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，可以通过热，高[H+] O，电压，蛛形二烯酸代谢物，辣椒素（热辣椒辣椒的刺激提取物）和信号脂质PI（4,5）P2（4,5）P2（PIP2）激活。 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。我们和其他人以前已经确定PIP2直接激活TRPV1。我们最近的工作表明，细胞内C末端结构域的近端部分包括PIP2结合位点的至少一部分。然而，无法控制天然膜的脂质组成，细胞和切除的斑块中的众多酶和其他蛋白质的存在，以及在细胞内特异性标记细胞内通道的困难，证明了严重的实验障碍，以通过PIP2和其他激活模态理解TRPV1的调节。我们已经开发了一种新的方法，可以在合成巨型Unilamelar囊泡（GUV）中以高密度重建纯化的TRPV1通道。在此提案中，我们将应用标准的贴片钳方法，贴片钳荧光法（PCF）和过渡金属离子FRET（TMFRET）来研究定义的脂质组成的GUV中纯化的TRPV1通道。在我们的Cysteineless TRPV1背景中设计的单半胱氨酸将用于具有荧光团的GUV中特定标记通道，从而完全消除了背景荧光问题。用于重建的GUV将包括结合过渡金属的合成脂质，这些脂质在我们开发的新型短距离TMFRET方法中充当短距离射液器。 PCF允许我们同时通过电生理学记录通道的功能，并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激活以及热量，Ca2+/CAM和ATP激活相关的细胞内N和C末端域的动力学。