Cholesterol Regulation of Endothelial K+ Channels

内皮 K 通道的胆固醇调节

• 批准号: 9263758
• 负责人: David Eddington
• 金额: $ 42.36万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2020-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9263758
• 关键词: Address; Affect; Animal Model; Antiatherogenic; ApoE knockout mouse; Apolipoprotein E; Arteries; Atherosclerosis; Binding; Binding Sites; Biochemical; Biological Assay; Biophysics; Biopsy; Blood Vessels; Blood flow; Cardiovascular Diseases; Cells; Cessation of life; Cholesterol; Crossbreeding; Data; Development; Dominant-Negative Mutation; Down-Regulation; Dyslipidemias; Endothelial Cells; Endothelium; Environment; Event; Family suidae; Fatty acid glycerol esters; Functional disorder; Funding; Goals; Grant; High Density Lipoproteins; Human; Impairment; Ion Channel; Knockout Mice; Lipoproteins; Low-Density Lipoproteins; Mediating; Membrane; Modeling; Molecular; Morbidity - disease rate; Motion; Mus; Mutation; Patients; Plasma; Play; Potassium Channel; Production; Regulation; Resistance; Risk Factors; Role; Site-Directed Mutagenesis; Sterols; Techniques; Testing; Thinness; Tissues; Transmembrane Domain; Vasodilation; Woman; endothelial dysfunction; hemodynamics; hypercholesterolemia; in vivo; insight; inward rectifier potassium channel; men; molecular dynamics; mouse model; novel therapeutic intervention; oxidized low density lipoprotein; prevent; public health relevance; sensor; subcutaneous; trafficking

DESCRIPTION (provided by applicant): Dyslipidemia-induced endothelial dysfunction is known to play a major role in the initiation of atherosclerosis. Our studies discovered that plasma hypercholesterolemia results in suppression of endothelial inwardly-rectifying K+ (Kir) channels, one of the major endothelial ion channels and a flow sensor. Our long term goal is to elucidate the mechanisms responsible for cholesterol-induced regulation of endothelial ion channels and determine the contribution of these mechanisms to endothelial dysfunction. During the two previous funding periods of this grant, we have provided the first mechanistic insights into cholesterol-induced suppression of Kir channels and demonstrated that it correlates with an impairment of flow-induced vasodilatation in vivo. In the current proposal, we extend these studies to address three new goals: In Aim 1, we elucidate further the molecular basis of cholesterol-induced suppression of Kir channels specifically focusing on identifying putative cholesterol binding sites and determining how cholesterol binding regulates channel gating. To achieve this goal, we will use a combination of Molecular Dynamics simulations, a state-of-the-art computational approach, with site-directed mutagenesis, and biophysical and biochemical approaches to determine the impact of these mutations on cholesterol sensitivity of Kir channels and on cholesterol-Kir binding. The analysis will be done for an array of different sterols that differ in their ability to bind to the channels and/or affect channel function. In Aim 2, we will extend our studies to determine the sensitivity of endothelial Kir channels to pro- and anti-atherogenic lipoprotein profiles and hemodynamic environments and test the impact of cholesterol-induced suppression of endothelial Kir channels on the imbalance between NO and ROS production. Specifically, we will test the hypothesis that Kir channels are suppressed by pro-atherogenic lipoproteins (LDL, oxLDL) and flow environment (disturbed flow) and rescued by anti-atherogenic lipoproteins (HDL) and laminar unidirectional flow. Furthermore, we will address the hypothesis that suppression of endothelial Kir results in the inhibition of NO release and increase in ROS production and that this mechanism contributes significantly to endothelial dysfunction. Finally, in Aim 3, we will test the hypothesis that cholesterol-induced suppression of Kir channels plays a major role in the impairment of flow-induced vasodilatation in isolated arteries, a hallmark of endothelial dysfunction. Specifically, we will first test the role of Kir channels in endothelial dysfunction in ApoE-/- knockout mice, one of the well-established animal models of atherosclerosis and then we will extend our studies to human arteries isolated from biopsies obtained from patients with pro (high LDL) and anti (high HDL)- atherogenic lipoprotein profiles. Molecular techniques will be employed in human arterial tissue to determine the mechanism of Kir channels contribution to flow induced vasodilatation a critical endothelium- dependent mechanism of blood flow regulation. We believe that taken together, these studies will make a significant contribution to the understanding of cholesterol regulation of ion channels and dyslipidemia- induced endothelial dysfunction.

描述(由申请人提供)：已知血脂异常引起的内皮功能障碍在动脉粥样硬化的启动中起主要作用。我们的研究发现，血浆高胆固醇血症导致内皮细胞内向整流钾通道(KIR)的抑制，KIR通道是主要的内皮离子通道之一，也是流量传感器。我们的长期目标是阐明胆固醇诱导内皮细胞离子通道调节的机制，并确定这些机制在内皮功能障碍中的作用。在这笔赠款的前两个资助期，我们首次提供了对胆固醇诱导的KIR通道抑制的机械性见解，并证明了它与体内血流诱导的血管扩张受损有关。在目前的提案中，我们将这些研究扩展到三个新的目标：在目标1中，我们进一步阐明了胆固醇诱导抑制KIR通道的分子基础，特别是集中在识别可能的胆固醇结合部位和确定胆固醇结合如何调节通道门控。为了实现这一目标，我们将结合使用分子动力学模拟、最先进的计算方法和定点突变，以及生物物理和生化方法来确定这些突变对KIR通道的胆固醇敏感性和胆固醇-KIR结合的影响。将对不同的甾醇阵列进行分析，这些不同的甾醇在与通道结合和/或影响通道功能的能力上不同。在目标2中，我们将扩大我们的研究范围，以确定内皮KIR通道对促动脉粥样硬化和抗动脉粥样硬化的脂蛋白谱和血流动力学环境的敏感性，并测试胆固醇诱导的内皮KIR通道抑制对NO和ROS产生失衡的影响。具体地说，我们将检验KIR通道被促动脉粥样硬化的脂蛋白(LDL，oxLDL)和流动环境(扰乱的流动)抑制，并被抗动脉粥样硬化的脂蛋白(HDL)和层流单向流动所拯救的假设。此外，我们将解释这样的假设，即抑制内皮KIR导致NO释放的抑制和ROS产生的增加，并且这一机制显著地促进了内皮功能障碍。最后，在目标3中，我们将检验这样一种假设，即胆固醇诱导的抑制 KIR通道在血流诱导的血管扩张的损伤中起主要作用，而血流诱导的血管扩张是内皮功能障碍的标志。具体地说，我们将首先测试KIR通道在ApoE/-基因敲除小鼠内皮细胞功能障碍中的作用，ApoE/-基因敲除小鼠是公认的动脉粥样硬化的动物模型之一，然后我们将把研究扩展到从高密度脂蛋白血症患者(高密度脂蛋白)和高密度脂蛋白血症患者的活检中分离出的人动脉。分子技术将在人体动脉组织中被用来确定KIR通道在血流诱导的血管扩张中的作用机制，这是血流调节的关键的内皮依赖机制。我们相信，这些研究将对理解胆固醇对离子通道的调节做出重大贡献。 和血脂异常引起的内皮功能障碍。