The accessibility of a novel reentrant loop of the glutamate transporter GLT-1 is restricted by its substrate
The accessibility of a novel reentrant loop of the glutamate transporter GLT-1 is restricted by its substrate
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DOI：
10.1074/jbc.275.13.9684
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发表时间：
2000-03-31
影响因子：
4.8
通讯作者：
Kanner, BI
中科院分区：
文献类型：
--
作者：
Grunewald, M;Kanner, BI
兴奋性神经递质谷氨酸通过几种相关的钠和钾偶联转运蛋白从突触间隙中清除。因此，它们限制了这种递质的神经毒性。基于单个半胱氨酸对大巯基试剂3-N-马来酰亚胺基（丙酰基）生物胞素的可接近性，我们提出了星形胶质细胞谷氨酸转运蛋白GLT-1的拓扑模型（格鲁内瓦尔德，M，，Bendahan，k和Kanner，B. I.（1998）Neuron 21，623-632）。由于几个意想不到的观察结果，我们研究了被工程化到被认为是细胞内的环中的19个半胱氨酸残基的拓扑布置。我们已经探讨了这些半胱氨酸的小和大巯基试剂的可及性。不渗透的亲水性巯基试剂[（2-三甲基铵）乙基]甲硫基磺酸盐仅在这些位置中的两个位置抑制转运活性，在G365 C处较弱，在A364 C处较强。谷氨酸盐及其不可转运的类似物二氢红藻氨酸盐显著保护A364 C转运蛋白免受该不渗透试剂的影响。使用生物素化的马来酰亚胺，我们发现，在用它测试的14个突变体中，只有A364 C可以从胞外侧接近它。这与我们以前的观察结果一起表明，环-包括氨基酸残基354，359，373和379-主要是细胞内的，但它的一个短区域形成了一个可重入的孔环样结构，其可及性是依赖于矿石的转运蛋白的构象。
The excitatory neurotransmitter glutamate is removed from the synaptic cleft by several related sodium-and potassium-coupled transporters. They thereby restrict the neurotoxicity of this transmitter. Based on the accessibility of single cysteines to the large sulfhydryl reagent 3-N-maleimidyl(propionyl)biocytin, we have proposed a topological model for the astroglial glutamate transporter GLT-1 (Grunewald, M,, Bendahan, k and Kanner, B. I. (1998) Neuron 21, 623-632), Because of several unexpected observations, we have investigated the topological disposition of 19 cysteine residues engineered into a loop proposed to be intracellular. We have probed the accessibility of these cysteines to small and large sulfhydryl reagents. The impermeant hydrophilic sulfhydryl reagent [(2-trimethylammonium)ethyl] methanethiosulfonate inhibits transport activity only at two of these positions, weakly at G365C and potently at A364C, Glutamate and its nontransportable analogue dihydrokainate markedly protect A364C transporters against this impermeant reagent. Using a biotinylated maleimide, we found that, among the 14 mutants tested with it, only A364C is accessible to it from the extracellular side. This, together with our previous observations, indicates that the loop-including amino acid residues 354, 359, 373, and 379-is largely intracellular, but a short region of it forms a reentrant pore-loop-like structure, the accessibility of which is dependent ore the conformation of the transporter.