Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which a conserved glutamate residue mediates proton symport.

人兴奋性氨基酸转运蛋白 3 (hEAAT3) 介导神经元、肠道和肾脏中的谷氨酸摄取。在这里，我们报告了 hEAAT3 在几种功能状态下的冷冻电镜结构，其中转运蛋白是空的，仅与耦合的钠离子结合，或完全加载三个钠离子、一个质子和底物天冬氨酸。这些结构表明，hEAAT3 通过一种电梯机制运作，涉及三个功能独立的亚基。当底物结合位点靠近细胞质时，它对底物的亲和力非常低，这可能有助于其释放并允许快速运输周转。钠离子和底物的耦合摄取机制在进化上遥远的家族中是保守的，并通过与 EAAT 中的质子耦合而得到增强。