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.

这个竞争性的更新应用程序寻求对 Pi 实验室的一项初步研究的持续支持 阐明全身麻醉分子机制的计算方法。研究领域 上一个资助期在解释一般行动时挑战了传统的结构-功能范式。 对离子通道蛋白的麻醉剂（Tang&Xu, PNAS, 99:16035-16040, 2002）并提出了替代方案 认为全身麻醉对蛋白质整体动态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作用的共同机制的基础 麻醉剂。为了检验这个中心假设，我们将采用 4x4 方法来整合 4 个互补状态 - 最先进的计算方法，具有 4 个级别的实验数据验证。我们将重点关注 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 (a7)5 nAChR，以及肌肉型鱼雷亚型 (alkplvQ nAChR)。 将同源建模、分子动力学 (MD) 模拟集成到完全水合的三元膜中 patch、粗粒度正则模式分析（NMA）和布朗动力学（BD）将用于生成和 验证 (alkpIvS、(a4)2(p2)3 和 (a7)5 nAChR 的高分辨率、封闭式和推定开放式结构模型 以(a1)2p1v5 nAChR的4-A解析结构为模板。灵活的配体对接或 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将 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。通过实验改变 nAChR 对麻醉剂敏感性的两组突变将被 测试了全球动态变化。结构的四个级别的实验验证将包括激动剂 结合亲和力、与低分辨率实验结构的拓扑匹配和孔残基可及性、I-V 曲线计算，以及阳离子/阴离子和单价/二价离子渗透率比率。我们大量的 初步结果支持以下四个具体目标：（1）利用现有的生成和验证 实验数据，神经元 (a4)2(p2)3 nAChR 的封闭通道和假定开放通道结构 （对挥发性麻醉剂过敏）和 (a7)5 nAChR（对挥发性麻醉剂不敏感）以及开放式 鱼雷的通道结构（al^plvfi nAChR（对挥发性麻醉剂敏感）；（2）执行广泛的、 在麻醉剂不存在和存在的情况下对野生型和突变体通道进行多种子 MD 模拟； (3) 至 使用完全平衡的模型进行正常模式分析以确定麻醉对全局动力学的影响 SA#2 中的结构作为输入； (4) 将麻醉对整体动态的影响与通道功能联系起来 对通过假定的开放通道的离子渗透进行布朗动力学计算。研究 将弥合对通道蛋白麻醉作用的实验和理论理解，从而 促进未来新型麻醉药物的设计，这些药物特异性更强，副作用更少。