1-Methyl-4-phenylpyridinium-induced down-regulation of dopamine transporter function correlates with a reduction in dopamine transporter cell surface expression

Abstract

The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP+) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP+-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP+produced a concentration- and time-dependent reduction in the uptake of [3H]dopamine. We observed a significant decrease in [3H]WIN 35428 binding in the intact cells with MPP+. The saturation analysis of the [3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (Bmax) with an increase in the dissociation equilibrium constant (Kd) after MPP+treatment. Furthermore, biotinylation assays confirmed that MPP+reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP+may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity.