Conformational Changes in Glutamate transporters

这是该项目的第六个年头，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。我们以前发现，细菌谷氨酸转运体显示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无关。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。我们对构象动力学的研究今年继续进行。我们最近报道，细胞外的GltPH环必须是完整的，才能有效地运输。今年，我们详细探讨了这种效应的机制，发现当34环被切断时，蛋白质保持底物亲和力，但最大转运显着减少。我们证明了这一效应与底物转位步骤的激活能有关，暗示着转位区域的活塞式运动中的环。我们的EPR研究也取得了进展，揭示了GltPh的apo形式在膜中是内向的，并随着蛋白质与底物结合而向外转移。

This was the sixth 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. We previously 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. Our studies on conformational dynamics continued this year. We recently reported that a extracellular loop of gltPH must be intact for effective transport. This year we probed the mechanism of this effect in detail and found that when the 34 loop is cut the proteins maintains substrate affinities but maximal transport is significantly reduced. We demonstrated that this effect relates to the activation energy of the substrate translocation step, implicating the loop in the piston like movement of the translocation domain. Our EPR studies have also progressed and reveal that the apo form of GltPh is inward-facing in the membrane and shifts to a more outward form as the protein binds substrate.