Peroxynitrite Inactivates the Human Dopamine Transporter by Modification of Cysteine 342: Potential Mechanism of Neurotoxicity in Dopamine Neurons

Abstract

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.