A cognate dopamine transporter-like activity endogenously expressed in a COS-7 kidney-derived cell line

The activity of the dopamine transporter is an important mechanism for the maintenance of normal dopaminergic homeostasis by rapidly removing dopamine from the synaptic cleft. In kidney-derived COS-7, COS-1 and HEK-293 but no t in other mammalian cell lines (CHO, Y1, Ltk(-)), we have characterized a putative functional dopamine transporter displaying a high affinity (K-m si milar to 250 nM) and a low capacity (similar to 0.1 pmol/10(5) cells/min) f or [H-3]dopamine uptake. Uptake displayed a pharmacological profile clearly indicative of the neuronal dopamine transporter, Estimated Ki values of nu merous substrates and inhibitors for the COS-dopamine transporter and the c loned human neuronal transporter (human dopamine transporter) correlate wel l with the exception of a few notable compounds, including the endogenous n eurotransmitter dopamine, the dopamine transporter inhibitor GBR 12,909 and the dopaminergic agonist apomorphine, As with native neuronal and cloned d opamine transporters, the uptake velocity was sodium-sensitive and reduced by phorbol ester pre-treatment, Two mRNA species of 3.8 and 4.0 kb in COS-7 cells were revealed by Northern blot analysis similar in size to that seen in native neuronal tissue, A reverse-transcribed PCR analysis confirmed th e existence of a processed dopamine transporter, However, no immunoreactive proteins of expected dopamine transporter molecular size or [H-3]WIN 35,42 8 binding activity were detected. A partial cDNA of similar to 1.3 kb, isol ated from a COS-1 cDNA library and encoding transmembrane domains 1-6, disp layed a deduced amino acid sequence homology of similar to 96% to the human dopamine transporter, Taken together, the data suggest the existence of a non-neuronal endogenous high affinity dopamine uptake system sharing strong functional and molecular homology to that of the cloned neuronal dopamine transporter. (C) 1999 Federation of European Biochemical Societies.