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J. Biol. Chem., Vol. 281, Issue 4, 2012-2023, January 27, 2006

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Tyr-95 and Ile-172 in Transmembrane Segments 1 and 3 of Human Serotonin Transporters Interact to Establish High Affinity Recognition of Antidepressants^*

L. Keith Henry, Julie R. Field, Erika M. Adkins¶, M. Laura Parnas||, Roxanne A. Vaughan||, Mu-Fa Zou**, Amy H. Newman**, and Randy D. Blakely1

From the Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee 37232, the ^¶Department of Pathology, Utah Medical Center, Salt Lake City, Utah 84112, the ^||Department of Biochemistry and Molecular Biology, University of North Dakota, Grand Forks, North Dakota 58203, and ^**NIDA-Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224

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 [¹²⁵I]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 (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.

Received for publication, May 9, 2005 , and in revised form, October 25, 2005.

^* This work was supported by a National Alliance for Research on Schizophrenia and Depression Young Investigator award (to L. K. H.), the Forest Research Institute, National Institutes of Health Grants DA07390 (to R. D. B.) and DA15175 (to R. A. V.), and by the NIDA-Intramural Research Program (to A. H. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Ste. 7140 MRBIII, Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, TN 37232-8548. Tel.: 615-936-3705; Fax: 615-936-3040; E-mail: randy.blakely{at}vanderbilt.edu.

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