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The Journal of Neuroscience, June 1, 2002, 22(11):4399-4405
Peroxynitrite Inactivates the Human Dopamine Transporter by
Modification of Cysteine 342: Potential Mechanism of Neurotoxicity in
Dopamine Neurons
Samuel U.
Park1,
Jasmine V.
Ferrer4, 5,
Jonathan A.
Javitch4, 5, and
Donald M.
Kuhn1, 2, 3
1 Department of Psychiatry and Behavioral
Neurosciences, 2 Center for Molecular Medicine and
Genetics, Wayne State University School of Medicine, and
3 The John D. Dingell Veterans Affairs Medical
Center, Detroit, Michigan 48201, and 4 Departments of
Pharmacology and Psychiatry and 5 Center for Molecular
Recognition, Columbia University College of Physicians and Surgeons,
New York, New York 10032
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
Vmax. 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.
Key words:
dopamine transporter; peroxynitrite; cysteine
residues; dopamine; Parkinson's disease; neurotoxic amphetamines
Copyright © 2002 Society for Neuroscience 0270-6474/02/22114399-07$05.00/0
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