp technique and its variations. These techniques have been instrumental in elucidating the behavior of ion channels in the cells. Based on this information, it has been possible to group and classify a vast number of ion channels. The functional classification of ion channels is described in this chapter. This initial functional classification has led to the current organized gene-based comprehensive IUPHAR database which allows us to navigate the complex families of ion channels. Patch-clamping has also been instrumental in defining the functional properties of detailed segments of the channel proteins through “structure–function” studies, thus pioneering the field of functional proteomics. Ion channels are involved in the etiology and progression of many diseases and patch-clamping has revealed the functional implications of genetic mutations that underlie the channelopathies.

Chapter 20 – Patch-Clamp Techniques

Effects of neurotoxic chemicals on central synaptic function can be assessed using electrophysiological recording techniques in freshly isolated brain slices. The three most commonly used electrophysiological recording methods in brain slices are extracellular microelectrode recording, intracellular microelectrode recording, and whole-cell patch clamp recording. This unit presents the basic procedures and applications of extra- and intracellular recordings in hippocampal slices and whole-cell recording in cerebellar slices.


Keywords:

Brain slices;
synaptic transmission;
extracellular recording;
intracellular recording;
whole-cell recording

Electrophysiological studies of neurotoxicants on central synaptic transmission in acutely isolated brain slices.

The development of the patch clamp technique has enabled investigators to directly measure gap junction conductance between isolated pairs of small cells with resolution to the single channel level. The dual patch clamp recording technique requires specialized equipment and the acquired skill to reliably establish gigaohm seals and the whole cell recording configuration with high efficiency. This chapter describes the equipment needed and methods required to achieve accurate measurement of macroscopic and single gap junction channel conductances. Inherent limitations with the dual whole cell recording technique and methods to correct for series access resistance errors are defined as well as basic procedures to determine the essential electrical parameters necessary to evaluate the accuracy of gap junction conductance measurements using this approach.

Establishment of the Dual Whole Cell Recording Patch Clamp Configuration for the Measurement of Gap Junction Conductance

This unit describes essential procedures to record the activity of and the effects of pharmacological manipulation upon tetrodotoxin-resistant (TTXr) sodium channels. Readers are guided through the process of assembling a functional |patch-clamp| electrophysiology apparatus and construction of software-controlled voltage protocols that address various aspects of channel gating. Guidelines are also presented for analysis and display of the data typically generated by these experiments. For investigators wishing to study native channels in their appropriate cellular environment, protocols describing the isolation of viable cells from the main tissues that commonly express TTXr channels are also included.


Keywords:

sodium channel;
TTX-resistant;
activation;
inactivation;
state-dependent;
use-dependent;
voltage-dependent;
open channel;
inactivated channel;
tonic block

Electrophysiological Analysis of Tetrodotoxin‐Resistant Sodium Channel Pharmacology

Patch clamping refers to a wide range of electrophysiological measurements, all of which have in common the use of patch pipets and the formation of gigaohm seals. The purpose of this unit is to describe the fabrication of patch pipets. The aspects of the pipet geometry that are important to different applications and the different procedures that have been found to most reliably and simply achieve these results are described. Parameters for glass selection are detailed in the beginning of the unit. Pulling patch and whole-cell pipets, elastomer coating, fire polishing, pipet filling, and pipet testing in an experimental setup are highlighted. Additional support protocols describe alternative ways to optimize pipet geometry and cleaning the glass before pulling. Considerations for noise and dynamic performance are emphasized as these two requirements for single-channel and whole-cell current measurements dictate how the pipets must be fabricated.

Fabrication of patch pipets.

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Fabrication of patch pipets.