PROTEIN-KINASE A PHOSPHORYLATION AND G-PROTEIN REGULATION OF PURIFIEDRENAL NA-MEMBRANES( CHANNELS IN PLANAR BILAYER)

Purified bovine renal epithelial Na+ channels incorporated into planar lipid bilayer membranes were used to evaluate the biophysical consequ ences of its phosphorylation by protein kinase A (PKA). We also studie d the effects of pertussis toxin-induced ADP-ribosylation on single ch annel activity of nonphosphorylated and PKA phosphorylated channels. P KA-induced phosphorylation resulted in a significant increase in singl e channel open probability (P-o) with no change in single channel cond uctance, as well as increased the probability of multiple channel open ings in the bilayer. Further, PKA conferred a voltage sensitivity to c hannel gating without affecting open channel conduction properties. PK A-phosphorylated Na+ channels were inhibited by subsequent ADP-ribosyl ation with pertussis toxin (PTX). Addition of guanosine 5'-3-O-(thio)t riphosphate reversed this inhibition. However, exposure of nonphosphor ylated Na+ channels to PTX increased channel open probability by a fac tor 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-as sociated Gi protein on function depend upon the previous phosphorylati on state of the protein.