Fig 2: Pretreatment with norBNI restores cocaine-induced increase in extracellular DA the DS of freely moving DAT Val559 mice. A) Cartoon displaying the placement of the microdialysis probe in the DS. The insert shows a coronal slice with the black the arrow indicating the position of the active dialysis probe.B) Male WT and DAT Val559 mice were injected with norBNI (10 mg/kg, i.p.) and cocaine (10 mg/kg, i.p.) and extracellular DA was measured using in vivo microdialysis in the DS. 20 min samples where collected and DA is expressed as fold increase of basal DA (i.e. the average from t=0 to 60 min). Arrows indicate times for systemic administration of norBNI and cocaine. N=5 animals per genotype, data are shown as mean and SD.C) Shown is the total area under the curve for the traces shown in (B). Data are shown as the mean and error bars indicate the SD. Ns= not significant, unpaired Student’s t-test with Welch’s correction.

Fig 3: KOR-agonism enhances phosphorylation of DAT at Thr53 in native preparations and augments DAT-mediated clearance in the NAc in vivo.A) Male rat striatal synaptosomes were treated with vehicle or 50 μM U0126 for 15 min at 30°C, followed by treatment with vehicle or 10 μM U69,593 for an additional 15 min 30°C. After treatment, samples were subjected to SDS-PAGE and western blotted for total and p-Thr53 DAT. Upper panel shows representative western blots. Lower panel shows quantification of p-Thr53 DAT staining as a percentage of basal levels. (n=4; one-way ANOVA, Tukey’s multiple comparisons test).B and C) Rats were injected s.c. with vehicle or U69,593 (0.32 mg/kg) and sacrificed at indicated times. Tissue from the dorsal (B) or ventral striatum (C) were western blotted for total and p-Thr53 DAT in duplicate. Signals from the U69,593 treated samples were compared to the time-matched control. The upper panels show representative western blots for the dorsal and ventral striatum with each time-matched control followed by U69,593 treatment. The lower panels show quantification of the p-Thr53 DAT staining as a percentage of the time-matched control (Two-tailed, unpaired t-test).D) Schematic representation of the electrode-micropipette assembly that was lowered into the NAc of anaesthetized rats to allow for in vivo chromatographic measurement of DA clearance rates.E) Representative oxidation currents converted to micromolar values observed upon pressure ejection of DA before (gray traces) and 2 min after intra-NAc injection of vehicle (black trace), U69,593 (890 μM, barrel concentration, red trace). The leftward-shift in the representative trace following U69,593 injection is indicative of increased DA clearance.F) U69,593 decreased the clearance time of exogenously applied DA in the NAc (T50 and T80; n= 7 observations per condition, two-tailed paired t-test) when compared to the pre-drug value. Bars indicate the mean and error reflect SD. Individual values from each animal are reflected by the corresponding symbols. *denotes P<0.05; **denotes P<0.01, ns = not significant.

Fig 4: Capacity to phosphorylate rDAT at Thr53 is required for rKOR activation to elevate transporter surface expression. EM4 cells were transfected with either WT DAT or DAT Ala53 plus rKOR, subjected to U69,593 and biotinylated as described in Methods.A: Treatment with U69,593 did not affect WT DAT band densities at ~55–60 kDa and ~85–90 kDa, derived from total protein lysates (n=3 independent observations per condition, Student’s two-tailed unpaired t-test).B: Incubation with U69,593 increased the WT DAT band densities at ~55–60 kDa and ~85–90 kDa, derived from biotinylated protein lysates (unpaired, two-tailed t-test). No effect was observed on the DAT Ala53 band densities at ~55–60 kDa (n=3 independent observations per condition, Student’s two-tailed unpaired t-test).C and D: Protein lysates were immunoblotted with p-Thr53 antibody. Bands marked with an asterisk indicate non-specific immunoreactivity.C: In total protein lysates, incubation with U69,593 increased the density of the p-Thr53 immunoreactive band at ~55–60 kDa for WT DAT (Student’s unpaired, two-tailed t-test). No effect was observed on the band densities at ~85–90 kDa. No specific bands were detected for the DAT Ala53 variant (n=3–5 independent observations per condition, Student’s two-tailed unpaired t-test).D: U69,593 augmented band densities at ~55–60 and ~85–90 kDa for biotinylated WT DAT (n=5 independent observations per condition, Student’s two-tailed unpaired t-test). No specific bands were detected for the DAT Ala53 variant.All bars represent the mean with error bars reflecting the SD. Individual quantifications are displayed as individual symbols. *=P<0.05; **=P<0.01; ****=P<0.0001. n.s.=not significant

Fig 5: mKOR antagonism reduces the elevated surface expression and Thr53 phosphorylation of DAT Val559 in the DS without affecting WT DAT in acute slices and with no effects in the VS.Acute coronal slices containing the DS (highlighted in area in A) or the VS (highlighted in area in D) were treated with the KOR antagonist norBNI (1 μM) for 20 min and DAT surface expression and phosphorylation at Thr53 were determined as described in Methods.B and C) In the DS, antagonism of mKOR reduced the enhanced surface expression (B) (n=8; two-way ANOVA, Šídák’s multiple comparisons test) and Thr-53 phosphorylation (C) (n=6; two-way ANOVA, Šídák’s multiple comparisons test) of tansporters in DAT Val559 mice. No effect was detected with WT DAT mice.E and F) Treatment with norBNI remained without effect on DAT surface expression (E) (n=6, two-way ANOVA, Šídák’s multiple comparisons test) and Thr-53 phosphorylation (F) (n= 7, two-way ANOVA, Šídák’s multiple comparisons test) in the VS.All bars show the mean and SD. Symbols reflect individual observations. ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001