Solubilization of High-Affinity,Guanine Nucleotide-Sensitive μ-Opioid Receptors from Rat Brain Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane-associated receptors with morphine during solubilization in the detergent 3-(3-cholamidopropyl)dimethyl]-1-propane sulfonate appeared to stabilize the μ-opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3-(3-cholamidopropyl)dimethyl]-1-propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound ³H]d -Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin (³H]DAMGO), a μ-selective opioid agonist, with high affinity (K_D = 1.90 ± 0.93 nM; B_max = 629 ± 162 fmol/mg of protein). Of the membrane-associated ³H]-DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific ³H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate). The solubilized receptor also bound ³H]diprenorphine, a nonselective opioid antagonist, with high affinity (K_D = 1.4 ± 0.39 nM, B_max = 920 ± 154 fmol/mg of protein). Guanosine 5′-O-(3-thiotriphosphate) did not diminish ³H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with ³H]diprenorphine for the solubilized binding sites; in contrast, d -Pen²,d -Pen⁵]-enkephalin, a δ-selective opioid agonist, and U50488H, a κ-selective opioid agonist, failed to compete with ³H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for ³H]diprenorphine binding sites better fit a two-site than a one-site model with K_Dhigh = 2.17 ± 1.5 nM, B_max = 648 ± 110 fmol/mg of protein and K_Dlow = 468 ± 63 nM, B_max = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate), the DAMGO displacement curve better fit a one- than a two-site model with K_D = 815 ± 33 nM, B_max = 965 ± 124 fmol/mg of protein.