title: G-protein regulation of outwardly rectified epithelial chloride channels incorporated into planar bilayer membranes.
abstract: Experiments were designed to test if immunopurified outwardly rectified chloride channels (ORCCs) and the cystic fibrosis transmembrane conductance regulator (CFTR) incorporated into planar lipid bilayers are regulated by G-proteins. pertussis toxin (PTX) (100 ng/ml) + NAD (1 mM) + ATP (1 mM) treatment of ORCC and CFTR in bilayers resulted in a 2-fold increase in single channel open probability (Po) of ORCC but not of CFTR. Neither PTX, NAD, nor ATP alone affected the biophysical properties of either channel. Further, PTX conferred a linearity to the ORCC current-voltage curve, with a slope conductance of 80 +/- 3 picosiemens (pS) in the +/- 100 mV range of holding potentials. PKA-mediated phosphorylation of these PTX + NAD-treated channels further increased the Po of the linear 80-pS channels from 0.66 +/- 0.05 to >0.9, and revealed the presence of a small (16 +/- 2 pS) linear channel in the membrane. PTX treatment of a CFTR-immunodepleted protein preparation incorporated into bilayer membranes resulted in a similar increase in the Po of the larger conductance channel and restored PKA-sensitivity that was lost after CFTR immunodepletion. The addition of guanosine 5'-3-O-(thio)triphosphate (100 mum) to the cytoplasmic bathing solutions decreased the activity of the ORCC and increased its rectification at both negative and positive voltages. ADP-ribosylation of immunopurified material revealed the presence of a 41-kDa protein. These results demonstrate copurification of a channel-associated G-protein that is involved in the regulation of ORCC function.
authors.affiliation: Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: I I
authors.initials: II
authors.lastname: Ismailov
authors.forename: B
authors.initials: B
authors.lastname: Jovov
authors.forename: C M
authors.initials: CM
authors.lastname: Fuller
authors.forename: B K
authors.initials: BK
authors.lastname: Berdiev
authors.forename: D A
authors.initials: DA
authors.lastname: Keeton
authors.forename: D J
authors.initials: DJ
authors.lastname: Benos
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology; D004848:Epithelium / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D008051:Lipid Bilayers; D008564:Membrane Potentials / Q000187:drug effects; D009243:NAD / Q000494:pharmacology; D037342:Pertussis Toxin; D010766:Phosphorylation; D014132:Trachea / Q000502:physiology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D018118:Chloride Channels; D008051:Lipid Bilayers; D010566:Virulence Factors, Bordetella; D009243:NAD; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins