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--d ependent neurotransmitter transporters responsible for the rapid clear ance of dopamine from synaptic clefts, The predicted primary sequence of DAT contains numerous consensus phosphorylation sites, In this repo rt we demonstrate that DATs undergo endogenous phosphorylation in stri atal synaptosomes that is regulated by activators of protein kinase C. Rat striatal synaptosomes were metabolically labeled with [P-32]ortho phosphate, and solubilized homogenates were subjected to immunoprecipi tation with am antiserum specific for DAT, Basal phosphorylation occur red in the absence of exogenous treatments, and the phosphorylation le vel was rapidly increased when synaptosomes were treated with the phos phatase inhibitors okadaic acid or calyculin, Treatment of synaptosome s with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also increased the level of phosphate incorporation. This occurr ed within 10 min and was dose-dependent between 0.1 and 1 mu M PMA, DA T phosphorylation was also significantly increased by two other protei n kinase C activators, (-)-indolactam V and 1-oleoyl-2-acetyl sn glyce rol. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate at 1 0 mu M was without effect, and PMA-induced phosphorylation was blocked by treatment of synaptosomes with the protein kinase C inhibitors sta urosporine and bisindoylmaleimide. These results indicate that DATs un dergo rapid in vivo phosphorylation in response to protein kinase C ac tivation and that a robust mechanism exists in synaptosomes for DAT de phosphorylation, Dopamine transport activity in synaptosomes was reduc ed by all treatments that promoted DAT phosphorylation, with comparabl e dose, time, and inhibitor characteristics. The change in transport a ctivity was produced by a reduction in V-max with no significant effec t on the K-m for dopamine, These results suggest that synaptosomal dop amine transport activity is regulated by phosphorylation of DAT and pr esent a potential mechanism for local neuronal control of synaptic neu rotransmitter levels and consequent downstream neural activity.