Tyr-95 and Ile-172 in Transmembrane Segments 1 and 3 of Human Serotonin Transporters Interact to Establish High Affinity Recognition of Antidepressants

Abstract

In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [ 125 I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/ I172M had a synergistic impact on (RS)-CIT recognition (ϳ10,000-fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/ I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT. SERT,2 like several other members of the SLC6A gene family, acts to remove neurotransmitter from the synapse following neurotransmission (1) and is a major target for treatment of mood disorders, including major depression, anxiety, post-traumatic stress, and obsessive-compulsive disorders (2). Agents that target SERT include tricyclic antidepressants and serotonin-specific reuptake inhibitors (SSRIs) that block 5HT binding and uptake. Despite its clinical significance, very little is understood concerning the structural aspects of SERT and how they relate to its function and antagonist recognition. The current data predict that SERT proteins are composed of 12 transmembrane-spanning segments, intracellular NH 2 and COOH termini and a large second extracellular...

Functional coding variation in recombinant inbred mouse lines reveals multiple serotonin transporter-associated phenotypes

“…Nonetheless, the reduced SERT activity in the GK lines is still adequate for acute potentiation of 5-HT signaling by an SSRI (citalopram). One might expect that reduced SERT activity might have resulted in altered citalopram responsiveness; however, the dosage of citalopram optimized for responses in the TST paradigm may simply be above that required to distinguish SSRI sensitivities in vivo, although it should also be noted that the variants are not located in the predicted binding pocket for citalopram (26,27). We also found equivalent citalopram recognition by GK/ER variants in our transfection studies (Fig.…”

Transgenic elimination of high-affinity antidepressant and cocaine sensitivity in the presynaptic serotonin transporter

“…Previously, our heterologous expression studies demonstrated that the M172 substitution produces significant reductions in the potency of multiple SSRIs, cocaine, and cocaine analogues to inhibit 5-HT transport (28), despite the retention of normal potency of substrates (e.g., 5-HT, 3,4-methylenedioxymethamphetamine). Competition studies that used forebrain synaptosomes from M172 mice produced very similar results ( Fig.…”

“…By using the pharmacological differences displayed by human and Drosophila melanogaster SERTs (hSERT and dSERT), we identified variation at a single residue in TM3 (I172 in human and mouse, M167 in fly) proximal to the proposed binding site for 5-HT (28). Remarkably, when we substituted M172 for I172 in hSERT and mouse SERT (mSERT) cDNAs, we observed a significant, 100-to 1,000-fold reduction in potency of many SERT antagonists in transfected cells without an impact on basal 5-HT transport (28). Conversely, dSERT I167 displayed increased potencies for these same drugs.…”

Synthesis and serotonin transporter activity of 1,3-bis(aryl)-2-nitro-1-propenes as a new class of anticancer agents

“…It has also been previously shown that the protonated amine of a series of sulfur-substituted -alkyl phenethylamines is essential for protein-ligand binding. Other commonly annotated significant binding residues include Ile172 and Tyr95, which allow for hydrophobic and aromatic interactions respectively 34 .…”

“…For this study the homology model of hSERT constructed by Jørgensen et al (2007) was used 33 . A number of previous studies have identified key binding residues involved in the binding of SERT ligands [33][34][35][36] . 5-HT, the natural substrate of SERT, and amphetamines such as 4-MTA interact with residues Ala96, Asp98 and Phe335 via binding of the protonated amine (Fig.…”