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Originally published In Press as doi:10.1074/jbc.M008483200 on October 31, 2000

J. Biol. Chem., Vol. 276, Issue 6, 4038-4045, February 9, 2001
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Functional Role of Critical Stripe Residues in Transmembrane Span 7 of the Serotonin Transporter
EFFECTS OF Na+, Li+, AND METHANETHIOSULFONATE REAGENTS*

Gunjan Kamdar, Kendall M. Y. Penado, Gary Rudnick, and Megan M. StephanDagger

From the Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510

Mutations at critical residue positions in transmembrane span 7 (TM7) of the serotonin transporter affect the Na+ dependence of transport. It was possible that these residues, which form a stripe along one side of the predicted alpha -helix, formed part of a water-filled pore for Na+. We tested whether cysteine substitutions in TM7 were accessible to hydrophilic, membrane-impermeant methanethiosulfonate (MTS) reagents. Although all five cysteine-containing mutants tested were sensitive to these reagents, noncysteine control mutants at the same positions were in most cases equally sensitive. In all cases, MTS sensitivity could be traced to changes in accessibility of a native cysteine residue in extracellular loop 1, Cys-109. Moreover, none of the TM7 cysteines reacted with the biotinylating reagent MTSEA-biotin when tested in the C109A background. It is thus unlikely that the critical stripe forms part of a water-filled pore. Instead, studies of the ion dependence of the reaction between Cys-109 and MTS reagents lead to the conclusion that TM7 is involved in propagating conformational changes caused by ion binding, perhaps as part of the translocation mechanism. The critical stripe residues on TM7 probably represent a close contact region between TM7 and one or more other TMs in the transporter's three-dimensional structure.

* This work was supported by a grant from the National Institute on Drug Abuse (to M. M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Tel.: 203-737-1601; Fax: 203-785-7670; E-mail: megan. stephan{at}yale.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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