SOLUBILIZATION OF HIGH-AFFINITY, GUANINE NUCLEOTIDE-SENSITIVE MU-OPIOID RECEPTORS FROM RAT-BRAIN MEMBRANES

High-affinity mu-opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with na ltrexone to increase the density of opioid receptors in brain membrane s. Occupancy of the membrane-associated receptors with morphine during solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-pr opane sulfonate appeared to stabilize the mu-opioid receptor. After re moval of free morphine by Sephadex G50 chromatography and adjustment o f the 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate concentratio n to 3 mM, the solubilized opioid receptor bound [H-3][D-Ala(2),N-Me-P he(4),Gly-ol(5)]-enkephalin ([H-3]DAMGO), a mu-selective opioid agonis t, with high affinity (K-D = 1.90 +/- 0.93 nM B-max = 629 +/- 162 fmol /mg of protein). Of the membrane-associated [H-3]DAMGO binding sites, 29 +/- 7% were recovered in the solubilized fraction. Specific [H-3]DA MGO binding was completely abolished in the presence of 10 mu M guanos ine 5'-O-(3-thiotriphosphate). The solubilized receptor also bound [H- 3]diprenorphine, a nonselective opioid antagonist, with high affinity (K-D = 1.4 +/- 0.39 nM, B-max = 920 +/- 154 fmol/mg of protein). Guano sine 5'-O-(3-thiotriphosphate) did not diminish [H-3]diprenorphine bin ding. DAMGO at concentrations between 1 nM and 1 mu M competed with [H -3]diprenorphine for the solubilized binding sites; in contrast, [D-Pe n(2),D-Pen(5)]-enkephalin, a delta-selective opioid agonist, and U5043 3H, a kappa-selective opioid agonist, failed to compete with [H-3]dipr enorphine for the solubilized binding sites at concentrations of <1 mu M. In the absence of guanine nucleotides, the DAMGO displacement curv e for [H-3]diprenorphine binding sites better fit a two-site than a on e-site model with K-Dhigh = 2.17 +/- 1.5 nM B-max = 648 +/- 110 fmol/m g of protein and K-Dlow = 468 +/- 63 nM B-max = 253 +/- 84 fmol/mg of protein. In the presence of 10 mu M guanosine 5'-O-(3-thiotriphosphate ), the DAMGO displacement curve better fit a one- than a two-site mode l with K-D = 815 +/- 33 nM, B-max = 965 +/- 124 fmol/mg of protein.