Binding and Orientation of Tricyclic Antidepressants within the Central Substrate Site of the Human Serotonin Transporter

TY - JOUR

T1 - Binding and Orientation of Tricyclic Antidepressants within the Central Substrate Site of the Human Serotonin Transporter

AU - Sinning, Steffen

AU - Musgaard, M

AU - Jensen, Marie

AU - Severinsen, K

AU - Celik, L

AU - Koldsø, H

AU - meyer, tine

AU - Bols, Mikael

AU - Jensen, Henrik H

AU - Schiøtt, Birgit

AU - Wiborg, Ove

PY - 2010/3/12

Y1 - 2010/3/12

N2 - 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 aTCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 Å 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 Asp98. Furthermore, the 7-position of the imipramine ring is found vicinal to Phe335, and the pocket lined by Ala173 and Thr439 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).

AB - 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 aTCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 Å 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 Asp98. Furthermore, the 7-position of the imipramine ring is found vicinal to Phe335, and the pocket lined by Ala173 and Thr439 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).

M3 - Journal article

SN - 0021-9258

VL - 285

SP - 8363

EP - 8374

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

ER -