Species-scanning mutagenesis of the serotonin transporter reveals residuesessential in selective, high-affinity recognition of antidepressants

The serotonin transporter (SERT) is a high-affinity sodium/chloride-depende nt neurotransmitter transporter responsible for reuptake of serotonin from the extracellular space. SERT is a selective target of several clinically i mportant antidepressants. In a cross-species analysis comparing human and b ovine SERTs, the kinetic parameters for serotonin uptake were found to be s imilar, however, the pharmacological profiles of the two transporters diffe r. Following transient expression in COS-1 cells, IC50 values were determin ed for several antidepressants and psychostimulants. The potencies of the a ntidepressants citalopram, fluoxetine, paroxetine and imipramine were sever al-fold higher at hSERT compared with bSERT. No species selectivity was obs erved for the antidepressants fluvoxamine, and sertraline or for the psycho stimulants cocaine, the cocaine analogue beta -carbomethoxy-3 beta-(4-iodop henyl)tropane, or for 3,4-methylenedioxymethamphetamine (MDMA). Analysis of six hSERT/bSERT chimeras and subsequent species-scanning mutagenesis of ea ch isoform revealed methionine-180, tyrosine-495, and phenylaianine-513 to be responsible for the increase in citalopram and paroxetine potencies at h SERT and methionine-180 and phenylalanine-513 to confer species selectivity at hSERT for fluoxetine and imipramine. Results were obtained by doing the forward, bovine to human, mutations and confirmed by doing the reverse mut ations. Citalopram analogues were used to define the roles of methionine-18 0, tyrosine-495, and phenylaianine-513 and to reveal molecular interactions with individual functional groups of citalopram. We suggest that methionin e-180 interacts with the heterocyclic nucleus of citalopram or stabilizes t he binding pocket and phenylalanine-513 to be a steric blocker of antidepre ssant recognition.