Regulation of the functional activity of the human dopamine transporter by protein kinase C

The role of protein kinase C (PKC) was examined in the regulation of dopamine transport in C6 glioma cells stably expressing the human dopamine transporter. The PKC activating phorbol esters phorbol 12-myristate 13-acetate (PMA) and 4 beta-12,13-dibutyrate phorbol-ester (PDBu) inhibited [H-3]dopamine uptake concentration dependently. These effects were attenuated by the PKC inhibitor staurosporine but were unaltered by another inhibitor, chelerythrine, or the phosphatase inhibitor okadaic acid. The potency of PMA in inhibiting [H-3]dopamine uptake was similar to that in inhibiting the binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane ([H-3]WIN 35,428), and again staurosporine, but not chelerythrine, weakened the effect of PMA. The reduction in dopamine transporter activity by PMA was caused by a decrease in the V-max value of [H-3]dopamine uptake, opposed by a smaller reduction in the K-m value, whereas the effect of PMA on [H-3]WIN 35,428 binding was caused by a reduction in the B-max value without a change in the Kd value. The lower K-m value in the presence of PMA was accompanied by a higher IC50 of dopamine in inhibiting [H-3]WIN 35,428 binding; the latter effect was attenuated by the co-presence of staurosporine. The results are discussed in the context of transporter loss from the cell surface, or a model with phosphorylation affecting the shared dopamine and WIN 35,428 binding domain on the transporter as well as affecting a part of the dopamine binding domain lying outside that for WIN 35,428. (C) 1997 Elsevier Science Inc.