Conformational Ensemble of Glutamate Transporters: Structure and Ionic Modulatio

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：
7736453
负责人：
Olga Boudker
金额：
$ 36.97万
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 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7736453
关键词：
Amino Acid Transporter Amino Acids Anions Binding Binding Sites Biochemical Brain Cell membrane Cessation of life Coupled Coupling Crystallization Cysteine Cytoplasm Data Development Disease Dissociation Engineering Epilepsy Excitatory Amino Acids Family Figs - dietary Ganglia Glutamate Transporter Glutamates Goals Homologous Gene Ions Learning Left Location Membrane Membrane Proteins Memory Mercury Mind Molecular Molecular Structure Morphology Motion Mutagenesis Myxoid cyst Nerve Degeneration Neuraxis Neurons Neurotransmitters Neutral Amino Acids Oxidation-Reduction Positioning Attribute Potassium Process Proteins Protons Publishing Pyrococcus horikoshii Research Resolution Retina Schizophrenia Sequence Alignment Site Site-Directed Mutagenesis Sodium Solutions Solvents Stroke Structure Synapses Synaptic Cleft Transportation Traumatic Brain Injury analog base cell type conformational conversion crosslink extracellular inhibitor/antagonist link protein molecular pump mutant neuronal excitability neurotransmission post stroke potassium ion public health relevance research study sodium ion solute stoichiometry uptake

项目摘要

DESCRIPTION (provided by applicant): The excitatory amino acid transporters (EAATs) clear glutamate from the synaptic cleft following rounds of neurotransmission and are responsible for the uptake of acidic and neutral amino acids by all cell types. Driven by pre-existing electrochemical gradients, EAATs couple substrate uptake to the co-transport of sodium ions and protons and to the counter-transport of potassium. Previous crystallographic studies on a bacterial homologue from Pyrococcus horikoshii, GltPh have revealed the transporter in the outward facing states with the substrate-binding site accessible to the extracellular solution and either occluded when bound to a substrate or exposed when bound to a blocker. These structures have explained how the substrate and sodium ions reach their binding sites from the extracellular solution but left unanswered how these solutes are translocated across the membrane and released into the cytoplasm. It is believed that a conformational transition of the transporter into an inward facing state, in which the substrate-binding site is accessible to the intracellular solution, is a prerequisite for the substrate dissociation into the cytoplasm. In the current application, we propose to use double cysteine mutagenesis and cross-linking to delineate the structural re- arrangements that occur upon transition of the transporter into the inward facing state and to probe the dynamics of the process. We further propose to determine the crystal structures of the transporter molecules covalently constrained in the inward facing state and possibly in other intermediate transport states by cysteine cross-linking. Finally, we will employ the site directed mutagenesis in conjuncture with biochemical and structural analyses to probe the location of the ion binding and permeation sites in GltPh and, by analogy, in EAATs PUBLIC HEALTH RELEVANCE: Glutamate transporters are membrane proteins responsible for the clearance of the neurotransmitter glutamate from the synapses following rounds of neurotransmission. Their malfunction is associated with numerous diseases and pathological states including neurodegeneration, epilepsy, schizophrenia, traumatic brain injury and stroke. We focus our study on the mechanism and atomic structure of these molecular pumps essential for the proper development and function of the central nervous system.

描述（由申请人提供）：兴奋性氨基酸转运蛋白（EAATS）在神经传递后从突触裂缝中清除谷氨酸，并负责通过所有细胞类型的酸性和中性氨基酸的摄取。在先前存在的电化学梯度的驱动下，Eaats夫妇夫妇对钠离子和质子的共转移以及钾的反转移。先前对来自Horikoshii的细菌同源物的晶体学研究，GLTPH揭示了在外部面向状态中的转运蛋白，其底物结合位点可接收到细胞外溶液的基质结合位点，并在与底物绑定时绑定时被咬合时，或在与阻止器结合时被绑定。这些结构已经解释了底物和钠离子如何从细胞外溶液中到达其结合位点，但没有得到解答，如何将这些溶质转移到整个膜上并释放到细胞质中。据信，转运蛋白向内向状态的构象转变，其中细胞内溶液可以访问底物结合位点，这是底物解离向细胞质的先决条件。在当前的应用中，我们建议使用双半胱氨酸诱变和交联，以描绘转运蛋白向内朝向状态过渡后发生的结构排列并探测该过程的动力学。我们进一步建议通过半胱氨酸交联，可以确定在面向状态以及可能在其他中间运输状态下的转运蛋白分子的晶体结构。 Finally, we will employ the site directed mutagenesis in conjuncture with biochemical and structural analyses to probe the location of the ion binding and permeation sites in GltPh and, by analogy, in EAATs PUBLIC HEALTH RELEVANCE: Glutamate transporters are membrane proteins responsible for the clearance of the neurotransmitter glutamate from the synapses following rounds of neurotransmission.它们的故障与许多疾病和病理状态有关，包括神经变性，癫痫，精神分裂症，脑损伤和中风。我们将研究重点放在这些分子泵的机理和原子结构上，对于中枢神经系统的正常发育和功能所必需。