Cholesterol Regulation of Endothelial K+ Channels

血脂异常引起的内皮功能障碍在动脉粥样硬化的发生中起着重要作用。我们的 研究发现，血浆高胆固醇血症会导致内皮细胞抑制- 整流钾(KIR)通道和KIR通道在内皮细胞对血流的反应中起主要作用。我们的龙 学期目标是阐明胆固醇诱导内皮细胞离子调节的机制。 并确定胆固醇抑制KIR对血管功能和血管功能的影响 动脉粥样硬化的发展。在前一次资助期间，我们发现了一种新的模式 通过多个动态接触的胆固醇-Kir2相互作用提供了Kir2.1发挥作用的直接证据 在血流诱导的血管扩张和NO释放中起关键作用，并表明高胆固醇血症诱导 血流诱导的血管扩张功能受损可归因于Kir2.1的抑制。在目前的提案中，我们 扩展这些研究以实现三个新目标：在目标1中，我们解决如何实现以下基本问题 胆固醇与我们已经确定的特定结合部位的结合转化为抑制 频道选通。具体地说，我们在计算研究的基础上提出了一个新的假设。 预测胆固醇结合解偶联通道内的特定残基，这对门控至关重要 进程。这一假说将使用多尺度分子动力学的组合来解决 模拟，一种最先进的计算方法，随后是定点突变，功能性 用高通量电生理、生化和中子分析通道功能 散射研究以评估胆固醇与Kir2通道的直接相互作用。在目标2中，我们将扩展我们的 确定Kir2.1在两种主要的内皮血流反应中的胆固醇抑制作用的研究：1) PECAM1/Src/VEGFR2/PI3K/Akt信号轴的激活与细胞骨架重构。这个目标是 基于我们的RNA测序分析，揭示了Kir2.1在流动敏感基因中的主要作用 表达包括PECAM1/VEGFR2机械传感器复合体的表达。具体来说，我们将 验证高胆固醇血症抑制内皮细胞KIR通道损害的假设 流动诱导的VEGFR2的激活和一种小的GTP酶RhoA的激活，并改变流动诱导的 细胞骨架重塑。最后，在目标3中，我们将确定内皮Kir2.1在病变形成中的作用 血脂异常的小鼠。我们已经证实，Kir2.1的全球缺失会夸大损伤 血脂异常ApoE-/-小鼠的形成。在拟议的研究中，我们将确定这种影响是否特定于 内皮细胞Kir2.1.此外，我们还将采用一种新的Kir2.1救援模型，转基因CRISPR 表达胆固醇不敏感Kir2.1突变体的小鼠。我们认为，这些研究放在一起 将对理解胆固醇对离子通道的调节做出重大贡献， 血脂异常引起的内皮功能障碍，以及病变形成的机制。

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 cholesterol regulation of ion channels, dyslipidemia-induced endothelial dysfunction, and the mechanisms of lesion formation.