Localization of cocaine analog [125I]RTI 82 irreversible binding to transmembrane domain 6 of the dopamine transporter

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

Research output: Contribution to journalArticlepeer-review

Abstract

The site of cocaine binding on the dopamine transporter (DAT) was investigated using the photoactivatable irreversible cocaine analog [ 125I]3β-(p-chlorophenyl)tropane-2β-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 Met271/272 or Met290 in TM5 to Met370/371 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 TMs4, 5, or 7 in the labeled fragments, with additional support for these conclusions obtained by epitopespecific immunoprecipitation and secondary digestion of CNBr fragments with endoproteinase Lys-C. The final localization of [125I]RTI 82 incorporation to rat DAT Met290-Lys 336 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.

Original languageEnglish (US)
Pages (from-to)8915-8925
Number of pages11
JournalJournal of Biological Chemistry
Volume282
Issue number12
DOIs
StatePublished - Mar 23 2007
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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