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

跨膜电压与 Ci-VSP 酶活性位点的通讯

• 批准号: 8718448
• 负责人: Daniel Montgomery Clemens
• 金额: $ 4.99万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-17 至 2017-02-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8718448
• 关键词: Active Sites; Address; Affect; Alzheimer' s Disease; Amino Acids; Binding; Biological Assay; Catalytic Domain; Cell membrane; Cell physiology; Cells; Characteristics; Charcot-Marie-Tooth Disease; Charge; Communication; Complement; Coupling; Cysteine; Data; Development; Disease; Drug Design; Electrodes; Electrostatics; Enzymes; Family; Fluorometry; Future; Goals; Grant; Hybrids; Hydrogen Bonding; Intervention; Ion Channel; Ion Pumps; Label; Malignant Neoplasms; Measurement; Measures; Mediating; Membrane; Membrane Protein Traffic; Membrane Proteins; Methods; Minor; Monitor; Motion; Movement; Mutation; Oculocerebrorenal Syndrome; Optics; Organism; PH Domain; Phosphate-Binding Proteins; Phosphatidylinositol Phosphates; Phosphoric Monoester Hydrolases; Phosphotransferases; Play; Proteins; Public Health; Reporter; Role; Signal Transduction; Stimulus; Structure; Substrate Specificity; Techniques; Testing; Time; Variant; Work; extracellular; flexibility; human disease; inorganic phosphate; insight; novel; patch clamp; public health relevance; research study; sensor; tensin; voltage; voltage clamp

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.

描述（由申请人提供）：磷脂酰肌醇磷酸盐（PIPS）是膜夹层的二级信使，通过对离子泵和频道的作用，可以调节各种细胞过程（例如膜运输和细胞骨架动力学）和生物电信信号。 PIPS和PIP结合蛋白的不正调与多种疾病有关，包括各种癌症，Lowe综合征和Charcot-Marie-Tooth病等发育疾病以及阿尔茨海默氏病。含电压传感器的磷酸酶（VSP）是电压控制的酶，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。跨膜电压 - 感应域（VSD）和胞质磷酸酶结构域（PD）。这两个结构域由17个氨基酸（带正电荷的细胞内接头）固定在一起，该氨基酸将膜去极化传达到催化PD，激活和调节磷酸酶活性。 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.为该项目开发了有关这些技术的新型变化，这将使衡量细胞内结构域运动的动作成为可能，从而可以直接监视VSD-PD耦合和PD门控。深入了解跨膜电压传达到VSP中的酶活性的机制将开辟药理干预的可能性，以调整其活性水平和底物特异性。靶向VSP可能是控制PIP信号传导并解决受PIPS和PIP结合蛋白影响的人类疾病的最佳方法。此外，参与信号级联的其他膜蛋白可能具有相似的机制，以将细胞外刺激传达到酶活性。本提案中介绍的技术，特别是在VCF和PCF中使用细胞内标签的技术，应适用于研究参与细胞内信号传导的其他膜蛋白。