Anesthetic Effects on Ion Channel Structures & Dynamics

描述（由申请人提供）：此申请是R01GM066358第9-13年的竞争性续期。该项目的重点是计算研究麻醉对离子通道的作用，长期目标是确定全身麻醉的分子机制。我们对1422和17个烟碱乙酰胆碱受体（nAChR）亚型的计算研究的一个重要发现是，麻醉剂与这两种nAChR亚型的结合是相似的，尽管它们对挥发性麻醉剂的功能敏感性不同。考虑到同一超家族中的许多麻醉敏感和不敏感受体具有高度的结构相似性，仅通过麻醉剂结合来区分对麻醉剂的功能敏感性是不可能的。随着最近生产全长17 nAChR及其细菌五聚体通道类似物GLIC的成功，光反应麻醉剂的可用性以及超级计算能力的重大进步，本继续应用建议测试中心假设，即只有那些能够改变受体先前存在的整体构象之间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距离静电相互作用。三个新的目标是：(1)生产高数量和高质量的全长17、1722和1422 nachr，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)利用光反应性麻醉标记和荧光猝灭技术确定Aim 1中产生的受体和突变体的麻醉结合位点。(3)通过先进的计算揭示麻醉作用于这些离子通道的潜在机制，包括长时间尺度（长达5个时间尺度）的分子动力学模拟，以发现在何处以及为什么只有一类特定的麻醉结合才能产生功能影响，表现为抑制或增强。提出的研究的创新体现在新颖的假设、综合的方法和多学科合作。这些研究的意义在于，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更小的新型麻醉剂提供了新的平台。

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.