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 microM rotenone and 0.9 microM 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 Kd for suppression by [ATP]i ([ATP]i causing half-maximal suppression of current through K+ATP channels) was markedly increased to 305 microM (n = 9) compared to patches excised from control myocytes not exposed to metabolic inhibitors (Kd = 46 microM, 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 (Kd = 120 microM, n = 6). In myocytes that were pretreated with 10 microM ryanodine in addition to removing extracellular Ca2+, the reduction in ATP sensitivity was completely prevented (Kd = 23 microM, n = 6). 4. In inside-out membrane patches excised from control non-metabolically inhibited myocytes, elevated free [Ca2+]i (2 microM) did not alter the sensitivity of the K+ATP channel to closure by [ATP]i, suggesting that in metabolically inhibited myocytes elevated [Ca2+]i acted indirectly. K+ATP channel run-down was found to increase the sensitivity of K+ATP channels to closure to [ATP]i (Kd = 16 microM, 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 Kd from 39 to 213 microM (n = 8) and 110 microM (n = 5), respectively. Calpain I had no apparent effect on the Kd.(ABSTRACT TRUNCATED AT 400 WORDS)