Molecular Studies of Human CNS Glutamate Transporters

描述（由申请人提供）：中枢神经系统中的兴奋性氨基酸转运蛋白（EAATs）维持细胞外谷氨酸浓度低于兴奋毒性水平，并有助于清除神经传递过程中释放的谷氨酸。在之前的资助期内，我们的实验室利用了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试剂来确定底物运输和离子渗透所需的结构特征。在这个竞争性更新应用中，我们计划使用引入的半胱氨酸对和交联试剂来评估运输周期中不同残基的接近性。研究将继续强调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模拟谷氨酸转运体构象动力学的实验相结合。高斯网络建模（GNM）和分子动力学（MD）模拟是非常适合研究大型多功能结构的技术，如离子通道和神经递质转运体。迄今为止，处理这种多聚体蛋白的方法仅限于原子相互作用或有限的亚纳秒时间范围，与我们在实验中观察到的现象相比，这太过局限或太快。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域的结构和流动性的结构-功能实验来测试。第三个目标将探索从蜘蛛毒液中纯化的神经保护化合物的作用机制，该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，即将未占据的载体重新定向到外部。该化合物选择性作用于主要胶质载体EAAT2，增加谷氨酸内流而不外排，为开发具有治疗潜力的EAATs变构激活剂提供了原理证明。临床和实验研究强调了理解兴奋性氨基酸转运体的结构、功能和动力学的重要性，这些研究涉及退行性疾病（如ALS、亨廷顿氏病、缺血诱导的神经毒性和阿尔茨海默氏痴呆症）中细胞外谷氨酸浓度的增加。

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.