title: Interactions of tryptamine derivatives with serotonin transporter species variants implicate transmembrane domain I in substrate recognition.
abstract: The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is responsible for the inactivation of synaptic 5-HT and is also a target for multiple psychostimulants. Despite the critical role of SERT in 5-HT inactivation and psychostimulant response, many aspects of the transporter's recognition of ligands are poorly defined. We took advantage of sequence divergence of SERT species variants to identify structural determinants of substrate recognition. Tryptamine derivatives with substitutions at the 4 and 7 positions on the phenyl ring, the indole nitrogen, and the beta position show up to 40-fold potency differences for inhibiting [(3)H]5-HT transport in cells transfected with either human or Drosophila melanogaster SERT cDNAs. Species selectivities of these derivatives were largely recapitulated in antagonist binding. Human/D. melanogaster SERT chimera studies implicated the first two SERT transmembrane domains (TMDs) in the potency of the indole nitrogen-substituted compounds N-isopropyltryptamine (NIT), 5-methoxy-N-isopropyltryptamine (5-MNIT), and the 7-substituted compound 7-benzyloxytryptamine (7BT). Potency differences of analogs with substitutions at the 4 and beta positions are influenced by sequences distal to this region. Within TMD I-II, species-scanning mutagenesis implicated a single residue (Y95 in human SERT, F90 in D. melanogaster SERT) in the recognition of NIT, 5-MNIT, and 7BT. Remarkably, this is the same site we established previously in species-specific recognition of the antagonists citalopram and mazindol. These findings support a critical role for TMD I residues in defining shared aspects of SERT substrate and antagonist recognition.
authors.affiliation: Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: E M
authors.initials: EM
authors.lastname: Adkins
authors.forename: E L
authors.initials: EL
authors.lastname: Barker
authors.forename: R D
authors.initials: RD
authors.lastname: Blakely
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016296:Mutagenesis; D009419:Nerve Tissue Proteins; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins; D013045:Species Specificity; D014363:Tryptamines / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D002352:Carrier Proteins; D029721:Drosophila Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C493230:SLC6A4 protein, human; C493233:SerT protein, Drosophila; D050486:Serotonin Plasma Membrane Transport Proteins; D014363:Tryptamines; C030820:tryptamine