Investigating the Structural Basis of Allosteric Coupling in Gaba A Receptors Using Increased Atmospheric Pressure

利用增加的大气压研究 Gaba A 受体中变构偶联的结构基础

• 批准号: 9818422
• 负责人: Ronald Alkana
• 金额: $ 40.1万
• 依托单位: University of Southern California
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-15 至 2005-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9818422&HistoricalAwards=false
• 关键词: Investigating; Structural; Basis; Allosteric; Coupling

Ronald L. AlkanaABSTRACTThe overall goal of the proposed work is to increase understanding of the basic mechanisms involved in communication between nerve cells in the brain. The proposal builds on previous findings demonstrating that increased atmospheric pressure (hyperbaric exposure) is a direct, highly selective blocker of a little explored, poorly defined process-allostenc coupling-that modulates the effectiveness of a family of neurotransmitters receptors in the brain, called ligand gated ion channels (LOICs), that play major roles in nerve-nerve communication. In particular, the proposed work focuses on one of these channels. This channel responds to gamma-aminobutyric acid (GABA) and is called the GABAA channel. When GABA binds to its receptor on the GABAA the channel opens a channel and allows chloride ions to enter the nerve cell. The resultant build-up of chloride ions in the nerve cell inhibits the cells action and decreases its excitablity. The GABAA system is the major inhibitory system in the mammalian brain.The effectiveness of GABA' 5 action in opening chloride ion channels can be modulated by several classes of compounds that act at distinct but interacting sites on the GABAA receptor. These sites include those for the benzodiazepines, barbiturates and neuroactive steroids. When one of these compounds (ligands) bind to their respective sites on the GABAA receptor, it causes a conformation change in the receptor that affects the ability of other ligands to bind or affect the receptor. This process, referred to as allosteric coupling between sites, modulates the effectiveness of the primary agonist-GAB A-as well as the effectiveness of other allosteric modulators.The molecular structures and functions of the portions of the GAB AA receptor that bind ligands has been extensively studied. In contrast, little attention has been devoted to understanding the elements that underlie allosteric coupling. The elements mediating coupling are difficult to study directly due to a lack of tools. Recent behavioral and biochemical findings in our laboratory suggest that hyperbaric exposure offers a new approach that can help in studying allosteric coupling. Moreover, this work with hyperbaric exposure suggests that there are fundamental, previously unrecognized, differences in the manner in which binding sites on the GABAA receptor are coupled and that hyperbaric exposure can be used to study these differences.The specific objective of the research to be undertaken is to test two hypotheses: Hypothesis 1:The different pattern of sensitivity to pressure antagonism among allosteric modulators of GABAA receptor function will provide new insights into the structural and functional determinants of coupling. The logic for this hypothesis is based on the assumption that the selectivity of pressure antagonism results from pressure's ability to block common physico-chemical changes underlying coupling and that the sensitivity of these physico-chemical changes to pressure reflect similarities in their underlying molecular structures. The hypothesis will be tested by systematically investigating four predictions regarding the selectivity of pressure antagonism based on known functional distinctions in coupling within and between different sites on the GABAA receptor using biochemical measures of GABAA receptor function in mouse brain cell membranes. Hypothesis 2:Differences in the structural and functional determinants of GABAA receptor coupling identified inABSTRACT DRAAT 12/4/98testing Hypothesis 1 reflect differences in the protein subunits that comprise the receptor. This hypothesis will be tested by determining the sensitivity to pressure antagonism of allosterically modulated events using biochemical and molecular biological techniques.The proposed work will increase knowledge regarding the manner in which the effectiveness of nerve cell transmission is controlled. The proposed work will lay the foundation for future studies that will use hyperbaric exposure in combination with molecular manipulations in recombinant cells to identify molecular components that mediate allosteric coupling. This information in turn will facilitate the development of molecular models of these structures. The proposed work will also lay the foundation for future investigations that will investigate coupling in other allosterically modulated channels (e.g., NMDA, 5HT3...). These studies could support known and/or could reveal previously unrecognized similarities between allosterically modulated ion channels. Finally, future studies will investigate whether differences in coupling mechanisms have currently unrecognized physiological and behavioral significance. Therefore, the proposed and future work should lead to important new insights regarding the role LGICs play in mediating and modulating brain function and behavior.

