Estradiol effects on the dopamine transporter - Protein levels, subcellular location, and function

Background: The effects of estrogens on dopamine (DA) transport may have important implications for the increased incidence of neurological disorders in women during life stages when hormonal fluctuations are prevalent, e.g. during menarche, reproductive cycling, pregnancy, and peri-menopause. Results: The activity of the DA transporter (DAT) was measured by the specific uptake of ³H-DA. We found that low concentrations (10^-14 to 10^-8M) of 17β-estradiol (E₂) inhibit uptake via the DAT in PC12 cells over 30 minutes, with significant inhibition taking place due to E₂ exposure during only the last five minutes of the uptake period. Such rapid action suggests a non-genomic, membrane-initiated estrogenic response mechanism. DAT and estrogen receptor-α (ERα) were elevated in cell extracts by a 20 ng/ml 2 day NGFβ treatment, while ERβ was not. DAT, ERα and ERβ were also detectable on the plasma membrane of unpermeabilized cells by immunocytochemical staining and by a fixed cell, quantitative antibody (Ab)-based plate assay. In addition, PC12 cells contained RNA coding for the alternative membrane ER GPR30; therefore, all 3 ER subtypes are candidates for mediating the rapid nongenomic actions of E₂. At cell densities above 15,000 cells per well, the E₂-induced inhibition of transport was reversed. Uptake activity oscillated with time after a 10 nM E₂ treatment; in a slower room temperature assay, inhibition peaked at 9 min, while uptake activity increased at 3 and 20-30 min. Using an Ab recognizing the second extracellular loop of DAT (accessible only on the outside of unpermeabilized cells), our immunoassay measured membrane vs. intracellular/nonvesicular DAT; both were found to decline over a 5-60 min E₂ treatment, though immunoblot analyses demonstrated no total cellular loss of protein. Conclusion: Our results suggest that physiological levels of E₂ may act to sequester DAT in intracellular compartments where the transporter's second extramembrane loop is inaccessible (inside vesicles) and that rapid estrogenic actions on this differentiated neuronal cell type may be regulated via membrane ERs of several types.