Voltage clamping of Xenopus laevis oocytes utilizing agarose-cushion electrodes

Abstract

Two-electrode voltage clamping of expressed ion channels in intact oocytes of the South African clawed frog Xenopus laevis has been refined to allow stable, low-resistance electrical access to the cytosol (50–800 kΩ). Glass microelectrodes were filled with a cushion of 1 % agarose at their tips to prevent KC1 leakage (agarose-cushion electrodes). Insertion of these electrodes into X. laevis oocytes yielded stable preparations for periods of more than l h with a stable input resistance of 1–4MΩ. Furthermore, a simple modification of the voltage-clamp circuit (charging compensator) is described that increases the flexibility of arrangements for differential recording of the membrane potential in order to subtract voltage drops across a series resistance. The result is a considerable increase in the practically attainable speed of the voltage clamp with the conventional two-electrode arrangement. The performance of the charging compensator was tested on an equivalent circuit that simulates the oocyte and electrodes. In addition, the combination of agarose-cushion electrodes and the charging compensator was tested on oocytes expressing Shaker H4 currents. The fidelity of the voltageclamp circuit was also verified by measuring the membrane potential with additional independent microelectrodes connected to a differential amplifier, independent of the two-electrode voltage clamp system. The system described here will be useful for ion channel studies in X. laevis oocytes requiring long-term recordings and/or measurements of large, fast ion currents.