A number of K⁺ channels play important roles in cell osmoregulation, and their activity is regulated during cell volume changes. Such regulation may be caused by the volume changes affecting cellular signalling pathways (e.g. cytosolic Ca⁺⁺, pH, ATP and cytoskeleton), or directly by volume-associated changes in the membrane tension. Here we describe a method to determine the contribution of each of these mechanisms to the regulation of the voltage-gated KCNQ1 channel, which was recently found to be dramatically regulated by small, reversible changes in cell volume when co-expressed with water channels in Xenopus laevis oocytes (Grunnet et al., 2003). We use cell-attached macropatches (diameter ~10 mm) in Xenopus oocytes expressing human KCNQ1 (without KCNE1) for non-stationary fluctuation analysis, in the absence and the presence of local membrane stretch (by suction). Preliminary values obtained for single-channel current (i ~0.46 pA at +90 mV without pressure and in symmetrical 110 mM K⁺), number of channels (N ~235), and maximal open state probability (P_o max ~ 0.66) compare with those of Yang & Sigworth (1998). The V_½ for conductance activation was 42.4 ± 6.6 mV (N = 7). Experiments with application of pressure to the macropatch are in progress for deducing which of the suggested mechanisms is responsible for the regulation of KCNQ1.

REFERENCES

Grunnet, M., Jespersen, T., MacAulay, N. K., Jørgensen, N., Schmitt, N., Pongs, P., Olesen, S.P.& Klaerke, D.A. 2003, J Physiol 549, 419-427..

Yang, Y. & Sigworth, F.J. 1998. J Gen Physiol 112, 665-678.