The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines

Abstract

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.

Fig. 1. 18 °C recycling blockade decreases DA transport and DAT surface expression in DAT-PC12 cells

A, DA uptake assay of DAT-PC12 cells preincubated at 37 °C (filled bar) or 18 °C (open bar) for 30 min. Data are expressed as % control uptake ± S.E. *, p < 0.005 (Student’s t test, n = 4). B, steady state cell surface biotinylation of DAT-PC12 cells. Inset, representative immunoblot showing surface DAT and TfR after incubation at 37 or 18 °C. Bar graph, averaged data expressed as % loss of 37 °C cell surface pool ± S.E. (n = 3, DAT; n = 2, TfR).

Fig. 2. Bafilomycin reduces the surface expression of TfR but not DAT in DAT-PC12 cells

A, representative immunoblot of cell surface DAT and TfR following 30-min treatment with vehicle or 0.5 μm bafilomycin A₁. B, quantitation of DAT and TfR surface levels following treatment with vehicle (open bars) versus 0.5 μm bafilomycin A₁ (filled bars). Data are expressed as % vehicle surface pool ± S.E. *, p < 0.005 compared with vehicle-treated cells (Student’s t test, n = 4).

Fig. 3. Relative DAT internalization rates are sensitive to PKC activation and expression level

A, representative immunoblot showing total cell surface DAT labeled at 0 °C (total), MesNa strip of 0 °C biotinylated cells (strip), and 10′ internalized DAT ± 1 μm PMA. B, DAT internalization was assessed at 37 °C in untreated (open bars) versus 1 μm PMA-treated (filled bars) DAT-PC12 cell lines. Data are expressed as % total surface DAT internalized in 10 min ± S.E. (n = 2-11). *, p < 0.01 compared with vehicle-treated cells (Student’s t test, n = 5-12). Inset, immunoblot of DAT expression in three stably transfected DAT-PC12 cell lines. Equivalent amounts of total cellular protein were loaded. C, time course of PMA effect on DA transport. Data are expressed as % specific DA uptake ± S.E. *, p < 0.01 (ANOVA with Dunnett’s post hoc analysis, n = 2).

Fig. 4. DAT constitutively recycles and PKC decreases DAT recycling rates

A, biotinylation for 45 min at 4 and 37 °C. Biotinylated (B) and nonbiotinylated (NB) proteins were separated by streptavidin pull-down and assessed by immunoblot for DAT, TfR, and the early endosomal marker rab5A. Similar results were obtained in three independent experiments. B, DAT-PC12 cells were biotinylated for 30 min at either 0 or 37 °C in the presence of vehicle or 1 μm PMA. Biotinylated and nonbiotinylated proteins were separated by streptavidin pull-down. A representative blot is shown. C, averaged DAT recycling data from 0 °C (hatched bars) versus 37 °C biotinylation in the presence of either vehicle (open bars) or 1 μm PMA (filled bars). Data are expressed as % maximal biotinylated protein ± S.E. for each cell line. *, p < 0.001 compared with 0 °C biotinylation (ANOVA with Bonferroni multiple comparisons test, n = 5-8); **, p < 0.05 compared with 37 °C vehicle treatment (ANOVA with Bonferroni multiple comparisons test, n = 5-8). D, averaged TfR recycling data from 0 °C (hatched bars) versus 37 °C biotinylation in the presence of either vehicle (open bars) or 1 μm PMA (filled bars). Data are expressed as % maximal biotinylated protein ± S.E. *, p < 0.01 compared with 0 °C biotinylation (ANOVA with Bonferroni multiple comparisons test, n = 2-8).

Fig. 5. Model for constitutive and PKC-regulated DAT trafficking

Dynamic DAT trafficking is a rheostat that fine-tunes DA signaling. Under basal conditions DAT continually cycles through the endocytic pathway. Upon PKC activation, DAT internalization is accelerated, and DAT recycling back to the cell surface is attenuated. Thus, DAT accumulates in recycling endosomes, and DAT surface pools are depleted.