The serotonin transporter (SERT), which belongs to a family of sodium/chloride-dependent transporters, is the major pharmacological target in the treatment of several clinical disorders, including depression and anxiety. In the present study we show that the dissociation rate, of the SERT double-mutant, N208Q/N217Q, which has been suggested to be unable to self-assemble into oligomeric complexes, is retarded to an extent similar to that found with the wild-type, raising the possibility that the allosteric mechanism is mediated within a single subunit. A species-scanning mutagenesis study comparing human and bovine SERT revealed that Met180, Tyr495 and Ser513 are important residues in mediating the allosteric effect, as well as contributing to high-affinity binding at the primary site.

The serotonin transporter (SERT), which belongs to a family of sodium/chloride-dependent transporters, is the major pharmacological target in the treatment of several clinical disorders, including depression and anxiety. Interaction with a low-affinity allosteric site on SERT modulates the ligand affinity at the high-affinity binding site. Serotonin (5-hydroxytryptamine) and certain SERT inhibitors possess affinity for both sites. In the present study, we report the characterization of a severely attenuated allosteric mechanism at the recently cloned chicken serotonin transporter (gSERT). A cross-species chimera study was performed, followed by species scanning mutagenesis. Residues important for the allosteric mechanism were mapped to the C-terminal part of SERT containing the transmembrane domains 10 to 12. We identified nine residues located in four distinct amino acid segments. The contribution of each segment and individual residues was investigated. Consequently, a gSERT mutant with a restored allosteric mechanism, as well as a human SERT (hSERT) mutant with a severely attenuated allosteric mechanism, was generated. The nine residues confer a functional allosteric mechanism for different combinations of ligands, suggesting that they contribute to a general allosteric mechanism at SERT. The finding of an allosteric mechanism at SERT is likely to be of physiological importance, in that serotonin was also found to act as an allosteric effector at duloxetine, RTI-55 and (S)-citalopram. Furthermore, the allosteric potency of 5-HT was found to be conserved for both hSERT and gSERT.Serotonergic neurotransmission is modulated by clearance of serotonin (5-hydroxytryptamine; 5-HT). The clearance of 5-HT from the synaptic cleft is maintained by the serotonin transporter (SERT). The transporter therefore affects the magnitude and duration of the signaling and thus plays a key role in the spatio-temporal fine-tuning of serotonergic neurotransmission SERT is a well established molecular target of both drugs of abuse (cocaine and amphetamines) and most high-affinity antidepressants. Multiple classes of antidepressants, including tricyclic antidepressants, 5-HT-selective reuptake inhibitors, and antidepressants with dual actions, are directed toward SERT. They enhance serotonergic neurotransmission by competitively inhibiting 5-HT reuptake with inhibitory constants in the low nanomolar range (Barker and Blakely, 1995;Owens et al., 1997;Tatsumi et al., 1997).Dissociation of the tricyclic imipramine from platelet membranes is attenuated in the presence of 5-HT Meyerson, 1982, 1985), suggesting that 5-HT acts at a site distinct from the imipramine binding site. Several high-affinity SERT inhibitors (citalopram, paroxetine, sertraline, imipramine) can also act as allosteric ligands (Plenge and Mellerup, 1985;Plenge et al., 1991). The affinity-modulating or allosteric site has been shown to be present at all the monoamine transporters (i.e., serotonin, dopamine, and norepinephrine transporters) (Plenge and Mellerup, 1...

No abstract