Reconstructing a Chloride-binding Site in a Bacterial Neurotransmitter Transporter Homologue

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. 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.