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

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Abstract
The present investigation tested the hypothesis that nitric oxide (NO) potentiates ATP-sensitive K+ (KATP) 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 KATP 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 KATP 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 KATP 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 KATPchannels. Protein phosphatase 2A (PP2A, 1 U/ml) reversed the PKG-mediated KATP 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 KATP channels in rabbit ventricular myocytes.

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