Cholesterol Regulation of Endothelial K+ Channels

内皮 K 通道的胆固醇调节

• 批准号: 9060393
• 负责人: David Eddington
• 金额: $ 41.86万
• 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 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9060393
• 关键词: Address; Affect; Animal Model; Antiatherogenic; ApoE knockout mouse; Apolipoprotein E; Arteries; Atherosclerosis; Binding; Binding Sites; Biochemical; Biological Assay; Biopsy; Blood Vessels; Blood flow; Breeding; Cardiovascular Diseases; Cells; Cessation of life; Cholesterol; Data; Development; Dominant-Negative Mutation; Down-Regulation; Dyslipidemias; Endothelial Cells; Endothelium; Environment; Event; Family suidae; Fatty acid glycerol esters; Functional disorder; Funding; Goals; Grant; Health; High Density Lipoproteins; Human; Impairment; Ion Channel; Kir2.1 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; Staging; Sterols; Techniques; Testing; Tissues; Transmembrane Domain; Vasodilation; Woman; base; endothelial dysfunction; hemodynamics; hypercholesterolemia; in vivo; insight; inward rectifier potassium channel; men; molecular dynamics; mouse model; novel therapeutic intervention; prevent; 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）：已知血脂异常诱导的内皮功能障碍在动脉粥样硬化的引发中起主要作用。我们的研究发现，血浆高胆固醇血症会抑制内皮内向整流 K+ (Kir) 通道，这是主要的内皮离子通道和流量传感器之一。我们的长期目标是阐明胆固醇诱导的内皮离子通道调节机制，并确定这些机制对内皮功能障碍的贡献。在该资助的前两个资助期间，我们首次对胆固醇诱导的 Kir 通道抑制提供了机制见解，并证明它与体内血流诱导的血管舒张受损相关。在当前的提案中，我们扩展了这些研究以实现三个新目标：在目标 1 中，我们进一步阐明了胆固醇诱导的 Kir 通道抑制的分子基础，特别侧重于识别假定的胆固醇结合位点并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、定点诱变以及生物物理和生化方法来确定这些突变对 Kir 通道胆固醇敏感性和胆固醇-Kir 结合的影响。将对一系列不同的甾醇进行分析，这些甾醇的结合通道和/或影响通道功能的能力不同。在目标 2 中，我们将扩展我们的研究，以确定内皮 Kir 通道对促动脉粥样硬化和抗动脉粥样硬化脂蛋白谱以及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 产生之间不平衡的影响。具体来说，我们将检验这样的假设：Kir 通道被促动脉粥样硬化脂蛋白（LDL、oxLDL）和流动环境（扰动流动）抑制，并被抗动脉粥样硬化脂蛋白（HDL）和层流单向流动拯救。此外，我们将提出这样的假设：抑制内皮 Kir 会导致 NO 释放的抑制和 ROS 产生的增加，并且这种机制显着导致内皮功能障碍。最后，在目标 3 中，我们将检验以下假设：胆固醇诱导的抑制 Kir 通道在孤立动脉中血流诱导的血管舒张受损中发挥着重要作用，这是内皮功能障碍的标志。具体来说，我们将首先在 ApoE-/- 基因敲除小鼠（一种成熟的动脉粥样硬化动物模型）中测试 Kir 通道在内皮功能障碍中的作用，然后我们将把我们的研究扩展到从具有亲（高 LDL）和抗（高 HDL）致动脉粥样化脂蛋白谱的患者的活检中分离出的人类动脉。分子技术将用于人体动脉组织，以确定 Kir 通道对血流诱导血管舒张的贡献机制，这是血流调节的关键内皮依赖性机制。我们相信，总的来说，这些研究将为理解离子通道的胆固醇调节做出重大贡献 和血脂异常引起的内皮功能障碍。