Interactions of Tryptamine Derivatives with Serotonin Transporter Species Variants Implicate Transmembrane Domain I in Substrate Recognition

  1. Erika M. Adkins1,
  2. Eric L. Barker2 and
  3. Randy D. Blakely1
  1. 1Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee (E.M.A., R.D.B.); and 2Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University School of Pharmacy, West Lafayette, Indiana (E.L.B.)

    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 β position show up to 40-fold potency differences for inhibiting [3H]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 compoundsN-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 β 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.

    Footnotes

    • Send reprint requests to: Randy D. Blakely, Ph.D., Rm 419, MRBII, Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, TN 37232-6420. E-mail:randy.blakely{at}mcmail.vanderbilt.edu

    • This work was supported in part by National Institutes of Health Grants MH12399 (E.M.A.) and DA07390 (R.D.B.).

    • Abbreviations:
      HT
      hydroxytryptamine
      SERT
      serotonin transporter
      MDMA
      methylenedioxymethamphetamine
      DAT
      dopamine transporter
      NET
      norepinephrine transporter
      dSERT
      Drosophila melanogaster serotonin transporter
      hSERT
      human serotonin transporter
      HEK
      human embryonic kidney
      TMD
      transmembrane domain
      7-MT
      7-methyltryptamine
      7-BT
      7-benzyloxytryptamine
      NIT
      N-isopropyltryptamine
      5-MNIT
      5-methoxy-N-isopropyltryptamine
      • Received September 7, 2000.
      • Accepted November 17, 2000.
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    1. Molecular Pharmacology March 1, 2001 vol. 59 no. 3 514-523
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