Fig. 9: Dpep(20-42)DM dissects the DM heterodimer and MOR monomer functions after M-agonist applications in cells, maintains morphine analgesia and reduces tolerance development in mice.

a Schematic figure showing that Dpep(20-42)DM reduces DM heterodimers, helping to dissect the DM heterodimer and MOR monomer functions, including signaling and internalization, upon M-agonist addition. b Time courses of MOR and DOR internalization before and after the addition of 0.5 µM morphine or DAMGO in the presence and absence of 1 µM DpepDM (mean ± SEM; 10 cells for each data point). MOR internalization was examined in MD-cells and M-cells, whereas DOR internalization was observed only in MD-cells c Schematic illustration showing immensely enhanced efficiencies of binding to internalization machineries such as GRKs and β-arrestins and of DM*^DAMGO heterodimer internalization. d (Top) Experimental design for observing Ca²⁺ mobilization after M-agonist addition. Ca²⁺ mobilization was monitored by Fluo-4 fluorescence intensity in M-, D-, and MD-cells. (Bottom) Typical (among 20 cells) Fluo-4 images and single-molecule images of DOR and MOR. e Ca²⁺ mobilization parametrized by using [F_Max-F_b]/F_b for Fluo-4 signals before and after the addition of morphine and DAMGO, in the presence and absence of Dpep(20-42)DM. Horizontal bars, crosses, boxes, and whiskers indicate median values, mean values, interquartile ranges (25–75%), and 10-90% ranges, respectively. * and ns represent significant (p < 0.05) and non-significant (p ≥ 0.05) differences, respectively (Tukey’s multiple comparison test). f Schematic illustration showing the osmotic pump implantation for intracerebroventricular injection of Dpep(20-42)DM and subcutaneous morphine injection in a mouse [Created in BioRender. Aladag, A. (2025) https://BioRender.com/0tevzb7]. g Tail flick test results on day 11, showing that morphine-induced analgesia was maintained by the continuous administration of Dpep(20-42)DM (10 µg/day). The analgesia effect lasted for at least 120 min. h Dpep(20-42) reduces morphine tolerance in mice. For g and h, * represents significant (p < 0.05) difference (two-way ANOVA test; the data show mean ± SEM; n = 17 DpepDM group, n = 8 control peptide group). All of the statistical parameters and analysis results including sample size n and p values are provided in Supplementary Data 2. Source data are provided as a Source Data file.