The regulation of ATP-sensitive potassium (K ATP ) channel activity is complex and a multitude of factors determine their open probability. Physiologically and pathophysiologically, the most important of these are intracellular nucleotides, with a long-recognized role for glycolytically derived ATP in regulating channel activity. To identify novel regulatory subunits of the K ATP channel complex, we performed a two-hybrid protein-protein interaction screen, using as bait the mouse Kir6.2 C terminus. Screening a rat heart cDNA library, we identified two potential interacting proteins to be the glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triose-phosphate isomerase. The veracity of interaction was verified by co-immunoprecipitation techniques in transfected mammalian cells. We additionally demonstrated that pyruvate kinase also interacts with Kir6.2 subunits. The physiological relevance of these interactions is illustrated by the demonstration that native Kir6.2 protein similarly interact with GAPDH and pyruvate kinase in rat heart membrane fractions and that Kir6.2 protein co-localize with these glycolytic enzymes in rat ventricular myocytes. The functional relevance of our findings is demonstrated by the ability of GAPDH or pyruvate kinase substrates to directly block the K ATP channel under patch clamp recording conditions. Taken together, our data provide direct evidence for the concept that key enzymes involved in glycolytic ATP production are part of a multisubunit K ATP channel protein complex. Our data are consistent with the concept that the activity of these enzymes (possibly by ATP formation in the immediate intracellular microenvironment of this macromolecular K ATP channel complex) causes channel closure.K ATP channels act as metabolic sensors of a large diversity of cell types by directly coupling their energy metabolism to cellular excitability. This function serves as a crucial regulatory mechanism in the responses of various cell types to their metabolic demand. For example, K ATP channels mediate insulin release from pancreatic ␤ cells, control the firing rate of glucose-responsive neurons in the ventromedial hypothalamus, and protect neurons during hypoxia. K ATP channels also have unique roles in the cardiovascular system. In the coronary vasculature, they participate in the maintenance of the coronary vascular tone, whereas in cardiac myocytes, K ATP channel modulation causes alterations in action potential duration and induction of arrhythmias during cardiac ischemia (1).The minimum requirement for the formation of a heterologously expressed K ATP channel appears to be the presence of two types of subunits, namely a pore-forming subunit (Kir6.x) belonging to the family of inward rectifying K ϩ channel subunits and a sulfonylurea receptor regulatory subunit, which is a member of the family of ABC-cassette proteins (2). However, ion channels are increasingly realized to be multisubunit macromolecular complexes (3-5). Recent evidence suggest that the K ATP channel pro...

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Prevailing data suggest that sarcolemmal ATP-sensitive (K ATP ) channels in the adult heart consist of Kir6.2 and SUR2A subunits, but the expression of other K ATP channel subunits (including SUR1, SUR2B, and Kir6.1) is poorly defined. The situation is even less clear for the immature heart, which shows a remarkable resistance to hypoxia and metabolic stress. The hypoxia-induced action potential shortening and opening of sarcolemmal K ATP channels that occurs in adults is less prominent in the immature heart. This might be due in part to the different biophysical and pharmacological properties of K ATP channels of immature and adult K ATP channels. Because these properties are largely conferred by subunit composition, it is important to examine the relative expression levels of the various K ATP channel subunits during maturation. We therefore used RNAse protection assays, reverse transcription-PCR approaches, and Western blotting to characterize the mRNA and protein expression profiles of K ATP channel subunits in fetal, neonatal, and adult mouse heart. Our data indicate that each of the K ATP channel subunits (Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B) is expressed in the mouse heart at all of the developmental time points studied. However, the expression level of each of the subunits is low in the fetal heart and progressively increases with maturation. Each of the subunits seems to be expressed in ventricular myocytes with a subcellular expression pattern matching that found in the adult. Our data suggest that the K ATP channel composition may change during maturation, which has important implications for K ATP channel function in the developing heart. ATP-sensitive K ϩ (K ATP ) channels are robustly expressed in the immature heart (1,2). However, K ATP channels in the immature heart may differ in several respects to those of adult cardiac tissue. In immature rabbit ventricle, K ATP channel open probability is roughly the same as in the adult, but they exhibit a significantly smaller single-channel conductance (~55 pS) and channel density, as defined by the number of open channels per patch (3). In the immature rat heart, K ATP channel density is also high, but the single-channel conductance and the mean open time are the same as in the adult (2). Reports describing a smaller unitary conductance in neonatal rat atrium (4) suggested that there may be regional differences. The immature rat heart K ATP channel has a lower open probability, and the channels are much more sensitive to block by intracellular ATP (2,5). In addition to these biophysical changes, there are reports that immature K ATP channels may have a different pharmacological profile. The neonatal atrial K ATP channel shows a unique functional and pharmacological profile resembling the pancreatic ␤ cell channel for its unusually high affinity for glibenclamide and diazoxide (4,5). These characteristics suggest that neonatal K ATP channels may differ in their molecular composition relative to the mature heart. K ATP channels are believed to be hetero-...

BackgroundElectrophysiological data suggest that cardiac KATP channels consist of Kir6.2 and SUR2A subunits, but the distribution of these (and other KATP channel subunits) is poorly defined. We examined the localization of each of the KATP channel subunits in the mouse and rat heart.ResultsImmunohistochemistry of cardiac cryosections demonstrate Kir6.1 protein to be expressed in ventricular myocytes, as well as in the smooth muscle and endothelial cells of coronary resistance vessels. Endothelial capillaries also stained positive for Kir6.1 protein. Kir6.2 protein expression was found predominantly in ventricular myocytes and also in endothelial cells, but not in smooth muscle cells. SUR1 subunits are strongly expressed at the sarcolemmal surface of ventricular myocytes (but not in the coronary vasculature), whereas SUR2 protein was found to be localized predominantly in cardiac myocytes and coronary vessels (mostly in smaller vessels). Immunocytochemistry of isolated ventricular myocytes shows co-localization of Kir6.2 and SUR2 proteins in a striated sarcomeric pattern, suggesting t-tubular expression of these proteins. Both Kir6.1 and SUR1 subunits were found to express strongly at the sarcolemma. The role(s) of these subunits in cardiomyocytes remain to be defined and may require a reassessment of the molecular nature of ventricular KATP channels.ConclusionsCollectively, our data demonstrate unique cellular and subcellular KATP channel subunit expression patterns in the heart. These results suggest distinct roles for KATP channel subunits in diverse cardiac structures.

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