Amphetamine-induced decreases in dopamine transporter surface expression are protein kinase C-independent

Abstract

Amphetamine (AMPH) is a potent dopamine (DA) transporter (DAT) inhibitor that markedly increases extracellular DA levels. In addition to its actions as a DAT antagonist, acute AMPH exposure induces DAT losses from the plasma membrane, implicating transporter-specific membrane trafficking in amphetamine's actions. Despite reports that AMPH modulates DAT surface expression, the trafficking mechanisms leading to this effect are currently not defined. We recently reported that DAT residues 587-596 play an integral role in constitutive and protein kinase C (PKC)-accelerated DAT internalization. In the current study, we tested whether the structural determinants required for PKC-stimulated DAT internalization are necessary for AMPH-induced DAT sequestration. Acute amphetamine exposure increased DAT endocytic rates, but DAT carboxy terminal residues 587-590, which are required for PKC-stimulated internalization, were not required for AMPH-accelerated DAT endocytosis. AMPH decreased DAT endocytic recycling, but did not modulate transferrin receptor recycling, suggesting that AMPH does not globally diminish endocytic recycling. Finally, treatment with a PKC inhibitor demonstrated that AMPH-induced DAT losses from the plasma membrane were not dependent upon PKC activity. These results suggest that the mechanisms responsible for AMPH-mediated DAT internalization are independent from those governing PKC-sensitive DAT endocytosis.

Fig. 1

Amphetamine decreases in DAT surface levels do not require PKC-sensitive DAT structural determinants. (A) Schematic illustrating the 587–596 region of wild type and 587–590(4A) DAT. (B) Cell surface biotinylation. PC12 cells stably transfected with the indicated DAT constructs were treated ±5 µM AMPH, 30 min, 37 °C and surface DAT levels were measured by cell surface biotinylation as described in Section 2. Top: representative immunoblot showing surface (S) and intracellular (I) fractions from biotinylated cells following treatment with ±5 µM AMPH. Bottom: averaged data. Bars represent the percent total DAT on the cell surface following drug treatment ± SEM. Asterisks indicate a significant difference from vehicle control for each construct: *p < 0.0001, **p < 0.007; Student’s t-test (n = 7–9).

Fig. 2

AMPH stimulates DAT endocytic rates but does not require DAT residues 587–590. Internalization assay. PC12 cells stably transfected with the indicated DAT cDNAs were biotinylated at 4 °C and were warmed for 10 min, 37 °C, ±5 µM AMPH. Residual surface biotin was stripped by reducing and internalized (biotinylated) proteins were identified by streptavidin affinity chromatography and immunoblot as described in Section 2. (A) Representative immunoblot showing total surface DAT at t = 0 min (T), strip controls (S) and amount of DAT internalized in the presence of either vehicle (V) or AMPH (A). (B) Averaged data. Bars represent percent total surface DAT ± SEM internalized in 10 min, ±5 µM AMPH. Asterisks indicate significant difference from vehicle-treated controls for each construct: *p < 0.03, **p < 0.002; Student’s t-test, n = 8.

Fig. 3

AMPH decreases DAT delivery to the cell surface, but does not slow global endocytic recycling. Endocytic recycling assay: surface proteins were biotinylated at 4 °C and proteins newly delivered to the cell surface were identified by biotinylating at 37 °C, ±5 µM AMPH for the indicated times as described in Section 2. Data are expressed as the percent total protein biotinylated at the indicated times ± SEM. (A) Representative immunoblot showing non-biotinylated (−) and biotinylated (+) DAT fractions obtained at the indicated times after shifting cells to 37 °C, ±5 µM AMPH. (B) Averaged data. Points represent percent total DAT biotinylated ± SEM from vehicle- (dashed line) and AMPH-treated (solid line) cells at the indicated timepoints. Zero timepoints for vehicle- and AMPH-treated cells are identical. Data were fit to a first order exponential equation as described in Section 2. The r² values: vehicle = 0.94, AMPH ¼ 0.98. (C) TfR recycling measured from the DAT immunoblots, stripped and re-probed with anti-TfR antibodies. Data from vehicle- (dashed line) and AMPH-treated (solid line) cells were fit to a first order exponential equation as described in Section 2. The r² values: vehicle = 0.99, AMPH = 0.99.

Fig. 4

PKC activity is not required for AMPH-stimulated DAT surface losses. (A) Cell surface biotinylation. DAT-PC12 cells were pretreated with either vehicle or 1 mM BIM I, 20 min, 37 °C, followed by treatment ±5 µM AMPH, 30 min, 37 °C proteins were labeled and isolated by cell surface biotinylation and streptavidin pulldown as described in Section 2. Top: representative immunoblot indicating surface (S) and intracellular (I) DAT fractions following treatment with either vehicle, 5 µM AMPH, 1 µM BIM or 1 µM BIM/5 µM AMPH. Bottom: average data. Bars represent percent vehicle surface levels ± SEM following the indicated drug treatments. Asterisks indicate significant difference from vehicle-treated cells: *p < 0.001, **p < 0.01; one-way ANOVA with Bonferroni’s multiple comparison test, n = 5. (B and C) BIM blocks PKC-induced DAT downregulation and surface losses. DAT-PC12 cells were pretreated with either vehicle or 1 µM BIM I, 20 min, 37 °C, followed by treatment with ±1 µM phorbol-12-myristate-13-acetate (PMA), 30 min, 37 °C. [³H]DA uptake was measured as described in Section 2 and surface proteins were labeled and isolated by cell surface biotinylation as described in Section 2. (B) DA uptake assay. Bars represent percent specific DA uptake ± SEM as compared to vehicle-treated cells. *Significant difference from vehicle p < 0.001, **significant difference from PMA p < 0.01; one-way ANOVA with Bonferroni’s multiple comparison test, n = 4. (C) Surface biotinylation. Top: representative immunoblot indicating surface (S) and intracellular (I) DAT fractions following the indicated treatments. Bottom: averaged data. Bars represent percent total surface DAT ± SEM following the indicated drug treatments. *Significant difference from vehicle p < 0.05, **significant difference from PMA p < 0.05; one-way ANOVA with Bonferroni’s multiple comparison test, n = 3.