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J Biol Chem. 2011 Jan 28;286(4):2834-42. doi: 10.1074/jbc.M110.186064. Epub 2010 Nov 29.

Reconstructing a chloride-binding site in a bacterial neurotransmitter transporter homologue.

Author information

1
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.

Abstract

In ion-coupled transport proteins, occupation of selective ion-binding sites is required to trigger conformational changes that lead to substrate translocation. Neurotransmitter transporters, targets of abused and therapeutic drugs, require Na(+) and Cl(-) for function. We recently proposed a chloride-binding site in these proteins not present in Cl(-)-independent prokaryotic homologues. Here we describe conversion of the Cl(-)-independent prokaryotic tryptophan transporter TnaT to a fully functional Cl(-)-dependent form by a single point mutation, D268S. Mutations in TnaT-D268S, in wild type TnaT and in serotonin transporter provide direct evidence for the involvement of each of the proposed residues in Cl(-) coordination. In both SERT and TnaT-D268S, Cl(-) and Na(+) mutually increased each other's potency, consistent with electrostatic interaction through adjacent binding sites. These studies establish the site where Cl(-) binds to trigger conformational change during neurotransmitter transport.

PMID:
21115480
PMCID:
PMC3024779
DOI:
10.1074/jbc.M110.186064
[Indexed for MEDLINE]
Free PMC Article

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