Abstract

Presynaptic dopamine D₂ receptors (D₂Rs) regulate dopamine transporter (DAT) activity in the brain. A potential mechanism was suggested by the observations that somatodendritic D₂R activation produces hyperpolarization and the velocity of DAT expressed in Xenopus laevis oocytes varies with changes in membrane potential. To investigate whether D₂R regulation of DAT function is voltage-dependent, we coexpressed the long isoform of the human (h) D₂R and the hDAT in oocytes. Most DAT substrates fully activate D₂Rs at concentrations used to measure uptake. Thus, DAT function was compared under conditions of maximal D₂R activation (0.1–10 μM DA) or maximal D₂R blockade (DA + 1 μM (−)-sulpiride). D₂R activation significantly increased [³H]DA uptake into unclamped oocytes expressing relatively lower velocities. Uptake measured with a saturating concentration of DA suggested a D₂R-induced increase in V _max. The D₂R-mediated enhancement of DA uptake was not associated with changes in resting membrane potential and was abolished by pertussis toxin pretreatment. Furthermore, in voltage-clamped oocytes, D₂R activation enhanced both DA uptake and DAT-mediated steady-state currents by as much as 70%. Activation of D₂Rs resulted in a 59% increase in cell surface binding of the cocaine analog [³H]WIN 35,428; this effect was also abolished by pertussis toxin pretreatment. Saturation experiments confirmed that D₂R activation was associated with an increased B _max and unchangedK _i for [³H]WIN 35,428. These results suggest that D₂R-induced up-regulation of DAT activity occurs via a voltage-independent mechanism that depends on G_i/o activation and a rapid increase in expression of functional DAT molecules at the cell surface.