Cholesterol Regulation of Endothelial K+ Channels

内皮 K 通道的胆固醇调节

• 批准号: 8721685
• 负责人: David Eddington
• 金额: $ 40.92万
• 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/8721685
• 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; 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; novel therapeutic intervention; 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开始中起主要作用。我们的研究发现，血浆高胆固醇血症导致内皮内皮抑制内皮的K+（KIR）通道，主要的内皮离子通道之一和流动传感器。我们的长期目标是阐明负责胆固醇诱导的内皮离子通道调节的机制，并确定这些机制对内皮功能障碍的贡献。在该赠款的前两个资金期间，我们为胆固醇引起的KIR通道的抑制提供了第一个机械见解，并证明它与流量诱导的体内血管静脉的损害相关。在当前的提案中，我们将这些研究扩展到了三个新目标：在AIM 1中，我们进一步阐明了胆固醇诱导的抑制KIR通道的分子基础，专门针对识别推定的胆固醇结合位点并确定胆固醇如何调节通道座的降解。为了实现这一目标，我们将结合分子动力学模拟，最先进的计算方法以及定位的诱变以及生物物理和生化方法来确定这些突变对KIR通道胆固醇敏感性以及对胆固醇 - 基尔 - 基尔 - 基尔 - 基尔 - 基尔 - 基尔（Cholesterol-Kir-Kir）结合的影响。分析将针对各种不同的固醇阵列进行，这些固醇与通道和/或影响通道功能的能力有所不同。在AIM 2中，我们将扩展研究，以确定内皮基尔KIR通道对促和抗动脉粥样硬化脂蛋白谱和血液动力学环境的敏感性，并测试胆固醇诱导的抑制内皮kiR通道对NO和ROS产生之间不平衡的影响。具体而言，我们将检验以下假设：KIR通道被促动脉粥样硬化脂蛋白（LDL，OXLDL）和流动环境（流动障碍）抑制，并被抗动脉粥样硬化的脂蛋白（HDL）和层层层次单向流动。此外，我们将解决以下假设：抑制内皮KIR会导致抑制无释放和ROS产生的增加，并且这种机制对内皮功能障碍产生了重大贡献。最后，在AIM 3中，我们将测试胆固醇诱导抑制的假设 KIR通道在孤立的动脉中流动诱导的血管舒张损伤（内皮功能障碍的标志）中起着重要作用。具体而言，我们将首先测试KIR通道在APOE - / - 敲除小鼠中的内皮功能障碍中的作用，这是动脉粥样硬化的良好动物模型之一，然后我们将我们的研究扩展到从从患有Pro（高LDL）和Anti（高HDL）和抗（高HDL）的患者的活检中分离出的人类动物的研究。分子技术将用于人类动脉组织中，以确定KIR通道对流动诱导的血管舒张的贡献的机理，一种关键的血液流动调节依赖性内皮的机制。我们认为，这些研究将对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。