Endothelial TRPV and Potassium Channels Regulate Vascular Function

描述(申请人提供)：内皮细胞(ECs)排列在所有血管中，具有独特的定位，可以检测神经体液和机械信号，并将它们转化为细胞内分子信号，诱导周围血管的松弛。EC Ca~(2+)在启动血管扩张信号中起着关键作用。越来越多的人意识到 对这种血管调节功能很重要的钙离子增加是局部发生的， 不是在全球范围内。内皮细胞局部钙升高的一个重要来源是通过内皮细胞膜上的瞬时受体电位香草酸(TRPV)通道的钙离子内流。我最近发现了肠系膜动脉内皮细胞通过单个TRPV4通道--钙小颗粒--的基本钙内流事件(Sonkusare et al.，Science，2012)，并进一步证明，合作开放每个EC中只有三个TRPV4通道，通过激活内皮细胞中的中、小电导、钙敏感的钾通道，可以产生最大的血管扩张。尽管已经有证据表明内皮细胞中存在TRPV1和V3通道，但它们的基本特性和生理调节器还没有被探索。通过TRPV1/V3/V4通道监测单个钙离子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。我的初步数据首次显示，用选择性激动剂激活TRPV1和V3通道会产生具有不同生物物理特性的火花，每种通道类型都不同。此外，生理调节剂，如Gq蛋白偶联受体激动剂和温度，可以不同地激活TRPV1/3/4通道。这项研究验证了TRPV1/V3/V4通道受到生理信号的不同调制，并通过不同的效应通路调节血管功能的假说，并应揭示内皮细胞局部血管扩张通讯的途径。因此，这些研究将为了解高血压、糖尿病和动脉粥样硬化等血管疾病中导致内皮功能障碍的病理机制奠定坚实的基础。在指导阶段，我将使用最先进的电生理学和高分辨率共聚焦钙离子成像，一种新的光遗传学方法，以及体内EC钙离子和动脉直径的成像来确定TRPV1/V3通道的不同生物物理特征，并研究GqPCR信号对TRPV4通道的调控。在这一阶段，我还将在我的咨询委员会的持续指导下，继续我的专业和科学职业发展。在独立阶段，我将使用钙离子成像、压力肌图术和EC膜片钳来研究TRPV1/V3通道通过流量、温度和GqPCR信号的差异激活，以及TRPV1/V3下游介导血管扩张到这些调节剂的通路。这个项目将有助于我继续进行技术、智力和专业培训，并将有助于我在学术研究所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.