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.

TRPV 1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 转化成疼痛信号因此，抑制TRPV 1是设计TRPV 1的主要策略之一。 下一代止痛药TRPV 1的特征之一是其多模式激活 配置文件;也就是说，能够检测和，显着，整合来自不同的信息， 引发动作电位的环境因素（如配体结合、pH和温度） 在感觉轴突的外周末端。从分子的角度来看，这种多峰性是 作为不同刺激的“传感器”的不同位点之间的变构偶联的结果 和激活门。本项目研究这种变构偶联的机制， 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。提出的三个基本问题 我们最近的一些研究结果将指导我们的调查。目标1针对 TRPV 1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，其预测已经部分得到验证 实验性的我们发现，水化和脱水的四个疏水口袋存在于 TRPV 1的结构影响S6中保守天冬酰胺残基的方向; 该侧链的旋转又导致孔的打开。我们将测试这个 假设与广泛的计算和实验。目标2建立在 观察到润湿/去湿现象显示出精确的温度依赖性， 从而提供了一种可行的热激活机制。这一目标是致力于定量 润湿/去湿热力学的表征和我们的实验测试 使用诱变的模型。最后，目标3研究了TRPV 1的基础机制 PIP 2的规则。我们的初步数据表明，这种脂质有利于构象转变 孔内衬S6螺旋从典型的β-螺旋到含有非典型构象的非典型构象， 螺旋的一部分。我们将使用计算的组合来测试这个假设。 建模、定点诱变、全细胞和切除的贴片电生理学。