The mechanism of allosteric modulation of glutamate transporters

谷氨酸转运蛋白变构调节机制

• 批准号: 10303051
• 负责人: Olga Boudker
• 金额: $ 56.75万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303051
• 关键词: Acute; Address; Affect; Allosteric Site; American; Amino Acid Transporter; Antibodies; Binding; Biological Assay; Brain; Carrier Proteins; Central Nervous System Diseases; Chemicals; Chronic; Communication; Complex; Computer Models; Cryoelectron Microscopy; Crystallization; Development; Drug Targeting; Elevator; Enhancers; Epilepsy; Excision; Excitatory Amino Acid Transporter 1; Excitatory Amino Acids; Fluorescence Resonance Energy Transfer; Future; Glutamate Transporter; Glutamates; Goals; Homologous Gene; Human; Injury; Knowledge; Lead; Measurement; Mediating; Methods; Microscopy; Modernization; Molecular; Molecular Conformation; Movement; Mutagenesis; Neurodegenerative Disorders; Neuronal Injury; Neurotransmitters; Pain; Peripheral; Pharmaceutical Chemistry; Pharmacology; Phylogenetic Analysis; Play; Population; Process; Property; Proteins; Research; Resolution; Role; Sampling; Specificity; Stroke; Structure; Synaptic Cleft; Techniques; Technology; Testing; Transmembrane Transport; Traumatic Brain Injury; Variant; Vesicle; X-Ray Crystallography; base; biophysical analysis; clinically relevant; conformational conversion; design; drug discovery; excitotoxicity; extracellular; improved; in vitro Model; in vivo Model; multidisciplinary; nanobodies; neuron loss; neurotoxic; novel strategies; painful neuropathy; particle; programs; scaffold; single molecule; small molecule; therapy development; thermostability; three dimensional structure; virtual screening

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.

摘要 兴奋性氨基酸转运蛋白（EAAT）清除突触释放的谷氨酸，维持细胞外 谷氨酸浓度低于神经毒性水平。特别是神经胶质谷氨酸转运体EAAT 2在神经胶质细胞中起着重要的作用。 在突触间隙谷氨酸清除中起主要作用。细胞内过量谷氨酸的清除 作为治疗神经变性疾病的临床相关手段， 导致神经元损伤和死亡。 我们设计了一个独特的研究计划，以了解变构调节的分子机制 最近发现的小化合物分子，包括长期寻找的谷氨酸转运蛋白激活剂， 运输为了实现这一目标，我们将获得和整合多学科知识的运输功能， 转运蛋白及其与变构蛋白复合物的3D结构和单分子动力学 化合物.反过来，这一知识将有助于理解的基本膜运输机制， 是人脑中最重要的兴奋性神经递质 我们将努力实现以下目标： 目的1：阐明人类EAAT中对变构重要的分子决定簇 调节剂活性我们将使用功能研究和计算方法来定义变构位点 在EAAT 2内介导化合物的作用。 目的2：确定人EAAT与别构调节剂复合物的结构。我们将确定 EAAT与正负变构调节剂复合的三维结构， 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EAAT的速率来调节EAAT的功能 运输过程中的构象转变我们将研究构象动力学及其 通过使用单分子FRET和其它光谱技术的变构调节剂的调节， 这些研究使用单囊泡/单转运蛋白测定来确定变构调节剂对 周转率和运输周期的时间安排。 影响：这项研究计划产生的信息将为药物发现开辟新的途径， 转运蛋白作为许多严重衰弱性CNS病症的重要药物靶标，例如创伤性 脑损伤、中风、癫痫、ALS和神经性疼痛，这些疾病共同影响了近5%的美国人， 人口