Solubilization of high-affinity, guanine nucleotide-sensitive μ-opioid receptors from 7315c cell membranes

High-affinity mu-opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane-associated receptors with morphine before their solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [3H]-etorphine with high affinity (KD = 0.304 +/- 0.06 nM; Bmax = 154 +/- 33 fmol/mg of protein). Of the membrane-associated [3H]etorphine binding sites, 40 +/- 5% were recovered in the solubilized fraction. Both mu-selective and non-selective enkephalins competed with [3H]etorphine for the solubilized binding sites; in contrast, delta- and kappa-opioid enkephalins failed to compete with [3H]etorphine for the solubilized binding sites at concentrations of < 1 microM. The mu-selective ligand [3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin also bound with high affinity (KD = 0.79 nM; Bmax = 108 +/- 17 fmol/mg of protein) to the solubilized material. Of the membrane-associated [3H][D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin binding sites, 43 +/- 3% were recovered in the solubilized material. Guanosine 5'-O-(3-thiotriphosphate), GTP, and guanosine 5'-O-(2-thiodiphosphate), but not adenylylimidodiphosphate, diminished [3H][D-Ala2,N-Me-Phe4,Gly5-ol] enkephalin binding in a concentration-dependent manner. Finally, mu-opioid receptors from rat brain membranes were also solubilized in a high-affinity, guanine nucleotide-sensitive state if membrane-associated receptors were occupied with morphine before and during their solubilization with the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate.