Diadenosine tetraphosphate-gating of cardiac K_ATP channels requires intact actin cytoskeleton

Abstract.

Diadenosine polyphosphates (ApnA) have been recently discovered in the heart, and their levels found to be regulated by ischemia. These signaling molecules are believed to regulate cellular processes that alarm a cell to metabolic stress. In particular, changes in cardiac diadenosine polyphosphates (ApnA) levels may contribute to the regulation of ATP-sensitive K⁺ (K_ATP) channel activity, an ion channel that couples the cellular metabolic state with membrane excitability. A feature of myocardial ischemia is the disruption of the actin cytoskeleton which critically regulates the behavior of K_ATP channels. Whether the integrity of actin microfilaments regulates the interaction of ApnA with K_ATP channels is not known. The inside-out configuration of the patch-clamp technique was applied to cardiomyocytes isolated from guinea-pig heart. Following patch excision, the prototype dinucleotide, diadenosine tetraphosphate (Ap4A), inhibited K_ATP channel opening. Treatment of the internal side of membrane patches with either cytochalasin B or DNase I, disrupters of the actin cytoskeleton, prevented Ap4A-induced inhibition of K_ATP channel opening. Application of purified actin to DNase-treated membrane patches restored the ability of Ap4A to close K_ATP channels. This study shows that inhibition of cardiac K_ATP channel by Ap4A, a putative alarmone, requires intact subsarcolemmal actin network. Such interaction between K_ATP channels, the cardiomyocyte cytoskeleton and intracellular Ap4A could affect different channel-dependent functions.