Communication of Trans-membrane Voltage to the Enzymatic Active Site of Ci-VSP

描述（由申请人提供）：磷脂酰肌醇磷酸（PIP）是膜限制的第二信使，通过其对离子泵和通道的作用调节各种细胞过程（如膜运输和细胞骨架动力学）和生物电信号。PIP和PIP结合蛋白的失调涉及几种疾病，包括各种癌症、发育疾病如Lowe综合征和Charcot-Marie-Tooth病以及阿尔茨海默病。含电压传感器的磷酸酶（VSP）是从PIP去除磷酸基团的电压控制的酶。VSP是由两个结构域组成的模块蛋白;跨膜电压敏感结构域（VSD）和胞质磷酸酶结构域（PD）。这两个结构域通过17个氨基酸的带正电荷的细胞内接头保持在一起，该接头将膜去极化传递给催化PD，从而激活和调节磷酸酶活性。该项目的目标是了解使用两种不同的混合光电生理技术将室间隔缺损感测到的跨膜电压传递到PD的机制，这些技术能够真实的监测特定蛋白片段的运动：完整细胞中的双电极电压钳荧光测定法（VCF）和切除斑块上的膜片钳荧光测定法（PCF）。这些技术的新变化正在开发中，这将使它成为可能，第一次，以测量细胞内域的运动，使VSD-PD耦合和PD门控可以直接监测。深入了解跨膜电压与VSPs酶活性之间的联系机制，将为药物干预调节其活性水平和底物特异性提供可能性。靶向VSP可能是控制PIP信号传导和解决受PIP和PIP结合蛋白误调节影响的人类疾病的最佳方式。此外，它是可能的，其他膜蛋白参与信号级联共享类似的机制，用于通信细胞外刺激酶活性。本提案中提出的技术，特别是在VCF和PCF中使用细胞内标记，应适用于参与细胞内信号传导的其他膜蛋白的研究。

DESCRIPTION (provided by applicant): Phosphatidylinositol phosphates (PIPs) are membrane-confined secondary messengers that regulate a diverse range of cellular processes (such as membrane trafficking and cytoskeletal dynamics) and bioelectrical signaling through their action on ion pumps and channels. Misregulation of PIPs and PIP binding proteins is implicated in several diseases including various cancers, developmental diseases such as Lowe syndrome and Charcot- Marie-Tooth disease, and Alzheimer's disease. Voltage-sensor-containing phosphatases (VSPs) are voltage- controlled enzymes that remove phosphate groups from PIPs. VSPs are modular proteins comprised of two domains; a membrane-spanning voltage-sensing domain (VSD) and a cytosolic phosphatase domain (PD). The two domains are held together by a 17 amino acid, positively charged intracellular linker that communicates membrane depolarization to the catalytic PD, activating and modulating phosphatase activity. The goal of this project is to understand the mechanism by which transmembrane voltage sensed by the VSD is communicated to the PD using two different hybrid optical-electrophysiological techniques that enable motions of specific protein segments to be monitored in real time: two-electrode voltage-clamp fluorometry (VCF) in intact cells and patch-clamp fluorometry (PCF) on excised patches. Novel variations on these techniques are being developed for this project that will make it possible, for the first time, to measure the motions of intracellular domains, so that VSD-PD coupling and PD gating can be monitored directly. Insight into the mechanism by which transmembrane voltage is communicated to enzymatic activity in VSPs will open up the possibility of pharmacological intervention to adjust their activity level and substrate specificity. Targeting VSPs may be an optimal way to control PIP signaling and address human diseases affected by misregulation of PIPs and PIP binding proteins. Additionally, it is likely that other membrane proteins involved in signaling cascades share similar mechanisms for communicating extracellular stimuli to enzymatic activity. The techniques presented in this proposal, specifically the use of intracellular labels in VCF and PCF, should be applicable to the study of other membrane proteins involved in intracellular signaling.