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 (P-o) 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.