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 (SDS-PAGE). 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 (IRCK1) and upon incubation of the cRNA-injected oocytes with tunicamycin, its molecular weight was decreased and at the same time IRCK1 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.(ABSTRACT TRUNCATED AT 250 WORDS)