The 4b-4c loop of excitatory amino acid transporter 1 containing four critical residues essential for substrate transport
The 4b-4c loop of excitatory amino acid transporter 1 containing four critical residues essential for substrate transport
复制标题
兴奋性氨基酸转运蛋白 1 的 4b-4c 环含有底物转运所必需的四个关键残基
DOI：
10.1080/07391102.2019.1664935
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发表时间：
2019-09-14
影响因子：
4.4
通讯作者：
Qu, Shaogang
中科院分区：
文献类型：
--
作者：
He, Suifen;Zhang, Wenlong;Qu, Shaogang
摘要在哺乳动物中，兴奋性氨基酸转运蛋白（EAAT）的4 b-4c环跨越了50多个氨基酸残基，这些氨基酸残基是Horikoshii火球菌谷氨酸转运蛋白同源物（GltPh）中不存在的。这部分插入是后生动物所特有的，也是EAAT定位所不可或缺的。兴奋性氨基酸转运体（EAAT）1是两种胶质细胞谷氨酸转运体之一，其负责有效地清除突触间隙中的谷氨酸以防止神经毒性和细胞死亡。尽管EAAT 1cryst（一种人类耐热EAAT 1）的晶体结构在2017年得到了解析，但EAAT 1cryst中4 b-4c环的结构-功能关系尚未阐明。为了研究4 b-4c环的作用，我们在突变体中进行了丙氨酸扫描诱变，并观察到T192 A，Y194 A，N242 A和G245 A突变体的转运活性显著降低。T192 A和Y194 A突变体的表面表达量甚至下降了80%以上，并且大部分滞留在细胞质中。然而，当T192和Y194被保守残基取代时，T192 S和Y194 F的转运活性和表面表达在很大程度上恢复，并且它们的动力学参数（Km值）也与野生型EAAT 1相当。相反，N242和G245被保守残基取代不能拯救摄取功能，这表明N242和G245在谷氨酸摄取过程中可能发挥不可替代的作用。这些结果表明，EAAT 1的4 b-4c环不仅影响谷氨酸摄取活性，而且还影响T192和Y194对EAAT 1的表面定位。作者：Ramaswamy H. Sarma
Abstract In the mammalians, the 4b-4c loop of excitatory amino acid transporters (EAATs) spans more than 50 amino-acid residues that are absent in glutamate transporter homologue of Pyrococcus horikoshii (GltPh). This part of insertion is unique for metazoans and indispensable to the localization of EAATs. The excitatory amino acid transporter (EAAT) 1 is one of the two glial glutamate transporters, which are responsible for efficiently clearing glutamate from the synaptic cleft to prevent neurotoxicity and cell death. Although the crystal structure of EAAT1cryst (a human thermostable EAAT1) was resolved in 2017, the structure-function relationship of the 4b-4c loop has not been elucidated in EAAT1cryst. To investigate the role of the 4b-4c loop, we performed alanine-scanning mutagenesis in the mutants and observed dramatically decreased transport activities in T192A, Y194A, N242A, and G245A mutants. The surface expression of T192A and Y194A mutants even decreased by more than 80%, and most of them were detained in the cytoplasm. However, when T192 and Y194 were substituted with conservative residues, the transport activities and the surface expressions of T192S and Y194F were largely recovered, and their kinetic parameters (Km values) were comparable to the wild-type EAAT1 as well. In contrast, N242 and G245 substituted with conservative residues could not rescue the uptake function, suggesting that N242 and G245 may play irreplaceable roles in the glutamate uptake process. These results indicate that the 4b-4c loop of EAAT1 may not only affect the glutamate uptake activity, but also influence the surface localization of EAAT1 by T192 and Y194. Communicated by Ramaswamy H. Sarma