Translocation of dopamine and binding of WIN 35,428 measured under identical conditions in cells expressing the cloned human dopamine transporter.

Abstract	Translocation of [3H]dopamine and binding of [3H]WIN 35,428 were measured in intact C6 glioma cells expressing the cloned human dopamine transporter (hDAT) under identical conditions of assay buffer (phosphate-Krebs) and temperature (25 degrees C) with uptake at initial velocity and binding at equilibrium. In the intact cells, [3H]dopamine uptake was a one-component process; in contrast, [3H]WIN 35,428 binding included both a high-affinity component, inhibitable by micromolar concentrations of dopamine, and a low-affinity component only partially inhibited by millimolar concentrations of dopamine. Binding (high-affinity) over uptake Ki ratios were on the average 2.3 for the inhibitors WIN 35,428, cocaine, GBR 12909, and BTCP. The potency of dopamine in inhibiting its own translocation was close to that in inhibiting [3H]WIN 35,428 binding consonant with a more rapid reorientation step of the DAT in the C6-hDAT system than in rat striatal synaptosomes. The similarity in turnover values of the DAT estimated in the current experiments with the C6-hDAT system and in our previous study on rat striatal synaptosomes, performed under comparable conditions, suggest that all DAT's inserted into the C6 cell membrane are functionally active.
Authors	M E Reith, C Xu, L Zhang, L L Coffey
Journal	Naunyn-Schmiedeberg's archives of pharmacology (Naunyn Schmiedebergs Arch Pharmacol) 1996 Aug-Sep Vol. 354 Issue 3 Pg. 295-304 ISSN: 0028-1298 [Print] Germany
PMID	8878059 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
Chemical References	Carrier Proteins Dopamine Plasma Membrane Transport Proteins Dopamine Uptake Inhibitors Membrane Glycoproteins Membrane Transport Proteins Nerve Tissue Proteins SLC6A3 protein, human Slc6a3 protein, rat (1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester Cocaine Dopamine
Topics	Analysis of Variance Animals Binding Sites Binding, Competitive Carrier Proteins (metabolism) Cells, Cultured (drug effects, metabolism) Cocaine (analogs & derivatives, antagonists & inhibitors, metabolism) Dopamine (pharmacokinetics) Dopamine Plasma Membrane Transport Proteins Dopamine Uptake Inhibitors (metabolism) Humans Membrane Glycoproteins Membrane Transport Proteins Nerve Tissue Proteins Rats

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