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

Elife. 2017 Jun 1;6:e25850. doi: 10.7554/eLife.25850.

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

Keywords: Aspartate transporter from Pyrococcus horikoshii; anion channeling; biophysics; computational biology; human excitatory amino acid transporter 1; structural biology; systems biology.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Anions / metabolism*
  • DNA Mutational Analysis
  • Excitatory Amino Acid Transporter 1 / chemistry*
  • Excitatory Amino Acid Transporter 1 / genetics
  • Excitatory Amino Acid Transporter 1 / metabolism*
  • Glutamic Acid / metabolism*
  • Models, Molecular
  • Patch-Clamp Techniques
  • Point Mutation

Substances

  • Anions
  • Excitatory Amino Acid Transporter 1
  • Glutamic Acid