Peroxynitrite inactivates the human dopamine transporter by modification of cysteine 342: potential mechanism of neurotoxicity in dopamine neurons.
Peroxynitrite (ONOO(-)) has been implicated as a causative factor in dopamine neuronal damage resulting from exposure to methamphetamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and it may be involved in the etiology of Parkinson's Disease. ONOO(-) causes a concentration-dependent and irreversible reduction in dopamine uptake by EM4 cells stably expressing the human dopamine transporter (hDAT). The effect of ONOO(-) is manifested as a reduction in V(max). Cysteine, dithiothreitol, glutathione, and N-acetyl-cysteine, reagents that interact directly with ONOO(-), prevent this inhibition, whereas a scavenger of hydroxyl radical (dimethylsulfoxide), hydrogen peroxide (catalase), and superoxide (superoxide dismutase) did not. Dopamine in the extracellular medium protects the hDAT from ONOO(-), whereas intracellular dopamine does not. Parachloromercuribenzoic acid and 2-aminoethyl methanethiosulfonate (MTSEA), which share with ONOO(-) the ability to modify cysteine sulfhydryls, also inhibit hDAT function. ONOO(-) treatment lowers cysteine-specific labeling of the hDAT by MTSEA-biotin, suggesting that ONOO(-) reacts with one or more cysteines in hDAT. A mutant of hDAT (X7C) in which all intracellular and extracellular loop cysteines were mutated was resistant to inhibition by ONOO(-). Sensitivity to ONOO(-) was restored in mutants of hDAT in which reduced cysteines were present only in the first (C135) and third (C342) intracellular loops (CD-DAT), or in which C342 alone had been reintroduced into X7C (X7C-M342C). These results indicate that the hDAT is inhibited by ONOO(-) through oxidation of cysteine 342. Our studies also substantiate the possibility that drugs known to decrease DAT function in vivo (e.g., methamphetamine and MPTP) may exert their effects through ONOO(-)-mediated oxidative stress.
Park
Samuel U
SU
Department of Psychiatry, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, and The John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48201, USA.
Ferrer
Jasmine V
JV
Javitch
Jonathan A
JA
Kuhn
Donald M
DM
D000975:Antioxidants / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D001710:Biotin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002460:Cell Line; D003545:Cysteine / Q000187:drug effects* / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism* / Q000493:pharmacokinetics; D050483:Dopamine Plasma Membrane Transport Proteins; D004305:Dose-Response Relationship, Drug; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008562:Membrane Glycoproteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000187:drug effects*; D020258:Neurotoxicity Syndromes / Q000209:etiology; D030421:Peroxynitrous Acid / Q000378:metabolism / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014162:Transfection
D000975:Antioxidants; D050483:Dopamine Plasma Membrane Transport Proteins; D016166:Free Radical Scavengers; D008562:Membrane Glycoproteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; C419543:N-biotinylaminoethyl methanethiosulfonate; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; D013439:Sulfhydryl Reagents; D030421:Peroxynitrous Acid; D001710:Biotin; D003545:Cysteine; D004298:Dopamine