ATP-sensitive K(+) (K(ATP)) channels in the heart are normally closed by high intracellular ATP, but are activated during ischemia to promote cellular survival. These channels are heteromultimers composed of Kir6.2 subunit, an inwardly rectifying K(+) channel core, and SUR2A, a regulatory subunit implicated in ligand-dependent regulation of channel gating. Here, we have shown that the muscle form (M-LDH), but not heart form (H-LDH), of lactate dehydrogenase is directly physically associated with the sarcolemmal K(ATP) channel by interacting with the Kir6.2 subunit via its N-terminus and with the SUR2A subunit via its C-terminus. The species of LDH bound to the channel regulated the channel activity despite millimolar concentration of intracellular ATP. The presence of M-LDH in the channel protein complex was required for opening of K(ATP) channels during ischemia and ischemia-resistant cellular phenotype. We conclude that M-LDH is an integral part of the sarcolemmal K(ATP) channel protein complex in vivo, where, by virtue of its catalytic activity, it couples the metabolic status of the cell with the K(ATP) channels activity that is essential for cell protection against ischemia.

Cardiac sarcolemmal ATP-sensitive K + (K ATP ) channels, composed of Kir6.2 and SUR2A subunits, couple the metabolic status of cells with the membrane excitability. Based on previous functional studies, we have hypothesized that creatine kinase (CK) may be a part of the sarcolemmal K ATP channel protein complex. The inside-out and whole cell patch clamp electrophysiology applied on guinea pig cardiomyocytes showed that substrates of CK regulate K ATP channels activity. Following immunoprecipitation of guinea-pig cardiac membrane fraction with the anti-SUR2 antibody, Coomassie blue staining revealed, besides Kir6.2 and SUR2A, a polypeptide at ∼48 kDa. Western blotting analysis confirmed the nature of putative Kir6.2 and SUR2A, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis identified p48 kDa as a muscle form of CK. In addition, the CK activity was found in the anti-SUR2A immunoprecipitate and the cross reactivity between an anti-CK antibody and the anti-SUR2A immunoprecipitate was observed as well as vice verse. Further results obtained at the level of recombinant channel subunits demonstrated that CK is directly physically associated with the SUR2A, but not the Kir6.2, subunit. All together, these results suggest that the CK is associated with SUR2A subunit in vivo, which is an integral part of the sarcolemmal K ATP channel protein complex. Keywordsheart; K ATP channels; SUR2A; Kir6.2 In the heart, creatine kinase (CK) is a major phosphotransfer system essential in supporting cardiac energy balance (1). To ensure communications between sites that generate, use, and sense ATP, cardiac cells rely on phosphotransfer networks that facilitate the transfer and distribution of energy-rich phosphoryls between cellular compartments in a kinetically and thermodynamically efficient manner mediated by CK (2).ATP-sensitive K + (K ATP ) channels belong to a group of intracellular ATP sensors and they couple the metabolic status of cell with membrane excitability (3). Numerous studies have demonstrated that potassium channel openers, drugs that promote opening of K ATP channels, decrease infarct size, mimic ischemic preconditioning, and improve functional and energetic recovery of cardiac muscle following ischemic and hypoxic insults (4,5). More recently, evidence has suggested that activation of both sarcolemmal and mitochondrial K ATP channels may promote cellular survival (4), which would agree with the idea that these channels may communicate through phosphotransfer reactions from an intracellular compartment to the cell UKPMC Funders Group Author ManuscriptUKPMC Funders Group Author Manuscript membrane (1). The structure of mitochondrial K ATP channels is still unknown, but the proteins constituting the sarcolemmal K ATP channel complex have been cloned recently (6-8). Sarcolemmal K ATP channels are heteromultimers composed of two structurally distinct proteins (Kir6.2 and SUR2A) (7). The Kir6.2 subunit was shown to form the inwardly rectifying K + channel core, p...

Chronic exposure to lower oxygen tension may increase cellular resistance to different types of acute metabolic stress. Here, we show that 24-h-long exposure to slightly decreased oxygen tension (partial pressure of oxygen (PO 2 ) of 100 mm Hg instead of normal 144 mm Hg) confers resistance against acute hypoxia/reoxygenation-induced Ca 2؉ loading in heart-derived H9c2 cells. The number of ATP-sensitive K ؉ (K ATP ) channels were increased in cells exposed to PO 2 ‫؍‬ 100 mm Hg relative to cells exposed to PO 2 ‫؍‬ 144 mm Hg. This was due to an increase in transcription of SUR2A, a K ATP channel regulatory subunit, but not Kir6.2, a K ATP channel poreforming subunit. PO 2 ‫؍‬ 100 mm Hg also increased the SUR2 gene promoter activity. Experiments with cells overexpressing wild type of hypoxia-inducible factor (HIF)-1␣ and dominant negative HIF-1␤ suggested that the HIF-1-signaling pathway did not participate in observed PO 2 -mediated regulation of SUR2A expression. On the other hand, NADH inhibited the effect of PO 2 ‫؍‬ 100 mm Hg but not the effect of PO 2 ‫؍‬ 20 mm Hg. LY 294002 and PD 184 352 prevented PO 2 -mediated regulation of K ATP channels, whereas rapamycin was without any effect. HMR 1098 inhibited the cytoprotective effect of PO 2 ‫؍‬ 100 mm Hg, and a decrease of PO 2 from 144 to 100 mm Hg did not change the expression of any other gene, including those involved in stress and hypoxic response, as revealed by Affymetrix high density oligonucleotide arrays. We conclude that slight hypoxia activates HIF-1␣-independent signaling cascade leading to an increase in SUR2A protein, a higher density of K ATP channels, and a cellular phenotype more resistant to acute metabolic stress.