Endothelial TRPV and Potassium Channels Regulate Vascular Function

描述（由申请人提供）：内皮细胞（EC）排列在所有血管中，具有独特的位置，可检测神经体液和机械信号，并将其转化为诱导周围平滑肌松弛的细胞内分子信号。EC Ca 2+在启动血管舒张信号中起关键作用。人们越来越认识到， 对于这种血管调节功能重要的Ca 2+增加是局部发生的， 不是全球性的。EC中这种局部Ca 2+增加的一个重要来源是通过EC膜上的TRPV（瞬时受体电位香草酸）通道的Ca 2+内流。我最近在来自完整肠系膜动脉的EC中发现了通过单个TRPV 4通道-Ca 2+火花-的基本Ca 2+内流事件（Sonkusare等人，Science，2012），并且进一步证明了每个EC中少至三个TRPV 4通道的协同打开通过激活EC中的中等电导和小电导、Ca 2+敏感性钾通道而引起最大血管舒张。尽管有证据表明TRPV 1和V3通道存在于EC中，但尚未探索其基本性质和生理调节剂。通过TRPV 1/V3/V4通道监测单一Ca 2+内流的能力是研究这些通道的生理和病理作用的有力工具。我的初步数据首次表明，TRPV 1和V3通道与选择性激动剂的激活产生的生物物理特性，是不同的每种通道类型的火花。此外，生理调节剂，如Gq蛋白偶联受体（GqPCR）激动剂和温度，差异激活TRPV 1/3/4通道。该研究验证了TRPV 1/V3/V4通道受生理信号差异调节的假设，并差异参与效应通路以调节血管功能，并应揭示EC中局部血管舒张通信的通路。因此，这些研究将为理解血管疾病如高血压、糖尿病和动脉粥样硬化中内皮功能障碍的病理机制奠定坚实的基础。在指导阶段，我将采用最先进的电生理学和高分辨率共聚焦Ca 2+成像，一种新的光遗传学方法和EC Ca 2+和动脉直径的体内成像，以确定TRPV 1/V3通道的不同生物物理特征，并研究GqPCR信号对TRPV 4通道的调节。在此期间，我还将继续在我的咨询委员会的指导下继续我的专业和科学职业发展。在独立阶段，我将使用Ca 2+成像，压力肌描记术和EC膜片钳研究流量，温度和GqPCR信号转导对TRPV 1/V3通道的差异激活，以及TRPV 1/V3下游介导这些调节剂的血管舒张的途径。这个项目将有助于我继续接受技术、智力和专业培训，并将帮助我在学术研究机构建立一个独立的研究实验室。

DESCRIPTION (provided by applicant): Endothelial cells (ECs), which line all blood vessels, are uniquely positioned to detect neurohumoral and mechanical signals and transform them into intracellular molecular signals that induce relaxation of the surrounding smooth muscle. EC Ca2+ plays a pivotal role in initiating a vasodilatory signal. There is a growing appreciation that increases in Ca2+ that are important for this vasoregulatory function are those that occur locally, not globally. An important source of such local Ca2+ increases in ECs is Ca2+ influx through TRPV (transient receptor potential vanilloid) channels on EC membranes. I recently discovered elementary Ca2+ influx events through single TRPV4 channels-Ca2+ sparklets-in ECs from intact mesenteric arteries (Sonkusare et al., Science, 2012), and further demonstrated that cooperative opening of as few as three TRPV4 channels per EC causes maximal vasodilation through activation of intermediate- and small- conductance, Ca2+-sensitive potassium channels in ECs. Although there is evidence for TRPV1 and V3 channels in ECs, their elementary properties and physiological modulators have not been explored. The ability to monitor unitary Ca2+ influx through TRPV1/V3/V4 channels is a powerful tool for investigating the physiological and pathological roles of these channels. My preliminary data show for the first time that activation of TRPV1 and V3 channels with selective agonists produces sparklets with biophysical properties that are distinct for each channel type. Moreover, physiological modulators, such as Gq protein-coupled receptor (GqPCR) agonists and temperature, differentially activate TRPV1/3/4 channels. The proposed research tests the hypothesis that TRPV1/V3/V4 channels are differentially modulated by physiological signals and differentially engage effector pathways to regulate vascular function, and should reveal pathways of local vasodilatory communication in ECs. As such, these studies will lay a solid foundation for understanding pathological mechanisms responsible for endothelial dysfunction in vascular disorders such as hypertension, diabetes, and atherosclerosis. During the mentored phase, I will employ state-of-the-art electrophysiology and high-resolution confocal Ca2+ imaging, a novel optogenetic approach and in vivo imaging of EC Ca2+ and arterial diameter to determine the distinct biophysical signatures of TRPV1/V3 channels and investigate the modulation of TRPV4 channels by GqPCR signaling. During this phase, I will also continue my professional and scientific career development with continued guidance from my advisory committee. During the independent phase, I will use Ca2+ imaging, pressure myography and EC patch-clamp to study differential activation of TRPV1/V3 channels by flow, temperature, and GqPCR signaling, and pathways downstream of TRPV1/V3 that mediate vasodilation to these modulators. This project will facilitate my continued technical, intellectual, and professional training, and will asist me in establishing an independent research laboratory at an academic research institute.