The Role of CIC-6 in Vascular Control of Blood Pressure

项目总结 近一半的美国成年人患有高血压，这使他们患中风、血管疾病的风险增加。 损伤、心脏病发作、心力衰竭和肾脏疾病。最近的全基因组关联研究表明 CLCN6基因的一些突变可以降低高血压和中风的风险。CLCN6编码 电压敏感氯离子通道6(ClC-6)。到目前为止，人们对ClC-6的功能或其作用知之甚少 在血压动态平衡方面。这项提议的总体目标是建立生理和分子上的 ClC-6对血管平滑肌细胞功能和血压的影响 指导阶段：初步数据表明，ClC-6在高尔基体中表达 VSMC。我假设VSMC中ClC-6的活性通过提供一种 钙摄取的电荷平衡，从而维持膜的电子中性性。这通过以下方式发生 高尔基体特异的钙-ATPase、SPCA1和ClC-6的缺失减少高尔基体钙蓄积量 和信号，这改变了VSMC的收缩能力。具体目标1.确定ClC-6在高尔基体钙离子中的作用 在VSMC中处理。我将利用平面脂质双层电生理学和复杂的钙成像来 测定ClC-6氯通道特性及其对高尔基体钙存储能力的影响。 此外，这些实验将为高尔基体特异性钙释放在VSMC中的作用提供第一个证据。 CA2对血管收缩和扩张刺激的反应，这是以前从未被研究过的。 独立阶段：该项目的这一阶段将制定一条独立的调查路线，以 ClC-6对VSMC功能的分子作用从这些实验中获得的洞察力将进一步解释 ClC-6调节血压的生理机制。我的初步数据显示 已证实ClC-6可预防或减缓高血压发展过程中的大动脉硬化。这就做 进一步检测ClC-6对细胞增殖、迁移、凋亡和细胞外基质蛋白的影响 在血压正常和高血压的血管中沉积。我假设CLC-6的损失将减少 分泌细胞外基质蛋白，减缓细胞增殖，从而降低高血压引起的 纤维化和中膜增厚。这些改变将导致动脉硬化和血管的破坏。 重塑，并减少外周血管阻力，为ClC-6如何缓和提供了理论基础 血压。特定目的2.阐明ClC-6对血管僵硬和血管重塑的影响 在高血压期间。解决这一假设的实验将包括一系列不同的实验， 包括脉搏波速度测量、组织学分析、迁移分析、肌造影术、隧道和 BrdU检测和转录分析。这些研究将使我们了解这种蛋白质的功能 通过在分子、细胞和系统水平上定义该通道在血管系统中的作用。

PROJECT SUMMARY Nearly half of the US adult population has hypertension, which puts them at increased risk for stroke, vascular damage, heart attack, heart failure, and kidney disease. Recent genome-wide association studies have linked a number of mutations in the gene, CLCN6, to reduced hypertension and stroke risk. CLCN6 encodes the voltage-sensitive chloride channel 6 (ClC-6). To date, very little is known of about the function ClC-6, or its role in blood pressure homeostasis. The overall goal of this proposal is to establish the physiological and molecular roles of ClC-6 on vascular smooth muscle cell (VSMC) function and blood pressure. Mentored Phase: Preliminary data have demonstrated that ClC-6 is expressed in the Golgi apparatus of VSMCs. I hypothesize that ClC-6 activity in VSMCs regulates luminal Golgi Ca2+ stores by providing a charge balance for Ca2+ uptake, thereby maintaining membrane electroneutrality. This occurs through an association with the Golgi-specific Ca2+-ATPase, SPCA1, and loss of ClC-6 reduces Golgi Ca2+ stores and signaling, which alters VSMC contractility. Specific Aim 1. Establish the role of ClC-6 on Golgi Ca2+ handling in VSMCs. I will utilize planar lipid bilayer electrophysiology and sophisticated Ca2+ imaging to determine ClC-6 chloride channel properties and their impact on the Ca2+ storage capacity of the Golgi. Furthermore, these experiments will provide the first evidence of the role of Golgi-specific Ca2+ release in VSMC Ca2+ handling in response to vasocontraction and dilation stimuli, which has never before been examined. Independent Phase: This phase of the project will develop an independent line of investigation into the molecular effects of ClC-6 on VSMC function. Insight gained from these experiments will further explain the physiological mechanism underlying ClC-6 regulation of blood pressure control. My preliminary data have established that ClC-6 prevents or slows large artery vessel stiffening during development of hypertension. I will further examine the effect of ClC-6 on cellular proliferation, migration, apoptosis, and extracellular matrix protein deposition in normotensive and hypertensive vessels. I hypothesize that loss of ClC-6 will reduce extracellular matrix protein secretion and slow cell proliferation, thereby reducing hypertension induced fibrosis and media thickening. These changes will result in abrogated arterial stiffening and vessel remodeling, and reduce peripheral vascular resistance, providing a rationale for how ClC-6 moderates blood pressure. Specific Aim 2. Elucidate the influence of ClC-6 on vascular stiffness and vessel remodeling during hypertension. Experiments to address this hypothesis will include a diverse range of experiments, including pulse-wave velocity measurements, histological analyses, migration assays, myography, TUNNEL and BrdU assays, and transcriptomic analysis. These studies will inform our understanding of the protein's function by defining the role of this channel in vasculature at the molecular, cellular, and systemic levels.