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.
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.
Sotiria
S
Tavoulari
Ahsan N
AN
Rizwan
Lucy R
LR
Forrest
Gary
G
Rudnick
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport / Q000502:physiology; D002712:Chlorides / Q000378:metabolism; D016937:Gram-Positive Endospore-Forming Rods / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D018377:Neurotransmitter Agents / Q000737:chemistry / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein
D001426:Bacterial Proteins; D002712:Chlorides; D018377:Neurotransmitter Agents; D050486:Serotonin Plasma Membrane Transport Proteins