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 scrutini ze the first possibility, at the molecular level, we used the human pore-fo rming alpha-subunit of the Ca2+-sensitive K+ channel, Hslo, and the alpha-i soform of cGMP-dependent protein kinase I. In cell-attached patches of oocy tes 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 similar to 20 mV and was rev ersed by alkaline phosphatase treatment after patch excision, In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 p atches. In contrast, and consistent with the intact cell experiments, purif ied kinase applied to the cytoplasmic side of reconstituted channels increa sed their open probability, This stimulatory effect was absent when heat-de natured kinase was used. Biochemical experiments show that the purified kin ase incorporates gamma-P-33 into the immunopurified Hslo band of similar to 125 kDa, Furthermore, in vivo phosphorylation largely attenuates this labe ling in back-phosphorylation experiments. These results demonstrate that th e Lu-subunit of large conductance Ca2+-sensitive K+ channels is substrate f or G-I alpha kinase in vivo and support direct phosphorylation as a mechani sm for PKG-I alpha-induced activation of maxi-K channels.