Cardiovascular drug target, TRPV2

阳离子通道TRPV2在心血管系统的发育和功能中是重要的。多 研究表明，它是门控响应机械刺激，但机制似乎是间接的， 目前还不清楚。据报道，靶向TRPV2的激活或抑制的药物具有有益的作用。 在动物模型和临床研究中， 药物的特异性和效力都很差。本项目的目标是更好地了解 TRPV2调节的结构和细胞基础，并开始开发更特异性和 有效的抑制剂和活化剂。具体目标1是确定磷酸肌醇的作用， 机械应力对纯化和重构状态下TRPV2活性的影响。磷酸肌醇组合物将是 操纵和通道活性与和不添加激动剂和抑制剂，或响应机械 刺激，将使用Ca2+指示剂染料或膜片钳电生理学测量。这些 实验将检验TRPV2是组成性活性通道并且受到负性抑制的假设。 磷脂酰肌醇（4，5）二磷酸（PIP2）的调节，以及相反的假设，PIP2是 刺激或改变通道选择性或磷酸肌醇作用是间接的。他们还将测试 假设TRPV2是直接机械敏感的。具体目标2是确定 磷酸肌醇和机械应力对TRPV2活性的影响。TRPV2将 在培养的细胞中表达，并使用Ca2+通量测定和膜片钳实验进行测定。表达 的重组酶工程响应细胞外配体，或膜电压，以及 荧光标记的磷酸肌醇结合结构域和抑制剂将用于操纵和监测 在膜的细胞质面上的磷酸肌醇。这些实验将检验这样一个假设， TRPV2在细胞环境中受细胞内磷酸肌醇和膜张力调节。具体 目的3：确定TRPV2调控的结构基础。TRPV2在脂质纳米材料中的结构将是 在存在磷酸肌醇以及激动剂和抑制剂的情况下，使用冷冻电子 显微镜诱变实验将靶向被认为在调节中起重要作用的残基， 磷酸肌醇或其它效应物。将测试突变对功能和结构的影响。这些 实验将进一步验证这一假设，即直接相互作用介导这些调节的影响。 分子，并确定那些这样做的结构基础。这些目标的实现将使 主要贡献，我们了解的一般原则，管理离子通道的调节， TRPV家族，阐明TRPV 2在正常生理和疾病中作用的机制基础，并帮助 以指导未来依赖TRPV2调节作为靶点的治疗努力。

The cation channel TRPV2 is important in development and function of the cardiovascular system. Multiple studies suggest it is gated in response to mechanical stimulus, but the mechanism appears to be indirect and is not yet known. Drugs targeting either activation or inhibition of TRPV2 have been reported to have beneficial effects on pathological cardiovascular symptoms in both animal models and clinical studies, but the available drugs have poor specificity and potency. The goal of this project is to develop a better understanding of the structural and cellular basis of TRPV2 regulation, and to begin the process of developing more specific and efficacious inhibitors and activators. Specific Aim 1 is to determine the effects of phosphoinositides and mechanical stress on TRPV2 activity in a purified and reconstituted state. Phosphoinositide composition will be manipulated and channel activity with and without added agonists and inhibitors, or in response to mechanical stimulus, will be measured using Ca2+-indicator dyes or with patch-clamp electrophysiology. These experiments will test the hypothesis that TRPV2 is a constitutively active channel and subject to negative regulation by phosphatidylinositol (4,5) bisphosphate (PIP2), as well as the counter hypotheses that PIP2 is stimulatory or alters channel selectivity or that effects of phosphoinositides are indirect. They will also test the hypothesis that TRPV2 is directly mechanosensitive. Specific Aim 2 is to determine the effects of phosphoinositides and mechanical stress on TRPV2 activity in the context of cellular membranes. TRPV2 will be expressed in cultured cells and assayed using Ca2+-flux assays and patch clamp experiments. Expression of recombinant enzymes engineered to respond to extracellular ligands, or to membrane voltage, as well as fluorescently tagged phosphoinositide binding domains and inhibitors, will be used to manipulate and monitor phosphoinositides on the cytoplasmic face of the membrane. These experiments will test the hypothesis that TRPV2 is regulated by intracellular phosphoinositides and membrane tension in a cellular context. Specific Aim 3: Determine the structural basis for regulation of TRPV2. The structure of TRPV2 in lipid nanodics will be determined in the presence of phosphoinositides as well as agonists and inhibitors using cryo-electron microscopy. Mutagenesis experiments will target residues proposed to play important roles in regulation by phosphoinositides or other effectors. Mutations will be tested for their effects on function and structure. These experiments will further test the hypothesis that direct interactions mediate the effects of these regulatory molecules and identify the structural basis for those which do so. Accomplishment of these aims will make a major contribution to our understanding of the general principles governing the regulation of ion channels of the TRPV family, clarify the mechanistic basis for the roles of TRPV2 in normal physiology and disease, and help to guide future therapeutic efforts relying on TRPV2 regulation as a target.