Mechanisms of TRPV1 Channel Regulation

TRPV1通道调节机制

• 批准号: 8638032
• 负责人: Sharona E Gordon
• 金额: $ 40.54万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8638032
• 关键词: Amino Acids; Arachidonic Acids; Binding; Binding Sites; C-terminal; Calmodulin; Capsaicin; Cells; Charge; Chemicals; Chili Pepper; Cysteine; DNA Sequence Rearrangement; Electrophysiology (science); Engineering; Enzymes; Event; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorometry; Goals; Head; Heating; Ion Channel; Ions; Label; Lipid Binding; Lipids; Maps; Measures; Mediating; Membrane; Methods; Modality; Modification; Molecular; Movement; Mutagenesis; N-terminal; Neurons; Pain; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Physiological; Proteins; Regulation; Scanning; Shapes; Signal Transduction; Site; Stimulus; TRPV1 gene; Techniques; Testing; Transition Elements; Work; acyl group; density; fluorophore; innovation; inorganic phosphate; novel; novel strategies; pain receptor; patch clamp; receptor; reconstitution; response; tool; unilamellar vesicle; voltage

DESCRIPTION (provided by applicant): TRPV1 ion channels are multimodal receptors that can be activated by heat, high [H+]o, voltage, arachidonic acid metabolites, capsaicin (the pungent extract of hot chili peppers), and the signaling lipid PI(4,5)P2 (PIP2). Ca2+/Calmodulin (Ca2+/CaM) and ATP may modulate its activity as well. Our long-term goal is to understand the molecular mechanism by which TRPV1 integrates these multiple physiological stimuli. We and others have previously established that PIP2 directly activates TRPV1. Our recent work indicates that the proximal part of the intracellular C-terminal domain comprises at least part of the PIP2 binding site. However, the inability to control the lipid composition of native membranes, the presence of myriad enzymes and other proteins in cells and excised patches, and the difficulty of specifically labeling intracellular domains of channels within cells have proven serious experimental barriers to understanding regulation of TRPV1 by PIP2 and other activation modalities. We have developed a novel approach to reconstitute purified TRPV1 channels at high density in synthetic Giant Unilamellar Vesicles (GUVs). In this proposal we will apply standard patch-clamp methods, Patch-Clamp Fluorometry (PCF), and Transition Metal Ion FRET (tmFRET) to study purified TRPV1 channels in GUVs of defined lipid composition. Single cysteines engineered into our cysteineless TRPV1 background will be used to site-specifically label channels in the GUVs with fluorophore, completely eliminating the background fluorescence problem. The GUVs used for reconstitution will include synthetic lipids that bind transition metals which act as short- distance FRET quenchers in the novel short-range tmFRET approach we have developed. PCF allows us to simultaneously record the function of the channel with electrophysiology and the rearrangement of the channel with fluorescence. These new tools will allow us to measure dynamics of the intracellular N- and C-terminal domains associated with PIP2 activation as well as with activation by heat, Ca2+/CaM, and ATP.

描述（由申请人提供）：TRPV1 离子通道是多模式受体，可被热、高 [H+]o、电压、花生四烯酸代谢物、辣椒素（辣椒的辛辣提取物）和信号脂质 PI(4 ,5)P2（画中画2）。 Ca2+/钙调蛋白 (Ca2+/CaM) 和 ATP 也可能调节其活性。我们的长期目标是了解 TRPV1 整合这些多种生理刺激的分子机制。我们和其他人之前已经确定 PIP2 直接激活 TRPV1。我们最近的工作表明细胞内 C 端结构域的近端部分至少包含部分 PIP2 结合位点。然而，无法控制天然膜的脂质组成、细胞和切除的斑块中存在无数酶和其他蛋白质，以及特异性标记细胞内通道的胞内结构域的困难，已经证明是理解 TRPV1 调节的严重实验障碍。 PIP2 和其他激活方式。我们开发了一种新方法，可以在合成巨型单层囊泡 (GUV) 中以高密度重建纯化的 TRPV1 通道。在本提案中，我们将应用标准膜片钳方法、膜片钳荧光测定法 (PCF) 和过渡金属离子 FRET (tmFRET) 来研究特定脂质成分的 GUV 中的纯化 TRPV1 通道。设计到我们的无半胱氨酸 TRPV1 背景中的单个半胱氨酸将用于用荧光团对 GUV 中的通道进行位点特异性标记，从而完全消除背景荧光问题。用于重构的 GUV 将包括结合过渡金属的合成脂质，过渡金属在我们开发的新型短程 tmFRET 方法中充当短程 FRET 猝灭剂。 PCF 使我们能够同时用电生理学记录通道的功能和用荧光记录通道的重排。这些新工具将使我们能够测量与 PIP2 激活以及热、Ca2+/CaM 和 ATP 激活相关的细胞内 N 端和 C 端结构域的动态。