HIGH-AFFINITY RECOGNITION OF SEROTONIN TRANSPORTER ANTAGONISTS DEFINED BY SPECIES-SCANNING MUTAGENESIS - AN AROMATIC RESIDUE IN TRANSMEMBRANE DOMAIN-I DICTATES SPECIES-SELECTIVE RECOGNITION OF CITALOPRAM AND MAZINDOL

Human and Drosophila melanogaster serotonin (5-HT) transporters (SERTs ) exhibit similar 5-HT transport kinetics and can be distinguished pha rmacologically by many, but not all, biogenic amine transporter antago nists. By using human and Drosophila SERT chimeras, major determinants of potencies of two transporter antagonists, mazindol and citalopram, were tracked to the amino-terminal domains encompassing transmembrane domains I and II. Species-scanning mutagenesis, whereby amino acid su bstitutions are made switching residues from one species to another, w as employed on the eight amino acids that differ between human and Dro sophila SERTs in this region, and antagonist potencies were reassessed in 5-HT uptake assays. A single mutation in transmembrane domain I of human SERT, Y95F, shifted both citalopram and mazindol to Drosophila SERT-Iike potencies, Strikingly, these potency changes were in opposit e directions suggesting Tyros contributes both positive and negative d eterminants of antagonist potency. To gain insight into how the Y95F m utant might influence mazindol potency, we determined how structural v ariants of mazindol responded to the mutation, Our studies demonstrate the importance of the hydroxyl group on the heterocyclic nucleus of m azindol for maintaining species-selective recognition of mazindol and suggest that transmembrane domain I participates in the formation of a ntagonist-binding sites for amine transporters.