Molecular Studies of Human CNS Glutamate Transporters

描述(申请人提供)：中枢神经系统中的兴奋性氨基酸转运体(EAAT)将细胞外谷氨酸浓度维持在兴奋性毒性水平以下，并有助于清除神经传递过程中释放的谷氨酸。在之前的赠款期间，我们的实验室鉴定并鉴定了五种不同的人类谷氨酸载体亚型，EAATs1-5。这些载体对底物的传输与两到三个钠离子、一个质子的共传输和一个钾离子的反向传输在热力学上是耦合的；因此，这个过程是电生的。然而，当施加衬底时，移动的电荷量比从耦合离子和衬底的通量预测的要大：我们还证明了在衬底施加过程中产生的电流来自非耦合的阴离子通量。利用一个高功能的无半胱氨酸版本的EAAT1，我们将继续鉴定使用半胱氨酸取代和巯基修饰试剂进行底物运输和离子渗透所需的结构和拓扑特征。研究现在将开始强调使用两种电极电压钳技术对非洲爪哇卵母细胞中与EAAT1突变相关的电流进行电生理学分析。此外，我们将使用哺乳动物EAAT的两个细菌同源物作为模型来确定谷氨酸转运体的结构，以期获得更高分辨率的结构信息。使用高分辨率冷冻电子显微镜技术，我们将成像脂质双层中的细菌载体，以确定大体结构特征，包括可能的寡聚体状态。荧光和电子顺磁共振(EPR)光谱实验将用于改进结构模型，并分析纯化细菌载体中与易位相关的构象变化。临床和实验研究强调了了解不同兴奋性氨基酸转运体的结构、功能和调控的重要性，这些研究表明，在ALS、亨廷顿病、缺血诱导的神经毒性和阿尔茨海默病等退行性疾病中，转运体异常堆积。

DESCRIPTION (provided by applicant): Excitatory amino acid transporters (EAATs) in the CNS maintain extracellular glutamate concentrations below excitotoxic levels and contribute to the clearance of glutamate released during neurotransmission. Over the previous grant period our laboratory identified and characterized five distinct human glutamate carrier subtypes, EAATs1-5. Transport of substrates by these carriers is thermodynamically coupled to the co-transport of two to three sodium ions, one proton, and the countertransport of a potassium ion; thus, this process is electrogenic. However, the amount of charge moved when substrates are applied is greater than would be predicted from the flux of coupled ions and substrate: we have also shown that this current elicited during substrate application arises from an uncoupled anion flux. Taking advantage of a highly functional cysteine-less version of EAAT1, we will continue to identify the structural and topological features required for substrate transport and ion permeation using cysteine substitutions together with sulfhydryl-modifying reagents. Studies will now begin to emphasize the electrophysiological analyses of currents associated with EAAT1 mutants using two electrode voltage clamp techniques in Xenopus oocytes. In addition, we will use two bacterial homologs of the mammalian EAATs as models for determining the structure of glutamate transporters with a view towards obtaining higher resolution structural information. Using high-resolution cryo-electron microscopic techniques we will image the bacterial carriers in lipid bilayers to determine gross structural features, including possible oligomeric state. Fluorescence and electron paramagnetic resonance (EPR) spectroscopy experiments will be used to refine structural models and analyze translocation-related conformational changes in purified bacterial carriers. The importance of understanding the structure, function, and regulation of different excitatory amino acid transporters is underscored by clinical and experimental studies, which have implicated abnormal transmitter reaccummulation in degenerative disorders such as ALS, Huntington's disease, ischemia-induced neurotoxicity, and Alzheimer's dementia.