PROTEIN-KINASE C-INDUCED CHANGES IN THE STOICHIOMETRY OF ATP BINDING ACTIVATE CARDIAC ATP-SENSITIVE K- A POSSIBLE MECHANISTIC LINK TO ISCHEMIC PRECONDITIONING( CHANNELS )

Activation of both ATP-sensitive K+ (K-ATP) channels and the enzyme pr otein kinase C (PKC) has been associated with the cardioprotective res ponse of ischemic preconditioning. We recently showed that at low cyto plasmic ATP (less than or equal to 50 mu mol/L), PKC inhibits K-ATP ch annel activity. This finding is surprising, as both K-ATP channels and PKC are activated during preconditioning. However, PKC also altered A TP binding to the channel, changing the Hill coefficient from approxim ate to 2 to approximate to 1. This apparent change in stoichiometry wo uld lead to a PKC-induced activation of K-ATP channels at more physiol ogical (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 studie d at millimolar internal ATP levels using excised inside-out membrane patches from rabbit ventricular myocytes. Application of purified cons titutively active PKC (20 nmol/L) to the intracellular surface of the patches produced an approximately threefold increase in the channel op en probability. The specific PKC inhibitor peptide PKC(19-31) prevente d this increase. Heat-inactivated PKC had no effect on K-ATP channel p roperties. K-ATP channel activity spontaneously returned to control le vels 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 2 A protein phosphatase (PP2A). In the presence of exogenous PP2A (7.5 n mol/L), PKC had no effect. We conclude that the PKC-induced increase i n K-ATP channel activity at millimolar ATP results from a crossing of the ATP concentration-response curves for inhibition of the phosphoryl ated and nonphosphorylated forms of the channel. This identifies a mec hanism by which PKC activates K-ATP channels at near physiological lev els of ATP and thus could link these two components in a signaling pat hway that induces ischemic preconditioning.