The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines.
The dopamine transporter (DAT) removes dopamine from the extracellular milieu and is potently inhibited by number of psychoactive drugs, including cocaine, amphetamines, and methylphenidate (Ritalin). Multiple lines of evidence demonstrate that protein kinase C (PKC) down-regulates dopamine transport, primarily by redistributing DAT from the plasma membrane to endosomal compartments, although the mechanisms facilitating transporter sequestration are not defined. Here, we demonstrate that DAT constitutively internalizes and recycles in rat pheochromocytoma (PC12) cells. Temperature blockades demonstrated basal internalization and reliance on recycling to maintain DAT cell surface levels. In contrast, recycling blockade with bafilomycin A1 significantly decreased transferrin receptor (TfR) surface expression but had no effect on DAT surface levels, suggesting that DAT and TfR traffic via distinct endosomal mechanisms. Kinetic analyses reveal robust constitutive DAT cycling to and from the plasma membrane, independent of transporter expression levels. In contrast, phorbol ester-mediated PKC activation accelerated DAT endocytosis and attenuated transporter recycling in a manner sensitive to DAT expression levels. These data demonstrate constitutive DAT trafficking and that PKC-mediated DAT sequestration is achieved by a combination of accelerated internalization and reduced recycling. Additionally, the differential sensitivity to expression level exhibited by constitutive and regulated DAT trafficking suggests that these two processes are mediated by independent cellular mechanisms.
Loder
Merewyn K
MK
Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts School of Medicine, 303 Belmont Street, Worcester, MA 01604, USA.
Melikian
Haley E
HE
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport; D019921:Biotinylation; D002460:Cell Line; D050483:Dopamine Plasma Membrane Transport Proteins; D004705:Endocytosis; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D018942:Macrolides; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000378:metabolism* / Q000502:physiology; D008954:Models, Biological; D009419:Nerve Tissue Proteins; D016716:PC12 Cells; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013696:Temperature; D013997:Time Factors; D014162:Transfection
D000900:Anti-Bacterial Agents; D050483:Dopamine Plasma Membrane Transport Proteins; D018942:Macrolides; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; C493216:Slc6a3 protein, rat; C057620:bafilomycin A; D011493:Protein Kinase C