Protein kinase C-induced changes in the stoichiometry of ATP binding activate cardiac ATP-sensitive K+ channels - A possible mechanistic link to ischemic preconditioning

Activation of both ATP-sensitive K+ (K-ATP) channels and the enzyme protein kinase C (PKC) has been associated with the cardioprotective response of ischemic preconditioning. We recently showed that at low cytoplasmic ATP (less than or equal to 50 mu mol/L), PKC inhibits K-ATP channel activity. This finding is surprising, as both K-ATP channels and PKC are activated during preconditioning. However, PKC also altered ATP binding to the channel, changing the Hill coefficient from approximate to 2 to approximate to 1. This apparent change in stoichiometry would lead to a PKC-induced activation of K-ATP channels at more physiological (millimolar) levels of ATP. The aim of the present study was to determine whether PKC activates cardiac K-ATP channels at millimolar levels of ATP. The effects of PKC on single K-ATP channels were studied at millimolar internal ATP levels using excised inside-out membrane patches from rabbit ventricular myocytes. Application of purified constitutively active PKC (20 nmol/L) to the intracellular surface of the patches produced an approximately threefold increase in the channel open probability. The specific PKC inhibitor peptide PKC(19-31) prevented this increase. Heat-inactivated PKC had no effect on K-ATP channel properties. K-ATP channel activity spontaneously returned to control levels after washout of PKC. This spontaneous reversal did not occur in the presence of 5 nmol/L okadaic acid, suggesting that the reversal of PKC's action is dependent on activity of a membrane-associated type 2A protein phosphatase (PP2A). In the presence of exogenous PP2A (7.5 nmol/L), PKC had no effect. We conclude that the PKC-induced increase in K-ATP channel activity at millimolar ATP results from a crossing of the ATP concentration-response curves for inhibition of the phosphorylated and nonphosphorylated forms of the channel. This identifies a mechanism by which PKC activates K-ATP channels at near physiological levels of ATP and thus could link these two components in a signaling pathway that induces ischemic preconditioning.