Anesthetic Effects on Ion Channel Structures & Dynamics

描述(由申请人提供)：本申请是R01GM066358的竞争续期，期限为9-13年。该项目专注于麻醉剂对离子通道作用的计算研究，长期目标是确定全身麻醉的分子机制。我们对1422和17亚型烟碱型乙酰胆碱受体(NAChRs)的计算研究中的一个重要发现是，无论这两种亚型对挥发性麻醉药的功能敏感性如何，与这两种nAChR亚型的麻醉剂结合是相似的。考虑到同一超家族中的许多麻醉敏感和不敏感受体具有很高的结构相似性，单靠麻醉剂结合不太可能区分麻醉药的功能敏感性。随着最近成功地生产全长17nAChR及其细菌五聚体通道类似物GLIC，光反应性麻醉药的可获得性以及超级计算能力的显著进步，这一继续应用提出了检验中心假设，即只有那些能够改变受体先前存在的全局构象之间的平衡的麻醉剂结合位点才会产生功能影响。形成这种结合位点的残基通常涉及远程静电相互作用。这三个新的目标是：(1)生产高质量的全长17,1722和1422 nAChRs，以及GLIC及其阴离子传导突变体，用于理论预测的功能测试。(2)用光反应性麻醉剂标记和荧光猝灭的方法确定Aim 1产生的受体和突变体中的麻醉剂结合部位。(3)通过高级计算揭示麻醉剂对这些离子通道作用的潜在机制，包括长时间尺度(高达5s时间尺度)的分子动力学模拟，以发现为什么只有亚类特定的麻醉剂结合才能产生功能影响，表现为抑制或增强。本研究的创新体现在研究假设的新颖性、研究方法的整合性和多学科合作等方面。这项研究的意义在于为量化低亲和力药物对蛋白质的作用提供了范式转换的概念框架，为未来合理设计副作用减少的新型麻醉药提供了新的平台。

DESCRIPTION (provided by applicant): This application is a competing renewal of R01GM066358 for Years 9-13. The project has focused on computational investigations of the anesthetic action on ion channels with the long-term goal of determining the molecular mechanism of general anesthesia. One of the important discoveries from our computational studies of the 1422 and 17 subtypes of nicotinic acetylcholine receptor (nAChRs) is that anesthetic binding to these two nAChR subtypes are similar regardless of their different functional sensitivity to volatile anesthetics. Given that many anesthetic-sensitive and insensitive receptors within the same superfamily share high structure similarity, it is unlikely that anesthetic binding alone can differentiate functional sensitivities to anesthetics. With the recent success in producing ful-length 17 nAChR and its bacterial pentameric channel analogue GLIC, the availability of photoreactive anesthetics and the significant advancement in supercomputing power, this continuation application proposes to test the central hypothesis that only those anesthetic binding sites capable of shifting the equilibriums among the preexisting global conformations of the receptor will have functional impact. The residues forming such binding sites often involve long-range electrostatic interaction. The three new aims are: (1) to produce high quantity and quality full-length 17, 1722, and 1422 nAChRs, as well as GLIC and its anion-conducting mutants, for functional tests of the theoretical predictions. (2) To determine the anesthetic binding sites in the receptors and mutants produced in Aim 1 using photoreactive anesthetic labeling and fluorescence quenching. (3) To reveal underlying mechanisms of anesthetic action on these ion channels through advanced computations, including long-timescale (up to 5s timescale) molecular dynamics simulations to discover where and why only a sub-class of specific anesthetic binding can have functional impact, manifested as either inhibition or potentiation. The innovation of the proposed research is reflected in the novel hypotheses, integrated aproaches, and multidisciplinary collaborations. The significance of the proposed studies is the paradigm-shifting conceptual framework to quantify the action of low-affinity drugs on proteins, providing a new platform for future rational design of novel anesthetics with reduced side effects.