Mechanisms of TRPV1 Channel Regulation

说明(申请人提供)：TRPV1离子通道是多模式受体，可被热、高[H+]O、电压、花生四烯酸代谢产物、辣椒素(辣椒素)和信号脂质PI(4，5)P2(PIP2)激活。Ca~(2+)/CaM(Ca~(2+)/CaM)和ATP对其活性也有调节作用。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证实，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至少包含PIP2结合位点的一部分。然而，无法控制天然膜的脂质组成，细胞和切除的补片中存在着无数的酶和其他蛋白质，以及难以专门标记细胞内通道的胞内域，这些都证明了理解PIP2和其他激活方式对TRPV1调控的严重实验障碍。我们开发了一种新的方法，在人工合成的巨大单层囊泡(GUV)中以高密度重建纯化的TRPV1通道。在这项提案中，我们将应用标准膜片钳方法、膜片钳荧光测定法(PCF)和过渡金属离子荧光测定法(TmFRET)来研究具有特定脂质组成的GUV中纯化的TRPV1通道。在我们的无半胱氨酸TRPV1背景中设计的单个半胱氨酸将被用来用荧光团定位标记GUV中的通道，完全消除背景荧光问题。用于重建的GUV将包括与过渡金属结合的合成脂类，在我们开发的新型短程tmFRET方法中，这些脂类充当短距离FRET猝灭剂。PCF可以用电生理同时记录通道的功能，用荧光同时记录通道的重排。这些新工具将使我们能够测量与PIP2激活以及热、钙/钙调素和三磷酸腺苷激活相关的细胞内N-末端和C-末端结构域的动态。

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.