The glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, and pyruvate kinase are components of the K(ATP) channel macromolecular complex and regulate its function.

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.

Expression of ATP-sensitive K+ channel subunits during perinatal maturation in the mouse heart.

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.

Immunolocalization of KATP channel subunits in mouse and rat cardiac myocytes and the coronary vasculature.

BACKGROUND: Electrophysiological 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. RESULTS: Immunohistochemistry 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. CONCLUSIONS: Collectively, 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.

K ATP channels of primary human coronary artery endothelial cells consist of a heteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits.

Functional ATP-sensitive potassium (K(ATP)) channels can be reconstituted by expression of various combinations of different pore-forming subunits (Kir6.1 and Kir6.2) and sulfonylurea receptor (SUR) subunits. Using dominant negative and gene knockout approaches, Kir6.2 subunits have been identified as required pore-forming components of plasmalemmal K(ATP) channels in ventricular myocytes. Previous data obtained in heterologous expression systems suggest that Kir6.1 and Kir6.2 subunits are capable of forming a functional heteromultimeric channel complex. However, until now the existence of such heteromultimeric Kir6.1/Kir6.2 complexes has not been demonstrated for native K(ATP) channels. The primary aim of this study was to identify the molecular composition of native K(ATP) channels in primary human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) from human origin. We specifically investigated the potential that heteromultimeric Kir6.1/Kir6.2 channels exist in these cells. Using reverse transcriptase-polymerase chain reaction, we detected the expression of Kir6.1, Kir6.2, and SUR2B in both cell types. Western blotting and immunoprecipitation assays demonstrated the presence of Kir6.1 protein in both HCAEC and HCASMC; however, Kir6.2 protein was only expressed in HCAEC. Interaction between Kir6.1 and Kir6.2 subunits was demonstrated by reciprocal co-immunoprecipitation of these two subunits in HCAEC. Furthermore, Kir6.1 and Kir6.2 were detected in the immunoprecipitate when using an anti-SUR2 antibody. Confocal microscopy imaging demonstrated Kir6.1 and Kir6.2 subunits to co-localize at the cell surface membrane in HCAEC. In conclusion, our data characterize the molecular composition of primary human coronary smooth muscle and endothelial cells. We demonstrate that human coronary endothelial K(ATP) channels consist of a heteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits.