Conformational Changes in Glutamate transporters

谷氨酸转运蛋白的构象变化

• 批准号: 7594705
• 负责人: Joseph A Mindell
• 金额: $ 42.78万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594705
• 关键词: Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Aspartate; Bacteria; Bacterial Proteins; Biochemical; Biological Models; Characteristics; Chloride Ion; Chlorides; Chromosome Pairing; Cysteine; Development; Disease; Fluorescence; Fluorescent Probes; Glutamate Transporter; Glutamates; Goals; Homologous Gene; Ions; Knowledge; Label; Lead; Localized; Methods; Modeling; Monitor; Movement; Mutagenesis; Neuraxis; Neurons; Neurotransmitters; Play; Process; Property; Proteins; Proteolysis; Radioactive Tracers; Reporting; Role; Role playing therapy; Scanning; Shapes; Site-Directed Mutagenesis; Structural Models; Synapses; Therapeutic Agents; Work; reconstitution; research study; uptake

This was the third year for this project, which is using biochemical and biophysical methods to examine conformational changes related to transport in glutamate transporters. These proteins are of critical importance in the central nervous system, where they play important roles in clearing neurotransmitters from synapses and in shaping the electrical activity of post-synaptic neurons. These transporters have been implicated as playing roles in a variety of diseases, including ALS, Alzheimers disease, and exitotoxicity. It is critical to understand the fundamental mechanisms by which there transporters function because such knowledge could lead to the development of therapeutic agents active against these proteins. We seek to analyze the dynamic movements of the functioning transporter on the way to a detailed understanding of its mechanism. Our approach is to analyze the details of transport in model glutamate transporters obtained from bacteria. These can be expressed and purified in large quantities and are amenable to biophysical methods not available for their mammalian cousins. Last year we discovered that the bacterial glutamate transporters display a chloride transport activity which is stoichiometrically uncoupled from glutamate uptake. This chloride transport activity is similar to one which is important in the mammalian transporters and suggests that the bacterial homologs provide an excellent structural model in which to study the process of chloride transport in these proteins. This year we completed our characterization of this chloride conductance demonstrating that it operates using similar parts of the protein as the eukaryotic glutamate transporters. We also found that, in contrast to the EAATs, no other ions besides Na+, Cl-, and the substrate are involved in the transport cycle. Our studies on conformational changes in these proteins began this year; using limited proteolysis we found evidence of a significant substrate-induced conformational change and performed preliminary experiments using cysteine-scanning mutagenesis and site-directed fluorescence labeling to localize the protein movement.

这是该项目的第三年，该项目使用生物化学和生物物理方法来研究与谷氨酸转运蛋白转运相关的构象变化。这些蛋白质在中枢神经系统中至关重要，它们在清除突触中的神经递质和塑造突触后神经元的电活动中发挥重要作用。这些转运蛋白已被认为在多种疾病中发挥作用，包括ALS、阿尔茨海默病和兴奋毒性。了解转运蛋白发挥作用的基本机制是至关重要的，因为这些知识可能导致开发对这些蛋白质有活性的治疗剂。我们试图分析的方式，以详细了解其机制的动态运动的功能运输。我们的方法是分析从细菌中获得的模型谷氨酸转运蛋白的运输细节。它们可以大量表达和纯化，并且适用于它们的哺乳动物表亲所不具备的生物物理方法。去年，我们发现细菌谷氨酸转运蛋白显示出与谷氨酸摄取化学计量分离的氯离子转运活性。这种氯离子转运活性类似于哺乳动物转运蛋白中的重要活性，并表明细菌同系物提供了一个极好的结构模型，用于研究这些蛋白质中氯离子转运的过程。今年，我们完成了对这种氯离子电导的表征，证明它使用与真核谷氨酸转运蛋白相似的蛋白质部分进行操作。我们还发现，在相反的EAAT，没有其他离子除了Na+，Cl-，和基板参与运输周期。我们对这些蛋白质构象变化的研究始于今年;使用有限的蛋白质水解，我们发现了显着的底物诱导的构象变化的证据，并进行了初步实验，使用半胱氨酸扫描诱变和定点荧光标记定位的蛋白质运动。