Molecular Mechanism of pH Regulation of TRPV1 Activation

描述（由申请人提供）：热传感器TRPV 1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。一种这样的刺激是由炎症、组织损伤和局部缺血引起的细胞外酸化。低pH值被认为通过直接作为通道激活剂和间接通过增强通道对其他刺激的反应来激活TRPV1。TRPV1的激活是如何被pH控制的，以及它与热、配体和内源性通道调节剂的激活的关系在很大程度上仍然是未知的。重要的是，TRPV1活性对各种物理和化学因素的高度独特的易感性使得该通道成为疼痛临床干预的有吸引力的靶点。我们研究的首要目标是通过阐明热，辣椒素和其他刺激物对其多模态激活的分子机制来了解TRPV1的细胞传感功能。在拟议的研究中，我们的目的是揭示TRPV1的细胞外H+调节的结构和机制的性质。由于H+诱导的TRPV1活性具有热依赖性和激动剂依赖性成分，因此本研究还将阐明热和激动剂如何控制TRPV1活性。我们通过光学、电生理和分子方法的结合来实现我们的目标。特别是，我们将应用补丁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直接相关的结构变化对通道激活的影响。使用这些方法，我们将解决的问题，如何在pH值，温度和浓度的激动剂的变化是由TRPV1，什么通道结构传达这些刺激，以及这些刺激如何收敛到控制TRPV1激活。这些问题的答案应该直接有利于开发新的临床工具来治疗TRPV1介导的神经元疼痛。

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.