1. ATP-sensitive K+ (K(ATP)+) channels are believed to make an important contribution to the increased cellular K+ efflux and shortening of the action potential duration (APD) during metabolic inhibition, hypoxia, and ischaemia in the heart. The mechanisms by which the activity of the K(ATP)+ channel is regulated during conditions of metabolic impairment are not completely clear. Extrinsic factors such as increased [ADP]i, acidosis, and stimulation of adenosine receptors appear to decrease the K(ATP)+ channel's sensitivity to closure by [ATP]i. The purpose of this study was to determine whether the K(ATP)+ channel itself is intrinsically altered by the processes associated with metabolic impairment. 2. Isolated guinea-pig ventricular myocytes were metabolically inhibited in glucose-free 1-8 mM Ca2+ Tyrode solution containing 9 mum rotenone and 0.9 muM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) while recording unitary currents through K(ATP)+ channels in cell-attached patches. When K(ATP)+ channel activity became maximal, the patch was excised (inside-out) into 150 mm K+ bath solution containing different ATP concentrations. The K(d) for suppression by [ATP]i ([ATP], causing half-maximal suppression of current through K(ATP)+ channels) was markedly increased to 305 muM (n = 9) compared to patches excised from control myocytes not exposed to metabolic inhibitors (K(d) = 46 mum, n = 28). 3. A [Ca2+]i-dependent process was involved in K(ATP)+ channel modification during metabolic inhibition. Removal of extracellular Ca2+ during metabolic inhibition led to an intermediate decrease in the ATP sensitivity of the K(ATP)+ channels (K(d) = 120 mum, n = 6). In myocytes that were pretreated with 10 mum ryanodine in addition to removing extracellular Ca 2+, the reduction in ATP sensitivity was completely prevented (K(d) = 23 muM, n = 6). 4. In inside-out membrane patches excised from control non-metabolically inhibited myocytes, elevated free [Ca 2+]i (2 mum) did not alter the sensitivity of the K(ATP)+ channel to closure by [ATP]i, suggesting that in metabolically inhibited myocytes elevated [Ca 2+]i acted indirectly. K(ATP)+ channel run-down was found to increase the sensitivity of K(ATP)+ channels to closure to [ATP]i (K(d) = 16 muM, n = 13). 5. Inside-out membrane patches excised from control non-metabolically inhibited myocytes were also exposed to various proteases, phospholipases and other reagents that may be activated during metabolic inhibition. Trypsin and chymotrypsin treatment increased the K(d) from 39 to 213 mum (n = 8) and 110 muM n = 5), respectively. Calpain I had no apparent effect on the K(d). Phospholipases A2, C and D were all found to modestly desensitize K(ATP)+ channels to closure by [ATP]i. Treatment of excised membrane patches with the free radical generating system, H2O2 + ferric chloride, or with the glycolytic inhibitor and sulfhydryl-modifying agent, iodoacetate, did not significantly affect the ATP sensitivity of the K(ATP)+ channel. 6. In conclusion, we have found that in isolated ventricular myocytes subjected to severe metabolic inhibition a Ca 2+ -dependent process intrinsically modified the K(ATP)+ channel to decrease its sensitivity to closure by [ATP]i. We speculate that this process may contribute to persistent cellular K+ loss and failure of APD to recover fully in reperfused myocardium after prolonged ischaemia.
