title: GTP-binding proteins regulate high conductance anion channels in rat bile duct epithelial cells.
abstract: Epithelial cells from the intrahepatic bile duct contribute to bile formation, but little is known of the cellular mechanisms responsible. In these studies, we have characterized the endogenous GTP-binding proteins (G proteins) present in these cells and evaluated their role in regulation of high conductance anion channels. G proteins were identified in purified plasma membranes of isolated bile duct epithelial cells using specific antisera on Western blots, and ion channel activity was measured in excised inside-out membrane patches using patch-clamp recording techniques. In patches without spontaneous channel activity, addition 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 probability. Activated channels were anion selective (gluconate/Cl- permeability ratio of 0.17 +/- 0.04) and had a unitary conductance of approximately 380 pS. Channel open probability was also increased by the nonhydrolyzable GDP analogue guanosine 5'-0-(2-thiodiphosphate) in 8/14 (57%) attempts. In contrast, channel open probability was rapidly and reversibly decreased by the nonhydrolyzable analogue of GTP 5' guanylylimidodiphosphate in 7/9 (78%) attempts. Western blotting with specific antisera revealed that both Gi alpha-2 and Gi alpha-3 were present in significant amounts, whereas Gi alpha-1 and Go alpha were not detected. These studies indicate that in bile duct epithelial cells, high conductance anion channels are inhibited, in a membrane-delimited manner, by PTX-sensitive G proteins.
authors.affiliation: Duke University Medical Center, Department of Medicine, Durham, North Carolina 27710.
authors.forename: J M
authors.initials: JM
authors.lastname: McGill
authors.forename: T W
authors.initials: TW
authors.lastname: Gettys
authors.forename: S
authors.initials: S
authors.lastname: Basavappa
authors.forename: J G
authors.initials: JG
authors.lastname: Fitz
mesh_terms: D000818:Animals; D000838:Anions / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism / Q000493:pharmacokinetics; D001653:Bile Ducts, Intrahepatic / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism / Q000493:pharmacokinetics; D004847:Epithelial Cells; D004848:Epithelium / Q000737:chemistry / Q000502:physiology; D019204:GTP-Binding Proteins / Q000032:analysis / Q000502:physiology*; D007473:Ion Channels / Q000502:physiology*; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D037342:Pertussis Toxin; D051381:Rats; D017207:Rats, Sprague-Dawley; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D000838:Anions; D001639:Bicarbonates; D002712:Chlorides; D007473:Ion Channels; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins