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 obse rvations that somatodendritic D2R activation produces hyperpolarization and the velocity of DAT expressed in Xenopus laevis oocytes varies with change s in membrane potential. To investigate whether D2R regulation of DAT funct ion 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 c oncentrations used to measure uptake. Thus, DAT function was compared under conditions of maximal D2R activation (0.1-10 muM DA) or maximal D2R blocka de (DA + 1 muM (2)-sulpiride). D2R activation significantly increased [H-3] DA uptake into unclamped oocytes expressing relatively lower velocities. U ptake measured with a saturating concentration of DA suggested a D2R-induce d increase in V-max. The D2R-mediated enhancement of DA uptake was not asso ciated with changes in resting membrane potential and was abolished by pert ussis toxin pretreatment. Furthermore, in voltage-clamped oocytes, D2R acti vation 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 surfa ce binding of the cocaine analog [H-3] WIN 35,428; this effect was also abo lished by pertussis toxin pretreatment. Saturation experiments confirmed th at 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 mole cules at the cell surface.