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J. Biol. Chem., Vol. 282, Issue 12, 8915-8925, March 23, 2007

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Localization of Cocaine Analog [¹²⁵I]RTI 82 Irreversible Binding to Transmembrane Domain 6 of the Dopamine Transporter^*

Roxanne A. Vaughan¹, Dhananjay S. Sakrikar, M. Laura Parnas, Steven Adkins, James D. Foster, Romain A. Duval, John R. Lever, Santosh S. Kulkarni^¶, and Amy Hauck-Newman^¶

From the Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203-9037, the Departments of Radiology, and Medical Pharmacology and Physiology, University of Missouri and the Harry S. Truman Veterans Administration Medical Center, Columbia, Missouri 65212, and the ^¶Medicinal Chemistry Section, Intramural Research Program, National Institute on Drug Abuse, Bethesda, Maryland 21224

The site of cocaine binding on the dopamine transporter (DAT) was investigated using the photoactivatable irreversible cocaine analog [¹²⁵I]3-(p-chlorophenyl)tropane-2-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([¹²⁵I]RTI 82). The incorporation site of this compound was mapped to transmembrane domains (TMs) 4–6 using epitope-specific immunoprecipitation of trypsin fragments and further localized using cyanogen bromide (CNBr), which hydrolyzes proteins on the C-terminal side of methionine residues. CNBr hydrolysis of [¹²⁵I]RTI 82-labeled rat striatal and expressed human DATs produced fragments of 5–10 kDa consistent with labeling between Met^271/272 or Met²⁹⁰ in TM5 to Met^370/371 in TM7. To further define the incorporation site, substitution mutations were made that removed endogenous methionines and inserted exogenous methionines in combinations that would generate labeled CNBr fragments of distinct masses depending on the labeling site. The results obtained were consistent with the presence of TM6 but not TMs 4, 5, or 7 in the labeled fragments, with additional support for these conclusions obtained by epitope-specific immunoprecipitation and secondary digestion of CNBr fragments with endoproteinase Lys-C. The final localization of [¹²⁵I]RTI 82 incorporation to rat DAT Met²⁹⁰–Lys³³⁶ and human DAT I291M to R344M provides positive evidence for the proximity of cocaine binding to TM6. Residues in and near DAT TM6 regulate transport and transport-dependent conformational states, and TM6 forms part of the substrate permeation pathway in the homologous Aquifex aeolicus leucine transporter. Cocaine binding near TM6 may thus overlap the dopamine translocation pathway and function to inhibit TM6 structural rearrangements necessary for transport.

Received for publication, November 16, 2006 , and in revised form, January 8, 2007.

^* This work was supported by National Institute on Drug Abuse DA15175 (to R. A. V.), North Dakota EPSCoR Doctoral Dissertation Assistantship (to M. L. P.), P20 RRO 16741 from the IDeA Network of Biomedical Research Excellence Program of the National Center for Research Resources, and the National Institute on Drug Abuse-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: Dept. of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, 501 N. Columbia Road, Grand Forks, ND 58203-9037. Tel.: 701-777-3419; Fax: 701-777-2382; E-mail: rvaughan{at}medicine.nodak.edu.

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