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.
Similar content being viewed by others
References
Dascal N (1987) The use of Xenopus oocytes for the study of ion channels. CRC Crit Rev Biochem 22/4:317–387
Dascal N, Lotan I (1992) Expression of exogenous ion channels and neurotransmitter receptors in RNA-injected Xenopus oocytes. In: Longstaff A, Revest P (eds) Methods in molecular biology, 13. Humana Press, Totowa, NJ, pp 205–225
Dascal N, Lim NF, Schreibmayer W, Wang W, Davidson N, Lester HA (1993) Expression of an atrial G-protein-activated potassium channel in Xenopus oocytes. Proc Natl Acad Sci USA 90:6596–6600
Finkel AS, Gage P (1984) Conventional voltage clamping with two intracellular microelectrodes. In: Smith TG Jr, Lecar H, Redman SJ, Gage PW (eds) Voltage and patch clamping with microelectrodes. American Physiological Society, Bethesda, ML, pp 47–92
Hodgkin AL, Huxley AF, Katz B (1952) Measurements of currentvoltage relations in the membrane of the giant axon of Loligo. J Physiol (Lond) 116:424–448
Iverson L, Tanouye AM, Lester HA, Davidson N, Ruby B (1988) A-type potassium channels expressed from Shaker locus cDNA. Proc Natl Acad Sci USA 85:5723–5727
Sigworth F (1980) The variance of sodium current fluctuations at the node of ranvier. J Physiol (Lond) 307:97–129
Smith TG Jr, Barker JL, Smith BM, Colburn TR (1980) Voltage clamping with microelectrodes. J Neurosci Methods 3:105–128
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schreibmayer, W., Lester, H.A. & Dascal, N. Voltage clamping of Xenopus laevis oocytes utilizing agarose-cushion electrodes. Pflugers Arch. 426, 453–458 (1994). https://doi.org/10.1007/BF00388310
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00388310