Molecular mechanism of TRPV1 activation

TRPV1激活的分子机制

• 批准号: 10009447
• 负责人: Vincenzo Carnevale
• 金额: $ 35.14万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-10 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10009447
• 关键词: Action Potentials; Address; Affect; Afferent Neurons; Amber; Amino Acids; Arginine; Asparagine; Axon; Binding; Biology; Calcium; Cations; Cells; Charge; Chemicals; Codon Nucleotides; Complex; Computational Biology; Computer Models; Coupling; Cryoelectron Microscopy; Data; Dehydration; Dependence; Drug Design; Electrophysiology (science); Environmental Risk Factor; Face; Free Energy; Hydration status; Hydrophobicity; Hyperalgesia; Hypersensitivity; Image; Investigation; Ion Channel; Ions; Lead; Ligand Binding; Lipid Binding; Lipids; Mechanics; Medical; Membrane; Microscopic; Microscopy; Modeling; Molecular; Molecular Conformation; Motion; Mutagenesis; Nature; Nociceptive Stimulus; Pain; Peripheral; Pharmaceutical Preparations; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Proteins; Publishing; Regulation; Research; Role; Rotation; Sampling; Sensory; Side; Site; Site-Directed Mutagenesis; Stimulus; Structure; Symptoms; Syndrome; TRPV1 gene; Techniques; Temperature; Testing; Therapeutic; Thermodynamics; United States; Vertebral column; Work; alpha helix; carbonyl group; chronic pain; cofactor; conformational conversion; cost; design; experimental study; flexibility; inorganic phosphate; molecular dynamics; next generation; novel; pain signal; sensor; small molecule; structured data; synergism; tool; unnatural amino acids

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.

TRPV1 是一种非选择性阳离子通道，在伤害性刺激的转导中发挥着至关重要的作用 转化为疼痛信号。因此，抑制 TRPV1 是设计的主要策略之一。 下一代抗痛药。 TRPV1 的特点之一是其多模式激活 轮廓;也就是说，能够检测并整合来自不同领域的信息。 启动动作电位的环境因素（例如配体的结合、pH 值和温度） 位于感觉轴突的外围末端。从分子的角度来看，这种多峰性是 作为不同刺激的“传感器”的不同位点之间变构耦合的结果 和激活门。该项目研究了这种变构耦合的机制 计算生物学（分子动力学、自由能计算）、最先进的化学 生物学（非天然氨基酸）和电生理学。提出三个基本问题 我们最近的一些研究结果将指导我们的调查。目标 1 解决了 TRPV1激活的分子机制。工作假设来自我们最近 已发表的计算工作，其预测已部分得到验证 实验性地。我们发现存在于四个疏水口袋的水合和脱水 TRPV1的结构影响S6中保守的天冬酰胺残基的方向；这 该侧链的旋转反过来又负责孔的打开。我们将测试这个 假设与大量的计算和实验。目标 2 建立在 观察到润湿/反润湿现象表现出精致的温度依赖性和 从而提供了一种可行的热激活机制。这一目标致力于定量 润湿/反润湿热力学的表征以及我们的实验测试 使用诱变的模型。最后，目标 3 研究 TRPV1 的基础机制 PIP2 的监管。我们的初步数据表明这种脂质有利于构象转变 孔内壁 S6 螺旋从规范的 α 螺旋到包含  螺旋的一段。我们将结合计算来检验这个假设 建模、定点诱变、全细胞和切除斑块电生理学。