Structural and Functional Underpinnings of Proton Coupling in Glutamate Transporters

项目摘要 谷氨酸是大脑中主要的兴奋性神经递质，对大脑中的几个关键功能是必需的。 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（EAAT）， 负责快速清除过量的谷氨酸，以防止兴奋性毒性，并在塑造突触 传输因此，EAAT功能障碍通常与神经病理学有关。我们的大多数 研究古细菌同系物，GltPh从Pyrococcus horikoshii的理解。在EAAT中， 底物转运与三个钠、一个质子和一个钾离子的反向转运相耦合， GltPh仅是钠偶联的，因此关于质子如何结合以及通过GltPh结合的机制的了解有限。 他们负责运输 细菌同系物，来自热坚芽孢杆菌的GltTBc，由质子的同向运输驱动，而不是 钠离子这些同源物具有相似的三聚体结构，但具有不同的离子偶联特异性。 没有GltTBc或该家族任何其他质子偶联转运蛋白的高分辨率结构。目标 我的第一个目标是获得一个高分辨率的结构GltTBc既确定潜在的质子化位点， 了解离子耦合过程中特异性的结构基础。为了确定质子在 GltTBc中的底物转运，目的2将阐明质子是否驱动谷氨酸结合或易位， 了解质子耦合的分子基础。 在这个奖学金下，我将有机会与威尔康奈尔大学的主要研究人员进行磋商， 相关核心研究设施。处于由威尔组成的“科学走廊”的中间 康奈尔大学，洛克菲勒研究所和纪念斯隆凯特琳癌症中心，将为我提供充足的 机会和资源，不仅可以提高我的技术技能，还可以让我展示和交流 向科学界展示我的成果这将有助于我的PI最近的进展，建立 分子生物物理学培训计划，提供另一种支持，社区和专业知识来源 在三个学院的校园里。

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.