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

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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.