The mechanism that promotes transition from the ATP-to the ADP-liganded state of ATP-sensitive K ؉ (K ATP ) channels and consequent channel opening in a cytosolic environment of high ATP concentration has yet to be understood. A mechanism examined here that could reverse the ATP-inhibited state is based on the action of adenylate kinase to catalyze phosphoryl transfer between ATP and AMP, resulting in transformation of ATP into ADP. In membrane patches excised from guinea pig cardiomyocytes, AMP alone did not affect channel behavior but increased the open probability of ATP-inhibited K ATP channels. This required MgCl 2 and a hydrolyzable form of ATP and was prevented by P 1 ,P 5 -di-adenosine-5 -pentaphosphate, an inhibitor of adenylate kinase. The single channel amplitude and kinetics of channel openings induced by the ADP-generating substrates of adenylate kinase, AMP and MgATP, were indistinguishable from the biophysical properties of the K ATP channel exhibited after addition of MgADP. In whole cell voltage-clamped cardiomyocytes, introduction of exogenous adenylate kinase along with millimolar MgATP and AMP induced a K ؉ current that was suppressed by a sulfonylurea blocker of K ATP channels. Enriched sarcolemmal membrane preparations were found to possess ATP⅐AMP phosphotransferase activity with properties attributable to an extramitochondrial isoform of adenylate kinase. These results indicate that adenylate kinase is a naturally occurring component of sarcolemmal membranes that could provide dynamic governance of K ATP channel opening through its phosphoryl transfer catalytic action in the microenvironment of the channel.ATP-sensitive K ϩ (K ATP ) 1 channels are involved in signaling networks that transduce cellular metabolic events into membrane potential changes and have been implicated in glucoseinduced insulin secretion in pancreatic ␤ cells or ischemiaassociated action potential shortening in heart muscle (1-5). Although the defining property of K ATP channels is their inhibition by intracellular ATP (5), which is readily demonstrable in excised membrane patches, as is the effect of ADP to reverse this ATP-inhibited state (1, 2, 6 -9), the mechanism by which opening of this channel is governed in situ has not been elucidated. The major question still to be resolved is how transition from the ATP-to the ADP-liganded state is accomplished.The common assumption that changes in the cytosolic concentrations of adenine nucleotides are the sole determinant of K ATP channel opening has been contested (2, 4, 6, 10 -13). In cardiac cells, the ATP concentration (ϳ5-10 mM) exceeds by over 100-fold the IC 50 value for K ATP channel closure (12, 13). This translates into a requirement for a change of two orders of magnitude in the intracellular ATP concentration, which is incompatible with cell viability, to achieve a mass action-induced change in the state of ATP-liganding and channel opening.Considering that altered concentrations of cytosolic ATP and/or ADP are not readily detectable nor correlated with predi...