ANESTHETIC EFFECTS ON ION CHANNEL STRUCTURES & DYNAMICS

对离子通道结构的麻醉作用

• 批准号: 8127591
• 负责人: PEI TANG
• 金额: $ 10.26万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8127591
• 关键词: Acetylcholine; Adverse effects; Affect; Affinity; Agonist; Anesthetics; Anions; Arts; Binding; Binding Proteins; Binding Sites; Budgets; Cations; Cereals; Characteristics; Cholesterol; Communication; Complex; Computer Simulation; Computing Methodologies; Data; Development; Docking; Doctor of Philosophy; Drug effect disorder; Electronics; Electrophysiology (science); Equilibrium; Event; Exhibits; Extracellular Domain; Factor X; Frequencies; Funding; Future; Gated Ion Channel; General Anesthesia; General anesthetic drugs; Global Change; Goals; Halothane; Homology Modeling; Hot Spot; Hydrophobicity; Interest Group; Ion Channel; Ion Channel Protein; Ions; Isoflurane; Knowledge; Laboratories; Learning; Ligands; Link; Manuals; Maps; Mediating; Membrane; Membrane Proteins; Microscopy; Modeling; Molecular; Molecular Conformation; Molecular Target; Mono-S; Motion; Movement; Muscle; Mutagenesis; Mutate; Mutation; Nature; Neurons; Nicotinic Receptors; Permeability; Photoaffinity Labels; Physiological; Point Mutation; Population; Positioning Attribute; Predisposition; Protein Dynamics; Protein Isoforms; Proteins; Published Comment; Publishing; Radial; Recommendation; Research; Research Activity; Research Personnel; Research Project Grants; Resolution; Roentgen Rays; Seeds; Side; Simulate; Site; Structural Models; Structure; Study Section; System; Testing; Time; Torpedo; United States National Institutes of Health; Validation; Variant; Vertebral column; Water; aqueous; base; computer studies; design; extracellular; flexibility; gramicidin A; insight; molecular dynamics; mutant; network models; novel; programs; protein function; protein structure; receptor; research study; simulation; structural biology

This competing renewal application seeks continued support for a primary research in the Pi's laboratory using the computational approaches to elucidating the molecular mechanisms of general anesthesia. Research in the previous funding period challenged the traditional structure-function paradigm in explaining the action of general anesthetics on ion channel proteins (Tang&Xu, PNAS, 99:16035-16040, 2002) and proposed an alternative viewpoint that the effects of general anesthetics on protein global dynamics on the timescale matching the characteristic time of protein function might underlie a common mechanism of action of general anesthetics. To test this central hypothesis, we will take 4x4 approach to integrate 4 complementary state- of-the-art computational methods with 4 levels of validation with experimental data. We will focus on the anesthetic-hypersensitive neuronal (04)2(32)3 nicotinic acetylcholine receptor (nAChR) and the anesthetic- insensitive (a7)5 nAChR, as well as the Torpedo isoform of the muscle-type (alkplvQ nAChR. A novel integration of homology modeling, molecular dynamics (MD) simulations in a fully hydrated ternary membrane patch, coarse-grained normal mode analysis (NMA), and Brownian dynamics (BD) will be used to generate and validate high-resolution, closed and putatively open structural models for (alkpIvS, (a4)2(p2)3 and (a7)5 nAChR on the basis of the 4-A resolution structure of the (a1)2p1v5 nAChR as a template. Flexible ligand docking or manual docking of anesthetics (halothane and isoflurane) at experimentally identified anesthetic-binding sites will be followed by MD equilibration to encode anesthetic effects on tertiary and quaternary structures. NMA and BD will then be used to quantify the gating-related low-frequency motions of the receptors and ion permeation across the channel. Two groups of mutations that changed nAChR's sensitivity to anesthetics experimentally will be tested for global dynamics changes. Four levels of experimental validation for structures will include agonist- binding affinity, topology matching to low-resolution experimental structures and pore residue accessibilities, I-V curve calculations, and cation/anion and mono-/di-valence ion permeability ratios. Our substantive amount of preliminary results supports the following four specific aims: (1) To generate and validate, using existing experimental data, the closed- and putative open-channel structures of the neuronal (a4)2(p2)3 nAChR (hypersensitive to volatile anesthetics) and (a7)5 nAChR (insensitive to volatile anesthetics) as well as the open- channel structure for Torpedo (al^plvfi nAChR (sensitive to volatile anesthetics); (2) To perform extensive, multi-seed MD simulations on wild type and mutant channels in the absence and presence of anesthetics; (3) To carry out normal mode analysis to determine anesthetic effects on global dynamics, using the fully equilibrated structures in SA#2 as input; and (4) To relate anesthetic effects on global dynamics to channel function by performing Brownian dynamics calculations of ion permeation through the putative open-channels. The research will bridge the experimental and theoretical understanding of anesthetic effects on channel proteins, thereby facilitating the future design of new and novel anesthetic drugs that are more specific with fewer side effects.

