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 oocytes and can be further phosphorylated upon treatment for a short time 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 whereas treatments for a short time with various cAMP analogs do not affect the channel function, prolonged treatment with 8-bromoadenosine 3',5'-cyclic monophosphorothioate ((Sp)-8-Br-cAMPS), a membrane-permeant cAMP analog, enhances the current amplitude. It also enhances the current amplitude through a mutant channel that cannot be phosphorylated by protein kinase A activation. The enhancement is inhibited in the presence of (Rp)-8-Br-cAMPS, a membrane-permeant protein kinase A inhibitor. Concomitant SDS-polyacrylamide gel electrophoresis analysis reveals that this treatment not only brings about phosphorylation of the wild-type channel, but also increases the amounts of both wild-type and mutant channel proteins; the latter effect can be inhibited by cycloheximide, a protein synthesis inhibitor. In the presence of cycloheximide, the (Sp)-8-Br-cAMPS treatment enhances only the wild-type current amplitudes and induces accumulation of wild-type channels in the plasma membrane of the oocyte. In summary, prolonged treatment with (Sp)-8-Br-cAMPS regulates RCK1 function via two pathways, a pathway leading to enhanced channel synthesis and a pathway involving channel phosphorylation that directs channels to the plasma membrane.
Levin
G
G
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat Aviv, Israel.
Keren
T
T
Peretz
T
T
Chikvashvili
D
D
Thornhill
W B
WB
Lotan
I
I
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D051662:Kv1.1 Potassium Channel; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D015221:Potassium Channels / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated; D014981:Xenopus
C489293:Kcna1 protein, rat; D009838:Oligodeoxyribonucleotides; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D051662:Kv1.1 Potassium Channel; D015124:8-Bromo Cyclic Adenosine Monophosphate; D017868:Cyclic AMP-Dependent Protein Kinases