The dopamine transporter (DAT) clears dopamine from the synaptic cleft thus playing a rote in regulating dopaminergic neuroactivity. Consensus phosphorylation sites are present in the DAT primary sequence and protein kinase C activators regulate dopamine uptake activity. The possibility that direct phosphorylation of DAT occurs was examined using rat striatal brain tissue. Freshly dissected rat striatal slices were incubated with 32P-orthophosphate, solubilized, immunoprecipitated with an antipeptide DAT antibody, and analyzed by SDS-PAGE electrophoresis and autoradiography. The results show phosphorylation of DAT based on several criteria. An 80 kDa 32P-labeled protein was immunoprecipitated which exactly co-migrated on gels with photoaffinity-labeled DAT. The 32P-labeled band was not immunoprecipitated with preimmune serum, and immunoprecipitation of the protein was blocked by the immunizing but not an irrelevant peptide. This is the first reported demonstration of neurotransmitter transporter phosphorylation in brain tissue and presents the possibility that DAT function undergoes acute and/or chronic regulation resulting in fine-tuning of neuronal activity. This work was supported by the NIDA Intramural Research Program.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology