Cell and molecular biology of dopamine transporter
Dopamine (DA) plays an important role in brain reward, both to natural reinforcers and addictive drugs. Removal of DA from the extracellular space and its transport back into DA neurons is an important mechanism controlling DA neurotransmission. This removal occurs via the DAT. DAT plays important roles in psychomotor stimu¬lant behav¬ioral activation and reward. By understanding how DAT activity is regulated, we will better appre¬ci¬ate its contribution to normal neurotransmission and to brain diseases like drug addic¬tion. Our current research is aimed at characterization of the mechanisms of endocytosis and intracellular trafficking of DAT. One set of projects involves structure-function studies of heterologously-expressed human DAT. We have performed extensive analysis of the molecular mechanisms of DAT endocytosis induced by PKC activation and identified key players involved. Our immediate goal is to elucidate the molecular mechanisms of PKC-dependent endocytosis and also elucidate the mechanisms that control constitutive PKC-independent endocytosis. The main focus in the nest several years will be on the development of in vitro and in vivo experimental models to study trafficking of endogenous DAT in dopaminergic neurons. To this end, we are working in two independent tracks. First, we are developing and characterizing midbrain-striatal organotypic cultures prepared from post-natal rats, and developing the reagents that can be used to manipulate DAT trafficking in these cultures. In the same time, we have generated the knock-in mice with epitope-tagged DAT that should allow quantitative analysis of DAT trafficking in vitro and in vivo in neurons obtained from these mice. Finally, the data obtained using mechanistic analyses will be further developed in experiments with the intact animals to analyze how changes in DAT trafficking at the synapse correlate with the behavior patterns and response of the drugs of abuse.
Vina-Vilaseca, A., and Sorkin, A. ¬Lysine63-linked polyubiquitination of the dopamine transporter requires WW3 and WW4 domains of Nedd4-2 and UBE2D ubiquitin-conjugating enzymes. J. Biol. Chem. 285, 7645-56, 2010.
Zahniser, N. R., and Sorkin, A. Trafficking of Dopamine Transporters in Psychostimulant Actions. Sem. Cell Dev. Biol. 20, 411-417, 2009.
Sorkina, T., Richards, T. L., Rao, A., Zahniser, N. R., and Sorkin, A. Negative Regulation of Dopamine Transporter Endocytosis by Membrane-Proximal N-Terminal Residues. J. Neuroscience. 9, 1361–1374, 2009.
Miranda, M., Dionne, K. R., Sorkina, T., and Sorkin, A. Three ubiquitin conjugation sites in the amino-terminus of the dopamine transporter mediate protein kinase C dependent endocytosis of the transporter. Mol. Biol. Cell. 18, 313-323, 2007.
Sorkina, T., Miranda, M., Dionne, K. R., Hoover, B.R., Zahniser N.R., and Sorkin, A. RNA Interference Screen Reveals an Essential Role of Nedd4–2 in Dopamine Transporter Ubiquitination and Endocytosis. J. Neuroscience. 26, 8195– 8205, 2006.