Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.

谷氨酸是哺乳动物大脑中一种关键的兴奋性神经递质，但过量的谷氨酸会导致许多神经疾病。几乎所有的细胞外谷氨酸都被属于 SLC1A 家族的神经胶质转运蛋白兴奋性氨基酸转运蛋白 2 (EAAT2) 回收。然而，在某些癌症中，EAAT2 表达增强，并通过代谢紊乱引起对治疗的抗性。尽管 EAAT2 发挥着重要作用，但尚未获得有关 EAAT2 的详细结构信息。在这里，我们报告了人类 EAAT2 在无底物和选择性抑制剂 WAY213613 结合状态下的冷冻电镜结构，分别为 3.2 Å 和 2.8 Å。EAAT2 形成一个三聚体，每个原聚体由运输结构域和支架结构域组成。除了谷氨酸结合位点外，运输结构域还拥有一个在运输周期中可能被破坏的空腔。WAY213613 占据 EAAT2 的谷氨酸结合位点和空腔以干扰其交替访问，其中灵敏度由空腔的内部环境定义。我们提供了 EAAT2 的分子特征及其选择性抑制机制的表征，这可能有助于 EAAT2 的基于结构的药物设计。