G-protein regulation of outwardly rectified epithelial chloride channels incorporated into planar bilayer membranes.

Academic Article

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.
  • Published In

    Keywords

  • Adenosine Diphosphate Ribose, Adenosine Triphosphate, Animals, Cattle, Cell Membrane, Chloride Channels, Cyclic AMP-Dependent Protein Kinases, Cystic Fibrosis Transmembrane Conductance Regulator, Epithelium, GTP-Binding Proteins, Guanosine 5'-O-(3-Thiotriphosphate), Kinetics, Lipid Bilayers, Membrane Potentials, NAD, Pertussis Toxin, Phosphorylation, Trachea, Virulence Factors, Bordetella
  • Author List

  • Ismailov II; Jovov B; Fuller CM; Berdiev BK; Keeton DA; Benos DJ
  • Start Page

  • 4776
  • End Page

  • 4780
  • Volume

  • 271
  • Issue

  • 9