Hydrogen peroxide induces loss of dopamine transporter activity: a calcium-dependent oxidative mechanism

H-2 O-2 dose dependently inhibited dopamine uptake in PC12 cells and in striatal synaptosomes. Treatment with H-2 O-2 resulted in a reversible reduction in V (max) , with no effect on its K (m) value. This suppressive effect of H-2 O-2 could be relieved by reducing agents (dithiothreitol and cysteine). Furthermore, an oxidizer (dithiodipyridine) also markedly suppressed the dopamine transporter (DAT). Oxidative stress therefore might contribute to the action of H-2 O-2 . H-2 O-2 appeared to modify DAT at both extracellular and intracellular sites because cumene-H-2 O-2 (a radical generator mostly restricted to plasma membranes) at high concentrations also slightly suppressed DAT activity and the intracellular overexpression of catalase ameliorated the inhibitory effect of H-2 O-2 . Internalization was unlikely to be involved because concanavalin A, which blocked endocytosis, did not prevent the H-2 O-2 -evoked inhibition of DAT activity. Interestingly, H-2 O-2 treatment evoked a Ca2+ influx in PC12 cells. Moreover, removal of external calcium by EGTA or reduction in the intracellular calcium level using BAPTA-AM reversed the inhibitory effect of H-2 O-2 . Conversely, depletion of intracellular calcium stores using thapsigargin did not affect the reduction in DAT activity by H-2 O-2 . Collectively, our results indicate that the DAT, one of the most important proteins controlling the dopaminergic system, is also a redox sensor. In addition, H-2 O-2 might suppress the DAT by a Ca2+ -dependent oxidative pathway.