Structural Rearrangements in GABA-A Receptors

描述(申请人提供)：伽马-氨基丁酸A型受体(GABAARs)介导大脑中的大部分突触抑制，并受各种临床重要药物的调节，如苯二氮类药物、巴比妥酸盐、类固醇、麻醉剂和抗惊厥药物。此外，GABAAR突变最近被认为与癫痫有关。我们研究计划的长期目标是从分子结构的角度了解GABAAR的功能。虽然最近的结晶学进展为GABAAR提供了有价值的结构模型，但要完全理解功能也需要蛋白质动力学的知识。GABAAR至少存在三种可相互转换的状态，具有不同的功能：非活动/关闭、活动/开放和不敏感/关闭。人们对这些结构转变过程中发生的蛋白质运动知之甚少，这些结构转变受神经递质和药物结合的调节。我们将使用荧光记录在非洲爪哇卵母细胞中表达的GABAARs中的位置特异性标记来研究激活、脱敏和药物调节的结构重排，因为它们实时发生。我们建议研究1)激动剂、部分激动剂和拮抗剂诱导的重排，2)整体蛋白质运动，3)戊巴比妥引起的结构变化，4)脱敏过程中的蛋白质运动。这些实验将借助最近阐明的原子级结构进行解释，以更深入地了解GABAARs及其近亲发挥作用的分子机制。如果不剖析调节其功能的蛋白质的运动，我们就不能指望预测药物或配体的作用，或者预测GABAAR致病突变的结果。这里提出的研究利用了一种创新的新方法，使我们能够了解GABAARs如何在健康和疾病状态下发挥作用。与公共健康相关：位于神经细胞膜上的配体门控离子通道的打开和关闭，调节整个大脑的信息流。这些通道的缺陷会导致多种疾病，如肌无力、多动症和癫痫。这些通道也是许多临床使用药物的靶点，包括肌肉松弛药、镇静催眠药、抗惊厥药、抗焦虑药和麻醉药。如果不知道这些通道在分子水平上是如何工作的，我们就不能指望预测药物的作用，设计更安全有效的药物，开发更好的治疗策略，或者预测致病突变的结果。这里提出的研究利用了一种创新的新方法，将增加我们对一种类型的离子通道GABAAR在健康和疾病中如何发挥作用的理解，并将为阐明其他相关的配体门控离子通道如何发挥作用建立可检验的假说。

DESCRIPTION (provided by applicant): Gamma-aminobutyric acid type A receptors (GABAARs) mediate the majority of synaptic inhibition in the brain and are modulated by a variety of clinically important drugs, such as benzodiazepines, barbiturates, steroids, anesthetics and anti-convulsants. Furthermore, GABAAR mutations have recently been linked to epilepsy. The long-term goal of our research program is to understand the function of the GABAAR in terms of its molecular structure. While recent crystallographic advances have provided valuable structural models of the GABAAR, achieving a full understanding of function also requires knowledge of protein dynamics. GABAARs exist in at least three interconvertible states with distinct functions: inactive/closed, active/open, and desensitized/closed. Very little is known about the protein motions that occur during these structural transitions, which are regulated by neurotransmitter and drug binding. We will use fluorescence recording of site-specific labels in GABAARs expressed in Xenopus oocytes to study the structural rearrangements underlying activation, desensitization, and drug modulation as they occur in real time. We propose to study 1) agonist, partial-agonist and antagonist induced rearrangements, 2) global protein motions, 3) pentobarbital induced structural changes and 4) protein motions during desensitization. The experiments will be interpreted with the aid of recently elucidated atomic- level structures to gain a deeper understanding of the molecular mechanisms underlying the function of GABAARs and their relatives. We cannot hope to predict the actions of a drug or ligand or predict the outcome of a disease-causing mutation in the GABAAR without dissecting the movements in the protein that mediate its function. The research proposed here utilizes an innovative new approach that will enable us to learn how GABAARs function in health and disease states. PUBLIC HEALTH RELEVANCE: The opening and closing of ligand-gated ion channels, which lie in the membranes of nerve cells, regulate information flow throughout the brain. Defects in these channels lead to wide variety of diseases, such as myasthenia, hyperekplexia and epilepsy. These channels are also the targets of a number of clinically used drugs, including muscle relaxants, sedative-hypnotics, anti-convulsants, anxiolytics and anesthetics. We cannot hope to predict the actions of a drug, design safer and more effective drugs, develop better therapeutic strategies or predict the outcome of a disease-causing mutation without knowledge of how these channels work at a molecular level. The research proposed here utilizes an innovative new approach that will increase our understanding of how one type of ion channel, the GABAAR, functions in health and disease and will establish testable hypotheses for elucidating how other related ligand-gated ion channels function.