The ion‐coupling mechanism of human excitatory amino acid transporters

• Published in: The EMBO journal 2022-01, Vol.41 (1), p.e108341-n/a
• Main Authors: Canul‐Tec, Juan C, Kumar, Anand, Dhenin, Jonathan, Assal, Reda, Legrand, Pierre, Rey, Martial, Chamot‐Rooke, Julia, Reyes, Nicolas
• Format: Article
• Language: English
• Subjects: Amino acids; Archaea; Binding Sites; Biochemistry, Molecular Biology; Brain; Ca2+/H+-exchanging ATPase; Calcium - metabolism; Calcium ions; Calcium transport; Cations, Divalent - metabolism; Cerebral blood flow; Coupling (molecular); Cryoelectron Microscopy; cryo‐EM; Crystallography; Cytoplasm; EMBO20; EMBO27; EMBO40; Energy sources; Excitatory Amino Acid Transporter 1 - chemistry; Excitatory Amino Acid Transporter 1 - metabolism; Excitatory Amino Acid Transporter 1 - ultrastructure; Excitatory amino acid transporters; Humans; Hydrogen; Ion Transport; Ischemia; Life Sciences; Models, Molecular; Molecular modelling; Neurotoxicity; Neurotransmission; neurotransmitter transport; Neurotransmitters; Occlusion; permeation and transport; Potassium; Protein Conformation; Protons; Residues; Sodium - metabolism; solute carrier; Structural Biology; Substrates; Synapses; Synaptic cleft; X‐ray crystallography
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.2021108341

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. Synopsis The mechanism by which excitatory amino acid transporters (EAATs) in the brain use ionic transmembrane gradients to ensure glutamate uptake from the synaptic cleft remains incompletely understood. This work combines structural data and thermodynamic analyses to describe a complete transport cycle of human EAAT1 in different ion bound conformations. EAAT1 Na + ‐coupling is conserved from archaea to humans. EAAT1 H + ‐coupling involves a conserved glutamate residue in the transport domain. Binding sites for glutamate and the counter‐transported K + overlap. Ca 2+ competes for a conserved Na + site and is thermodynamically coupled to neurotransmitter binding. Graphical Abstract Comprehensive structural and thermodynamic analyses of the synaptic glutamate transporter EAAT1 highlights overlapping binding sites and unexpected competition by Ca 2+ .