Data reporting
No statistical methods were used to predetermine sample size. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.
Materials
Alvimopan, naloxone, mitragynine pseudoindoxyl, DAMGO and loperamide were purchased from Cayman Chemicals and MedChemExpress. Detergents were purchased from Anatrace. The radioligand [3H]naltrexone (specific activity 48.19 Ci per mmol) was generously provided by the National Institute on Drug Abuse Drug Supply Program. All other general laboratory reagents and chemicals were purchased from Millipore Sigma, unless otherwise stated.
Constructs
For recombinant protein expression, full-length human MOR was subcloned into a modified pFastBac1 vector with an N-terminal Flag, 10xHis-tag and thermostabilized b 562 RIL (ref. 54) along with a C-terminal TwinStrep tag55 and an extra 10xHis-tag. Both N-terminal and C-terminal tags are flanked by HRV-3C protease cleavage sites to allow tag removal. For the G-protein-free ‘inactive’ MOR structure, two point mutations M264L and K2696.24R in ICL3 were incorporated, to allow Nb6 binding56.
For all BRET assays, transient transfections of wild-type full-length human MOR were performed using the pcDNA 3.1(−) vector in human embryonic kidney 293F (HEK293F) mammalian cells. These receptor constructs were expressed under the cytomegalovirus promoter and included an N-terminal haemagglutinin signal peptide followed by a Flag tag. For TRUPATH24 and GloSensor assays, the MOR construct was untagged and contained no fusion elements. In the RG-BRET assay, Rluc8 was fused immediately after the C terminus of the wild-type MOR construct.
In TRUPATH assays24, a tricistronic vector encoding Gα i1 , Gβ 1 and Gγ 2 was used, in which GFP2 was fused to the N terminus of Gγ 2 and Rluc8 was inserted after residue G90 of the Gα i1 subunit23. For RG-BRET assays, a Gα i1 β 1 γ 2 construct was used with GFP2 fused to the N terminus of Gγ 2 . The pOZITX-S1 plasmid, used as a control in these assays, was obtained from J. Javitch (Addgene plasmid no. 184925).
Cell culture and transfections
HEK293F cells were cultured in FreeStyle293 Expression Medium (Gibco) at 37 °C with 5% CO 2 and shaking at 110 rpm. For transfections, cells were seeded 1 day before and transfected at a cell density of 1.0 × 106 cells per ml using polyethyleneimine (PEI) with a DNA:PEI ratio of 1:2. For RG-BRET assays, MOR–Rluc8 and Gα i /Gβ 1 /Gγ 2 –GFP2 constructs were transfected at a 1:2 ratio. TRUPATH BRET assays used MOR and Gα i1 –Rluc8/Gβ 1 /Gγ 2 –GFP2 transfected at a 1:1 ratio. When pOZITX-S1 was used, Gα i /Gβ 1 /Gγ 2 –GFP2 and pOZITX-S1 plasmids were transfected at a 1:1 ratio. For MOR–Gα i -mediated cAMP inhibition assays, cells were cotransfected with wild-type human MOR, along with a split-luciferase-based cAMP biosensor (GloSensor, Promega) at a 1:1 ratio.
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