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.

血脂异常诱导的内皮功能障碍在动脉粥样硬化的开始中起主要作用。我们的研究发现，血浆高胆固醇血症会导致内皮抑制内皮纠正K+（KIR）通道，并且KIR通道在对流动的内皮反应中起着重要作用。我们的漫长术语目标是阐明负责胆固醇诱导的内皮离子调节的机制通道并确定胆固醇诱导的KIR抑制对血管功能的影响动脉粥样硬化的发展。在这笔赠款的前几个资金期间，我们发现了一种新模式通过多个动态接触的胆固醇-KIR2相互作用的相互作用提供了直接证据，表明Kir2.1扮演A 在流动诱导的血管舒张中至关重要的作用，无释放，表明高胆固醇诱导的流动诱导的血管舒张的损害可以归因于KIR2.1抑制。在当前的提议中，我们扩展这些研究以解决三个新目标：在AIM 1中，我们解决了如何解决的基本问题胆固醇与我们已经确定的特定结合位点结合，转化为抑制作用通道门控。具体而言，我们根据计算研究解决了一个新的假设预测胆固醇结合在通道内的特定残基，对于门控至关重要过程。该假设将使用多尺度分子动力学的组合来解决模拟是一种最先进的计算方法，然后进行站点定向的诱变，功能性通过高吞吐量电生理学以及生化和中子来分析通道功能散射研究以评估与KIR2通道的直接胆固醇相互作用。在AIM 2中，我们将扩展我们的研究确定Kir2.1胆固醇在两个主要的内皮流动反应中的作用：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，该分析揭示了KIR2.1在流敏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，我们会的检验以下假设，即高胆固醇疾病抑制内皮的KIR通道损害流动诱导的VEGFR2激活以及小的GTPase，RhoA和变化流动诱导的激活细胞骨架重塑。最后，在AIM 3中，我们将确定内皮Kir2.1在病变形成中的作用血脂性小鼠的。我们已经确定，kir2.1的全球缺陷夸大了病变血脂血症APOE - / - 小鼠中的形成。在拟议的研究中，我们将确定效果是否特定于内皮kir2.1。此外，我们还将采用一种新模型Kir2.1 Rescue，一种转基因CRISPR 表达胆固醇不敏感的KIR2.1突变体的小鼠。我们认为这是这些研究将对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以及病变形成的机制。

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。