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

The effects of 17 beta-estradiol (E-2) on dopamine (DA) transport could explain gender and life-stage differences in the incidence of some neurological disorders. We tested the effects of E-2 at physiological concentrations on DA efflux in nerve growth factor-differentiated rat pheochromocytoma cells that express estrogen receptors (ER) alpha, ER beta, and G-protein coupled receptor 30 (GPR30), and DA transporter (DAT). DAT efflux was determined as the transporter-specific loss of H-3-DA from pre-loaded cells; a 9-15 min 10(-9) M E-2 treatment caused maximal DA efflux. Such rapid estrogenic action suggests a non-genomic response, and an E-2-dendrimer conjugate (limited to non-nuclear actions) caused DA efflux within 5 min. Efflux dose-responses for E-2 were non-monotonic, also characteristic of non-genomic estrogenic actions. ER alpha siRNA knockdown abolished E-2-mediated DA efflux, while ER beta knockdown did not, and GPR30 knockdown increased E-2-mediated DA efflux (suggesting GPR30 is inhibitory). Use of ER-selective agonists/antagonists demonstrated that ER alpha is the predominant mediator of E-2-mediated DA efflux, with inhibitory contributions from GPR30 and ER beta. E-2 also caused trafficking of ER alpha to the plasma membrane, trafficking of ER beta away from the plasma membrane, and unchanged membrane GPR30 levels. Therefore, ER alpha is largely responsible for non-genomic estrogenic effects on DAT activity.