Two-electrode voltage clamp of Xenopus oocytes under high hydrostatic pressure

Abstract

Functional assays of cloned ion channels and other transport systems under various hydrostatic pressures provide information on the apparent changes in protein volume occurring during conformational rearrangements. Thus, they are valuable tools in the detailed study of the molecular steps underlying the functioning of such proteins. Here we present details of a set-up which can be used for two-electrode voltage-clamp experiments on Xenopus oocytes, commonly used for heterologous protein expression, at hydrostatic (oil) pressures as high as 60 MPa (≈600 atm.). The advantages of this set-up over pneumatic systems include the minimization of compression/decompression-induced temperature changes, and an increased safety of handling due to the small volume (<10 ml) of compression medium (oil) required. The performance of the system is illustrated using experimental data on the effects of high pressure on currents recorded from oocytes expressing a Shaker potassium channel mutant. This set-up is suitable for the investigation of all electrically measurable transport systems expressed in Xenopus oocytes.