Voltage clamping of Xenopus laevis oocytes utilizing agarose-cushion electrodes.
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 k omega). Glass microelectrodes were filled with a cushion of 1% agarose at their tips to prevent KCl leakage (agarose-cushion electrodes). Insertion of these electrodes into X. laevis oocytes yielded stable preparations for periods of more than 1 h with a stable input resistance of 1-4 M omega. 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 voltage-clamp 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.
Schreibmayer
W
W
Division of Biology, California Institute of Technology, Pasadena 91125.
Lester
H A
HA
Dascal
N
N
D000818:Animals; D003600:Cytosol / Q000502:physiology; D004558:Electric Stimulation; D005260:Female; D007473:Ion Channels / Q000502:physiology; D008839:Microelectrodes; D008954:Models, Biological; D009865:Oocytes / Q000502:physiology*; D012685:Sepharose; D014982:Xenopus laevis / Q000502:physiology*
D007473:Ion Channels; D012685:Sepharose