Protein kinase C-mediated phosphorylation and functional regulation of dopamine transporters in striatal synaptosomes

Dopamine transporters (DATs) are members of a family of Na+- and Cl--dependent neurotransmitter transporters responsible for the rapid clearance of dopamine from synaptic clefts, The predicted primary sequence of DAT contains numerous consensus phosphorylation sites, In this report we demonstrate that DATs undergo endogenous phosphorylation in striatal synaptosomes that is regulated by activators of protein kinase C. Rat striatal synaptosomes were metabolically labeled with [P-32]orthophosphate, and solubilized homogenates were subjected to immunoprecipitation with am antiserum specific for DAT, Basal phosphorylation occurred in the absence of exogenous treatments, and the phosphorylation level was rapidly increased when synaptosomes were treated with the phosphatase inhibitors okadaic acid or calyculin, Treatment of synaptosomes with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also increased the level of phosphate incorporation. This occurred within 10 min and was dose-dependent between 0.1 and 1 mu M PMA, DAT phosphorylation was also significantly increased by two other protein kinase C activators, (-)-indolactam V and 1-oleoyl-2-acetyl sn glycerol. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate at 10 mu M was without effect, and PMA-induced phosphorylation was blocked by treatment of synaptosomes with the protein kinase C inhibitors staurosporine and bisindoylmaleimide. These results indicate that DATs undergo rapid in vivo phosphorylation in response to protein kinase C activation and that a robust mechanism exists in synaptosomes for DAT dephosphorylation, Dopamine transport activity in synaptosomes was reduced by all treatments that promoted DAT phosphorylation, with comparable dose, time, and inhibitor characteristics. The change in transport activity was produced by a reduction in V-max with no significant effect on the K-m for dopamine, These results suggest that synaptosomal dopamine transport activity is regulated by phosphorylation of DAT and present a potential mechanism for local neuronal control of synaptic neurotransmitter levels and consequent downstream neural activity.