Substrates dissociate dopamine transporter oligomers

Abstract

Substrate-induced endocytic trafficking of dopamine transporter (DAT) has been observed, but little is known about the regulation of DAT oligomerization by substrate. The present study investigates the effect on substrates on DAT oligomerization and explores a potential link with the presence of DAT at the cell surface in human embryonic kidney cells transiently or stably expressing N-terminal tagged DAT constructs. Dopamine (100 microM) or amphetamine (2-10 microM) reduced Myc-DAT coimmunoprecipitated along with Flag-DAT (oligomeric DAT) in tandem with a reduction in surface DAT determined by biotinylation. Dopamine (10-1000 microM) and amphetamine (0.2-200 microM) reduced DAT oligomerization as assessed by cross-linking with copper sulfate phenanthroline or Cu2+. Inhibition of endocytosis by 10 microM phenylarsine oxide or 450 mM sucrose counteracted the effect of 10 microM DA or 2 microM amphetamine in reducing DAT cross-linking. In addition to overall similarities between the results with the two cross-linking agents and between the results with the two different endocytosis inhibitors, some differences were noted as well, likely related to the efficiency of the cross-linking process and the sulfhydryl-reactive properties of phenylarsine oxide, respectively. The present results are the first to indicate regulation of oligomerization of an solute carrier family 6 transporter, the DAT, by substrates that act at DAT. In addition, the present study opens up the possibility of an important linkage between oligomerization of DAT and endocytic or other modulatory mechanisms impacting surface DAT.

Fig. 1

Coimmunoprecipitation for detection of DAT oligomers. Cells were transiently transfected with vector, separately with Flag-DAT or Myc-DAT, or with Flag- and Myc-DAT combined. After immunprecipitation (IP) with anti-Flag antibody (A, B, C), immunoblotting (IB) of gels was carried out with anti-Myc antibody (A), anti-DAT antibody (B), or anti-Flag antibody (C). In experiments where IP was omitted, IB was done with anti-Myc antibody (D) or anti-DAT antibody (E). Lanes show results for a representative experiment (out of 3) with indication of molecular weights based on migration of marker proteins.

Fig. 2

Distribution of DAT bands on gels from immunoprecipitated, biotinylated, and cross-linked samples. Cells were transiently (A, B, D) or stably (C) transfected with a mixture of Flag- and Myc-DAT and analyzed by biotinylation (A), immunoprecipitation (IP) with anti-Flag antibody (B, D), or Cu²⁺ cross-linking (C) on 8% gels (A, B) or 4–12% gels (C, D). Pretreatments were with cocaine (COC), DA, or amphetamine (AMP) as indicated. Lanes show results for a representative experiment (out of 4) with indication of molecular weights based on migration of marker proteins. For quantitative analysis of all 4 experiments, see Fig. 3.

Fig. 3

Efect of DAT ligands on coimmunoprecipitation and surface expression of DAT. Coimmunoprecipitated (Co-IP) (top panels) and biotinylated (bottom panels) DATs came from cells as detailed in Fig. 2, pretreated with cocaine (COC), DA, or amphetamine (AMP) as indicated below the bars. Data are expressed as means ± SEM (vertical bar) of 4 experiments. * P<0.05 compared with 100% (one sample t-test).

Fig. 4

Treatment of cells with substrates and cross-linkers assessed by different antibodies. Cells were stably transfected with Myc-DAT and used for cross-linking with CuP or Cu²⁺ after (A, B) or before (C) treatment with vehicle, DA, or amphetamine (AMP) as indicated below the lanes. Western blotting was with anti-Myc antibody (A) or anti-DAT antibody (B, C). Lanes show results for a representative experiment (out of 3) with indication of molecular weights based on migration of marker proteins.

Fig. 5

Time course and concentration range for substrates affecting cross-linking. Cells were stably transfected with Myc-DAT and used for cross-linking with CuP or Cu²⁺ after pretreatment with DA or amphetamine. A. Typical PAGE experiment. Pretreatment time and concentration was as indicated below the lanes. Western blotting was with anti-Myc antibody. Lanes show results for a representative experiment (out of 3–4) with indication of molecular weights based on migration of marker proteins. B. Quantitative analysis of all time-course experiments. Data for CuP- (top panels) and Cu²⁺- (bottom panels) cross-linked DATs are expressed as means ± SEM (vertical bar) of 3–4 experiments. Pretreatment time (min) was as indicated below the bars; DA was at 100 μM and amphetamine at 2 μM. * P<0.05 compared with 0 min (Dunnett test). C. Quantitative analysis of all concentration-range experiments. Data for CuP- (top panels) and Cu²⁺- (bottom panels) cross-linked DATs are expressed as means ± SEM (vertical bar) of 3–4 experiments. Pretreatment time was 30 min. Substrate concentration was as indicated below the bars. * P<0.05 compared with 0 μM (Dunnett test).

Fig. 6

Effect of substrate on cross-linking and surface residency of DAT. Cells were stably transfected with Myc-DAT. A. Cross-linking was with CuP or Cu²⁺ after pretreatment with endocytosis blocker PAO or sucrose (SUC) (indicated above lanes) and with DA (10 μM) or amphetamine (AMP, 2 μM) (indicated below lanes). Vehicle instead of CuP or Cu²⁺ is indicated as “Control”; vehicle instead of PAO or SUC is indicated as “Buffer”; vehicle instead of AMP or DA in indicated as “Vehicle”. Either total DAT (top panels) or biotinylated surface DAT (bottom panels) was assessed by Western blotting with anti-Myc antibody. Lanes show results for a representative experiment (out of 3) with indication of molecular weights based on migration of marker proteins. B and C. Effect of PAO and SUC on total DAT (hatched bars) and surface DAT (open bars) with CuP (B) or Cu²⁺ (C) as cross-linker. Pretreatment was as indicated below the bars and data were analyzed for the ratio of DAT dimers (180 kDa) over monomers (85 kDa). Data are expressed as means ± SEM (vertical bar) of 3 experiments. * P<0.05 compared with corresponding Vehicle (Dunnett test).