3h dtg

(−)-Cocaine hydrochloride, haloperidol, (+)-pentazocine, 1,3-di(2-tolyl)guanidine (DTG), dextromethorphan hydrobromide, (+)- and (−)-N-allylnormetazocine (NANM, SKF10047), ifenprodil (+)-tartrate, BD1063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine) dihydrochloride, and GBR12909 dihydrochloride were obtained from Sigma-Aldrich, Inc. (St. Louis, MO) or Tocris Bioscience (Minneapolis, MN). [³H]DTG (48 – 53 Ci/mmol) and [³H](+)-pentazocine (35 – 37 Ci/mmol) were obtained from PerkinElmer, Inc. (Waltham, MA). E-N-1-(3′-iodoallyl)-N′-4-(3″,4″-dimethoxyphenethyl)-piperazine (E-IA-DM-PE-PIPZE) and [¹²⁵I]E-IA-DM-PE-PIPZE (ca. 2000 Ci/mmol) were prepared as previously described (Lever et al., 2012 (link)). [¹²⁵I]RTI-121 (3β-(4-iodophenyl)tropan-2β-carboxylic acid isopropyl ester) was prepared (ca. 2000 Ci/mmol) as previously described (Lever et al., 1996 (link)). Sterile bacteriostatic saline (0.9% NaCl, 0.9% benzyl alcohol; w/v) was used in formulations for animal studies. Other chemicals and solvents were the best available commercial grade, and were used as received.

Compounds were purchased from the following sources: 4-PPBP, BD1063, (+)-igmesine, PRE-084 from Tocris Biosciences (Minneapolis, MN); butamirate, carbetapentane, and NE-100 were from Santa Cruz Biotechnology (Dallas, TX); and donepezil oxeladin, promethazine was from Sigma-Aldrich (St. Louis, MO). Radioligands were purchased from PerkinElmer: ([³H] - (+)-pentazocine ((+)-Pentazocine, [RING-1,3-³H], 33.9 Ci/mmol, NET1056), [³H]-DTG (1,3-Di-o-tolylguanidine, [p-RING-³H]-, 50 Ci/mmol, NET986) (Saint Louis, MO). All compounds were prepared in DMSO at concentrations ranging from 10–100 mM. Stocks were then diluted 1:1000 (v/v) in the final assay solution.

Saturation binding was performed with a method similar to that of Chu and Ruoho⁵⁶ . Briefly, membrane samples from Expi293 cells (Thermo Fisher Scientific) expressing wild-type or mutant σ₂ receptor, prepared as described above, were thawed, homogenized with a glass dounce, and diluted in 50 mM Tris, pH 8.0. Binding reactions were done in 100 μL, with 50 mM Tris pH 8.0, [³H]-DTG (PerkinElmer), and supplemented with 0.1% bovine serum albumin to minimize non-specific binding. To assay non-specific binding, equivalent reactions containing 10 μM haloperidol were performed in parallel. Competition assays were performed in a similar fashion with 10 nM [³H]-DTG and the indicated concentration of the competing ligand. Samples were shaken at 37 °C for 90 min. Afterward, the reaction was terminated by massive dilution and filtration over a glass microfiber filter with a Brandel harvester. Filters were soaked with 0.3% polyethyleneimine for at least 30 min before use. Radioactivity was measured by liquid scintillation counting. Data analysis was done in GraphPad Prism 9.0, with K_i values calculated by Cheng-Prusoff correction using the experimentally measured probe dissociation constant.