Cholesterol regulation of endothelial K+ channels

描述（由申请人提供）：该项目的长期目标是确定膜脂组成在内皮离子通道调节中的作用。我们最近的研究表明，两种主要类型的内皮离子通道，即内校正K+通道（Kir）和容量调节CI-通道（VRAC）受到细胞胆固醇升高的强烈抑制，而细胞胆固醇是动脉粥样硬化发展的主要危险因素。本研究的重点是Kir通道，它在静态和血流动力学条件下对内皮膜电位的设定起主导作用，并且在内皮介导的血管张力控制的调节中也起重要作用。在初步研究中，我们已经表明细胞胆固醇的增加会导致活跃的Kir通道数量的减少。为了理解这种效应背后的机制，我们建议：(1)定义导致这种效应的Kir通道的结构决定因素。具体来说，确定胆固醇诱导的Kir抑制是否由已知的调节Kir的细胞内信号通路介导，以及Kir通道对胆固醇的敏感性是由细胞质尾部介导还是由通道的跨膜结构域介导。对两者的区分将有力地表明胆固醇和Kir之间的相互作用是直接的还是由其他蛋白质介导的。结构分析将使用两种互补的策略进行：不同的Kirs和嵌合蛋白的比较分析，以及定点突变。(2)确定活性Kir通道数量的减少是由于Kir通道蛋白表达受到抑制，其对质膜的靶向被破坏，还是异常地划分到富含胆固醇的脂质结构域。Western blot分析、定量PCR、荧光标记Kir蛋白的3D成像和富含胆固醇的脂质结构域的分离将被利用。(3)确定细胞胆固醇的增加是否会抑制暴露于生理水平剪切应力下的Kir通道。胆固醇对Kir活性的影响将在明确的剪切应力条件下进行评估，使用为此目的开发的新型“微创血流”设备。

DESCRIPTION (provided by applicant): The long-term goal of this project is to determine the role of membrane lipid composition in the regulation of endothelial ion channels. Our recent studies have shown that two major types of endothelial ion channels, the inwardly-rectifying K+ channels (Kir) and volume-regulated CI- channels (VRAC) are strongly suppressed by the elevation of cellular cholesterol, a major risk factor for the development of atherosclerosis. This study focuses on Kir channels that play the dominant role in setting endothelial membrane potential under static and hemodynamic conditions and are also known to play an important role in the regulation of endothelium-mediated control of vascular tone. In preliminary studies, we have shown that an increase in cellular cholesterol results in a decrease in the number of active Kir channels. To understand the mechanisms underlying this effect, we propose: (1) To define the structural determinants of the Kir channel that are responsible for this effect. Specifically, to determine whether cholesterol-induced suppression of Kir is mediated by intracellular signaling pathways that are known to regulate Kir, and whether the sensitivity of the Kir channels to cholesterol is mediated by the cytosolic tails or by the transmembrane domains of the channels. Discrimination between the two will give a strong indication of whether the interaction between cholesterol and Kir is direct or mediated by other proteins. Structural analysis will be performed using two complementary strategies: comparative analysis of different Kirs and chimeric proteins, and site-directed mutagenesis. (2) To determine whether a decrease in the number of active Kir channels is due to the suppression of Kir channel protein expression, disruption of its targeting to the plasma membrane or abnormal partition into cholesterol-rich lipid domains. Western blot analysis, quantitative PCR, 3D imaging of fluorescently-tagged Kir proteins and isolation of cholesterol-rich lipid domains will be utilized. (3) To determine whether an increase in cellular cholesterol suppresses Kir channels exposed to physiological levels of shear stress. The effect of cholesterol on Kir activity will be evaluated under well-defined shear stress conditions using a novel "Minimally Invasive Flow" device that was developed for this purpose.