Conformationally Sensitive Proximity Between the TM3–4 Loop and Hairpin Loop 2 of the Glutamate Transporter EAAT2 Revealed by Paired-Cysteine Mutagenesis
Conformationally Sensitive Proximity Between the TM3–4 Loop and Hairpin Loop 2 of the Glutamate Transporter EAAT2 Revealed by Paired-Cysteine Mutagenesis
复制标题
成对半胱氨酸诱变揭示谷氨酸转运蛋白 EAAT2 的 TM3-4 环和发夹环 2 之间构象敏感的邻近性
DOI：
10.1021/acschemneuro.0c00645
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发表时间：
2021
影响因子：
5
通讯作者：
Shaogang Qu
中科院分区：
文献类型：
--
作者：
Ji Wang;Shaogang Qu
兴奋性氨基酸转运蛋白（EAAT）通过将谷氨酸从突触间隙转运到相邻的神经胶质细胞和神经元来维持细胞外神经递质浓度低于神经毒性水平。尽管来自堀越火球菌的古菌 EAAT 同源物 GltPh 和人类谷氨酸转运蛋白 EAAT1 晶体的晶体结构已得到解析，但跨膜 3-4 (TM3-4) 环的转运机制及其在转运过程中的结构重排仍知之甚少。为了探索TM3-4环在运输循环中的空间位置和功能，我们在不含半胱氨酸的EAAT2（CL-EAAT2）中在TM3-4环和发夹环2（HP2）之间设计了一对半胱氨酸残基。我们观察到，氧化交联剂 Cu(II)(1,10-菲咯啉)3(CuPh) 对双取代 A167C/G437C 突变体的转运具有显着的抑制作用，而 dl-二硫苏糖醇 (DTT) 逆转了交联 A167C/G437C 对转运活性的影响，如通过 d-[3H]-天冬氨酸摄取测定的。此外，我们发现 CuPh 在该突变体中的作用是由于转运蛋白分子中二硫键的形成。此外，dl-苏型-β-苄氧基天冬氨酸（TBOA）减弱了A167C/G437C突变体中CuPh介导的抑制作用，而l-谷氨酸或KCl增强了CuPh介导的抑制作用，表明A167C和G437C半胱氨酸在面向外的构型中相距较远，而在面向内的构型中相距较近。总而言之，我们的研究结果提供了证据表明 TM3-4 环和 HP2 在运输周期中改变了空间邻近性。
Excitatory amino acid transporters (EAATs) serve to maintain extracellular neurotransmitter concentrations below neurotoxic levels by transporting glutamate from the synaptic cleft into apposed glia and neurons. Although the crystal structures of the archaeal EAAT homologue fromPyrococcus horikoshii, GltPh, and the human glutamate transporter, EAAT1cryst, have been resolved, the transport mechanism of the transmembrane 3–4 (TM3–4) loop and its structural rearrangement during transport have remained poorly understood. In order to explore the spatial position and function of the TM3–4 loop in the transport cycle, we engineered a pair of cysteine residues between the TM3–4 loop and hairpin loop 2 (HP2) in cysteine-less EAAT2 (CL-EAAT2). We observed that the oxidative cross-linking reagent Cu(II)(1,10-phenanthroline)3(CuPh) had a significant inhibitory effect on transport in the disubstituted A167C/G437C mutant, whereasdl-dithiothreitol (DTT) reversed the effect of cross-linking A167C/G437C on transport activity, as assayed byd-[3H]-aspartate uptake. Furthermore, we found that the effect of CuPh in this mutant was due to the formation of disulfide bonds in the transporter molecule. Moreover,dl-threo-β-benzyloxyaspartic acid (TBOA) attenuated, whilel-glutamate or KCl enhanced, the CuPh-mediated inhibitory effect in the A167C/G437C mutant, suggesting that the A167C and G437C cysteines were farther apart in the outward-facing configuration and closer in the inward-facing configuration. Taken together, our findings provide evidence that the TM3–4 loop and HP2 change spatial proximity during the transport cycle.