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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.