The dopamine transporter (DAT) has been identified as the principal brain receptor site best correlated with the rewarding and euphoric properties of cocaine. Euphoric responses to rapid administration of cocaine can be much more prominent than those that follow slower rates of administration. In previous FYs, investigators in this Branch have found that activators of protein kinase C (PKC) modulate dopamine transport in transiently-expressing COS cells. In this FY, we have identified the DAT as a phosphoprotein. We found evidence for rapid phosphorylation/ dephosphorylation cycling in expressing cell lines and in brain synaptosomes. Phosphorylation displays many parallels to functional reductions in DAT Vmax values identified in parallel experiments, and occurs largely on serine residues. Studies of site directed mutants in potential phosphoacceptor sites are beginning to identify the involved residues. These data increase evidence that PKC activation enhances levels of DAT phosphorylation, and makes phosphorylation an increasingly-strong candidate mechanism for rapid adaptations in dopaminergic systems relevant to cocaine-induced euphoria.
