Human members of the solute carrier 1 (SLC1) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of their functions is associated to neurodegenerative disorders and cancer. Here we present the first crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound. The structures show novel architectural features of the human transporters, including intra- and extracellular domains with potential roles in transport function, as well as regulation by lipids and post-translational modifications. The coordination of the inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry, reveal an allosteric mechanism of inhibition, whereby the transporter is locked in the outward-facing states of the transport cycle. Our results provide unprecedented insights into the molecular mechanisms of function and pharmacology of human SLC1 transporters.

Human excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate in the brain and are essential to maintain excitatory neurotransmission. Our understanding of the EAATs’ molecular mechanisms has been hampered by the lack of stability of purified protein samples for biophysical analyses. Here, we present approaches based on consensus mutagenesis to obtain thermostable EAAT1 variants that share up to ~95% amino acid identity with the wild type transporters, and remain natively folded and functional. Structural analyses of EAAT1 and the consensus designs using hydrogen-deuterium exchange linked to mass spectrometry show that small and highly cooperative unfolding events at the inter-subunit interface rate-limit their thermal denaturation, while the transport domain unfolds at a later stage in the unfolding pathway. Our findings provide structural insights into the kinetic stability of human glutamate transporters, and introduce general approaches to extend the lifetime of human membrane proteins for biophysical analyses.

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 2+ , are coupled to neurotransmitter binding. Ca 2+ competes for a conserved Na + site, suggesting a regulatory role for Ca 2+ 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.

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