(A) Representative immunoblots of surface hDAT (top left), total hDAT (top right) and corresponding Na-K ATPase (bottom left) and actin (bottom right) loading controls. hDAT expression was normalized to hDAT WT. hDAT R445C displayed significantly reduced surface (p<0.0001; n = 4, in triplicate) and total glycosylated (#) expression relative to hDAT WT (p<0.0001; n = 4, in triplicate). (B) Average 3[H]DA saturation curves of DA uptake measured in hDAT WT (black) or hDAT R445C (blue) cells (n = 3, in triplicate). Curves were fit to Michaelis-Menten kinetics to derive Km and Vmax. DA uptake for hDAT R445C was significantly reduced compared with hDAT WT at every DA concentration measured (F(6,64) = 52.4, p<0.0001), as were the kinetic constants, Km and Vmax (p<0.0001). (C) Representative traces of amperometric currents (DA efflux) recorded in response to AMPH application (left; 10 μM, indicated by arrow) from hDAT WT (black) and hDAT R445C (blue) cells loaded with DA (2 mM, 10 min) with whole-cell pipette. Quantitation of peak current amplitudes illustrated a significant reduction in DA efflux recorded from hDAT R445C compare to hDAT WT (right; p=0.008; n = 6–7). (D) Representative immunoblots of total hDAT (top) and actin loading controls (bottom), where glycosylated (#) and non-glycosylated (*) hDAT is highlighted. Ratio of mature (glycosylated) to immature (non-glycosylated) DAT (mDAT/iDAT) expression following CQ treatment was normalized to vehicle condition for hDAT WT and hDAT R445C cells (n = 4, in duplicate). Incubating hDAT R445C cells with CQ (1 mM, 4 hr) significantly increased the ratio of mDAT/iDAT (F(1,20) = 18.0), p=0.003. CQ also significantly increased mDAT/iDAT expression in hDAT WT cells (p=0.04). (E) Diagramed is the flight initiation assay used to determine take-off times for hDAT WT and hDAT R445C Drosophila (left). hDAT R445C and hDAT WT Drosophila were fed CQ (3 mM, 72 hr) or vehicle supplemented diet. Quantitation showed a significant reduction in the time to initiate flight in hDAT R445C flies (F(1,29) = 8.7, p=0.04) in response to CQ compared with vehicle conditions (right). Time for flight initiation was comparable in hDAT WT flies exposed to CQ and vehicle supplemented diet (p>0.05). Data represent mean ± SEM. Student’s t-test (A) and (C); Two-way ANOVA with Bonferroni’s multiple comparison test: (B), and (D–E).