TRANSLOCATION OF DOPAMINE AND BINDING OF WIN-35,428 MEASURED UNDER IDENTICAL CONDITIONS IN CELLS EXPRESSING THE CLONED HUMAN DOPAMINE TRANSPORTER

Translocation of [H-3]dopamine and binding of [H-3]WIN 35, 428 were me asured in intact C6 glioma cells expressing the cloned human dopamine transporter (hDAT) under identical conditions of assay buffer (phospha te-Krebs) and temperature (25 degrees C) with uptake at initial veloci ty and binding at equilibrium. In the intact cells, [H-3]dopamine upta ke was a one-component process; in contrast, [H-3]WIN 35,428 binding i ncluded both a high-affinity component, inhibitable by micromolar conc entrations of dopamine, and a low-affinity component only partially in hibited by millimolar concentrations of dopamine. Binding (high-affini ty) over uptake K-i ratios were on the average 2.3 for the inhibitors WIN 35,428, cocaine, CBR 12909, and BTCP. The potency of dopamine in i nhibiting its own translocation was close to that in inhibiting [H-3]W IN 35, 428 binding consonant with a more rapid reorientation step of t he DAT in the C6-hDAT system than in rat striatal synaptosomes. The si milarity in turnover values of the DAT estimated in the current experi ments with the C6-hDAT system and in our previous study on rat striata l synaptosomes, performed under comparable conditions, suggest that al l DAT's inserted into the C6 cell membrane are functionally active.