Site-directed alkylation of multiple opioid receptors. I. Binding selectivity.

We report a method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites. We used radioligands that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands, sufentanil for mu sites, [D-Ala2, D-Leu5] enkephalin for delta sites, and dynorphin A for kappa sites. After enrichment for mu receptors, [3H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [3H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but we found no evidence for residual mu or delta sites in kappa-enriched membranes. We used this method to identify ligands that are highly selective for each of the three types of sites: Tyr-D-Ala-Gly-(Me)Phe-Gly-ol, sufentanil, and morphiceptin for mu sites; (D- Pen2 , D- Pen5 ]enkephalin and [D- Pen2 ,L- Pen5 ]enkephalin for delta sites; and tifluadom and U50 ,488 for kappa sites.