Mechanism of activation by cGMP-dependent protein kinase of large Ca(2+)-activated K+ channels in mesangial cells.
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.
Stockand
J D
JD
Division of Renal Diseases and Hypertension, University of Texas Medical School at Houston 77030, USA.
Sansom
S C
SC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002227:Carbazoles; D002478:Cells, Cultured; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003995:Dibutyryl Cyclic GMP / Q000494:pharmacology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005920:Glomerular Mesangium / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D019319:Okadaic Acid / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D047428:Protein Kinase Inhibitors; D011758:Pyrroles / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology
D000470:Alkaloids; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D015221:Potassium Channels; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; C073601:KT 5823; D019319:Okadaic Acid; D003995:Dibutyryl Cyclic GMP; C057416:KT 5720; D000255:Adenosine Triphosphate; D017869:Cyclic GMP-Dependent Protein Kinases; D019311:Staurosporine; D002118:Calcium