The present investigation tested the hypothesis that the activation of protein kinase G (PKG) leads to a phosphorylation of Ca2+-activated potassium channel (KCa channel) and is involved in the activation of KCa channel activity in cerebral arterial smooth muscle cells of the rabbit. Single-channel currents were recorded in cell-attached and inside-out patch configurations of patch-clamp techniques. Both molsidomine derivative 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1, 50 micrometer) and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP, 100 micrometer), a membrane-permeable analogue of cGMP, increased the KCa channel activity in the cell-attached patch configuration, and the effect was removed upon washout of the drugs. In inside-out patches, single-channel current amplitude was not changed by SIN-1 and 8-pCPT-cGMP. Application of ATP (100 micrometer), cGMP (100 micrometer), ATP+cGMP (100 micrometer each), PKG (5 U/ microliter), ATP (100 micrometer)+PKG (5 U/ microliter), or cGMP (100 micrometer)+PKG (5 U/ microliter) did not increase the channel activity. ATP (100 micrometer)+cGMP (100 micrometer)+PKG (5 U/ microliter) added directly to the intracellular phase of inside-out patches increased the channel activity with no changes in the conductance. The heat-inactivated PKG had no effect on the channel activity, and the effect of PKG was inhibited by 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP, 100 micrometer), a potent inhibitor of PKG or protein phosphatase 2A (PP2A, 1 U/ml). In the presence of okadaic acid (OA, 5 nM), PP2A had no effect on the channel activity. The KCa channel activity spontaneously decayed to the control level upon washout of ATP, cGMP and PKG, and this was prevented by OA (5 nM) in the medium. These results suggest that the PKG-mediated phosphorylations of KCa channels, or some associated proteins in the membrane patch increase the activity of the KCa channel, and the activation may be associated with the vasodilating action.
Adenosine Triphosphate ; Cyclic GMP-Dependent Protein Kinases ; Membranes ; Molsidomine ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Okadaic Acid ; Patch-Clamp Techniques ; Phosphorylation ; Potassium Channels* ; Potassium* ; Protein Phosphatase 2 ; Signal Transduction*