Membrane potential assay

MOR-mediated activation of potassium channels was assayed using a membrane-potential dye from Molecular Devices (FLIPR Membrane Potential Assay kit) as previously described (71). On the day before the experiment, HEK293-GIRK4-MOR cells were plated into clear-bottomed, black-walled 96-well plates (Corning) precoated with poly-d-lysine for cell adherence. Cells were plated in L-15 media (Gibco, Thermo Fisher Scientific) supplemented with 1% (v/v) FBS. The following day, cell medium was aspirated, and plates were treated with either 200 nM β-CNA hydrochloride (Sigma-Aldrich) or Hepes-buffered salt solution (HBSS) vehicle control for 20 min. Testing of each compound in untreated and β-CNA–treated conditions was carried out in parallel. All experiments were carried out in duplicate. Cells were washed twice with HBSS after vehicle or β-CNA treatment and incubated for 1 hour in 50% L-15 media and 50% FLIPR dye reconstituted in HBSS in a final volume of 180 μl per well. All test compounds were serially diluted in a vehicle of 1% (v/v) dimethyl sulfoxide (DMSO), bovine serum albumin (BSA; 0.1 mg/ml) in HBSS at 10× concentrations in a 96-well compound plate (Corning) such that final assay concentrations of solvent were 0.1% DMSO and BSA (0.01 mg/ml). Plates were read in a FlexStation 3 (Molecular Devices). Two-minute baselines were taken for each well before compound addition. Then, 20 μl of 10× concentrated compound was added and read for 3 min. Peak effect was calculated by taking the minimum raw fluorescence value of the postaddition measurement period, expressed as a percentage of preaddition baseline and vehicle corrected, and normalized to the maximal response to DAMGO without β-CNA treatment.