Conformational Changes in Glutamate transporters

这是该项目的第三年，该项目使用生化和生物物理方法来检查与谷氨酸转运体转运相关的构象变化。这些蛋白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形成突触后神经元的电活动中起着重要作用。这些转运蛋白被认为在多种疾病中发挥作用，包括ALS、阿尔茨海默病和体外毒性。了解这些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针对这些蛋白质的治疗药物。我们试图在详细了解其机制的过程中分析功能转运体的动态运动。我们的方法是分析从细菌中获得的模型谷氨酸转运体的运输细节。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无法使用的生物物理方法。去年我们发现细菌谷氨酸转运体显示出一种氯化物转运活性，这种活性在化学计量上与谷氨酸摄取不耦合。这种氯离子转运活性与哺乳动物转运蛋白中的重要转运活性相似，这表明细菌同源物为研究这些蛋白质中的氯离子转运过程提供了一个很好的结构模型。今年，我们完成了对这种氯离子电导的表征，证明它使用了与真核谷氨酸转运蛋白相似的蛋白质部分。我们还发现，与eaat相比，除了Na+， Cl-和底物外，没有其他离子参与转运循环。我们对这些蛋白质构象变化的研究是从今年开始的；通过有限的蛋白质水解，我们发现了底物诱导构象变化的证据，并使用半胱氨酸扫描诱变和定点定向荧光标记进行了初步实验，以定位蛋白质运动。

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.