The roles of membrane estrogen receptor subtypes in modulating dopamine transporters in PC-12 cells

The effects of 17β-estradiol (E₂) on dopamine (DA) transport could explain gender and life-stage differences in the incidence of some neurological disorders. We tested the effects of E₂ at physiological concentrations on DA efflux in nerve growth factor-differentiated rat pheochromocytoma cells that express estrogen receptors (ER) α, ERβ, and G-protein coupled receptor 30 (GPR30), and DA transporter (DAT). DAT efflux was determined as the transporter-specific loss of ³H-DA from pre-loaded cells; a 9-15 min 10^-9 M E₂ treatment caused maximal DA efflux. Such rapid estrogenic action suggests a non-genomic response, and an E₂-dendrimer conjugate (limited to non-nuclear actions) caused DA efflux within 5 min. Efflux dose-responses for E₂ were non-monotonic, also characteristic of non-genomic estrogenic actions. ERα siRNA knockdown abolished E₂-mediated DA efflux, while ERβ knockdown did not, and GPR30 knockdown increased E₂-mediated DA efflux (suggesting GPR30 is inhibitory). Use of ER-selective agonists/antagonists demonstrated that ERα is the predominant mediator of E₂-mediated DA efflux, with inhibitory contributions from GPR30 and ERβ. E₂ also caused trafficking of ERα to the plasma membrane, trafficking of ERβ away from the plasma membrane, and unchanged membrane GPR30 levels. Therefore, ERα is largely responsible for non-genomic estrogenic effects on DAT activity.