ANESTHETIC EFFECTS ON ION CHANNEL STRUCTURES & DYNAMICS

这一竞争性续签申请寻求对PI实验室的初步研究的持续支持，该实验室使用 阐明全身麻醉分子机制的计算方法。在美国的研究 过去的资助期对传统的结构-功能范式在解释将军的行为上提出了挑战 麻醉药对离子通道蛋白的影响(唐、徐，99：16035-16040,2002年)，并提出了一种替代方案 认为全麻药对蛋白质整体动力学的影响与时间尺度匹配 蛋白质功能的特征时间可能是通用的作用机制的基础 麻醉剂。为了验证这一中心假设，我们将采用4x4方法来积分4个互补状态- 最先进的计算方法，具有4个级别的验证与实验数据。我们将重点关注 麻醉剂-超敏神经元(04)2(32)3烟碱型乙酰胆碱受体(NAChR)和麻醉剂- 不敏感(A7)5 nAChR，以及肌肉类型的鱼雷亚型(alkplvQ nAChR.一本小说 全水化三元膜的同源建模与分子动力学(MD)模拟的集成 将使用补丁、粗粒度正态模式分析(NMA)和布朗动力学(BD)来生成和 验证(alkpIVS，(A4)2(P2)3和(A7)5 nAChR)的高分辨率、封闭和假定开放的结构模型 以(A1)2p1v5 nAChR的4-A拆分结构为模板。灵活的配体对接或 将麻醉剂(氟烷和异氟醚)手动对接在实验确定的麻醉剂结合部位将 然后进行MD平衡，以编码三级和四级结构的麻醉效果。NMA和BD 然后将被用来量化与门控相关的受体的低频运动和离子渗透 频道。在实验上改变nAChR对麻醉药敏感性的两组突变将是 测试全球动态变化。四个层次的结构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/阴离子和一价/二价离子渗透率。我们的实质性金额为 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)3nAChR的封闭和可能的开放通道结构 (对挥发性麻醉药过敏)和(A7)5 nAChR(对挥发性麻醉药不敏感)以及开放的 鱼雷通道结构(对挥发性麻醉剂敏感)；(2)为了广泛执行， 麻醉剂存在和不存在时野生型和突变型通道的多种子分子动力学模拟 进行正常模式分析，以确定麻醉对全球动力学的影响，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经络功能联系起来 对离子在假定的明渠中的渗透进行布朗动力学计算。这项研究 将为麻醉剂对通道蛋白影响的实验和理论理解架起桥梁，从而 促进未来设计更具特效性和副作用更少的新型麻醉药。

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.