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是一种完全参与伤害性刺激的非选择性阳离子通道 变成疼痛信号。因此，抑制TRPV1是设计的主要策略之一 下一代抗生物药物。 TRPV1的标志之一是其多峰激活 轮廓;也就是说，检测和显着整合了潜水员的信息的能力 环境因素（例如配体的结合，pH和温度的结合）启动动作电位 在感觉轴突的外围末端。从分子的角度来看，这种多年性是 不同位点之间的变构耦合的结果是作为潜水员刺激的“传感器” 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。提出了三个基本问题 通过我们最近的一些研究结果将指导我们的调查。 AIM 1解决 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，其预测部分已经进行了验证 实验。我们发现，在 TRPV1的结构会影响构成Sparagine住所的方向； 该侧链的旋转又负责孔的打开。我们将测试这个 假设具有广泛的计算和实验。 AIM 2建立在 观察到润湿/脱水现象显示出独特的温度依赖性和 这个目标专门用于定量 润湿/脱水热力学的表征和对我们的实验测试的表征 使用诱变的模型。最后，AIM 3调查了TRPV1支撑的机制 PIP2调节。我们的初步数据表明，这种脂质有利于构象过渡 从规范螺旋到非典型构象的孔衬里S6螺旋的 -helix的片段。我们将使用计算的组合检验该假设 建模，位置定向诱变，全细胞和优秀的斑块电生理学。