Faculty Bibliography

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

Sorcin is a penta-EF hand Ca2+-binding protein that associates with both cardiac ryanodine receptors and L-type Ca2+ channels and has been implicated in the regulation of intracellular Ca2+ cycling. To better define the function of sorcin, we characterized transgenic mice in which sorcin was overexpressed in the heart. Transgenic mice developed normally with no evidence of cardiac hypertrophy and no change in expression of other calcium regulatory proteins. In vivo hemodynamics revealed significant reductions in global indices of contraction and relaxation. Contractile abnormalities were also observed in isolated adult transgenic myocytes, along with significant depression of Ca2+ transient amplitudes. Whole cell ICa density and the time course of activation were normal in transgenic myocytes, but the rate of inactivation was significantly accelerated. These effects of sorcin on L-type Ca2+ currents were confirmed in Xenopus oocyte expression studies. Finally, we examined the expression of sorcin in normal and failing hearts from spontaneous hypertensive heart failure rats. In normal myocardium, sorcin extensively co-localized with ryanodine receptors at the Z-lines, whereas in myopathic hearts the degree of co-localization was markedly disrupted. Together, these data indicate that sorcin modulates intracellular Ca2+ cycling and Ca2+ influx pathways in the heart