The mechanism of allosteric modulation of glutamate transporters

摘要 兴奋性氨基酸转运体(EAATs)清除突触释放的谷氨酸并维持细胞外 谷氨酸浓度低于神经毒性水平。尤其是胶质谷氨酸转运蛋白EAAT2 在突触裂隙的谷氨酸清除中起主要作用。清除细胞内多余的谷氨酸与此密切相关 作为一种临床相关的手段来治疗因谷氨酸过量而导致兴奋性毒性的神经退行性疾病 会导致神经元损伤和死亡。 我们设计了一个独特的研究程序来了解变构调节的分子机制 最近发现的小分子化合物对谷氨酸转运体的影响，包括长期寻找的 运输。为了实现这一目标，我们将获得并整合运输功能的多学科知识， 转运蛋白及其与变构配合物的三维结构和单分子动力学 化合物。反过来，这些知识将有助于理解膜的基本转运机制。 人脑中最重要的兴奋性神经递质。 我们将致力于实现以下目标： 目的1：阐明人类EAAT中对变构起重要作用的分子决定因素 调节器活性。我们将使用功能研究和计算方法来定义变构位置 在EAAT2中，介导化合物的影响。 目的2：确定人EAATs与变构调节剂的络合物的结构。我们将决定 具有正负变构调节剂的EAAT的三维结构有待解开 它们协调的原子细节。 目的3：确定变构调节剂是否通过改变速率来调节EAAT的功能 运输基础上的构象转变。我们将研究构象动力学及其 使用单分子FRET和其他光谱技术和耦合的变构调制器的调制 这些研究使用单囊泡/单转运体试验来确定变构调节剂对 周转率和运输周期的时间。 影响：该研究计划中产生的信息将为药物发现开辟新的途径，因为 转运体是许多严重削弱中枢神经系统疾病的重要药物靶点，如创伤性 脑损伤、中风、癫痫、肌萎缩侧索硬化症和神经病理性疼痛，这些疾病加起来影响了近5%的美国人 人口。

Abstract Excitatory amino acid transporters (EAATs) remove synaptically released glutamate and maintain extracellular glutamate concentrations below neurotoxic levels. Particularly the glial glutamate transporter EAAT2 plays a major role in glutamate clearance in synaptic clefts. Removal of excess cellular glutamate is strongly implicated as a clinically relevant means to treat neurodegenerative diseases where excitotoxicity due to excess glutamate contributes to neuronal injury and death. We have designed a unique research program to understand the molecular mechanisms of allosteric modulation of glutamate transporters by recently discovered small-compound molecules, including long-sought activators of transport. To reach this goal, we will obtain and integrate multidisciplinary knowledge of the transport function, 3D structures, and single-molecule dynamics of the transporters and their complexes with the allosteric compounds. In turn, this knowledge will aid understanding the basic membrane transport mechanisms of the most important excitatory neurotransmitter in the human brain. We will pursue the following aims: Aim 1: Elucidate the molecular determinants within the human EAATs that are important for allosteric modulator activity. We will use functional studies and computational approaches to define the allosteric site within EAAT2 that mediate the effects of the compounds. Aim 2: Determine the structures of human EAATs in complex with allosteric modulators. We will determine the three-dimensional structures of EAATs in complex with positive and negative allosteric modulators to unravel the atomic details of their coordination. Aim 3: Establish whether allosteric modulators modulate the function of EAATs though altering the rates of conformational transitions underlying transport. We will examine the conformational dynamics and its modulation by allosteric modulators using single-molecule FRET and other spectroscopic techniques and couple these studies with single-vesicle/single-transporter assays to determine the effects of allosteric modulators on turnover rates and the timing of transport cycles. IMPACT: Information generated in this research program will open new avenues for drug discovery as these transporters serve as important drug targets for many severe debilitating CNS conditions, such as traumatic brain injury, stroke, epilepsy, ALS and neuropathic pain, that collectively affect nearly 5% of the American population.