Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants.

Henry LK, Field JR, Adkins EM, Parnas ML, Vaughan RA, Zou MF, Newman AH, Blakely RD
J Biol Chem. 2006 281 (4): 2012-23

PMID: 16272152 · DOI:10.1074/jbc.M505055200

In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [(125)I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/I172M had a synergistic impact on (RS)-CIT recognition ( approximately 10,000-fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT.

MeSH Terms (41)

Adrenergic Uptake Inhibitors Amino Acid Sequence Animals Antidepressive Agents Binding, Competitive Binding Sites Blotting, Western Cadmium Cell Line Cell Membrane Citalopram Clomipramine Cocaine Cysteine Dopamine Uptake Inhibitors Fluoxetine HeLa Cells Humans Immunoprecipitation Isoleucine Kinetics LLC-PK1 Cells Mazindol Methionine Mice Models, Chemical Molecular Sequence Data Mutation N-Methyl-3,4-methylenedioxyamphetamine Nomifensine Protein Binding Protein Structure, Tertiary Protein Transport Radiopharmaceuticals Receptors, Serotonin Serotonin Serotonin Uptake Inhibitors Species Specificity Stereoisomerism Substrate Specificity Tyrosine

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