Mechanism of activation by cyclic gmp-dependent protein kinase (PKG) of large ca²⁺-acttvated k⁺ channels BK(Ca)J in human mesangial cells (MC)

We previously showed that cGMP-elevating vasodilators modulate MC tone by regulating the feedback, hyperpolarizing signal carried by BK(Ca). Patch clamp and molecular methods were used to elucidate the cGMP-dependent mechanism for activating BK(Ca). In cell attached patches, activation by cGMP of BK(Ca) was prevented by staurosporine, implicating a protein kinase in the signal transduction pathway. In inside-out patches, BK(Ca) was activated by Mg-ATP plus cGMP but was not affected by the separate addition of either agent Cyclic AMP plus Mg-ATP failed to activate BK(Ca). Activation by cGMP plus Mg-ATP of BK(Ca) was blocked by KT5823, a specific inhibitor of PKG but not by KT5720, a specific inhibitor of PKA. Exogenous PKG but not PKA activated BK(Ca). PKG activated BK(Ca) by decreasing the long closed time constant from 192 to 22 ms, resulting in a decrease in the half activation potential, a measure of voltage threshold, by 60 mV. The gating charge and Hill coefficient, measurements of voltage and calcium sensitivity, respectively, were not affected. Using RT-PCR to amplify cDNA products of hsia, a consensus PKG phosphorylation site was established on BK(Ca). In conclusion, cGMP-dependent protein kinase phosphorylates directly and activates mesangial BK(Ca) by decreasing the activation threshold through a reduction of the duration of the long closed state.