REGULATION OF HIGH-CONDUCTANCE ANION CHANNELS BY G-PROTEINS AND 5-HT1A RECEPTORS IN CHO CELLS

Abstract

This study addresses the mechanisms responsible for regulation of high-conductance anion channels by GTP binding proteins in Chinese hamster ovary (CHO) cells. Single-channel currents were measured in inside-out membrane patches using patch-clamp techniques. Anion-selective channels with a unitary conductance of 381 +/- 8 pS activated spontaneously in 48% of excised patches. In patches with no spontaneous channel activity, addition of GppNHp, a nonhydrolyzable analogue of GTP, activated channels in 8 of 12 studies, and in patches with spontaneous channel activity, GppNHp increased open probability in 4 of 4 experiments. In contrast, GDPbetaS, a nonhydrolyzable GDP analogue, inhibited both spontaneous and GppNHp-induced channel activity. In patches without spontaneous channel activity, addition of cholera toxin activated channels in five of eight studies. Interestingly, pertussis toxin had a similar effect, activating channels in five of seven previously quiescent patches. To further evaluate the possible role of inhibitory G proteins in channel regulation, activity was measured in cell-attached patches in cells transfected with the serotonin 5-HT1A receptor, which is coupled to effector mechanisms through a pertussis toxin-sensitive G protein. Stimulation of 5-HT1A-transfected cells with the receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin caused a transient decrease in open probability in either standard or high-potassium solutions. In aggregate, these findings suggest that both cholera and pertussis toxin-sensitive G proteins contribute to regulation of high-conductance anion channels in CHO cells.