ATP-sensitive K⁺channel activation by nitric oxide and protein kinase G in rabbit ventricular myocytes

Abstract

The present investigation tested the hypothesis that nitric oxide (NO) potentiates ATP-sensitive K + (K ATP ) channels by protein kinase G (PKG)-dependent phosphorylation in rabbit ventricular myocytes with the use of patch-clamp techniques. Sodium nitroprusside (SNP; 1 mM) potentiated K ATP channel activity in cell-attached patches but failed to enhance the channel activity in either inside-out or outside-out patches. The 8-(4-chlorophenylthio)-cGMP Rp isomer (Rp-CPT-cGMP, 100 μM) suppressed the potentiating effect of SNP. 8-(4-Chlorophenylthio)-cGMP (8-pCPT-cGMP, 100 μM) increased K ATP channel activity in cell-attached patches. PKG (5 U/μl) added together with ATP and cGMP (100 μM each) directly to the intracellular surface increased the channel activity. Activation of K ATP channels was abolished by the replacement of ATP with ATPγS. Rp-pCPT-cGMP (100 μM) inhibited the effect of PKG. The heat-inactivated PKG had little effect on the K ATP channels. Protein phosphatase 2A (PP2A, 1 U/ml) reversed the PKG-mediated K ATP channel activation. With the use of 5 nM okadaic acid (a PP2A inhibitor), PP2A had no effect on the channel activity. These results suggest that the NO-cGMP-PKG pathway contributes to phosphorylation of K ATP channels in rabbit ventricular myocytes.