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

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.