Cholesterol Regulation of Endothelial K+ Channels

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发生中起主要作用。我们的研究发现，血浆高胆固醇血症导致抑制内皮细胞内向整流K+（Kir）通道，一个主要的内皮细胞离子通道和流量传感器。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。在该资助的前两个资助期内，我们提供了对胆固醇诱导的Kir通道抑制的第一个机制性见解，并证明了它与体内血流诱导的血管舒张损伤相关。在目前的建议中，我们扩展这些研究，以解决三个新的目标：在目标1，我们进一步阐明胆固醇诱导的抑制Kir通道的分子基础，特别是专注于确定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，我们将使用分子动力学模拟的组合，一个国家的最先进的计算方法，与定点诱变，生物物理和生物化学方法，以确定这些突变对胆固醇敏感性的Kir通道和胆固醇-Kir结合的影响。将对结合通道和/或影响通道功能的能力不同的一系列不同甾醇进行分析。在目标2中，我们将扩展我们的研究，以确定内皮Kir通道的敏感性，促动脉粥样硬化和抗动脉粥样硬化脂蛋白的配置文件和血液动力学环境和测试的影响，胆固醇诱导的抑制内皮Kir通道之间的不平衡NO和ROS的生产。具体来说，我们将测试的假设，即Kir通道被抑制的促动脉粥样硬化脂蛋白（LDL，oxLDL）和流动环境（扰动流）和救援的抗动脉粥样硬化脂蛋白（HDL）和单向层流。此外，我们将解决的假设，抑制内皮细胞Kir的结果在抑制NO的释放和增加ROS的生产，这种机制有助于显着内皮功能障碍。最后，在目标3中，我们将检验胆固醇诱导的抑制胰岛素分泌的假设。 Kir通道在离体动脉中血流诱导的血管舒张损伤中起主要作用，这是内皮功能障碍的标志。具体来说，我们将首先测试Kir通道在ApoE-/-敲除小鼠内皮功能障碍中的作用，ApoE-/-敲除小鼠是一种成熟的动脉粥样硬化动物模型，然后我们将我们的研究扩展到从具有亲（高LDL）和抗（高HDL）致动脉粥样硬化脂蛋白谱的患者获得的活检中分离的人类动脉。分子生物学技术将应用于人动脉组织，以确定Kir通道对血流诱导的血管舒张的作用机制，这是血流调节的关键内皮依赖性机制。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。

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.