Cyclic GMP-dependent protein kinase activates cloned BKCa channels expressed in mammalian cells by direct phosphorylation at serine 1072

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and result s in smooth muscle relaxation, However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels . The present study was designed to clarify PKG-mediated modulation of chan nels at the molecular level. The cDNA encoding the alpha-subunit of the lar ge conductance Ca2+-activated K+ channel, cslo-alpha, was expressed in HER2 93 cells. Whole cell and single channel characteristics of cslo-alpha exhib ited functional features of native large conductance Ca2+-activated K+ chan nels in smooth muscle cells. The NO-donor sodium nitroprusside increased ou tward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) of cslo-a lpha channels 3.3-fold without affecting unitary conductance. The stimulato ry effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823 . Direct application of PKG-I alpha to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-alpha in which Ser -1072 (the only optimal consensus sequence for PKG phosphorylation) was rep laced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG act ivates cslo-alpha by direct phosphorylation at serine 1072.