REGULATION OF RCK1 CURRENTS WITH A CAMP ANALOG VIA ENHANCED PROTEIN-SYNTHESIS AND DIRECT CHANNEL PHOSPHORYLATION

We have recently shown that the rat brain Kv1.1 (RCK1) voltage-gated K + channel is partially phosphorylated in its basal state in Xenopus oo cytes and can be further phosphorylated upon treatment for a short tim e with a cAMP analog (Ivanina, T., Perts, T., Thornhill, W, B., Levin, G., Dascal, N., and Lotan, I. (1994) Biochemistry 33, 8786-8792), In this study, we show, by two-electrode voltage clamp analysis, that whe reas treatments for a short time with various cAMP analogs do not affe ct the channel function, prolonged treatment with 8-bromoadenosine 3', 5'-cyclic monophosphorothioate ((S-p)-8-Br-cAMPS), a membrane-permeant cAMP analog, enhances the current amplitude, It also enhances the cur rent amplitude through a mutant channel that cannot be phosphorylated by protein kinase A activation. The enhancement is inhibited in the pr esence of (R(p))-8-Br-cAMPS, a membrane-permeant protein kinase A inhi bitor. Concomitant SDS-polyacrylamide gel electrophoresis analysis rev eals that this treatment not only brings about phosphorylation of the wild-type channel, but also increases the amounts of both wild-type an d mutant channel proteins; the latter effect can be inhibited by cyclo heximide, a protein synthesis inhibitor. In the presence of cyclohexim ide, the (Sp)-8-Br-cAMPS treatment enhances only the wild-type current amplitudes and induces accumulation of wild-type channels in the plas ma membrane of the oocyte, In summary, prolonged treatment with (S-p)- 8-Br-cAMPS regulates RCK1 function via two pathways, a pathway leading to enhanced channel synthesis and a pathway involving channel phospho rylation that directs channels to the plasma membrane.