Structural and Functional Underpinnings of Proton Coupling in Glutamate Transporters

谷氨酸转运蛋白中质子偶联的结构和功能基础

• 批准号: 10314136
• 负责人: Yessenia Lopez
• 金额: $ 4.6万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2023-09-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10314136
• 关键词: Affinity; Amino Acid Transporter; Architecture; Aspartate; Bacillus; Binding; Binding Sites; Biological Assay; Biophysics; Brain; Calorimetry; Chlorides; Communities; Consult; Coupled; Coupling; Cryoelectron Microscopy; Dependence; Electron Microscopy; Energy-Generating Resources; Excitatory Amino Acids; Family; Fellowship; Functional disorder; Glutamate Transporter; Glutamates; Goals; Homologous Gene; Image; In Vitro; Institution; Ion Cotransport; Ions; Kinetics; Knowledge; Link; Liposomes; Memorial Sloan-Kettering Cancer Center; Modeling; Molecular; Molecular Conformation; Mutagenesis; Neuroglia; Neurons; Neurotransmitters; Orthologous Gene; Outcome; Play; Potassium; Protein Conformation; Protons; Pyrococcus horikoshii; Research Personnel; Resolution; Resources; Role; Science; Shapes; Site; Sodium; Source; Specificity; Structure; Synaptic Cleft; Synaptic Transmission; Technical Expertise; Testing; Titrations; Total Internal Reflection Fluorescent; Training Programs; Work; comparative; cryogenics; excitotoxicity; expectation; experimental study; extracellular; insight; neurological pathology; neurotransmission; neurotransmitter uptake; potassium ion; prevent; protonation; reconstitution; research facility; single-molecule FRET; sodium ion; stoichiometry; uptake

PROJECT ABSTRACT Glutamate is the primary excitatory neurotransmitter in the brain and is necessary for several crucial brain functions. Mammalian glutamate transporters, or excitatory amino acid transporters (EAATs), are responsible for rapidly clearing excessive glutamate to prevent excitotoxicity and in shaping synaptic transmission. Therefore, EAATs dysfunction is often linked to neurological pathologies. Most of our understanding resulted from studying archaeal homologue, GltPh from Pyrococcus horikoshii. In EAATs, substrate transport is coupled to three sodiums, one proton, and the countertransport of a potassium ion, while GltPh is exclusively sodium-coupled, so there is limited insight as to how protons bind, and the mechanism by which they drive transport. Bacterial homologue, GltTBc from Bacillus caldotenax, is driven by symport of protons rather than sodium ions. These homologues share a similar trimeric structure but have different ion-coupling specificity. There is no high-resolution structure for GltTBc or any other proton-coupled transporter of this family. The goal of my first Aim is to obtain a high-resolution structure of GltTBc to both identify potential protonation sites and understand the structural basis of specificity during ion coupling. To determine the role of protons during substrate transport in GltTBc, Aim 2 will elucidate whether protons drive glutamate binding or translocation and understand the molecular basis of proton coupling. Under this fellowship, I will have access to consult with leading researchers from Weill Cornell and associated core research facilities. Being in the middle of the “corridor of science”, which is composed of Weill Cornell, The Rockefeller Institution, and Memorial Sloan Kettering Cancer Center, will provide me with ample opportunities and resources to not only enhance my technical skills, but allow me to present and communicate my work to the scientific community. This will be aided by my PI’s recent advancements in establishing the Molecular Biophysics Training Program, which provides another source of support, community, and expertise within the Tri-Institiutional campus.

项目摘要 谷氨酸是大脑中的主要兴奋神经递质，对于几种至关重要 大脑功能。哺乳动物谷氨酸转运蛋白或兴奋性氨基酸转运蛋白（EAATS）是 负责快速清除多余的谷氨酸以防止兴奋性毒性和塑造突触 传播。因此，EAAT功能障碍通常与神经病理学有关。我们的大多数 理解是由于研究了horikoshii的古细菌同源物，GLTPH而产生的。在老鹰中， 底物运输偶联到三个钠，一个质子和钾离子的反通道，而 GLTPH仅是钠耦合的，因此对质子的结合方式和通过 他们驱动运输。 细菌同源物，caldotenax的glttbc，由质子的同义词而不是 钠离子。这些同源物具有相似的三聚体结构，但具有不同的离子耦合特异性。 该家族的GLTTBC或任何其他质子耦合转运蛋白没有高分辨率结构。目标 我的第一个目的是获得GLTTBC的高分辨率结构，以识别潜在的质子化位点和 了解离子耦合过程中特异性的结构基础。确定质子在 GLTTBC中的底物运输，AIM 2将阐明质子是驱动谷氨酸结合还是易位和 了解质子耦合的分子基础。 根据这项奖学金，我将可以访问Weill Cornell和 相关的核心研究设施。位于由威尔（Weill）组成的“科学走廊”中间 洛克菲勒机构康奈尔（Cornell）和纪念斯隆·凯特林（Sloan Kettering Cancer Center）将为我提供足够的 机会和资源不仅可以增强我的技术技能，还可以允许我介绍和交流 我给科学界的工作。我的PI最近在建立该公司的进步方面有助于 分子生物物理学培训计划，该计划提供了另一个支持，社区和专业知识的来源 在三个国家的校园内。