2-ELECTRODE VOLTAGE-CLAMP OF XENOPUS OOCYTES UNDER HIGH HYDROSTATIC-PRESSURE

Functional assays of cloned ion channels and other transport systems u nder various hydrostatic pressures provide information on the apparent changes in protein volume occurring during conformational rearrangeme nts. Thus, they are valuable tools in the detailed study of the molecu lar 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 protei n expression, at hydrostatic (oil) pressures as high as 60 MPa (approx imate to 600 atm.). The advantages of this set-up over pneumatic syste ms include the minimization of compression/decompression-induced tempe rature changes, and an increased safety of handling due to the small v olume ( < 10 mi) of compression medium toil) required. The performance of the system is illustrated using experimental data on the effects o f high pressure on currents recorded from oocytes expressing a Shaker potassium channel mutant. This set-up is suitable for the investigatio n of all electrically measurable transport systems expressed in Xenopu s oocytes. (C) 1998 Elsevier Science B.V. All rights reserved.