The transoocyte voltage clamp: a non-invasive technique for electrophysiological experiments with Xenopus laevis oocytes.

摘要

We developed a non-invasive technique for electrophysiological investigations of ion transport proteins endogenously or heterologously expressed in Xenopus laevis oocytes. We named this technique the transoocyte voltage clamp (TOVC). Whereas in the classical two-microelectrode voltage-clamp (TEVC) technique, the oocyte is impaled with two glass microelectrodes, we mount the egg in a modified Ussing chamber as used for transepithelial electrophysiological studies. The oocyte is introduced in a container that is positioned between the two chamber halves. Proper fixation of the oocyte in the aperture of the container is accomplished under a stereo binocular microscope and the electrical seal between the oocyte and the container is achieved with silicon grease. The new method allows measurement of transoocyte currents and conductances as well as the recording of membrane impedance and the fluctuation analysis of ion currents. We studied a K+ channel that resembles the inward rectifier K+ channel endogenously expressed in Xenopus laevis oocytes. K+ currents were obtained by exposing one side of the oocyte to K(+)-containing solutions and by the application of different voltages. Adding Cs+ and Ba2+ inhibited these currents. The analysis of the fluctuation in current demonstrated a Lorentzian component in the power density spectrum. With the transoocyte voltage clamped to zero, the corner frequency (fc) was 61+/-1.7 Hz. Imposed positive transoocyte potentials caused a downward shift of fc. These findings are consistent with previous data obtained using the TEVC technique, and extend the characterization of the channel with kinetic data obtained from noise analysis.