Activation of the extraneuronal monoamine transporter (EMT) from rat expressed in 293 cells

1 The extraneuronal monoamine transporter from rat (EMTr) was heterologously expressed by stable transfection in human embryonic kidney 293 cells and characterized in radiotracer experiments. 2 EMTr-mediated uptake of 1-methyl-4-phenylpyridinium (MPP+) was saturable, with a K-m of 151 mumol l(-1) and V-max of 7.5 nmol min(-1) mg protein(-1). 3 Compared to the human orthologue EMTh (gene symbol SLC22A3), EMTr was about two orders of magnitude more resistant to most inhibitors, including disprocynium24 and corticosterone. 4 Strikingly, inhibitors and substrates at low concentration stimulated EMTr-mediated transport above control level with MPP+ and noradrenaline as substrate, but not with cimetidine. Results were confirmed with EMT from mouse. 5 With different IC50-values for different substrates, the standard method to calculate K-i-values is not applicable. 6 Our experiments suggest that activation is not caused by changes in membrane potential or trans-stimulation. Since the extent of activation depends markedly on the chemical structure of the monitored substrate, involvement of a receptor-mediated signalling pathway or recruitment of transporter reserve are implausible. 7 To explain activation, we present a kinetic model which assumes two binding sites for substrate or inhibitor per transporter entity, possibly resulting from the assembly of homodimers. 8 Activation explains previous reports about inhibitor-insensitive catecholamine transport in rat brain. 9 We speculate that activation may serve to keep the transporter working for specific substrates in the face of inhibitors.