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研究与谷氨酸转运体运输相关的构象变化。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方面发挥着重要作用。这些转运蛋白被认为与多种疾病有关，包括肌萎缩侧索硬化症、阿尔茨海默病和兴奋性毒性。了解这些转运蛋白发挥作用的基本机制是至关重要的，因为这样的知识可能会导致开发出针对这些蛋白质的有效治疗剂。我们试图分析在详细了解其机制的过程中起作用的转运体的动态运动。我们的方法是分析从细菌获得的谷氨酸转运体模型中的运输细节。它们可以大量表达和纯化，并适用于它们的哺乳动物近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体表现出一种氯离子转运活性，这种活性在化学计量上与谷氨酸摄取是分离的。这种氯离子转运活性与哺乳动物转运蛋白中的一种重要活性相似，表明细菌同系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，我们完成了对氯离子电导的表征，证明了它使用与真核细胞谷氨酸转运蛋白相似的蛋白质部分进行操作。我们还发现，与EAATs相反，除了Na、Cl-和底物外，没有其他离子参与输运循环。我们对这些蛋白质的构象变化的研究始于今年；利用有限的蛋白质分解，我们发现了底物诱导的显着构象变化的证据，并使用半胱氨酸扫描突变和定点荧光标记进行了初步实验，以定位蛋白质的运动。

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.