Cardiovascular drug target, TRPV2

阳离子通道TRPV2在心血管系统的发育和功能中起重要作用。多重 研究表明，它是对机械刺激的反应，但其机制似乎是间接的和 目前还不清楚。据报道，针对TRPV2激活或抑制的药物具有 在动物模型和临床研究中对病理性心血管症状的影响，但可用的 药物的特异性和效力都很差。这个项目的目标是更好地了解 TRPV2调控的结构和细胞基础，并开始开发更具体和 有效的抑制剂和激活剂。具体目标1是确定磷脂酰肌醇和 机械应力对纯化和重组状态下TRPV2活性的影响。磷脂酰肌醇的组成将是 在有或没有添加激动剂和抑制剂的情况下，或在对机械刺激的反应中，操纵和引导活性 刺激，将使用钙离子指示剂染料或膜片钳电生理测量。这些 实验将检验这样的假设，即TRPV2是一个结构性活跃的通道，并受到负值的影响 磷脂酰肌醇(4，5)二磷酸(PIP2)的调节，以及PIP2是 刺激或改变通道选择性，或肌醇磷脂的作用是间接的。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 肌醇磷脂和机械应力对细胞膜中TRPV2活性的影响。TRPV2将 在培养的细胞中表达，并通过钙离子通量分析和膜片钳实验进行检测。表达式 重组酶被设计成对细胞外配体或膜电压以及 荧光标记的磷脂酰肌醇结合结构域和抑制物将用于操纵和监测 膜的细胞质表面有磷脂酰肌醇。这些实验将检验这一假设 TRPV2在细胞内受磷脂酰肌醇和膜张力的调节。特定的 目的3：确定TRPV2调控的结构基础。脂类纳米粒中TRPV2的结构将是 用冷冻电子在有磷脂酰肌醇以及激动剂和抑制剂存在的情况下测定 显微镜。突变实验将针对被认为在调控中发挥重要作用的残基 磷脂酰肌醇或其他效应物。突变将被测试其对功能和结构的影响。这些 实验将进一步检验这样的假设，即直接相互作用调节这些调节的效果。 并确定那些这样做的分子的结构基础。这些目标的实现将使 对我们理解离子通道调节的一般原则有重大贡献。 TRPV家族，阐明TRPV2在正常生理和疾病中作用的机制基础，并有助于 以TRPV2调控为靶点，指导未来的治疗努力。

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.