Dissection of an allosteric mechanism on the serotonin transporter: a cross-species study.

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.

Characterization of an allosteric citalopram-binding site at the serotonin transporter.

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 [3H]S-citalopram from human SERT, is retarded by the presence of serotonin, as well as by several antidepressants, when present in the dissociation buffer. Dissociation of [3H]S-citalopram from SERT is most potently inhibited by S-citalopram followed by R-citalopram, sertraline, serotonin and paroxetine. EC50 values for S- and R-citalopram are 3.6 +/- 0.4 microm and 19.4 +/- 2.3 microm, respectively. Fluoxetine, venlafaxine and duloxetine have no significant effect on the dissociation of [3H]S-citalopram. Allosteric modulation of dissociation is independent of temperature, or the presence of Na+ in the dissociation buffer. Dissociation of [3H]S-citalopram from a complex with 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.