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

• Published in: eLife 2017-06, Vol.6
• Main Authors: Cheng, Mary Hongying, Torres-Salazar, Delany, Gonzalez-Suarez, Aneysis D, Amara, Susan G, Bahar, Ivet
• Format: Article
• Language: English
• Subjects: anion channeling; Anions - metabolism; Aspartate transporter from Pyrococcus horikoshii; Biophysics and Structural Biology; Computational and Systems Biology; DNA Mutational Analysis; Excitatory Amino Acid Transporter 1 - chemistry; Excitatory Amino Acid Transporter 1 - genetics; Excitatory Amino Acid Transporter 1 - metabolism; Glutamate; Glutamic Acid - metabolism; human excitatory amino acid transporter 1; Ion transport; Models, Molecular; Observations; Patch-Clamp Techniques; Physiological aspects; Point Mutation; Transport proteins
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.25850

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, S, transiently accessible as EAAT1 reconfigures from substrate/ion-loaded into a substrate-releasing conformer. Opening of the anion permeation path in this S is controlled by the elevator-like movement of the substrate-binding core, along with its wall that simultaneously lines the anion permeation path ( ); and repacking of a cluster of hydrophobic residues near the extracellular vestibule ( ). Moreover, our results demonstrate that stabilization of 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.