PHOSPHORYLATION BY PROTEIN-KINASE-A OF RCK1 K+ CHANNELS EXPRESSED IN XENOPUS-OOCYTES

Phosphorylation-mediated regulation of voltage-gated K+ channels has been implicated in numerous electrophysiological studies; however, complementary biochemical studies have so far been hampered by the failure to isolate and characterize any K+ channel proteins of distinct molecular identity. We used the Xenopus oocyte expression system to study the biosynthesis and phosphorylation by protein kinase A (PKA) of rat brain RCK1 (Kv1.1) K-+ channel protein. RCK1 protein was isolated by immunoprecipitation from oocytes injected with RCK1 cRNA and analyzed by SDS-polyacrylamide gel electrophoresis (SDSPAGE). The channel protein was expressed in the form of several polypeptides. The 57-kDa polypeptide, usually the major constituent, resided both in the cytosol and in the plasma membrane. Its levels were correlated with RCK1 current amplitudes (I-RCK1) and upon incubation of the cRNA-injected oocytes with tunicamycin, its molecular weight was decreased and at the same time I-RCK1 was reduced. These results suggest that the membranal 57-kDa polypeptides represent functional channels that are N-glycosylated. Furthermore, a study of the phosphorylation of the RCK1 polypeptides revealed that the 57-kDa polypeptide was specifically targeted for phosphorylation by PKA. It could be phosphorylated in vitro by the catalytic subunit of PKA (PKA-CS). In its native state in intact oocytes, the 57-kDa polypeptide was partially phosphorylated and could be further phosphorylated in vivo by addition of a membrane-permeant cAMP analog. Site-directed mutagenesis demonstrated that phosphorylation of a single site on the C-terminus of the channel molecule fully accounts for these phosphorylations. This work thus characterizes biochemically what appears to be a functional K-+ channel polypeptide, demonstrates its phosphorylation by PKA in vitro and in intact cells, and localizes the phosphorylation site on the molecule.