Solubilization and Characterization of σ‐Receptors from Guinea Pig Brain Membranes

Abstract

The σ-receptor, a distinct binding site in brain tissue that may mediate some of the psychotomimetic properties of benzomorphan opiates and phencyclidine, has been sol-ubilized using the ionic detergent sodium cholate. Binding assays were performed with the solubilized receptor using vacuum filtration over polyethyleneimine-treated glass fiber filters. The pharmacological specificity of the solubilized binding site for σ-receptor ligands is nearly identical to the membrane-bound form of the receptor, with the order of potencies for displacement of the selective σ-ligand [³H]di-o-tolylguanidine ([³H]DTG) closely correlated. The stereoselectivity for (+)-benzomorphan opiate enantiomers was retained by the solubilized receptor. The soluble receptor retained high affinity for binding of [³H]DTG (K_D= 28 ± 0.5 nM) and (+)-[³H]3-(3-hydroxyphenyl)-N-(l-propyl)piperi-dine {(+)-[³H]3-PPP} (K_D= 36 ± 2 nM). Photoaffinity labeling of the solubilized receptor by [³H]p-azido-DTG, a σ-selective photoaffinity label, resulted in labeling of a 29-kilodalton polypeptide identical in size to that labeled in intact membranes. Estimation of the Stokes radius of the [³H]DTG binding site was obtained by Sepharose CL-6B chromatography in the presence of 20 mM cholate and calculated to be 8.7 nm. This value was identical to the molecular size found for the binding sites of the σ-selective ligands (+)-[³H]3-PPP and (+)-[³H]SKF-10,047, supporting the hypothesis that all three ligands bind to the same macro-molecular complex.