Dopamine D-2 receptor regulation of the dopamine transporter expressed in Xenopus laevis oocytes is voltage-independent

Presynaptic dopamine D-2 receptors (D(2)Rs) 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 D(2)Rs at concentrations used to measure uptake. Thus, DAT function was compared under conditions of maximal D2R activation (0.1-10 muM DA) or maximal D2R blockade (DA + 1 muM (2)-sulpiride). D2R activation significantly increased [H-3] 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 D(2)Rs resulted in a 59% increase in cell surface binding of the cocaine analog [H-3] 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 unchanged K-i for [H-3] 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.