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

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.

Author information

1
From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel and.
2
the Computational Structural Biology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892.
3
the Computational Structural Biology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892 lucy.forrest@nih.gov.
4
From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel and baruch.kanner@mail.huji.ac.il.

Abstract

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

PMID:
28213519
PMCID:
PMC5392685
DOI:
10.1074/jbc.M117.775189
[Indexed for MEDLINE]
Free PMC Article

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