Membrane Protein Mechanisms Unit, Institut Pasteur, Paris, France.
Membrane Protein Mechanisms Group, European Institute of Chemistry and Biology, University of Bordeaux, Pessac, France.
EMBO J. 2022 Jan 4;41(1):e108341. doi: 10.15252/embj.2021108341. Epub 2021 Nov 8.
Excitatory amino acid transporters (EAATs) maintain glutamate gradients in the brain essential for neurotransmission and to prevent neuronal death. They use ionic gradients as energy source and co-transport transmitter into the cytoplasm with Na and H , while counter-transporting K to re-initiate the transport cycle. However, the molecular mechanisms underlying ion-coupled transport remain incompletely understood. Here, we present 3D X-ray crystallographic and cryo-EM structures, as well as thermodynamic analysis of human EAAT1 in different ion bound conformations, including elusive counter-transport ion bound states. Binding energies of Na and H , and unexpectedly Ca , are coupled to neurotransmitter binding. Ca competes for a conserved Na site, suggesting a regulatory role for Ca in glutamate transport at the synapse, while H binds to a conserved glutamate residue stabilizing substrate occlusion. The counter-transported ion binding site overlaps with that of glutamate, revealing the K -based mechanism to exclude the transmitter during the transport cycle and to prevent its neurotoxic release on the extracellular side.
兴奋性氨基酸转运体（EAATs）在大脑中维持谷氨酸梯度对于神经传递和防止神经元死亡至关重要。它们利用离子梯度作为能量来源，与 Na 和 H 一起将递质共转运到细胞质中，同时将 K 反向转运以重新启动运输循环。然而，离子偶联运输的分子机制仍不完全清楚。在这里，我们展示了人类 EAAT1 在不同离子结合构象下的 3D X 射线晶体学和 cryo-EM 结构，以及热力学分析，包括难以捉摸的反向转运离子结合态。Na 和 H 的结合能，以及出人意料的 Ca ，与神经递质结合相偶联。Ca 与一个保守的 Na 位点竞争，表明 Ca 在突触处的谷氨酸转运中起调节作用，而 H 结合到一个保守的谷氨酸残基上，稳定底物的封闭。反向转运离子结合位点与谷氨酸的结合位点重叠，揭示了基于 K 的机制，在运输循环中排除递质，并防止其在细胞外侧的神经毒性释放。
