Cholesterol Regulation of Endothelial K+ Channels

内皮 K 通道的胆固醇调节

基本信息

批准号：
10836797
负责人：
Irena Levitan
金额：
$ 8.26万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-06 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10836797
关键词：
Address Aorta Apolipoprotein E Atherosclerosis Binding Binding Sites Biochemical Blood Vessels Cardiovascular Diseases Cell Line Cells Cessation of life Cholesterol Clustered Regularly Interspaced Short Palindromic Repeats Complex Cytoskeleton Data Dependovirus Descending aorta Development Down-Regulation Dyslipidemias Electrophysiology (science)Endothelial Cells Endothelium Functional disorder Funding Gene Expression Genetic Goals Grain Grant High Density Lipoproteins Human Hydrophobicity Impairment In Vitro Individual Injury Ion Channel KDR gene Kir2.1 channel Lesion Low-Density Lipoproteins Mediating Modeling Molecular Monomeric GTP-Binding Proteins Morbidity - disease rate Mus Mutation Neutrons PECAM1 gene PIK3CG gene Phenotype Phospholipids Phosphorylation Plasma Play Potassium Channel Process Proteins Radiolabeled Regulation Research Risk Factors Role Signal Transduction Site-Directed Mutagenesis Testing Thinness Transgenic Model Transgenic Organisms Translating Transmembrane Domain Vasodilation Woman atheroprotective computer studies endothelial dysfunction gain of function hypercholesterolemia in vivo insight inward rectifier potassium channel loss of function men molecular dynamics molecular scale mouse model mutant novel novel therapeutic intervention overexpression predictive modeling response simulation transcriptome sequencing

项目摘要

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.

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向内抑制纠正 K+ (Kir) 通道，并且 Kir 通道在内皮细胞对血流的反应中发挥着重要作用。我们的长长期目标是阐明胆固醇诱导的内皮离子调节机制通道并确定胆固醇诱导的 Kir 抑制对血管功能的影响和动脉粥样硬化的发展。在本次资助的前期资助期间，我们发现了一种新模式通过多个动态接触来研究胆固醇-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舒张和 NO 释放中起着至关重要的作用，并表明高胆固醇血症诱导血流引起的血管舒张受损可归因于 Kir2.1 抑制。在当前的提案中，我们扩展这些研究以解决三个新目标：在目标 1 中，我们解决了如何胆固醇与我们已经确定的特定结合位点的结合转化为抑制作用的通道选通。具体来说，我们根据我们的计算研究提出了一个新的假设预测胆固醇结合会解偶联通道内的特定残基，这对于门控至关重要过程。该假设将通过结合多尺度分子动力学来解决模拟，一种最先进的计算方法，然后是定点突变，功能通过高通量电生理学、生化和中子分析通道功能散射研究评估胆固醇与 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 胆固醇抑制在两种主要内皮血流反应中的作用的研究：1) PECAM1/Src/VEGFR2/PI3K/Akt 信号轴的激活和 2) 细胞骨架重塑。这个目标是基于我们的 RNA 测序分析揭示了 Kir2.1 在流敏感基因中的主要作用表达包括 PECAM1/VEGFR2 机械传感器复合物的表达。具体来说，我们将检验以下假设：高胆固醇血症条件下抑制内皮 Kir 通道会损害流诱导的 VEGFR2 激活和小 GTP 酶 RhoA 的激活，并改变流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小鼠。我们已经确定 Kir2.1 的整体缺乏会夸大病变血脂异常 ApoE-/- 小鼠中的形成。在拟议的研究中，我们将确定该效果是否特定于内皮Kir2.1。此外，我们还将采用Kir2.1救援的新模型，即转基因CRISPR 表达胆固醇不敏感的 Kir2.1 突变体的小鼠。我们相信，综合起来，这些研究将为理解离子通道的胆固醇调节做出重大贡献，血脂异常引起的内皮功能障碍及其病变形成机制。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The effect of cellular cholesterol on membrane-cytoskeleton adhesion.

细胞胆固醇对膜细胞骨架粘附的影响。

DOI：
10.1242/jcs.001370
发表时间：
2007
期刊：
Journal of cell science
影响因子：
4
作者：
Sun,Mingzhai;Northup,Nathan;Marga,Francoise;Huber,Tamas;Byfield,FitzroyJ;Levitan,Irena;Forgacs,Gabor
通讯作者：
Forgacs,Gabor

On, in, and under membrane.

膜上、膜内和膜下。