Interrelation of dopamine transporter oligomerization and surface presence as studied with mutant transporter proteins and amphetamine

Abstract

Our previous work suggested a role for oligomerization in regulating dopamine transporter (DAT) internalization, with d-amphetamine dissociating DAT oligomers and monomers being endocytosed. This model was put to detailed testing in the present work with the use of DAT constructs differentially tagged with Myc or Flag, reversal of tags in co-immunoprecipitation and cross-linking assays, and application of antibodies against different tags in biotinylation experiments. Upon pairing wild-type (WT) DAT with W84L mutant, effects of d-amphetamine on oligomerization (decrease) but not surface DAT are observed. Internalization of W84L monomers appears to be slow as inferred from the inability of d-amphetamine to reduce surface Myc upon co-expressing Flag-WT with Myc-W84L but not Myc-WT with Flag-W84L, and from the sluggish Myc-W84L endocytosis rate (both with or without d-amphetamine). Results obtained for D313N, D345N, or D436N mutants can all be accommodated by a model in which D-amphetamine is unable to dissociate mutant protomers from oligomers (tetramers or higher-order assemblies) that contain them; this interpretation is confirmed in experiments with both tag reversal in co-expression and antibody reversal in western blotting. Upon co-transfecting Myc- and Flag-tagged constructs, resulting tetramers can be calculated to be composed of different species (MycMycMycMyc, MycMycMycFlag, MycMycFlagFlag, MycFlagFlagFlag, and FlagFlagFlagFlag), but it is shown that outcomes predicted by models based on MycMycFlagFlag oligomers are not changed in a major way by the occurrence of the additional species.

Fig. 1

Various combinations of WT and D436N subjected to immunoprecipitation (IP) with anti-Flag antibody or directly worked up as total lysates or as biotinylated surface fractions, and to immunoblotting (IB) with either anti-Flag or anti-Myc antibody. Cells were treated for 30 min at 37°C with 2 μM d-amphetamine (AMP) or vehicle. Proteins in panels A to D were separated on 8% SDS-PAGE gels, and proteins in panels E and F were separated on 4–12% NuPAGE gels.

Fig. 2

Effect of d-amphetamine (amph) on coimmunoprecipitation and surface expression of WT and W84L DAT. Treatment with d-amphetamine was for 30 min at 37°C at 2 μM (open bars) or 20 μM (striped bars) and results are expressed as % of those from vehicle treatment. Myc was coimmunoprecipitated with Flag (A), and surface DAT (B) was assessed with the antibody indicated at the bottom of the figure. The used combinations of differentially tagged constructs are indicated below the bars for each condition. Gel bands from representative experiments are positioned above the bars; for each co-expression condition, bands corresponding to drug treatments are lined up with bars, and band from vehicle treatment is placed to the left of those (there is no vehicle bar). Data are means ± SEM (vertical bar) of 3 – 4 experiments. * P < 0.05 compared with 100% (one sample two-tailed t-test).

Fig. 3

Endocytosis of WT and W84L DAT in presence and absence of d-amphetamine (amph) (20 μM) for 20 min at 37°C. Control incubation for 20 min at 4°C (disallowing endocytosis) provided the amount of surface DAT available at the start of the endocytosis measurement, and endocytosed DAT was expressed as % of this initial amount (A). Representative gel bands are shown for the various incubation conditions applied to WT and W84L (B). Data are means ± SEM (vertical bar) of 3 experiments. * P < 0.05 compared with Vehicle and ^$ P < 0.05 compared with corresponding WT (two-tailed t-test).

Fig. 4

Effect of d-amphetamine (amph) on coimmunoprecipitation and surface expression of WT and D313N DAT. Data are means ± SEM (vertical bar) of 3 – 4 experiments. All other details are as in Fig. 2.

Fig. 5

Effect of d-amphetamine (amph) on coimmunoprecipitation and surface expression of WT and D345N DAT. Because coimmunoprecipitation effects on WT were as strong with 2 μM as with 20 μM of amphetamine, subsequent combinations of DAT constructs were tested only at the lower 2 μM dose. Representative bands from different experiments are lined up horizontally. Data are means ± SEM (vertical bar) of 3 – 6 experiments. All other details are as in Fig. 2.

Fig. 6

Effect of d-amphetamine (amph) on coimmunoprecipitation and surface expression of WT and D436N DAT. Data are means ± SEM (vertical bar) of 3 – 6 experiments. All other details are as in Fig. 5.

Fig. 7

Effect of dopamine (DA) and d-amphetamine (amph) on coimmunoprecipitation and surface expression of WT, D345N, and D436N DAT. Treatment with DA (100 μM, open bars) or amph (20 μM, striped bars) was for 30 min at 37°C. Data are means ± SEM (vertical bar) of 4 – 6 experiments. All other details are as in Fig. 2.

Fig. 8

Models for wild-type DAT (WT) (Models 1, 2, 4) and W84L (Model 3). See Discussion section for description of the models, their predicted outcomes, and experimental observations. OLIGO is oligomer, MONO is monomer, F stands for Flag, and M is Myc. Upon co-expressing Flag- with Myc-DAT, Myc was coimmunoprecipitated with Flag (CoIP M with F).

Fig. 9

Models for differentially tagged wild-type (WT) and W84L mutant (Mu). Mu containing oligomers can be dissociated just as purely WT containing oligomers but Mu monomers cannot internalize. Other details are as in Fig. 8.

Fig. 10

Models for differentially tagged wild-type (WT) and D313N, D345N, or D436N mutant (Mu). d-Amphetamine is unable to dissociate Mu protomers from oligomers that contain them. Other details are as in Fig. 8.

Fig. 11

Models for Myc-tagged wild-type (WT) and Flag-tagged D345N mutant (Mu). Models are shown for tetramers with composition other than fifty-fifty composition as shown in Fig. 9 (i.e. other than Myc-Myc-Flag-Flag). d-Amphetamine is unable to dissociate Mu protomers from oligomers that contain them. N/A: this species does not contribute to measurement as it cannot be detected with the particular combination of tags and antibodies used. Other details are as in Fig. 8.