The ion-coupling mechanism of human excitatory amino acid transporters

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 Ca²⁺ , are coupled to neurotransmitter binding. Ca²⁺ competes for a conserved Na⁺ site, suggesting a regulatory role for Ca²⁺ 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.