Molecular Mechanism of pH Regulation of TRPV1 Activation

描述（由申请人提供）：热传感器TRPV1通道是一种多模态受体，在介导各种有害刺激引起的神经元疼痛中起关键作用。其中一种刺激是由炎症、组织损伤和缺血引起的细胞外酸化。低pH值被认为通过作为通道激活剂直接激活TRPV1，并通过增强通道对其他刺激的反应间接激活TRPV1。TRPV1活化如何受pH控制，以及它与热、配体和内源性通道调节剂活化的关系，在很大程度上仍然未知。重要的是，TRPV1活性对多种物理和化学因素的高度独特的易感性使该通道成为临床干预疼痛的一个有吸引力的靶点。我们研究的首要目标是通过阐明TRPV1在热、辣椒素和其他刺激下多模态激活的分子机制来了解其细胞感知功能。在本研究中，我们旨在揭示细胞外H+调控TRPV1的结构和机制。由于H+诱导的TRPV1活性具有热依赖性和激动剂依赖性成分，本研究还将揭示热和激动剂如何控制TRPV1活性。我们通过结合光学、电生理和分子方法来实现我们的目标。特别是，我们将采用斑块荧光法直接观察通道蛋白的结构变化或调节分子的结合，使用荧光团作为分子传感器。同时荧光和电记录允许结构变化与其对通道激活的影响直接相关。使用这些方法，我们将解决TRPV1如何感知pH、温度和激动剂浓度的变化，哪些通道结构传递这些刺激，以及这些刺激如何汇聚以控制TRPV1的激活等问题。这些问题的答案将直接有利于开发治疗TRPV1介导的神经性疼痛的新临床工具。

DESCRIPTION (provided by applicant): The heat sensor TRPV1 channel is a polymodal receptor that plays a key role in mediating neuronal pain caused by various noxious stimuli. One such stimulus is extracellular acidification caused by inflammation, tissue damage and ischemia. Low pH is thought to activate TRPV1 both directly by serving as a channel activator and indirectly by potentiating the channel's response to other stimuli. How TRPV1 activation is controlled by pH as well as its relation to activation by heat, ligands, and endogenous channel modulators remains largely unknown. Importantly, the highly unique susceptibility of TRPV1 activity to a variety of physical and chemical factors makes the channel an attractive target for clinical intervention of pain. The overarching goal of our research is to understand the cellular sensing function of TRPV1 by elucidating molecular mechanisms underlying its polymodal activation by heat, capsaicin and other stimuli. In the proposed study we aim to reveal the structural and mechanistic nature of extracellular H+ regulation of TRPV1. As H+-induced TRPV1 activity has heat-dependent and agonist-dependent components, this investigation will also shed light on how heat and agonist control TRPV1 activity. We approach our goal through a combination of optical, electrophysiological, and molecular methods. In particular, we will apply a patch fluorometry approach to directly observe structural changes in the channel protein or the binding of regulatory molecules, using fluorophores as molecular sensors. Simultaneous fluorescent and electrical recordings permit direct correlation of structural changes to their effects on channel activation. Using these methods, we will address questions on how changes in pH, temperature, and the concentration of agonists are sensed by TRPV1, what channel structures convey these stimuli, and how these stimuli converge to control TRPV1 activation. Answers to these questions should directly benefit the development of new clinical tools for treating TRPV1- mediated neuronal pain.