AJP - Cell Physiology, Vol 272, Issue 4 C1222-C1231, Copyright © 1997 by American Physiological Society
G proteins activate ionic conductances at multiple sites in T84 cells
L. Izu, M. Li, R. DeMuro and M. E. Duffey
Department of Physiology, School of Medicine, State University of New York at Buffalo, 14214, USA.
We examined the role of G proteins in activation of ionic conductances in
isolated T84 cells during cholinergic stimulation. When cells were whole
cell voltage clamped to the K+ equilibrium potential (E(K)) or Cl-
equilibrium potential (E(Cl)) under standard conditions, the cholinergic
agonist, carbachol, induced a large oscillating K+ current but only a small
inward current. Addition of the GDP analogue, guanosine
5'-O-(2-thiodiphosphate), to pipettes blocked the ability of carbachol to
activate the K+ current. Addition of the nonhydrolyzable GTP analogue,
guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), to pipettes stimulated
large oscillating K+ and inward currents. This occurred even when Ca2+ was
absent from the bath but not when the Ca2+ chelator, ethylene
glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, was added to
pipettes. When all pipette and bath K+ was replaced with Na+ and cells were
voltage clamped between E(Na) and E(Cl), GTPgammaS activated oscillating
Na+ and Cl- currents. Finally, addition of inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3] to pipettes 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+.
