Dopamine transporter regulation by amphetamine: Phosphorylation and trafficking events.

Abstract

The dopamine transporter (DAT), the site of action of the psychostimulant drug amphetamine (AMPH), undergoes highly dynamic and complex regulation. There is extensive investigation of the compensatory effects of persistent amphetamine administration and protein kinase C on the down-regulation of the influx function of DAT. However, we find that amphetamine and protein kinase C have rapid and robust effects on the efflux function of DAT. This thesis describes the effects of amphetamine and protein kinase C on rapid DAT trafficking and DAT-mediated efflux of dopamine. In the first study, using classical pharmacological inhibitors, I determined that classical PKC isozymes are essential for AMPH-stimulated DA efflux. A highly selective inhibitor permitted identification of the important isoform as being PKCbeta. Using hDAT HEK cells that transiently overexpress either PKCalpha, PKCbeta_I, or PKCbeta_II, it was determined that overexpression of PKCbeta_II potentiated AMPH-stimulated DA efflux. In the second study, the AMPH-elicited rapid effects (<1min) on DAT trafficking was examined. Using biotinylation methods, I determined that AMPH recruits DAT to the cell surface within 30s. This increase in surface DAT corresponds to an increase in AMPH-stimulated DA efflux with no change in [<super>3</super>H]DA uptake. The AMPH-stimulated increase in cell surface DAT appears to be dependent on PKCbeta activity. In the third study, using a mutant DAT in which the first 22 N-terminal amino acids were truncated, I determined that deletion of the N-terminal of DAT results in an 80% loss in AMPH-stimulated efflux without impairing [<super> 3</super>H]DA uptake when compared to wildtype. A collaborative effort with Dr. Jonathan Javitch, Columbia University, demonstrated that phosphorylation of N-terminal serines was crucial for efflux. These results establish there are separate structural determinants for uptake <italic>versus</italic> efflux functions of DAT and that the regulation is through phosphorylation. In summary, the work in this thesis has delineated the isoform of protein kinase C that regulates AMPH-stimulated DA efflux, proven that there are separate structural determinants in the protein regulating inward and outward efflux and made the original observation that AMPH elicits rapid functional effects on trafficking of DAT. These findings are important for DA- and DAT-related pathologies such as schizophrenia and substance abuse.