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) + NAP (1 mM) + ATP (1 mM) treatment of ORCC and CFTR in bilayers resulted in a 2-fold increase in single channel open probability (P(o)) 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 P(o) 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 P(o) 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 μM) 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.
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    Digital Object Identifier (doi)

    Author List

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

  • 4776
  • End Page

  • 4780
  • Volume

  • 271
  • Issue

  • 9