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J. Biol. Chem., Vol. 279, Issue 22, 22926-22933, May 28, 2004

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Analysis of Transmembrane Domain 2 of Rat Serotonin Transporter by Cysteine Scanning Mutagenesis^*

Yuichiro Sato, Yuan-Wei Zhang, Andreas Androutsellis-Theotokis, and Gary Rudnick¶

From the Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066

The second transmembrane domain (TM2) of neurotransmitter transporters has been invoked to control oligomerization and surface expression. This transmembrane domain lies between TM1 and TM3, which have both been proposed to contain residues that contribute to the substrate binding site. Rat serotonin transporter (SERT) TM2 was investigated by cysteine scanning mutagenesis. Six mutants in which cysteine replaced an endogenous TM2 residue had low transport activity, and two were inactive. Most of the reduction in transport activity was due to decreased surface expression. In contrast, M124C and G128C showed increased activity and surface expression. Random mutagenesis at positions 124 and 128 revealed that hydrophobic residues at these positions also increased activity. When modeled as an -helix, positions where mutation to cysteine strongly affects expression levels clustered on the face of TM2 surrounding the leucine heptad repeat conserved within this transporter family. 2-(Aminoethyl)-methanethiosulfonate hydrobromide (MTSEA)-biotin labeled A116C and Y136C but not F117C, M135C, or Y134C, suggesting that these residues may delimit the transmembrane domain. None of the cysteine substitution mutants from 117 through 135 were sensitive to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) or MTSEA. However, treatment with MTSEA increased 5-hydroxytryptamine transport by A116C. Activation of A116C by MTSEA was observed only in mutants containing Cys to Ile mutation at position 357, suggesting that modification of Cys-116 activated transport by compensating for a disruption in transport in response to Cys-357 replacement. The reactivity of A116C toward MTSEA was substantially increased in the presence of substrates but not inhibitors. This increase required Na⁺ and Cl^–, and was likely to result from conformational changes during the transport process.

Received for publication, November 7, 2003 , and in revised form, February 23, 2004.

^* This work was supported by grants from the National Institute on Drug Abuse (to G. R.). 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.

Present address: National Research Institute of Brewing, Kagamiyama 3-7-1, Higashihiroshima, Japan.

Present address: Laboratory of Molecular Biology, NINDS, National Institutes of Health, 36 Convent Dr., Bethesda, MD 20892.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, Yale University School of Medicine, 333 Cedar St., P. O. Box 3333, New Haven, CT 06510. Tel.: 203-785-4548; Fax: 203-737-2027; E-mail: gary.rudnick{at}yale.edu.

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