Modulation of dopamine transporter functions by protein kinase C

The effects of phosphorylation on the functions of the bovine dopamine transporter (DAT) expressed in CV-1 cells were investigated. The cells expressing DAT stably were prepared by transfection of CV-1 cells (monkey kidney cell line) with plasmid pRc/CMV-DAT which is an expression vector pRc/CMV-based plasmid containing bovine DAT cDNA (Usdin et al., 1991). DAT functional assays followed the preincubations of DAT expressing cells with various modulators of protein kinases, such as phorbol 12-myristrate 13-acetate (PMA), staurosphorin, calphostin C, forskolin, and EGTA, on the concentration- or time-dependent manners. Dopamine uptake into DAT expressing cells was severely decreased by PMA pretreatment, and increased by staurosphorin or calphostin C, Similar effects were observed on the uptakes of 1-methyl-4-phenylpyridinium (MPP(+)) into DAT-expressing cells. However, DAT functions were not altered by forskolin or EGTA, These results suggest that the functions of DAT are modulated by protein kinase C (PKC).