Two-Electrode Voltage Clamp
Two-electrode voltage clamp (TEVC) is a conventional electrophysiological technique used to artificially control the membrane potential (V m) of large cells to study the properties of electrogenic membrane proteins, especially ion channels. It makes use o
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Introduction Ion channels of cell membranes may be gated by the membrane potential (Vm) and/or by specific chemicals. Those belonging to the superfamily of voltage-dependent ion channels are gated by the Vm directly, whereas others, classified as ligand-gated ion channels, are gated by their binding with specific chemicals called ligands. On the other hand, voltage-dependent ion channels can be subject to the modulation by chemical factors such as neurotransmitters, hormones, intracellular messengers or exogenous drugs; and some ligand-gated ion channels (such as NMDA receptors) are also affected by the change in Vm. Thus, to study the voltage dependent characteristics of ion channels readily, or to distinguish between the effects of Vm versus chemicals, an experimental procedure is needed to control the Vm (i.e., to change Vm in a desired pattern or set it at a desired level). This procedure, known as voltage clamp, was first designed by Cole and Marmont and improved by Hodgkin, Huxley and Katz for application to giant axons of squids as twoelectrode voltage clamp (TEVC) in late 1940s (1). It utilizes two intracellular electrodes, one to monitor Vm and the other to inject
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_6, © Springer Science+Business Media, LLC 2013
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a current to adjust Vm to desired values (the injected current being equal to the membrane current). Although the use of TEVC is limited to giant axons or large cells such as skeletal muscle cells, much of our understanding of the basic biophysical properties of ion channels has been acquired by using this approach. It was based on this method in combination with experiments using extracellular electrodes that Neher and Sakmann developed the currently widely used single electrode patch clamp technique (2). Nowadays popular though the latter is, it’s only suitable for clamping relatively small cells, but unfit for large cells; for the large currents would cause a significant voltage drop across the recording electrode that cannot be compensated to an acceptable extent. Thus, TEVC is still irreplaceable in voltage clamping large cells, especially Xenopus oocytes which are often used for exogenous expression of ion channels or receptors. The Xenopus oocyte is a convenient expression system widely employed to study the structure and function of ion channels and receptors using TEVC in combination with various molecular biological approaches, since its membrane has low expression of endogenous channels and receptors. The basic principle of TEVC recording from an oocyte is schematically depicted in Fig. 1. Vm is monitored by connecting the voltage electrode (electrode 1) to the input of a voltage follower (A1), which has very high input impedance and draws negligible
Fig. 1 Conventional two-electrode voltage clamp (TEVC) on an oocyte (see text for detail)
Two-Electrode Voltage Clamp
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current from the cell. The output of A1, the potential at
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