Fig. 4: mKOR agonism induces enhanced mDAT surface expression and phosphorylation at mDAT Thr53 while mKOR antagonism normalizes enhanced surface expression and Thr53 phosphorylation of mDAT Val559 in acute brain slices prepared from male mice containing the DS or VS.
A–D Acute slices containing the DS and/or the VS were exposed to 10 µM of U69,693 or vehicle for 7 min and surface expression and phosphorylation of p-Thr53 DAT was assessed. A, B In the DS, treatment with U69,593 increased surface DAT levels (A) (n = 9; two-way ANOVA, Å Ãdák’s multiple comparisons test) as well as phosphorylation of mDAT at Thr53 (B) (n = 10; two-way ANOVA, Å Ãdák’s multiple comparisons test). Higher basal phosphorylation levels at Thr53 were detected for the DAT Val559 when compared to WT DAT in the DS (n = 11; two-way ANOVA, Å Ãdák’s multiple comparisons test). C In the VS, KOR-agonist treatment increased surface mDAT levels independent of genotype as compared to vehicle treatment (n = 8; two-way ANOVA, Å Ãdák’s multiple comparisons test). D Treatment with U69,593 enhanced WT mDAT phosphorylation at Thr53, but remained without effect on DAT Val559 phosphorylation. E–H Acute coronal slices containing the DS or the VS were treated with the KOR antagonist norBNI (1 µM) for 20 min and DAT surface expression and phosphorylation at Thr53 were determined. E, F In the DS, antagonism of mKOR reduced the enhanced surface expression E (n = 8; two-way ANOVA, Å Ãdák’s multiple comparisons test) and Thr-53 phosphorylation F (n = 6; two-way ANOVA, Å Ãdák’s multiple comparisons test) of tansporters in DAT Val559 mice. No effect of norBNI was detected on WT DAT. G, H Treatment with norBNI remained without effect on DAT surface expression G (n = 6, two-way ANOVA, Å Ãdák’s multiple comparisons test) and Thr-53 phosphorylation H (n = 7, two-way ANOVA, Å Ãdák’s multiple comparisons test) in the VS. All bars show the mean and SD. Symbols reflect individual observations. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001.
