Abstract
Two-electrode voltage-clamp (TEV) of Xenopus laevis oocytes is easily applied for the rapid screening of ion channel function, in particular in pharmacological experiments. However, conventional TEV hardware is not straightforwardly operated by technical personnel because adjustment of the electronics requires considerable practical experience. Moreover, the faithful interpretation of experimental data is often compromised by an incomplete control of all physical parameters determining the voltage-clamp performance. We therefore designed and implemented a hardware/software combination with a built-in 16-bit DA/AD USB interface board providing complete software control of a TEV amplifier featuring full digital calibration and tuning as well as automatic operation via electrophysiological data acquisition software. By means of automated features, such as offset compensation, filter setting, software adjustable amplifier controls (type of controller, gain, response time), and electrode resistance measurements, TEV experiments can be performed in a highly reproducible manner while monitoring the complete set of amplifier control settings. Direct software access to stimulation bandwidth, clamp mode, gain, and response time allows for the objective and automated optimization of voltage-clamp parameters. Two methods for obtaining optimized clamp control parameters will be discussed. An automatic hardware transient compensation increases the dynamic range, particularly important when assaying voltage-gated ion channels. The method is applied for the recording of currents mediated by voltage-gated potassium and sodium channels. In addition to the voltage recording electrode and the current injecting electrode, we implemented a third electrode that injects current in parallel to the second electrode. According to Baumgartner et al. (Biophys. J. 77:1980-1991, 1999), this additional compensation electrode placed in the extracellular space corrects for local current flows and helps improving the voltage clamp performance in big cells such as Xenopus oocytes. Additional operation modes of a third electrode will be presented.
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