title: Engineered zinc-binding sites confirm proximity and orientation of transmembrane helices I and III in the human serotonin transporter.
abstract: The human serotonin transporter (hSERT) regulates neurotransmission by removing released serotonin (5-HT) from the synapse. Previous studies identified residues in SERT transmembrane helices (TMHs) I and III as interaction sites for substrates and antagonists. Despite an abundance of data supporting a 12-TMH topology, the arrangement of the TMHs in SERT and other biogenic amine transporters remains undetermined. A high-resolution structure of a bacterial leucine transporter that demonstrates homology with SERT has been reported, thus providing the basis for the development of a SERT model. Zn2+-binding sites have been utilized in transporters and receptors to define experimentally TMH proximity. Focusing on residues near the extracellular ends of hSERT TMHs I and III, we engineered potential Zn2+-binding sites between V102 or W103 (TMH I) and I179-L184 (TMH III). Residues were mutated to either histidine or cysteine. TMH I/III double mutants were constructed from functional TMH I mutants, and Zn2+ sensitivity was assessed. Dose-response assays suggest an approximately twofold increase in sensitivity to Zn2+ inhibition at the hSERT V102C/M180C and approximately fourfold at the V102C/I179C mutant compared to the hSERT V102C single mutant. We propose that the increased sensitivity to Zn2+ confirms the proximity and the orientation of TMHs I and III in the membrane. Homology modeling of the proposed Zn2+-binding sites using the coordinates of the Aquifex aeolicus leucine transporter structure provided a structural basis for interpreting the results and developing conclusions.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University School of Pharmacy and Pharmaceutical Sciences, West Lafayette, Indiana 47907, USA.
authors.forename: Kellie J
authors.initials: KJ
authors.lastname: White
authors.forename: Philip D
authors.initials: PD
authors.lastname: Kiser
authors.forename: David E
authors.initials: DE
authors.lastname: Nichols
authors.forename: Eric L
authors.initials: EL
authors.lastname: Barker
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D040681:Structural Homology, Protein; D015032:Zinc
chemical_list: D008565:Membrane Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; D015032:Zinc