Central Laboratory and Department of Neurology, Shunde Hospital, Southern Medical University (the First People's Hospital of Shunde Foshan), Foshan, Guangdong, China.
Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou, Guangdong, China.
J Biomol Struct Dyn. 2020 Aug;38(12):3599-3609. doi: 10.1080/07391102.2019.1664935. Epub 2019 Sep 15.
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 (Glt). 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 EAAT1 (a human thermostable EAAT1) was resolved in 2017, the structure-function relationship of the 4b-4c loop has not been elucidated in EAAT1. 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 (K 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.
在哺乳动物中，兴奋性氨基酸转运体（EAATs）的 4b-4c 环跨越超过 50 个氨基酸残基，而谷氨酸转运体同源物（Glt）中不存在这些残基。该插入部分是后生动物所特有的，对 EAATs 的定位是不可或缺的。兴奋性氨基酸转运体（EAAT）1 是两种神经胶质谷氨酸转运体之一，负责从突触间隙有效清除谷氨酸，以防止神经毒性和细胞死亡。尽管 2017 年已经解析了 EAAT1（一种人类耐热型 EAAT1）的晶体结构，但 EAAT1 中 4b-4c 环的结构-功能关系尚未阐明。为了研究 4b-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 T192A、Y194A、N242A 和 G245A 突变体的转运活性显著降低。T192A 和 Y194A 突变体的表面表达甚至降低了 80%以上，并且大部分被滞留在细胞质中。然而，当 T192 和 Y194 被保守残基取代时，T192S 和 Y194F 的转运活性和表面表达得到了很大的恢复，其动力学参数（K 值）与野生型 EAAT1 相当。相比之下，用保守残基取代 N242 和 G245 不能挽救摄取功能，这表明 N242 和 G245 在谷氨酸摄取过程中可能起着不可替代的作用。这些结果表明，EAAT1 的 4b-4c 环不仅可能影响谷氨酸摄取活性，而且可能通过 T192 和 Y194 影响 EAAT1 的表面定位。由 Ramaswamy H. Sarma 交流。
