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

基本信息

批准号：
9093846
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
项目类别：
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/9093846
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 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 Traumatic injury 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

项目摘要

DESCRIPTION (provided by applicant): 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

描述（由申请人提供）：谷氨酸转运蛋白将神经递质从突触裂缝中泵送到神经胶质细胞和神经元的细胞质中，以抗浓度梯度，通过以跨膜化神经桥的形式利用跨跨膜层的能量，以达到百万倍的梯度。具体而言，它们将每个分子的谷氨酸分子摄入到三个钠离子和质子的同学以及钾离子的替代。它们的功能障碍与一系列神经系统疾病以及创伤性损伤和中风后的广泛脑损伤有关。谷氨酸转运蛋白的完整运输周期涉及底物和交通离子的同义离子的结合，转运蛋白的外部面向构象，转运蛋白的异构化，将底物和离子的释放和离子释放到细胞质中，并将其触发的孔子与转运剂的返回结合到返回型号。有关该家族的结构信息来自对细菌同源物GLTPH的晶体学研究，该研究将天冬氨酸的吸收与三种钠离子的交配相结合，但不是其他离子的运动。在以前的资助时期，我们主要集中在GLTPH的大规模构象转变上，该转变是底物和耦合离子的跨膜易位的基础。现在，我们试图在结构和机械级别研究与膜和离子结合并释放在膜的两侧的事件，这些事件是转运蛋白功能特异性的核心。我们进一步建议采用诱变来重新构建GLTPH偶联与哺乳动物转运蛋白中观察到的质子和钾离子。最后，我们建议通过模仿该家族质子耦合成员的离子结合位点的氨基酸组成来实现从使用钠梯度到使用质子来驱动转运的特异性转换。通过将这些研究与骨质质子耦合细菌谷氨酸转运蛋白的结构研究相结合，我们旨在了解如何对保守蛋白结构和结构机制进行适应以允许功能多样化。我们的长期目标是实现这些转运蛋白的催化循环的完整机械描述，并获得对其功能特性的合理控制