这一竞争性的更新申请旨在继续支持Pi实验室的一项主要研究， 阐明全身麻醉分子机制的计算方法。的研究 上一个资助期挑战了传统的结构-功能范式， 麻醉剂对离子通道蛋白的作用（Tang&Xu，PNAS，99：16035-16040，2002），并提出了一种替代方案 认为全身麻醉药对蛋白质全局动力学的影响在时间尺度上与 蛋白质功能的特征时间可能是一般蛋白质的共同作用机制的基础。 麻醉剂为了验证这个中心假设，我们将采用4x 4方法来整合4个互补状态- 最先进的计算方法，通过实验数据进行4级验证。重点抓好 麻醉-超敏神经元（04）2（32）3烟碱乙酰胆碱受体（nAChR）和麻醉- 不敏感的（a7）5 nAChR，以及肌肉型的鱼雷亚型（alkplvQ nAChR.一种新型 在完全水合的三元膜中集成同源建模、分子动力学（MD）模拟 补丁，粗粒度正常模式分析（NMA）和布朗动力学（BD）将用于生成和 验证（a4）2（p2）3和（a7）5 nAChR的高分辨率、封闭和开放结构模型 以（a1）2 p1 v5 nAChR的4-A分辨结构为模板，柔性配体对接或 将麻醉剂（氟烷和异氟烷）手动对接在实验确定的麻醉剂结合位点， 随后进行MD平衡以编码对三级和四级结构的麻醉作用。NMA和BD 然后将用于量化受体的门控相关低频运动和离子渗透 频道。两组改变nAChR对麻醉剂敏感性的突变将在实验中进行研究。 测试全球动力学的变化结构的四级实验验证将包括激动剂- 结合亲和力，拓扑匹配低分辨率实验结构和孔残基辅助，I-V 曲线计算和阳离子/阴离子和一价/二价离子渗透率比。我们大量的 初步结果支持以下四个具体目标：（1）生成和验证，使用现有的 实验数据，神经元（a4）2（p2）3 nAChR的闭合和假定的开放通道结构 （对挥发性麻醉剂过敏）和（a7）5 nAChR（对挥发性麻醉剂不敏感）以及开放- Torpedo（对挥发性麻醉剂敏感）的通道结构;（2）为了进行广泛的， 在不存在和存在麻醉剂的情况下对野生型和突变型通道的多种子MD模拟;（3） 进行正常模式分析，以确定麻醉对全局动力学的影响，使用完全平衡的 SA#2中的结构作为输入;以及（4）通过以下方式将麻醉对全局动力学的影响与通道功能联系起来： 执行通过假定的开放通道的离子渗透的布朗动力学计算。研究 将连接麻醉剂对通道蛋白影响的实验和理论理解，从而 促进未来设计新的和新颖的麻醉药物，其更具有特异性和更少的副作用。