The dopamine transporter (DAT) has been identified as a principal brain receptor site previously 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 years, investigators in this Branch have found that activators of protein kinase C (PKC) modulate dopamine transport in transiently-expressing COS cells. In the current year, we have followed previous observations that identified DAT as a phosphoprotein to identify efects of several PKC, MAP kinase, MEKkinase and IP3 kinase agents on dopamine upatke rates. Studies of site directed mutants in potential phosphoacceptor sites identify N-terminal mutants as important for MAP kinase, IP3 kinase and for PKC regulation. These studies document that the DAT regulation can occur as internalization of the tranpsorter or as downregulation of the function of the transporter that remains expressed on cell surfaces, using activation of distince kinase pthways. Coexpression with KEPI also alters DAT function. These data increase evidence that PKC and MAP kinase regulation of DAT occurs through multiple direct- and indirect mechanisms. Work during this year has also provided insights into functions of the human allelic variants in the DAT 5' flanking region. Human variants and haplotypes from this region have beeen identified by genomic resequencing. Large constructions containing a number of variants in each of the two most prominent haplotypes have been expressed in two cell expression systems as luciferase fusion constructions. Interestingly, these results provide evidence for a 30-50% haplotype-dependent difference in levels of DAT expression. In vivo PET and postmortem studies appear to confirm these observations in vivo. This work appears to complement studies of regulation at a protein phospphorylation level to provide a more complete picture of the state- and trait-dependent features of regulation of this important determinant of dopaminergic functions including those important for drug reward.
