GTP-BINDING PROTEINS REGULATE HIGH-CONDUCTANCE ANION CHANNELS IN RAT BILE-DUCT EPITHELIAL-CELLS

Epithelial cells from the intrahepatic bile duct contribute to bile fo rmation, but little is known of the cellular mechanisms responsible. I n these studies, we have characterized the endogenous GTP-binding prot eins (G proteins) present in these cells and evaluated their role in r egulation of high conductance anion channels. G proteins were identifi ed in purified plasma membranes of isolated bile duct epithelial cells using specific antisera on Western blots, and ion channel activity wa s measured in excised inside-out membrane patches using patch-clamp re cording techniques. In patches without spontaneous channel activity, a ddition of cholera toxin to the cytoplasmic surface had no effect (n = 10). Addition of pertussis toxin caused an activation of channels in 13/34 (38%) attempts, as detected by an increase in channel open proba bility. Activated channels were anion selective (gluconate/Cl- permeab ility ratio of 0.17 +/- 0.04) and had a unitary conductance of approxi mately 380 pS. Channel open probability was also increased by the nonh ydrolyzable GDP analogue guanosine 5'-0-(2-thiodiphosphate) in 8/14 (5 7%) attempts. In contrast, channel open probability was rapidly and re versibly decreased by the nonhydrolyzable analogue of GTP 5' guanylyli midodiphosphate in 7/9 (78%) attempts. Western blotting with specific antisera revealed that both G(i)alpha-2 and G(i)alpha-3 were present i n significant amounts, whereas G(i)alpha-1 and G(o)alpha were not dete cted. These studies indicate that in bile duct epithelial cells, high conductance anion channels are inhibited, in a membrane-delimited mann er, by PTX-sensitive G proteins.