Journal of Biological Chemistry
Volume 286, Issue 4, 28 January 2011, Pages 2834-2842
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Membrane Biology
Reconstructing a Chloride-binding Site in a Bacterial Neurotransmitter Transporter Homologue*

https://doi.org/10.1074/jbc.M110.186064Get rights and content
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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.

Amino Acid Transport
Chloride Transport
Neurotransmitter Transport
Protein Structure
Serotonin Transporters
Chloride-binding Site
Ion-coupled Transport

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*

This work was supported, in whole or in part, by National Institutes of Health Grants GM075347 and DA007259 (to G. R.). This work was also supported by an Autism Speaks postdoctoral fellowship (to S. T.) and by the Deutsche Forschungsgemeinschaft Collaborative Research Center 807 “Transport and Communication across Biological Membranes” (to L. R. F.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.