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Molecular mechanism of TRPV1 activation

TRPV1激活的分子机制

基本信息

批准号：
10457897
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
项目类别：
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/10457897
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 synergism tool unnatural amino acids

项目摘要

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。