Molecular Studies of Human CNS Glutamate Transporters

描述(申请人提供)：中枢神经系统中的兴奋性氨基酸转运体(EAAT)将细胞外谷氨酸浓度维持在兴奋性毒性水平以下，并有助于清除神经传递过程中释放的谷氨酸。在之前的资助期间，我们的实验室利用高功能的无半胱氨酸版本的EAAT1，利用半胱氨酸取代和巯基修饰剂来鉴定底物运输和离子渗透所需的结构特征。在这个竞争性的更新应用中，我们计划使用引入的半胱氨酸对和交联剂来评估运输周期中不同残基的接近程度。研究将继续强调对EAAT功能的动力学、生化、药理学和电生理分析。在第二个目标中，这些方法将与使用计算方法的实验相结合，以模拟谷氨酸转运体的构象动力学。高斯网络建模(GNM)和分子动力学(MD)模拟是研究离子通道和神经递质转运体等大型多功能结构的理想技术。到目前为止，治疗这种多聚体蛋白质的方法一直被限制在原子相互作用或有限的亚纳秒时间范围内，与我们实验中观察到的现象相比，这些时间范围太局部化或太快。这两种互补方法的使用提供了一种识别关键相互作用的稳健方法，然后可以通过旨在改变感兴趣领域的结构和移动性的结构功能实验进行测试。第三个目标是探索从蜘蛛毒液中提纯的一种神经保护化合物的作用机制，这种化合物似乎通过改变运输周期中一个较少研究的过渡步骤来增强运输活性，即将未被占用的载体重新定向到外部。这种化合物选择性地作用于主要的神经胶质载体EAAT2，增加谷氨酸的内流而不是外流，为开发具有治疗潜力的EAATs的变构激动剂提供了原理证据。临床和实验研究强调了了解兴奋性氨基酸转运体的结构、功能和动力学的重要性，这些研究表明，在ALS、亨廷顿病、缺血诱导的神经毒性和阿尔茨海默病等退行性疾病中，细胞外谷氨酸浓度增加。

DESCRIPTION (provided by applicant): Excitatory amino acid transporters (EAATs) in the CNS maintain extracellular glutamate concentrations below excitotoxic levels and contribute to the clearance of glutamate released during neurotransmission. Over the previous funding period our laboratory took advantage of a highly functional cysteineless version of EAAT1, to identify the structural features required for substrate transport and ion permeation using cysteine substitutions together with sulfhydryl modifying reagents. In this competing renewal application we plan to assess proximity of different residues during the transport cycle using introduced cysteine pairs and crosslinking reagents. Studies will continue to emphasize kinetic, biochemical, pharmacological and electrophysiological analyses of EAAT function. In a second aim these approaches will be combined with experiments using computational methods to model the conformational dynamics of glutamate transporters. Gaussian network modeling (GNM) and molecular dynamic (MD) simulations are techniques ideally suited for the study of large, multifunctional structures such as ion channels and neurotransmitter transporters. To date, methods that treat such multimeric proteins have been restricted to atomic interactions or limited, sub- nanosecond time ranges, which are too localized or fast compared to the phenomena that are observable in our experiments. The use of these two complementary methods provide a robust way of identifying critical interactions, which then can be tested by structure-function experiments designed to alter the structure and mobility of the domain of interest. A third aim will explore the mechanism of action of a neuroprotective compound purified from a spider venom, which appears to enhance transport activity by altering a less studied transition step in the transport cycle, the reorientation of the unoccupied carrier to the outside. This compound, which acts selectively on the major glial carrier EAAT2, increases glutamate influx but not efflux, and provides proof of principle for the development of allosteric activators of EAATs with therapeutic potential. The importance of understanding the structure, function, and dynamics of excitatory amino acid transporters is underscored by clinical and experimental studies, which have implicated increases in extracellular glutamate concentration in degenerative disorders such as ALS, Huntington's disease, ischemia-induced neurotoxicity, and Alzheimer's dementia.