Molecular identity and hypercholesterolemia modulation of inwardly-rectifying potassium channels in endothelial cells
Abstract
Inwardly rectifying K+ (Kir) channels are responsible for maintaining endothelial membrane potential and play a key role in endothelium-dependent vasorelaxation. In this dissertation, several complementary strategies were applied to determine the Kir2 subunit composition of human aortic endothelial cells (HAECs). It was further demonstrated, for the first time, that endothelial Kir channels are suppressed by hypercholesterolemic levels of lipoproteins in vitro and by serum hypercholesterolemia in vivo. Moreover, the sensitivity of endothelial Kir channels to hemodynamic shear force was also determined and the shear-sensitivity of endothelial Kir channel under hypercholesterolemia conditions was investigated. Message RNA detection and quantitation showed specific expression of all four Kir2.x subunits in HAECs. Western blot and functional analyses, along with application of dominant-negative constructs, suggest that Kir2.2 and Kir2.1 are primary determinants of endogenous K+ conductance in HAECs under resting conditions and that Kir2.2 provides the dominant conductance in these cells. Activation of endothelial Kir channels by a laminar shear force furthermore underscores the key role of Kir channels in endothelium-dependent, flow-mediated regulation of vascular tone. The modulation of endothelial Kir channels by hypercholesterolemic environments was determined in this dissertation since plasma hypercholesterolemia is a major risk factor for development of endothelial dysfunction but little is known about the underlying molecular mechanism. Exposing human aortic endothelial cells to acetylated low-density lipoprotein or very low density lipoprotein resulted in a time- and concentration-dependent decrease in Kir current that correlated with the degree of cholesterol loading. The suppression was fully reversible by cholesterol depletion. A decrease in Kir current resulted in depolarization of endothelial membrane potential. Most important, the flow sensitivity of Kir currents was also impaired by cholesterol loading. Furthermore, this study showed that hypercholesterolemia in vivo also strongly suppresses endothelial Kir currents and causes a shift in endothelial membrane potential, as determined by comparing the currents in aortic endothelial cells freshly isolated from healthy or hypercholesterolemic pigs. As a whole, this dissertation addresses mechanistically the key determinants of endothelial membrane potential and illustrates one of the potential key mechanisms underlying hypercholesterolemia-induced endothelial dysfunction. ^
Subject Area
Biology, Cell|Engineering, Biomedical
Recommended Citation
Yun Fang,
"Molecular identity and hypercholesterolemia modulation of inwardly-rectifying potassium channels in endothelial cells"
(January 1, 2006).
Dissertations available from ProQuest.
Paper AAI3225455.
http://repository.upenn.edu/dissertations/AAI3225455