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Localization of cocaine analog [125I]RTI 82 irreversible binding to transmembrane domain 6 of the dopamine transporter.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2007 Mar 23; Vol. 282 (12), pp. 8915-25. Date of Electronic Publication: 2007 Jan 25. - Publication Year :
- 2007
-
Abstract
- The site of cocaine binding on the dopamine transporter (DAT) was investigated using the photoactivatable irreversible cocaine analog [125I]3beta-(p-chlorophenyl)tropane-2beta-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 approximately 5-10 kDa consistent with labeling between Met(271/272) or Met(290) 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 Met(290)-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.
- Subjects :
- Animals
Cocaine pharmacology
Cyanogen Bromide pharmacology
Dopamine Uptake Inhibitors pharmacology
Epitopes chemistry
Humans
Methionine chemistry
Protein Binding
Protein Structure, Tertiary
Protein Transport
Rats
Trypsin chemistry
Azides pharmacology
Cocaine analogs & derivatives
Dopamine Plasma Membrane Transport Proteins chemistry
Iodine Radioisotopes pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 282
- Issue :
- 12
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 17255098
- Full Text :
- https://doi.org/10.1074/jbc.M610633200