罗纳德湖Alkana摘要这项工作的总体目标是增加对大脑神经细胞之间通信的基本机制的理解。该提案建立在以前的研究结果的基础上，表明增加的大气压力（高压暴露）是一种直接的，高度选择性的阻滞剂，可以抑制一种很少探索的，定义不清的过程-allostenc耦合-调节大脑中一个神经递质受体家族的有效性，称为配体门控离子通道（LOIC），在神经-神经通信中发挥主要作用。特别是，拟议的工作重点关注其中一个渠道。该通道响应于γ-氨基丁酸（GABA），被称为GABAA通道。当GABA与GABAA上的受体结合时，通道打开通道并允许氯离子进入神经细胞。由此产生的氯离子在神经细胞中的积聚抑制了细胞的活动并降低了其兴奋性。GABAA系统是哺乳动物脑中的主要抑制系统。GABA ′ 5在开放氯离子通道中的作用的有效性可以通过作用于GABAA受体上不同但相互作用的位点的几类化合物来调节。这些位点包括苯二氮卓类、巴比妥类和神经活性类固醇。当这些化合物（配体）之一结合到GABAA受体上的它们各自的位点时，它引起受体的构象变化，影响其他配体结合或影响受体的能力。该过程称为位点之间的变构偶联，调节主要激动剂-GABAA-的有效性以及其它变构调节剂的有效性。相比之下，很少有人关注了解的要素，变构耦合。由于缺乏工具，很难直接研究介导耦合的元素。我们实验室最近的行为和生化研究结果表明，高压暴露提供了一种新的方法，可以帮助研究变构偶联。此外，这项高压暴露的研究表明，GABAA受体结合位点的偶联方式存在根本性的、以前未被认识到的差异，高压暴露可用于研究这些差异。研究的具体目标是检验两个假设：假设1：在GABAA受体功能的变构调节剂中，对压力拮抗作用的不同模式的敏感性将为结构调节提供新的见解。和耦合的功能决定因素。这种假设的逻辑是基于这样的假设，即压力拮抗作用的选择性来自于压力阻止耦合基础上的常见物理化学变化的能力，并且这些物理化学变化对压力的敏感性反映了其基础分子结构的相似性。该假设将通过系统地研究四个预测的压力拮抗作用的选择性的基础上已知的功能差异耦合内和不同的网站之间的GABAA受体，使用生化措施的GABAA受体功能在小鼠脑细胞膜。假设2：GABAA受体偶联的结构和功能决定因素的差异在1998年4月12日的实验中得到证实。假设1反映了组成受体的蛋白质亚基的差异。这一假设将通过使用生物化学和分子生物学技术确定变构调制事件对压力拮抗作用的敏感性来进行测试。拟议的工作将增加关于神经细胞传递的有效性被控制的方式的知识。拟议的工作将奠定基础，为未来的研究，将使用高压暴露与重组细胞中的分子操作相结合，以确定介导变构偶联的分子组分。这些信息反过来将促进这些结构的分子模型的发展。所提出的工作也将为未来的研究奠定基础，这些研究将研究其他变构调制通道中的耦合（例如，NMDA、5HT3.）。这些研究可以支持已知的和/或可以揭示以前未被识别的变构调节离子通道之间的相似性。最后，未来的研究将调查偶联机制的差异是否具有目前尚未认识到的生理和行为意义。因此，拟议的和未来的工作应该导致重要的新见解LGIC在调解和调节大脑功能和行为中发挥的作用。