Substrate transport and anion permeation proceed through distinct pathways in glutamate transporters.

Abstract

Advances in structure-function analyses and computational biology have enabled a deeper understanding of how excitatory amino acid transporters (EAATs) mediate chloride permeation and substrate transport. However, the mechanism of structural coupling between these functions remains to be established. Using a combination of molecular modeling, substituted cysteine accessibility, electrophysiology and glutamate uptake assays, we identified a chloride-channeling conformer, <i>iCh</i>S, transiently accessible as EAAT1 reconfigures from substrate/ion-loaded into a substrate-releasing conformer. Opening of the anion permeation path in this <i>iCh</i>S is controlled by the elevator-like movement of the substrate-binding core, along with its wall that simultaneously lines the anion permeation path (<i>global</i>); and repacking of a cluster of hydrophobic residues near the extracellular vestibule (<i>local</i>). Moreover, our results demonstrate that stabilization of <i>iCh</i>S by chemical modifications favors anion channeling at the expense of substrate transport, suggesting a mutually exclusive regulation mediated by the movement of the flexible wall lining the two regions.