Conformational Changes in Glutamate transporters

这是该项目的第二年，该项目将使用生化和生物物理方法来检查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在多种疾病中发挥作用，包括肌萎缩侧索硬化症、阿尔茨海默病、S病和兴奋毒性。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。在过去的一年里，我们发现细菌的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。我们还建立了其他离子，这些离子是耦合的(和非偶联的)在细菌蛋白质中运输。在接下来的一年里，我们将开始使用半胱氨酸扫描突变和定点荧光标记来确定分子的哪些部分在运输过程中移动，并详细描述这些移动。

This was the second year for this project, which will use 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, Alzheimer?s 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. In the past 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. We have also established the other ions which are coupled (and those not coupled) to transport in the bacterial proteins. In the upcoming year, we will start to use cysteine-scanning mutagenesis and site-directed fluorescence labeling to determine which parts of the molecule move during the transport process and characterize these movements in detail.