Figure 2
hDAT A559V does not transport AMPH. (a) Uptake was performed with 10 nM AMPH for 5 min in cells transfected with either empty vector (pcDNA3), hDAT or hDAT A559V. hDAT cells exhibited robust AMPH uptake blocked by pretreatment with 10 μM cocaine (*P<0.05, one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test, n=3). hDAT A559V cells exhibited uptake not significantly different from the empty vector control (P>0.05, one-way ANOVA followed by Dunnett’s multiple comparison test, n=3). inset: representative immunoblot for DAT demonstrates comparable levels of transporter expression between hDAT and hDAT A559V cells. (b) hDAT or hDAT A559V cells were treated with AMPH (10 μM) for the indicated time points. Top, representative immunoblot of DAT biotinylated proteins (‘surface DAT’) and total cellular lysate (‘total DAT’) following AMPH (10 μM) treatment. Bottom, quantification of biotinylated DAT protein fractions following AMPH treatment. The ratio of surface to total DAT protein is plotted normalized to time point 0 (no AMPH treatment). AMPH decreases surface DAT in hDAT cells, but not in hDAT A559V cells (**P<0.01, *P<0.05, one-way ANOVA followed by Dunnett’s multiple comparison test, n=6). (c) hDAT or hDAT A559V cells were treated with AMPH (10 nM) for the indicated time points. Top, representative immunoblot of DAT biotinylated fraction (‘surface DAT’) and total cellular lysate (‘total DAT’). Bottom, quantification of biotinylated DAT protein fractions following AMPH treatment. The ratio of surface to total DAT protein is plotted normalized to time point 0 (no AMPH treatment). The 10 nM concentration of AMPH decreases surface DAT in hDAT cells, but not in hDAT A559V cells (*P<0.05, Student’s t-test, n=4). AMPH, amphetamine; DAT, dopamine transporter; hDAT, human dopamine transporter.
