IDENTIFICATION OF A SINGLE AMINO-ACID, PHENYLALANINE-586, THAT IS RESPONSIBLE FOR HIGH-AFFINITY INTERACTIONS OF TRICYCLIC ANTIDEPRESSANTS WITH THE HUMAN SEROTONIN TRANSPORTER

When assayed in parallel using transfected mammalian cells, human and rat serotonin transporters (SERTs) exhibit consistent differences in p otency for tricyclic antidepressants but not for 5-hydroxytryptamine, cocaine, or nontricyclic serotonin transporter-selective reuptake inhi bitors. Previously, using chimeric proteins, we determined that domain s or residues distal to transmembrane domain 11 (amino acid 531) dicta te the increased sensitivity of human SERT to imipramine. Using an add itional chimera and site-directed mutagenesis, we have determined that a single amino acid, F586, is responsible for increased sensitivity t o imipramine, desipramine, and nortriptyline. Thus, mutation of wild-t ype rat SERT (V586) to the human SERT identity F586, but no other dive rgent amino acids between human and rat SERTs, selectively increased t ricyclic antidepressant potency. A reciprocal reduction in potency was observed when human SERT F586 was converted to the cognate rat SERT r esidue (V586). Interactions with other SERT antagonists, including par oxetine and cocaine, as well as the SERT substrates 5-hydroxytryptamin e and d-amphetamine were unaffected by interconversion of this residue . Phenylalanine conversion in the human norepinephrine transporter at the homologous position failed to alter tricyclic inhibition of catech olamine uptake, revealing a SERT-specific context for use of the aroma tic side chain at this position. Additional constraints on aromaticity at rat SERT position 586 were revealed by conversion of rat SERT V586 to Y586, which failed to replicate the effect of the F586 mutation. I n addition, conversion to V586D, but not V586R, increased tricyclic po tency to that of human SERT and additionally increased potency for coc aine but not paroxetine. These results implicate distal domains and a single residue in TMD 12 in the formation of high affinity SERT antago nist binding sites.