Binding competition assay between the human μ-opioid receptor expressed in human embryonic kidney 293 cells and the µ-opioid water-soluble variants. Inhibition of the native μ-opioid receptor constitutive signal from the interaction with naltrexone in the presence of increasing concentrations of wsMUR-TMs in sodium phosphate buffer is demonstrated which is essentially in a similar pattern, indicating a similar affinity of naltrexone for both of the variants. The K_d values are 67 ± 4 nM for wsMUR-TM and 70 ± 7 nM for wsMUR-TM_v2. The K_d value indicates the average value of three measurements. The error bars in the figure are obtained as the SD of three independent measurements. ΔF is used for the comparison of different runs of the same assay which reflects the signal to background of the assay. ΔF = [(Ratio_sample − Ratio_backgroud)/Ratio_backgroud](%). As we described previously for the homogeneous time-resolved fluorescence-binding assay,⁷  “Ratio” was the ratio of fluorescence emission at 665 and 620 nm, and K_d values were determined by fitting the dose–response curves using Prism (GraphPad Software, San Diego, CA).The K_d value indicates the average value of three measurements. wsMUR-TM = first variant of the water-soluble human μ-opioid receptor transmembrane portion; wsMUR-TM_v2 = modified (second) variant of the water-soluble human μ-opioid receptor transmembrane portion.