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Free keywords:
Amino Acid Transport; Chloride Transport; Neurotransmitter Transport; Protein Structure; Serotonin Transporters; Chloride-binding Site; Ion-coupled Transport
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.