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 d opamine transport in C6 glioma cells stably expressing the human dopam ine transporter. The PKC activating phorbol esters phorbol 12-myristat e 13-acetate (PMA) and 4 beta-12,13-dibutyrate phorbol-ester (PDBu) in hibited [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 okad aic acid. The potency of PMA in inhibiting [H-3]dopamine uptake was si milar to that in inhibiting the binding of 2 beta-carbomethoxy-3 beta- (4-fluorophenyl)tropane ([H-3]WIN 35,428), and again staurosporine, bu t not chelerythrine, weakened the effect of PMA. The reduction in dopa mine 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 ca used by a reduction in the B-max value without a change in the Kd valu e. The lower K-m value in the presence of PMA was accompanied by a hig her IC50 of dopamine in inhibiting [H-3]WIN 35,428 binding; the latter effect was attenuated by the co-presence of staurosporine. The result s are discussed in the context of transporter loss from the cell surfa ce, or a model with phosphorylation affecting the shared dopamine and WIN 35,428 binding domain on the transporter as well as affecting a pa rt of the dopamine binding domain lying outside that for WIN 35,428. ( C) 1997 Elsevier Science Inc.