Molecular Studies of Human CNS Glutamate Transporters

描述（由申请方提供）：CNS中的兴奋性氨基酸转运蛋白（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期间释放的谷氨酸。在之前的资助期间，我们的实验室鉴定并表征了五种不同的人类谷氨酸载体亚型，EAATs 1 -5。这些载体对底物的转运与两到三个钠离子、一个质子的共转运和一个钾离子的反转运相耦合;因此，这个过程是产电的。然而，当施加基板移动的电荷量是大于将从耦合离子和基板的通量预测：我们还表明，在基板应用引起的电流产生从一个未耦合的阴离子通量。利用EAAT 1的高功能半胱氨酸版本，我们将继续确定使用半胱氨酸取代与巯基修饰试剂一起进行底物转运和离子渗透所需的结构和拓扑特征。研究将开始，强调与EAAT 1突变体在非洲爪蟾卵母细胞中使用双电极电压钳技术相关的电流的电生理分析。此外，我们将使用两个细菌同源的哺乳动物EAAT模型确定谷氨酸转运蛋白的结构，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，我们将在脂质双层中对细菌载体进行成像，以确定总体结构特征，包括可能的寡聚状态。荧光和电子顺磁共振（EPR）光谱实验将用于完善结构模型，并分析纯化的细菌载体中与易位相关的构象变化。了解不同兴奋性氨基酸转运蛋白的结构、功能和调节的重要性已被临床和实验研究所强调，这些研究涉及退行性疾病如ALS、亨廷顿病、缺血诱导的神经毒性和阿尔茨海默氏痴呆中的异常递质再活化。

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.