Molecular mechanism of TRPV1 activation

TRPV1是一种非选择性阳离子通道，在伤害性刺激的转导中起关键作用 转化为疼痛信号。因此，抑制TRPV1是设计的主要策略之一 下一代止痛药。TRPV1的特征之一是它的多模式激活 配置文件；即检测并显著集成来自不同来源的信息的能力 启动动作电位的环境因素(如配体的结合、pH和温度) 在感觉轴突的外周末端。从分子的角度来看，这种多态是 不同部位之间的变构偶联作为不同刺激的“传感器”的结果 和激活门。本项目研究了这种变构偶联的机理。 计算生物学(分子动力学、自由能计算)、最新化学 生物学(非天然氨基酸)和电生理学。提出了三个基本问题 通过我们最近的一些研究成果将指导我们的调查。目标1针对的是 TRPV1激活的分子机制。工作假说来自于我们最近 已发表的计算工作，其预测已部分得到证实 试验性的。我们发现四个疏水口袋的水合和脱水存在于 TRPV1的结构影响S6中保守的天冬酰胺残基的取向； 这个侧链的旋转反过来又负责毛孔的打开。我们将对此进行测试 有大量计算和实验的假说。AIM 2建立在 观察到润湿/除湿现象显示出精确的温度依赖关系和 从而为热激活提供了一种可行的机制。这一目标致力于量化 润湿/去湿热力学的表征和对我们的实验测试 使用诱变技术的模型。最后，目标3研究了支持TRPV1的机制 由PIP2进行监管。我们的初步数据表明，这种脂类有利于构象转变。 将孔衬里S6螺旋从正则-螺旋转变为包含 -螺旋片段。我们将使用计算的组合来检验这一假设 建模、定点突变、全细胞和切除的斑块电生理学。

TRPV1 is a non-selective cation channel crucially involved in transduction of nociceptive stimuli into pain signals. Consequently, inhibition of TRPV1 is one of the major strategies for designing next generation anti-pain drugs. One of the hallmarks of TRPV1 is its polymodal activation profile; that is, the ability to detect and, remarkably, integrate the information from diverse environmental factors (e.g. binding of ligands, pH and temperature) to initiate an action potential in the peripheral ends of sensory axons. From a molecular point of view, this polymodality is the result of the allosteric coupling between distinct sites acting as "sensors" for the diverse stimuli and the activation gate. This project investigates the mechanism of this allosteric coupling using computational biology (molecular dynamics, free energy calculations), state-of-the-art chemical biology (non-natural amino acids) and electrophysiology. Three fundamental questions raised by some of our recent research findings will guide our investigation. Aim 1 addresses the molecular mechanism of activation of TRPV1. The working hypothesis comes from our recently published computational work whose predictions have been, in part, already verified experimentally. We found that hydration and dehydration of four hydrophobic pockets present in the structure of TRPV1 affect the orientation of a conserved asparagine residue in S6; the rotation of this side chain is, in turn, responsible for the opening of the pore. We will test this hypothesis with an extensive set of calculations and experiments. Aim 2 Builds on the observation that wetting/dewetting phenomena show exquisite temperature dependence and thus provide a viable mechanism for heat activation. This aim is devoted to the quantitative characterization of the wetting/dewetting thermodynamics and to the experimental testing of our model using mutagenesis. Finally, Aim 3 investigates the mechanism underpinning TRPV1 regulation by PIP2. Our preliminary data suggest that this lipid favor a conformational transition of the pore lining S6 helix from a canonical -helix to a non-canonical conformation containing a segment of -helix. We will test this hypothesis using the combination of computational modeling, site directed mutagenesis, whole cell and excised patch electrophysiology.