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. Author manuscript; available in PMC: 2021 Jun 3.
Published in final edited form as: Mol Psychiatry. 2019 Dec 3;26(8):4417–4430. doi: 10.1038/s41380-019-0620-0

Fig. 2. R443A substitution reduced PIP2 binding and inhibited AMPH-induced DA efflux in vitro.

Fig. 2

a hDAT WT or hDAT R443A (containing a His-eGFP tag on the N-terminus) were purified from cellular extracts. Solubilized hDAT WT or hDAT R443A proteins were incubated with a water-soluble analog of PIP2 conjugated to an orange fluorophore (BODIPY® TMR-PIP2). Specific binding was quantified as a ratio of PIP2 fluorescence to eGFP. Minimal PIP2 binding was measured in the presence of His-eGFP only (0.16 ± 0.05 AU) relative to hDAT WT (1.38 ± 0.12 AU) or hDAT R443A (0.90 ± 0.24 AU) (F(2,9) = 55.11, p < 0.0001; n = 4). hDAT R443A displayed a 34.8 ± 9.9% reduction in PIP2 binding compared with hDAT WT (p = 0.007). b Top: average 3[H]DA saturation curves of DA uptake measured in hDAT WT (closed squares) or hDAT R443A (open squares) cells (n = 4, in triplicate). Curves were fit to Michaelis–Menten kinetics to derive Km and Vmax. DA uptake for hDAT R443A was comparable to hDAT WT at every DA concentration measured (F(1,120) = 1.40, p > 0.05), as were the kinetic constants, Km and Vmax (p > 0.05). c Left: representative traces of amperometric currents (DA efflux) recorded from hDAT WT (top) and hDAT R443A (bottom) cells, in response to AMPH application (10 μM, indicated by arrow). Right: quantitation of peak current amplitudes. hDAT R443A display a 50.6 ± 15.1% decrease in AMPH-induced DA efflux relative to hDAT WT (p = 0.003; n = 14). d Left: representative immunoblots of surface hDAT (top), total (glycosylated and nonglycosylated) hDAT (middle), and actin as loading control (bottom). Right: hDAT expression is quantified as a ratio of surface to total glycosylated hDAT normalized to hDAT WT. hDAT R443A and hDAT WT had comparable expression (p > 0.05; n = 6, in triplicate). Data are presented as mean ± SEM. One-way ANOVA with Bonferroni’s multiple comparisons test: a; two-way RM ANOVA with Bonferroni’s multiple comparison test: b; Student’s t test: b, d; Wilcoxon matched-pairs signed rank test: c.