Conformational Changes in Glutamate transporters

这是该项目的第一年，该项目将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ALS、阿尔茨海默病和兴奋性毒性。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。为此，我们已经成功地表达和纯化了原核谷氨酸转运蛋白，这是一个近亲的哺乳动物蛋白。使用细菌蛋白质使我们能够分析分子的某些部分，这些部分很难或不可能用目前的哺乳动物表达系统进行评估。我们已经证明表达的转运蛋白是功能性的。我们将使用半胱氨酸扫描诱变和定点荧光标记，以确定哪些部分的分子在运输过程中移动，并详细描述这些运动。

This was the first 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 excitotoxicity. 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. Toward this end, we have successfully expressed and purified a prokaryotic glutamate transporter, which is a close cousin of the mammalian protein. Using the bacterial protein allows us to analyze parts of the molecule which would be difficult or impossible to assess with current mammalian expression systems. We have demonstrated that the expressed transporters are functional. We will 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.