An Extra Amino Acid Residue in Transmembrane Domain 10 of the γ-Aminobutyric Acid (GABA) Transporter GAT-1 Is Required for Efficient Ion-coupled Transport

J Biol Chem. 2017 Mar 31;292(13):5418-5428. doi: 10.1074/jbc.M117.775189. Epub 2017 Feb 17.


The GABA transporter GAT-1 mediates electrogenic transport of its substrate together with sodium and chloride. It is a member of the neurotransmitter:sodium:symporters, which are crucial for synaptic transmission. Compared with all other neurotransmitter:sodium:symporters, GAT-1 and other members of the GABA transporter subfamily all contain an extra amino acid residue at or near a conserved glycine in transmembrane segment 10. Therefore, we studied the functional impact of deletion and replacement mutants of Gly-457 and its two adjacent residues in GAT-1. The glycine replacement mutants were devoid of transport activity, but remarkably the deletion mutant was active, as were mutants obtained by deleting positions on either side of Gly-457. However, the inward rectification of GABA-induced transport currents by all three deletion mutants was diminished, and the charge-to-flux ratio was increased by more than 2.5-fold, both of which indicate substantial uncoupled transport. These observations suggest that the deletions render the transporters less tightly packed. Consistent with this interpretation, the inactive G457A mutant was partially rescued by removing the adjacent serine residue. Moreover, the activity of several gating mutants was also partially rescued upon deletion of Gly-457. Structural modeling showed that the stretch surrounding Gly-457 is likely to form a π-helix. Our data indicate that the "extra" residue in transmembrane domain 10 of the GABA transporter GAT-1 provides extra bulk, probably in the form of a π-helix, which is required for stringent gating and tight coupling of ion and substrate fluxes in the GABA transporter family.

Keywords: amino acid transport; chloride transport; membrane transport; neurotransmitter transport; sodium transport.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Intramural

MeSH terms

  • Amino Acids
  • Conserved Sequence / genetics
  • GABA Plasma Membrane Transport Proteins / chemistry*
  • GABA Plasma Membrane Transport Proteins / genetics
  • GABA Plasma Membrane Transport Proteins / metabolism
  • Glycine / genetics*
  • HeLa Cells
  • Humans
  • Ion Transport*
  • Mutagenesis, Site-Directed*
  • Protein Conformation
  • Protein Domains
  • Structure-Activity Relationship


  • Amino Acids
  • GABA Plasma Membrane Transport Proteins
  • Glycine

Associated data

  • PDB/2A65
  • PDB/3TT1
  • PDB/3TT3
  • PDB/4XP4
  • PDB/5I6X