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J. Biol. Chem., Vol. 280, Issue 35, 30807-30813, September 2, 2005

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Cysteine-scanning Mutagenesis of Serotonin Transporter Intracellular Loop 2 Suggests an -Helical Conformation^*

Yuan-Wei Zhang and Gary Rudnick

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

Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2-carbomethoxy-3-(4-[¹²⁵I]iodophenyl)tropane (-CIT) in intact cells expressing these mutants, but it inactivated -CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an -helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity.

Received for publication, April 14, 2005 , and in revised form, June 30, 2005.

^* This work was supported by grants from the National Institute on Drug Abuse. 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.

To whom correspondence should be addressed: Dept. of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520-8066. Tel.: 203-785-4548; Fax: 203-785-7670; E-mail: gary.rudnick{at}yale.edu.

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