G proteins activate ionic conductances at multiple sites in t84 cells. Izu, Leighton, Ming Li, Rob Demuro, and Michael E. Duffey. Department of Physiology, School of Medicine, State University of New York at Buffalo, Buffalo, New York 14214, U.S.A.
APStracts 3:0364C, 1996.
We used the colonic secretory epithelial cell line, T84, to examine the role of G proteins in activation of ionic conductances during cholinergic stimulation. We whole-cell voltage-clamped isolated T84 cells to EK& or ECl- under standard conditions so that K& and inward currents could be examined. Consistent with our previous results (Devor, D.C., S.M. Simasko, and M.E. Duffey. Am. J. Physiol. 258: C318-C326, 1990.), the cholinergic agonist, carbachol, induced a large oscillating K& current (225+/-47 pA, n=10) but only a small inward current. Addition of the GDP analog, GDP[beta]S, to pipettes blocked the ability of carbachol to activate the K& current. Addition of the non-hydrolyzable GTP analog, GTP[tau]S, to pipettes stimulated large oscillating K& and inward currents. Stimulation of these currents occurred even when Ca2& was absent from the bath, but they were blocked by addition of the Ca2& chelator, EGTA, to pipettes. We also determined the identity of the conductances that underlie the inward current by replacing all K& in pipettes and cell bath with Na&, lowering bath Na& concentration, and voltage-clamping cells between ENa& and ECl-. Under these conditions, GTP[tau]S activated both oscillating Na& and Cl- currents. Finally, we added Ins(1,4,5)P3 to pipettes. This addition activated large oscillating K& currents but only small inward currents. These results suggest that a carbachol-induced release of Ca2& from intracellular stores is activated by a G protein through the phospholipase C- Ins(1,4,5)P3 signaling pathway. In addition, this or another G protein activates Cl- current by directly gating Cl- channels to increase their sensitivity to Ca2&. 

Received 11 July 1995; accepted in final form 7 November 1996.
APS Manuscript Number C417-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 31 December 1996