Anesthetic Sites in Transmembrance Peptides by NMR

通过 NMR 确定跨膜肽中的麻醉位点

• 批准号: 7667104
• 负责人: PEI TANG
• 金额: $ 35.23万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7667104
• 关键词: Agonist; Anesthesia procedures; Anesthetics; Arts; Binding; Breathing; Characteristics; Communities; Data; Environment; Equilibrium; Funding; General Anesthesia; General anesthetic drugs; Goals; Halothane; Hypersensitivity; Indium; Investigation; Ion Transport; Isoflurane; Lead; Ligands; Link; Lipids; Measurable; Membrane; Molecular; Molecular Biology; Molecular Conformation; Motion; NIH Program Announcements; NMR Spectroscopy; Neurons; Neurotransmitters; Nicotinic Receptors; Nuclear Magnetic Resonance; Peptides; Pharmacology; Point Mutation; Population; Principal Investigator; Property; Proteins; Publishing; Research; Research Personnel; Resolution; Side; Site; Structure; Synaptic Transmission; Techniques; Time; Transmembrane Domain; Vesicle; Water; base; design; fluorescence imaging; insight; interfacial; mimetics; mutant; programs; protein structure; public health relevance; receptor; receptor sensitivity; research study; response; solid state nuclear magnetic resonance; theories; Agonist; Anesthesia procedures; Anesthetics; Arts; Binding; Breathing; Characteristics; Communities; Data; Environment; Equilibrium; Funding; General Anesthesia; General anesthetic drugs; Goals; Halothane; Hypersensitivity; Indium; Investigation; Ion Transport; Isoflurane; Lead; Ligands; Link; Lipids; Measurable; Membrane; Molecular; Molecular Biology; Molecular Conformation; Motion; NIH Program Announcements; NMR Spectroscopy; Neurons; Neurotransmitters; Nicotinic Receptors; Nuclear Magnetic Resonance; Peptides; Pharmacology; Point Mutation; Population; Principal Investigator; Property; Proteins; Publishing; Research; Research Personnel; Resolution; Side; Site; Structure; Synaptic Transmission; Techniques; Time; Transmembrane Domain; Vesicle; Water; base; design; fluorescence imaging; insight; interfacial; mimetics; mutant; programs; protein structure; public health relevance; receptor; receptor sensitivity; research study; response; solid state nuclear magnetic resonance; theories

DESCRIPTION (provided by applicant): This is a competing renewal of R01GM056257 for its 10th-13th years. The ultimate goal of the study is to elucidating the molecular mechanisms of general anesthesia. The research in the previous funding periods has led the investigators to realize that protein global dynamics (i.e., the slow modes of motion at tertiary and quaternary structural levels) is one of the most crucial elements in the manifestation of neurotransmitter-gated receptors' sensitivity to general anesthetics. Modulation of protein global dynamics may underlie a common molecular mechanism of anesthetic action. The approaches in this competing renewal aim at quantifying the anesthetic-induced changes in protein structure and dynamics at or near atomic resolution, using modern molecular biology and various biophysical techniques, notably the state-of-the-art high-resolution and solid- state nuclear magnetic resonance (NMR) spectroscopy. The central hypothesis is that critical amphipathic residues of ligand-gated near the membrane interface are constantly modulated by interactions with interfacial water and lipid molecules. This modulation produces a steady-state control of the channel dynamics, which in turn governs the transport properties of the channel. Anesthetics can preferentially target these pivotal residues and offset the balance of the steady-state channel dynamics by changing water or lipid association with the critical residues, thereby altering the characteristics of synaptic transmission through the channel. Substantive published as well as new preliminary results support the following three specific aims: (1) to determine and identify the structure differences between the anesthetic-supersensitive (14)2(22)3 and anesthetic-insensitive (17)5 neuronal nicotinic acetylcholine receptor (nAChR) transmembrane (TM) domains so that their different sensitivities to volatile anesthetics (halothane and isoflurane) can be understood on a structural basis; (2) to quantify the global dynamics of the (14)2(22)3 and (17)5 nAChR TM domains in membrane-mimetic environments in the absence and presence of anesthetics (halothane and isoflurane); and (3) to correlate channel global dynamics with ion transport in the presence and absence of anesthetics (a) by determining the dynamics of channel mutants having altered sensitivity to anesthetics, such as the double mutants of 14 (I268M and V274S) and 22 (V262M and D268S) with the corresponding residues in the TM2-TM3 loop of 17 subunit, and (b) by determining channel formation and ion transport rate of the wild type and mutants of the (14)2(22)3 and (17)5 nAChRs in membrane vesicles using fluorescence imaging and NMR exchange experiments. The results of this investigation will lead to a new paradigm for the analysis and interpretation of general anesthetic action on neuronal proteins. PUBLIC HEALTH RELEVANCE: This research will advance our understanding on how and where general anesthetics act at the molecular level. It will also solve the structure of the transmembrane domain of a class of neurotransmitter-gated receptor channels, for which no high-resolution structure is currently available. The research will eventually lead to better design of safer and more effective new anesthetic drugs.

描述（由申请人提供）：这是其10-13年的R01GM056257的竞争续约。该研究的最终目标是阐明全身麻醉的分子机制。前一家资金期间的研究使研究人员意识到，蛋白质全球动力学（即，第三纪和第四纪结构水平的慢速运动模式）是神经递质受体受体表现出的最关键元素之一，对一般麻醉剂的敏感性。蛋白质全球动力学的调节可能是麻醉作用的常见分子机制。这种竞争性更新中的方法旨在使用现代分子生物学和各种生物物理技术来量化麻醉诱导的蛋白质结构和动力学的变化，尤其是最先进的高分辨率高分辨率和固态核磁共振共振（NMR）。中心假设是，在膜界面附近配体门控的临界两亲性残基受到与界面水和脂质分子相互作用的不断调节。该调制产生对通道动力学的稳态控制，从而控制通道的传输特性。麻醉药可以优先针对这些关键残基，并通过改变与临界残基的水或脂质关联来抵消稳态通道动力学的平衡，从而改变通过通道突触传播的特征。已发表的实质性和新的初步结果支持以下三个具体目的：（1）确定和确定麻醉性 - 启发性敏感的结构差异（14）2（22）3和麻醉敏感的（17）5神经元素乙酰乙基乙基胆碱受体（NACHR）透射（NACHR）透射（TM）的影响，以使其与他们的静脉触及的静脉（TM）相互作用，以使其与众不同。异氟烷）可以从结构上理解； （2）在没有麻醉药（Halothane和isoflurane）的情况下，在膜模拟环境中量化（14）2（22）3和（17）5 NACHR TM域的全局动力学； （3）通过确定对麻醉剂的敏感性改变的通道突变体的动力学，例如14（I268m和V274s和V262m和D268S）与14（I268m和V268s）与17（v262m和d268s）与17的c中的14（i268m和v268s），将通道全球动力学与离子传输相关联（a）（a）（a），例如14（I268m和V274s）的双突变体（I268m和V274s）和17 cy n of sub sun 33。使用荧光成像和NMR交换实验确定（14）2（22）3和（17）5 NACHR的野生型和突变体的通道形成和离子传输速率。这项研究的结果将导致新的范式，用于分析和解释对神经元蛋白的全身麻醉作用。公共卫生相关性：这项研究将促进我们对一般麻醉在分子层的作用和何处的理解。它还将解决一类神经递质门控受体通道的跨膜结构域的结构，目前没有高分辨率结构。这项研究最终将导致更好地设计更安全，更有效的新麻醉药。