Abstract
The human serotonin transporter (hSERT) regulates the spatial and temporal actions of serotonin (5-HT) neurotransmission by removing 5-HT from the synapse. Previous studies have identified residues in the third transmembrane helix (TMH) that may be important for substrate translocation or antagonist recognition. We identified hSERT residues in TMH III that are divergent from Drosophila SERT and used species-scanning mutagenesis to generate reciprocal mutants. Transport inhibition assays suggest that the potency of substituted amphetamines was decreased for the hSERT mutants A169D, I172M, and S174M. In addition, there was a loss of potency for several antidepressants and 3-phenyltropane analogs for the I172M mutant. These results suggest that residues in TMH III may contribute to antagonist recognition. We carried out comparative molecular field analyses using selectivity fields to directly visualize the mutation-induced effects of antagonist potency for antidepressants, 3-phenyltropane analogs, and amphetamines. The hSERT I172M selectivity field analysis for the 3-phenyltropane analogs revealed that electrostatic interactions resulted in decreased potency. The amphetamine and antidepressant selectivity field analyses reveal the observed decreases in potencies for the hSERT I172M mutant are due to a change in tertiary structure of the hSERT protein and are not due to disruption of a direct binding site. Finally, the hSERT mutant A169D displayed altered kinetics for sodium binding, indicating that this residue may lie near the putative sodium binding site. A SERT homology model developed from the Aquifex aeolicus leucine transporter structure provides a structural context for further interpreting the results of the TMH III mutations.
Footnotes
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This study was supported by a Graduate Assistance in Areas of National Need Fellowship (to C.C.W.) and by the National Institutes of Health Grants DA018682 (to E.L.B. and D.E.N.) and DA05477 (to F.I.C.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.136200.
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ABBREVIATIONS: SERT, serotonin transporter; 5-HT, 5-hydroxytryptamine, serotonin; TCA, tricyclic antidepressant; TMH, transmembrane helix; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate; MDMA, 3,4-methylenedioxy-N-methyl-amphetamine; hSERT, human SERT; CoMFA, comparative molecular field analysis; KRH, Krebs-Ringer-HEPES; dSERT, Drosophila SERT; PBS-CM, phosphate-buffered saline supplemented with calcium and magnesium; MDA, 3,4-methylenedioxyamphetamine; N-dipropyl-indan, 3,4-methylenedioxy-2-N-N-dipropylindan; AMMT, 1-aminomethyl-6-methoxytetralin; DiFMDA, 1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)propan-2-amine; 2-Me-MDA, 2-methyl-3,4-methylenedioxyamphetamine; DCA, 3,4-dichloroamphetamine; RTI-31, 3β-(4-chorophenyl)tropane-2β-carboxylic acid methyl ester tartrate; RTI-32, 3β-(4-methylphenyl)tropane-2β-carboxylic acid methyl ester tartrate; RTI-55, 3β-(4-iodophenyl)tropane-2β-carboxylic acid methyl ester tartrate; RTI-83, 3β-(4-ethylphenyl)tropane-2β-carboxylic acid methyl ester tartrate; RTI-112,(-)-3β-(3-methyl-4-chlorophenyl)tropane-2β-carboxylic acid methyl ester tartrate; RTI-121, 3β-(4-iodophenyl)-tropane-2β-carboxylic acid isopropyl ester hydrochloride; RTI-142, (-)-N-nor-3β-(4-fluorophenyl)tropane-2β-carboxylic acid methyl ester; RTI-147, 3β-(4-chlorophenyl)tropane-2β-pyrrolidinecarboxamide hydrochloride; RTI-311, N-allyl-N-nor-3β-(4-iodophenyl)tropane-2β-carboxylic acid methyl ester tartrate.
- Received January 4, 2008.
- Accepted March 18, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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