A catalytically inactive mutant of type I cGMP-dependent protein kinase prevents enhancement of large conductance, calcium-sensitive K+ channels by sodium nitroprusside and cGMP

The activation of large conductance, calcium-sensitive K+ (BKCa) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. In HEK 293 cells transiently transfected with BKCa channels, we observed that the NO donor sodium nitroprusside and the membrane-permeable analog of cGMP, dibutyryl cGMP, were both able to enhance BKCa channel activity 4-5-fold in cell-attached membrane patches. This enhancement correlated with an endogenous cGMP-dependent protein kinase activity and the presence of the alpha isoform of type I cGMP-dependent protein kinase (cGKI). We observed that cotransfection of cells with BKCa channels and a catalytically inactive (dead) mutant of human cGKI alpha prevented enhancement of BKCa channel in response to either sodium nitroprusside or dibutyryl cGMP in a dominant negative fashion. In contrast, expression of wild-type cGKI alpha supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKI alpha were found to associate with BKCa channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKI alpha was able to directly phosphorylate immunoprecipitated BKCa channels, suggesting that cGKI alpha -dendent phosphorylation of BKCa channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKI alpha alone is sufficient to promote the enhancement of BKCa channels in situ after activation of the NO/cGMP signaling pathway.