Interactions of tryptamine derivatives with serotonin transporter species variants implicate transmembrane domain I in substrate recognition

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is responsible for the inactivation of synaptic 5-HT and is also a target for multiple ps ychostimulants. 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 SER T species variants to identify structural determinants of substrate recogni tion. 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 [H-3]5-HT transport in cells transf ected with either human or Drosophila melanogaster SERT cDNAs. Species sele ctivities of these derivatives were largely recapitulated in antagonist bin ding. Human/D. melanogaster SERT chimera studies implicated the first two S ERT transmembrane domains (TMDs) in the potency of the indole nitrogen-subs tituted compounds N-isopropyltryptamine (NIT), 5-methoxy-N-isopropyltryptam ine (5-MNIT), and the 7-substituted compound 7-benzyloxytryptamine (7BT). P otency differences of analogs with substitutions at the 4 and beta position s are influenced by sequences distal to this region. Within TMD I-II, speci es-scanning mutagenesis implicated a single residue (Y95 in human SERT, F90 in D. melanogaster SERT) in the recognition of NIT, 5-MNIT, and 7BT. Remar kably, this is the same site we established previously in species-specific recognition of the antagonists citalopram and mazindol. These findings supp ort a critical role for TMD I residues in defining shared aspects of SERT s ubstrate and antagonist recognition.