Anesthetic Effects on Ion Channel Structures & Dynamics

全身麻醉的分子机制仍不清楚。本研究的目的是阐明离子通道动力学在挥发性麻醉药作用中的重要作用。以我们的实验结果(主要来自核磁共振波谱)为指导，我们将使用大规模分子动力学(MD)模拟来研究由于与麻醉剂和非麻醉剂(非固定剂)相互作用而引起的跨膜通道动力学的变化，这些药物在结构上与麻醉剂相似，但特别是没有任何麻醉作用。本研究的两个模型通道是甘草素A(Ga)和由人甘氨酸受体(GlyR)α-1亚单位的第二和第三跨膜结构域组成的同五配基通道复合体。由伊利诺伊大学开发的NAMD2程序将用于并行计算。其具体目的是：(1)用从头算量子力学和分子动力学计算确定含氟挥发性麻醉剂和非麻醉剂分子的结构和性质，并模拟含有线性、循环麻醉剂和非麻醉剂的全水合膜系统中长达10 ns的离子通道动力学；(2)研究操控离子输运对麻醉剂引起的通道动力学变化的影响，并定性分析麻醉剂引起的操纵力变化；(3)研究存在和不存在麻醉剂-非麻醉剂对时GlyR TM2+TM3通道转向门控运动的通道动力学响应；(4)在GlyR的TM2区测定麻醉药强制结合和解除结合的GlyR通道动力学响应；(5)针对特定目标1-4中的所有模拟结果，通过分析均方根偏差(RMSD)和波动(RMSD)、自相关函数和广义序参数(S2)来量化通道和脂质动力学，并研究界面水、界面脂质和空腔动力学对麻醉剂诱导的通道动力学变化的影响。需要检验的中心假设是麻醉剂通过深刻改变通道动力学以及通道与脂质和界面水的关系来影响跨膜通道功能。这项研究的结果将极大地推动以下三个领域的科学研究：(1)大规模并行计算在生物学问题上的应用，特别是麻醉剂与膜相关蛋白的相互作用；(2)详细阐明剩余动态贡献(通过构象熵变化)对脂类和跨膜通道之间的界面结合的贡献；以及(3)基于动力学-功能关系的全身麻醉蛋白质理论的发展。

The molecular mechanisms of general anesthesia remain unknown. The goal of the proposed studies is to elucidate the important role of ion channel dynamics in the action of volatile anesthetics. Guided by our experimental results, mostly from NMR spectroscopy, we will use large-scale molecular dynamics (MD) simulations to investigate changes in transmembrane channel dynamics due to interaction with anesthetics and nonanesthetics (nonimmobilizers), which are structurally similar to the anesthetics but are peculiarly devoid of any anesthetic effects. Two model channels chosen for this study are gramicidin A (gA) and a homopentameric channel complex composed of the second and third transmembrane domains of the alpha-1 subunits of human glycine receptor (GlyR). The NAMD2 program, developed at the University of Illinois, will be used for parallel computing. The specific aims are: (1) to determine the structures and properties of fluorinated volatile anesthetic and nonanesthetic molecules by ab initio quantum mechanics and MD calculations, and to simulate ion channel dynamics up to 10 ns in fully hydrated membrane systems containing linear and cyclic anesthetics and nonanesthetics; (2) to study the steered ion transport effects on the changes in channel dynamics due to anesthetics and, conversely, to qualitatively analyze the anesthetic-induced changes in the steering force; (3) to investigate the channel dynamics responses to the steered gating movement of the GlyR TM2+TM3 channel in the presence and absence of anesthetic- nonanesthetic pairs; (4) to determine GlyR channel dynamics response to the forced binding and unbinding of anesthetics at the critical anesthetic-sensitive mutation site, S267, in the TM2 of GlyR; and (5) for all the simulation results in Specific Aims 1-4, to quantify the channel and lipid dynamics by analyzing root-mean-square deviation (RMSD) and fluctuations (RMSD), the autocorrelation functions, and generalized order parameters (S2), and to investigate the effects of interfacial water, interfacial lipids, and cavity dynamics on anesthetic-induced changes in channel dynamics. The central hypothesis to be tested is that anesthetics affect transmembrane channel function by profoundly changing the channel dynamics and the channel's association with lipids and interfacial water. The results from the proposed study will significantly advance the science in the following three areas: (1) large-scale parallel computing applications to biological problems, particularly anesthetic interaction with membrane-associated proteins; (2) detailed elucidation of residual dynamic contribution (through conformational entropy change) to the interfacial association between lipids and transmembrane channels; and (3) the development of a protein theory of general anesthesia on the basis of dynamics-function relationships.