Conformational Ensemble of Glutamate Transporters: Structure and IonicModulation

谷氨酸转运蛋白的构象整体：结构和离子调制

• 批准号: 8697890
• 负责人: Olga Boudker
• 金额: $ 37.08万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8697890
• 关键词: Active Biological Transport; Affinity; Amino Acid Transporter; Amino Acids; Architecture; Aspartate; Bacteria; Binding; Binding Sites; Biochemical; Biological Models; Brain; Brain Injuries; Cations; Coupled; Couples; Coupling; Cytoplasm; Dependence; Elevator; Energy-Generating Resources; Ensure; Environment; Event; Excitatory Amino Acids; Family; Family member; Functional disorder; Funding; Glutamate Transporter; Glutamates; Goals; Hand; Health; Heart; Homologous Gene; Human; Injury; Ion Cotransport; Ions; Light; Lipid Bilayers; Mediating; Membrane; Methodology; Molecular; Molecular Conformation; Movement; Mutagenesis; Neuroglia; Neurons; Neurotransmitters; Nutrient; Potassium; Probability; Process; Property; Proteins; Protons; Publishing; Pump; Reaction; Research; Resolution; Sampling; Series; Side; Signal Transduction; Site; Sodium; Specificity; Stroke; Structure; Synapses; Synaptic Cleft; Testing; X-Ray Crystallography; antiport; base; biophysical techniques; bone; conformational conversion; design; member; molecular pump; nervous system disorder; neurotransmission; novel; potassium ion; prevent; reconstitution; scaffold; sodium ion; tool; uptake

Project Summary Glutamate transporters pump the neurotransmitter from the synaptic cleft into the cytoplasm of glial cells and neurons against concentration gradients reaching a million-fold by harnessing the energy stored in the form of the trans-membrane electrochemical gradients of ions. Specifically, they couple uptake of each molecule of glutamate to the symport of three sodium ions and a proton and to the antiport of a potassium ion. Their dysfunction is associated with a range of neurological disorders and the extensive brain damage following traumatic injury and stroke. The complete transport cycle of glutamate transporters involves binding of the substrate and the symported ions to the outward facing conformation of the transporter, isomerization of the transporter into the inward facing conformation, the release of the substrate and ions into the cytoplasm, binding of the counter-transported potassium ion and the return of the transporter into the outward facing state. The structural information on this family comes from the crystallographic studies on a bacterial homologue, GltPh, which couples aspartate uptake to the symport of three sodium ions, but not to the movements of other ions. During our previous funded period, we have primarily focused on the large-scale conformational transitions of GltPh that underlie the trans-membrane translocation of the substrate and coupled ions. We now seek to investigate at the structural and mechanistic level the events associated with the substrate and ions binding and release on the two sides of the membrane that are at the heart of the functional specificity of the transporters. We further propose to employ mutagenesis to reconstitute in GltPh coupling to protons and potassium ions observed in the mammalian transporters. Finally, we propose to effectuate in GltPh a switch of specificity from employing sodium gradients to using protons to drive transport by mimicking the amino acid composition of the ion-binding sites of the proton-coupled members of the family. By combining these studies with the structural studies on the bone fide proton coupled bacterial glutamate transporters, we aim to understand how the conserved protein architecture and the structural mechanisms are adapted to allow functional diversification. Our long-term goal is to achieve a complete mechanistic description of the catalytic cycle of these transporters and to gain rational control over their functional properties.

项目摘要 谷氨酸转运蛋白将神经递质从突触left裂到神经胶质细胞的细胞质和 针对浓度梯度的神经元通过利用以 离子的跨膜电化学梯度。具体而言，它们融合了每个分子的吸收 谷氨酸到三个钠离子和一个质子和钾离子的替代物的同步。他们的 功能障碍与一系列神经系统疾病以及大脑损伤有关 创伤性伤害和中风。谷氨酸转运蛋白的完整运输周期涉及结合 底物和与转运蛋白的外部构象相称的离子，异构化的异构化 转运到向内构象中，底物和离子释放到细胞质中， 反传输钾离子的结合以及转运蛋白返回到外部状态。 有关该家族的结构信息来自有关细菌同源物的晶体学研究， GLTPH，将天冬氨酸的吸收与三个钠离子的交配相结合，但不适合其他动作 离子。在以前的资助时期，我们主要集中于大规模构象 GLTPH的过渡是基于底物和耦合离子的跨膜易位的基础。我们现在 寻求在结构和机械级别研究与底物和离子相关的事件 在膜的两侧结合并释放，这是该功能特异性的核心 运输商。我们进一步提议采用诱变来重新建立GLTPH耦合到质子和 在哺乳动物转运蛋白中观察到的钾离子。最后，我们建议在gltph a开关中实施 从采用钠梯度到使用质子来驱动传输的特异性通过模仿氨基酸 家族质子耦合成员的离子结合位点的组成。通过结合这些研究 通过对骨骼质子耦合细菌谷氨酸转运蛋白的结构研究，我们旨在 了解如何对保守的蛋白质结构和结构机制进行调整以允许 功能多样化。我们的长期目标是实现催化的完整机械描述 这些转运蛋白的循环并获得对其功能特性的合理控制。