Molecular Studies of Human CNS Glutamate Transporters

描述（由申请人提供）：中枢神经系统中的兴奋性氨基酸转运蛋白（EAATs）维持细胞外谷氨酸浓度低于兴奋毒性水平，并有助于清除神经传递过程中释放的谷氨酸。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，EAATs1-5。这些载体对底物的运输在热力学上与两到三个钠离子、一个质子的共运输和一个钾离子的反运输耦合；因此，这个过程是电致的。然而，当施加衬底时移动的电荷量大于从耦合离子和衬底的通量预测的量：我们还表明，在衬底施加期间引起的电流是由不耦合的阴离子通量引起的。利用高功能的无半胱氨酸版本EAAT1，我们将继续使用半胱氨酸取代和巯基修饰试剂来确定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蟾卵母细胞中与EAAT1突变体相关的电流进行电生理分析。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。使用高分辨率低温电子显微镜技术，我们将对脂质双层中的细菌载体进行成像，以确定总体结构特征，包括可能的低聚状态。荧光和电子顺磁共振（EPR）光谱实验将用于完善结构模型和分析纯化细菌载体的易位相关构象变化。临床和实验研究强调了理解不同兴奋性氨基酸转运蛋白的结构、功能和调控的重要性，这些研究涉及退行性疾病（如ALS、亨廷顿氏病、缺血诱导的神经毒性和阿尔茨海默氏痴呆症）中的异常递质再积累。

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.