title: Binding and orientation of tricyclic antidepressants within the central substrate site of the human serotonin transporter.
abstract: Tricyclic antidepressants (TCAs) have been used for decades, but their orientation within and molecular interactions with their primary target is yet unsettled. The recent finding of a TCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 A above the central site has prompted debate about whether this vestibular site in the bacterial transporter is applicable to binding of antidepressants to their relevant physiological target, the human serotonin transporter (hSERT). We present an experimentally validated structural model of imipramine and analogous TCAs in the central substrate binding site of hSERT. Two possible binding modes were observed from induced fit docking calculations. We experimentally validated a single binding mode by combining mutagenesis of hSERT with uptake inhibition studies of different TCA analogs according to the paired mutation ligand analog complementation paradigm. Using this experimental method, we identify a salt bridge between the tertiary aliphatic amine and Asp(98). Furthermore, the 7-position of the imipramine ring is found vicinal to Phe(335), and the pocket lined by Ala(173) and Thr(439) is utilized by 3-substituents. These protein-ligand contact points unambiguously orient the TCA within the central binding site and reveal differences between substrate binding and inhibitor binding, giving important clues to the inhibition mechanism. Consonant with the well established competitive inhibition of uptake by TCAs, the resulting binding site for TCAs in hSERT is fully overlapping with the serotonin binding site in hSERT and dissimilar to the low affinity noncompetitive TCA site reported in the leucine transporter (LeuT).
authors: Laboratory of Molecular Neurobiology, Centre for Psychiatric Research, Aarhus University Hospital, Skovagervej 2, 8240 Risskov, Denmark.
authors: Steffen
authors: S
authors: Sinning
authors: Maria
authors: M
authors: Musgaard
authors: Marie
authors: M
authors: Jensen
authors: Kasper
authors: K
authors: Severinsen
authors: Leyla
authors: L
authors: Celik
authors: Heidi
authors: H
authors: Koldsø
authors: Tine
authors: T
authors: Meyer
authors: Mikael
authors: M
authors: Bols
authors: Henrik Helligsø
authors: HH
authors: Jensen
authors: Birgit
authors: B
authors: Schiøtt
authors: Ove
authors: O
authors: Wiborg
mesh_terms: D000409:Alanine / Q000235:genetics; D000929:Antidepressive Agents, Tricyclic / Q000737:chemistry* / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D002478:Cells, Cultured; D006801:Humans; D007099:Imipramine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D007930:Leucine / Q000235:genetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D012701:Serotonin / Q000493:pharmacokinetics; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection; D014316:Tritium
chemical_list: D000929:Antidepressive Agents, Tricyclic; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D014316:Tritium; D001224:Aspartic Acid; D012701:Serotonin; D010649:Phenylalanine; D007930:Leucine; D000409:Alanine; D007099:Imipramine