Comparative Molecular Field Analysis Using Selectivity Fields Reveals Residues in the Third Transmembrane Helix of the Serotonin Transporter Associated with Substrate and Antagonist Recognition

Abstract

The human serotonin transporter (hSERT) regulates the spatial and temporal actions of serotonin (5-HT) neurotransmission by removing 5-HT from the synapse. Previous studies have identified residues in the third transmembrane helix (TMH) that may be important for substrate translocation or antagonist recognition. We identified hSERT residues in TMH III that are divergent from Drosophila SERT and used species-scanning mutagenesis to generate reciprocal mutants. Transport inhibition assays suggest that the potency of substituted amphetamines was decreased for the hSERT mutants A169D, I172M, and S174M. In addition, there was a loss of potency for several antidepressants and 3-phenyltropane analogs for the I172M mutant. These results suggest that residues in TMH III may contribute to antagonist recognition. We carried out comparative molecular field analyses using selectivity fields to directly visualize the mutation-induced effects of antagonist potency for antidepressants, 3-phenyltropane analogs, and amphetamines. The hSERT I172M selectivity field analysis for the 3-phenyltropane analogs revealed that electrostatic interactions resulted in decreased potency. The amphetamine and antidepressant selectivity field analyses reveal the observed decreases in potencies for the hSERT I172M mutant are due to a change in tertiary structure of the hSERT protein and are not due to disruption of a direct binding site. Finally, the hSERT mutant A169D displayed altered kinetics for sodium binding, indicating that this residue may lie near the putative sodium binding site. A SERT homology model developed from the Aquifex aeolicus leucine transporter structure provides a structural context for further interpreting the results of the TMH III mutations.

Mutational Mapping and Modeling of the Binding Site for (S)-Citalopram in the Human Serotonin Transporter

“…2 and Table 1). In particular, we found Ile-172 highly sensitive to mutation, which is in accordance with earlier findings (13,39 Table 1. Non-functional mutants are indicated on the right.…”

“…7C). The role of Phe-341 for (S)-citalopram inhibition have not previously been examined, whereas Ile-172 is well characterized as a major determinant for binding of several inhibitors (13,39). We propose that mutations such as I172M and F341Y cause dramatic changes in (S)-citalopram affinity by reshaping the hydrophobic bulge formed by these residues such that the ability of the fluorophenyl and cyanophtalane groups to bind in their respective sub-pockets is decreased.…”

“…This role of Ile-172 is consistent with a recent study by Walline et al (39) of the effect of I172M on the inhibitory potency of amphetamine analogs in hSERT. Here, it was suggested that the effects of I172M on potency of amphetamine analogs is probably not caused by disruption of direct inhibitor-protein interactions, but rather may result from a change in the tertiary structure of the inhibitor binding pocket (39).…”

Molecular determinants for selective recognition of antidepressants in the human serotonin and norepinephrine transporters

“…Generally, these prototypical SERT/NET inhibitors have low sensitivity toward N7 (Table S1), indicating that they have distinct binding modes compared with citalopram within the S1 site, or that they bind to a distinct site (11,17,35). Reboxetine was unaffected by N7 (Table S1), and combined with a recent study showing that reboxetine possesses a noncompetitive mode of NET inhibition (23), this finding indicates that the NRI binds to a site different from the S1 site.…”

Molecular Basis for Selective Serotonin Reuptake Inhibition by the Antidepressant Agent Fluoxetine (Prozac)

“…1) Wang et al, 2013). Combined with biochemical studies showing that most SSRIs inhibit SERT in a competitive manner (Graham et al, 1989;Koe et al, 1990;Apparsundaram et al, 2008), and several residues located within the S1 site of SERT and NET have been shown to be important for binding of SSRIs (Barker et al, 1999;Henry et al, 2006;Mason et al, 2007;Walline et al, 2008;Andersen et al, 2009Andersen et al, , 2010Koldsø et al, 2010;Sørensen et al, 2012), LeuBAT and Drosophila DAT seem to represent improved structural frameworks for studying the molecular pharmacology of human transporters compared with LeuT. Fluoxetine has been cocrystallized together with both LeuT and LeuBAT, and these studies have provided ambiguous insight into the binding mechanism of this important SSRI drug. Whereas fluoxetine binds to the S2 site in LeuT, it binds to the S1 site in LeuBAT ( Fig.…”