The large conductance, voltage-dependent, and calcium-sensitive K+ channel, Hslo, is a target of cGMP-dependent protein kinase phosphorylation in vivo.
Native large conductance, voltage-dependent, and Ca2+-sensitive K+ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human pore-forming alpha-subunit of the Ca2+-sensitive K+ channel, Hslo, and the alpha-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by approximately 20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates gamma-33P into the immunopurified Hslo band of approximately 125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the alpha-subunit of large conductance Ca2+-sensitive K+ channels is substrate for G-Ialpha kinase in vivo and support direct phosphorylation as a mechanism for PKG-Ialpha-induced activation of maxi-K channels.
Alioua
A
A
Department of Anesthesiology, UCLA, Los Angeles, CA 90095-1778, USA.
Tanaka
Y
Y
Wallner
M
M
Hofmann
F
F
Ruth
P
P
Meera
P
P
Toro
L
L
D000818:Animals; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008051:Lipid Bilayers; D010766:Phosphorylation; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D024681:Potassium Channels, Calcium-Activated; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008051:Lipid Bilayers; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; C016276:8-bromocyclic GMP; D011494:Protein Kinases; D006152:Cyclic GMP