Activation of both ATP-sensitive K+ (KATP) 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 (< or = 50 mumol/L), PKC inhibits KATP channel activity. This finding is surprising, as both KATP channels and PKC are activated during preconditioning. However, PKC also altered ATP binding to the channel, changing the Hill coefficient from approximately 2 to approximately 1. This apparent change in stoichiometry would lead to a PKC-induced activation of KATP channels at more physiological (millimolar) levels of ATP. The aim of the present study was to determine whether PKC activates cardiac KATP channels at millimolar levels of ATP. The effects of PKC on single KATP 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 KATP channel properties. KATP 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 KATP 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 KATP channels at near physiological levels of ATP and thus could link these two components in a signaling pathway that induces ischemic preconditioning.