Institute of Biological Information Processing (IBI-1), Molekular- und Zellphysiologie, and JARA-HPC, Forschungszentrum Jülich, Jülich, Germany.
Institut de Biologie Structurale (IBS), Université Grenoble Alpes-CEA-CNRS, 38000 Grenoble, France.
Sci Adv. 2020 Nov 18;6(47). doi: 10.1126/sciadv.aba9854. Print 2020 Nov.
Excitatory amino acid transporters (EAATs) harness [Na], [K], and [H] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na ions, [Na] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na:substrate coupling in the EAAT homolog Glt A lipidic cubic phase x-ray crystal structure of wild-type, Na-only bound Glt at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na-stabilized gate opening and electrostatic coupling of aspartate to Na binding provides a constant Na:substrate transport stoichiometry over a broad range of neurotransmitter concentrations.
兴奋性氨基酸转运体（EAATs）利用 [Na+]、[K+] 和 [H+] 梯度，从突触间隙快速有效地去除谷氨酸。由于每个谷氨酸与三个 Na+离子共转运，因此 [Na+] 梯度是谷氨酸摄取的主要驱动力。我们结合全原子分子动力学模拟、荧光光谱和 X 射线晶体学，研究了 EAAT 同系物 GltA 中的 Na:底物偶联。野生型脂质立方相 X 射线晶体结构中 Na 仅结合的 Glt 的分辨率为 2.5-Å，揭示了完全开放的、向外的状态，为随后的底物结合做好了准备。模拟和动力学实验表明，只有两个 Na 离子结合到 Na1 和 Na3 位点，才能通过构象选择样机制确保完全打开 HP2 门，并通过静电吸引实现高亲和力的底物结合。Na 稳定的门打开和天冬氨酸与 Na 结合的静电偶联的结合，在广泛的神经递质浓度范围内提供了恒定的 Na:底物转运计量比。
