Protein kinase A phosphorylation and G protein regulation of purified renal Na+ channels in planar bilayer membranes.

Purified bovine renal epithelial Na+ channels incorporated into planar lipid bilayer membranes were used to evaluate the biophysical consequences of its phosphorylation by protein kinase A (PKA). We also studied the effects of pertussis toxin-induced ADP-ribosylation on single channel activity of nonphosphorylated and PKA-phosphorylated channels. PKA-induced phosphorylation resulted in a significant increase in single channel open probability (Po) with no change in single channel conductance, as well as increased the probability of multiple channel openings in the bilayer. Further, PKA conferred a voltage sensitivity to channel gating without affecting open channel conduction properties. PKA-phosphorylated Na+ channels were inhibited by subsequent ADP-ribosylation with pertussis toxin (PTX). Addition of guanosine 5'-3-O-(thio)triphosphate reversed this inhibition. However, exposure of nonphosphorylated Na+ channels to PTX increased channel open probability by a factor of 3-5. These results demonstrate that a cAMP-dependent pathway is an important regulatory element for amiloride-sensitive Na+ channels and that the effects of PTX-induced ADP-ribosylation of the channel-associated Gi protein on function depend upon the previous phosphorylation state of the protein.