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（PIPs）是膜受限的次级信使，通过其对离子泵和通道的作用调节多种细胞过程（如膜运输和细胞骨架动力学）和生物电信号。PIP和PIP结合蛋白的失调与多种疾病有关，包括各种癌症、发育性疾病，如Lowe综合征和Charcot- Marie-Tooth病，以及阿尔茨海默病。含有电压传感器的磷酸酶（VSPs）是一种电压控制的酶，可以从pip中去除磷酸基团。vsp是由两个结构域组成的模块化蛋白质；一个跨膜电压感应域（VSD）和一个细胞质磷酸酶域（PD）。这两个结构域由一个17个氨基酸组成的带正电的细胞内连接体连接在一起，该连接体将膜去极化传递给催化PD，激活和调节磷酸酶活性。该项目的目标是了解VSD感知的跨膜电压传递给PD的机制，使用两种不同的混合光学-电生理技术，可以实时监测特定蛋白质片段的运动：完整细胞的双电极电压钳荧光测定法（VCF）和切除斑块的片钳荧光测定法（PCF）。该项目正在开发这些技术的新变体，这将首次使测量细胞内结构域的运动成为可能，从而可以直接监测VSD-PD耦合和PD门控。深入了解跨膜电压对VSPs酶活性的影响机制，将为调节其活性水平和底物特异性的药理干预提供可能。靶向VSPs可能是控制PIP信号和解决由PIP和PIP结合蛋白失调影响的人类疾病的最佳途径。此外，参与信号级联反应的其他膜蛋白可能共享类似的机制，将细胞外刺激传递给酶活性。本提案中提出的技术，特别是在VCF和PCF中使用细胞内标记，应该适用于研究参与细胞内信号传导的其他膜蛋白。

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.