STRUCTURE OF NA+ DEPENDENT GLUTAMATE TRANSPORTERS

高亲和力，钠依赖性谷氨酸转运蛋白负责 维持谷氨酸浓度低于神经毒性水平， 涉及与ALS相关的神经病理学变化，急性 局部缺血和中风。阐述了 运输机制将提供有关承运人如何贡献的见解， 疾病和神经退行性变，并可能建议可能的治疗方法 干预措施。拟议的研究旨在进一步解决 并开始阐明结构相关性 底物和抑制剂结合以及氯离子渗透。一是 FLAG表位的膜取向，插入到亲水环中， 将使用间接免疫荧光法测定蛋白质 在透性化和非透性化转染的COS细胞中的显微镜。 第二，单个半胱氨酸残基将被置于这些结构域中，并且其 使用膜渗透剂测定非洲爪蟾卵母细胞的膜取向 和不渗透的巯基反应性化合物。最后，被替代者 半胱氨酸可及性方法（SCAM）将用于检查是否 位于推定TMD 8中的残基与底物和抑制剂相互作用 或者参与氯离子的渗透。这些研究将 集中在一个高度保守的结构域， 底物和抑制剂结合。24个连续残基将被 在先前生成的功能性无半胱氨酸 运输和运输属性和电流与每个 将在卵母细胞中评估突变体。巯基反应MTS 将使用试剂来鉴定有助于 抑制剂和离子与载体的底物相互作用。

High affinity, sodium-dependent glutamate transporters are responsible for maintaining glutamate concentrations below neurotoxic levels and have been implicated in the neuropathological changes associated with ALS, acute ischemia and stroke. Elucidating the structural features underlying the mechanism of transport will provide insight on how the carriers contribute to disease and neurodegeneration and may suggest possible therapeutic interventions. The proposed studies aim to further resolve the transmembrane topology and to begin to elucidate the structural correlates of substrate and inhibitor binding and chloride ion permeation. First, the membrane orientation of a FLAG epitope, inserted into hydrophilic loops in the protein, will be determined using indirect immunofluorescence microscopy in permeabilized and nonpermeabilized transfected COS cells. Second, single cysteine residues will be placed in these domains and their membrane orientation determined in Xenopus oocytes using membrane-permeant and impermeant sulfhydryl-reactive compounds. Finally, the Substituted Cysteine Accessibility Method (SCAM) will be used to examine whether residues located in putative TMD 8 interact with substrates and inhibitors or are involved in the permeation of chloride ions. These studies will focus on a highly conserved domain which has already been implicated in substrate and inhibitor binding. Twenty-four consecutive residues will be replaced by cysteine in a previously-generated functional cysteineless transporter and the transport properties and currents associated with each of the mutants will be assessed in oocytes. Sulfhydryl-reactive MTS reagents will be used to identify residues that contribute to the interactions of substrates in inhibitors and ions with the carrier.