Mechanism of activation by cGMP-dependent protein kinase of large Ca(2+)-activated K+ channels in mesangial cells.

Abstract

The patch clamp method was employed to establish the mechanism of regulation by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) of large Ca(2+)-activated K+ channels (BKCa) in human mesangial cells. Dibutyryl cGMP (DBcGMP) significantly increased open probability (Po) of BKCa in the absence but not in the presence of staurosporine in cell-attached patches. In excised patches, BKCa was activated by simultaneous addition of MgATP plus cGMP but not cAMP plus MgATP. Activation by cGMP plus MgATP was blocked by KT-5823, an inhibitor of PKG, but not by KT-5720, an inhibitor of cAMP-dependent protein kinase (PKA). Thus a cGMP-specific endogenous kinase is associated with mesangial BKCa. In excised patches, exogenous PKG but not PKA or protein kinase C activated BKCa. The half-activation potential (V1/2), defined as the potential at which the Po = 0.5 with 1 microM Ca2+, was -34 and 42 mV for activated and inactivated BKCa, respectively; however, the gating charge (Zg), a measure of voltage sensitivity, was not affected by PKG. Similarly, the Ca1/2 (free Ca2+ concentration required to activate to Po = 0.5 at 40 mV) decreased from 1.74 to 0.1 microM on addition of PKG, but the Hill coefficient, a measure of Ca2+ sensitivity, was not affected. Activation of BKCa by PKG was heterogeneous with two populations: the majority (67%) activated by PKG and the minority unaffected. It is concluded that an endogenous PKG activates BKCa by decreasing the Ca2+ and voltage activation thresholds independently of sensitivities.