ATP-sensitive potassium channels (KATP) in the heart act as metabolic sensors that open when cellular energy levels fall and are thought to play a role in cardioprotection. KATP are comprised of four pore-forming Kir subunits and four sulphonylurea subunits, the isoforms that comprise the sarclemmal KATP in ventricular myocytes are predominantly kir6.2 and SUR2A although Kir6.1/SUR2B subunits have been detected. As current pharmacological tools do not allow distinction between the roles of each KATP channel subunit we have developed adenoviral shRNA and dominant negative (dn) constructs targeting Kir6.2. Single ventricular myocytes were isolated from adult male Wistar rats, infected with adenovirus and cultured. Whole-cell patch clamp was used to quantify knockdown of KATP current. After 24h current from cells infected with dnKir6.2 had been reduced to 46% of current observed in control cells (control 19.9±1.3sem pA/pF n=5; dnKir6.2 9.2±1.7 pA/pF; student t-test p<0.05), comparatively current from cells incubated with Kir6.2 shRNA had been reduced to 57% of that from control cells (control 9.4±1.3 pA/pF, n=11; shRNA 5.4±0.9 pA/pF, n=14 p<0.05). Effective Ca2+ homeostasis is a critical determinate of the ability of cardiac myocytes to recover from ischemia and reperfusion (I/R). Myocytes were continuously perfused with Tyrode buffer and their contraction synchronized by field stimulation at 1Hz. Intracellular Ca2+ ([Ca2+]i) was monitored using fura-2 during simulated I/R injury; a metabolic insult of 4 min perfusion with cyanide (2mM) and iodoacetic acid (1mM) in substrate-free Tyrode followed by reperfusion with Tyrode for 10 min. Downregulation of Kir6.2 by both shRNA and dnKir6.2 resulted in higher [Ca2+]i after I/R (fura-2 ratio; control, 0.59±0.04 n=14; kir6.2 shRNA, 0.83±0.06 n=23; dnKir6.2, 0.93±0.06 n=24; p<0.05 vs controls). To investigate any effect of Kir6.2 downregulation on the protective effect of ischemic preconditioning (IPC) we used an ischemic pelleting technique to simulate ischemia in isolated myocytes. Ischemia was achieved by pelleting cells under a layer of mineral oil for 60 min followed by reperfusion for 60 min. IPC was induced by 10 min pelleting and 10 min reperfusion prior to prolonged ischemia. IPC increased survival in myocytes infected with control shRNA from 41±4% (n=768) to 58±3% (n=717; p<0.01). The survival of myocytes expressing Kir6.2 shRNA was not significantly increased by IPC (ischemia, 32±4% n=825; preconditioning 36±3% n=770). These results show that both constructs, dnKir6.2 and Kir6.2 shRNA downregulated KATP current, and increased Ca2+ loading following simulated I/R. Unlike the shRNA, dnKir6.2 reduces KATP current without reducing channel number. Therefore, our results are consistent with functional Kir6.2 containing KATP channels having a direct role in the cardioprotective response to metabolic stress.