Molecular Mechanism of pH Regulation of TRPV1 Activation

描述(申请人提供)：热传感器TRPV1通道是一种多模式受体，在介导各种伤害性刺激引起的神经元疼痛中发挥关键作用。其中一种刺激是炎症、组织损伤和缺血引起的细胞外酸化。低pH被认为通过作为通道激活剂直接激活TRPV1，也通过增强通道对其他刺激的反应间接激活TRPV1。TRPV1的激活是如何由pH控制的，以及它与热、配体和内源性通道调节剂激活的关系在很大程度上仍不清楚。重要的是，TRPV1活性对各种物理和化学因素的高度独特敏感性使该通道成为临床治疗疼痛的有吸引力的靶点。我们研究的主要目标是通过阐明热、辣椒素和其他刺激激活TRPV1多峰的分子机制来了解TRPV1的细胞传感功能。在这项拟议的研究中，我们旨在揭示TRPV1细胞外H+调节的结构和机制。由于H+诱导的TRPV1活性具有热依赖和激动剂依赖的成分，本研究也将有助于阐明热和激动剂如何控制TRPV1的活性。我们通过光学、电生理和分子方法的组合来实现我们的目标。特别是，我们将应用斑块荧光计量学方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。同时的荧光和电子记录允许结构变化与它们对通道激活的影响直接相关。使用这些方法，我们将解决以下问题：TRPV1如何感知pH、温度和激动剂浓度的变化，什么通道结构传递这些刺激，以及这些刺激如何融合来控制TRPV1的激活。对这些问题的回答将直接有助于开发新的临床工具来治疗TRPV1介导的神经性疼痛。

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.