Dyslipidemia-induced endothelial dysfunction plays a major role in the initiation of atherosclerosis. Our studies discovered that plasma hypercholesterolemia results in suppression of endothelial inwardly- rectifying K+ (Kir) channels and that Kir channels play a major role in endothelial response to flow. Our long term goal is to elucidate the mechanisms responsible for cholesterol-induced regulation of endothelial ion channels and determine the impact of cholesterol-induced suppression of Kir on vascular function and atherosclerosis development. During the previous funding period of this grant, we discovered a new mode of cholesterol-Kir2 interactions via multiple dynamic contacts, provided direct evidence that Kir2.1 plays a crucial role in flow-induced vasodilation and NO release, and showed that hypercholesterolemia-induced impairment of flow-induced vasodilation can be attributed to Kir2.1 suppression. In the current proposal, we extend these studies to address three new goals: In Aim 1, we address the fundamental question of how cholesterol binding to the specific binding sites that we have already identified translates into the inhibition of channel gating. Specifically, we address a novel hypothesis based on our computational studies predicting that cholesterol binding uncouples specific residues within the channels, crucial for the gating process. This hypothesis will be addressed using a combination of multi-scale Molecular Dynamics simulations, a state-of-the-art computational approach, followed by site-directed mutagenesis, functional analysis of the channel function by high throughput electrophysiology, and biochemical and neutron scattering studies to evaluate direct cholesterol interactions with Kir2 channels. In Aim 2, we will extend our studies to determine the role of cholesterol suppression of Kir2.1 in two major endothelial flow responses: 1) activation of PECAM1/Src/VEGFR2/PI3K/Akt signaling axis and 2) cytoskeleton remodeling. This aim is based on our RNA sequencing analysis that revealed a major role of Kir2.1 in flow-sensitive gene expression including the expression of PECAM1/VEGFR2 mechanosensor complex. Specifically, we will test the hypothesis that suppression of endothelial Kir channels by hypercholesterolemic conditions impairs flow-induced activation of VEGFR2 and activation of a small GTPase, RhoA, and alters flow-induced cytoskeletal remodeling. Finally, in Aim 3, we will determine the role of endothelial Kir2.1 in lesion formation of dyslipidemic mice. We have already established that the global deficiency of Kir2.1 exaggerates lesion formation in dyslipidemic ApoE-/- mice. In the proposed study, we will determine if the effect is specific for endothelial Kir2.1. Furthermore, we will also employ a new model of Kir2.1 rescue, a transgenic CRISPR mouse that expresses a cholesterol-insensitive Kir2.1 mutant. We believe that taken together, these studies will make a significant contribution to the understanding of c...