Distinct Subtypes of the Opioid Receptor with Allosteric Interactions in Brain Membranes

Abstract: The binding isotherms of opioid receptors in rat brain membranes with [³H]D‐Ala²‐D‐Leu⁵‐enkephalin ([³H]DADLE), [³H]dihydromorphine ([³H]DHM), and [³H]etorphine were analysed to show the effects of Mg²⁺, Na⁺, and guanine nucleotides. Four opioid receptor subtypes of δ, K, μ₁, and μ₂ specificities were differentiated, where necessary with the aid of specific displacing ligands. Both a guanine nucleotide [guanosine‐5′‐(β, γ‐imido)triphosphate] and the cations (Na⁺, Mg²⁺) affect the affinity state of all four subtypes of the receptor. The opioid binding behaviour is found on detailed inspection to be complex, with cases of “half‐of‐the‐sites” reactivity and of cooperativity. By their behaviour under the various ionic conditions noted, it was concluded that these subtypes are distinct, without the need to assume interconvertibility by such agents. The evidence suggests that the formation of heterologous K–δ or μ₁–μ₂ receptor complexes is required for stabilization of the high‐affinity conformational state of the receptor. Important effects of cations in increasing the binding and regulating the equilibria of receptor association‐dissociation were observed when these studies were conducted, not in the Tris‐HCl buffer commonly used in opioid binding assays, but in N‐tris[hydroxymethyl]‐methyl‐2‐aminoethanesulphonate (K⁺) buffer (TES‐KOH; 10 mM, pH 7.5): it was found that ionic species of Tris can substitute for divalent cations. Dithiothreitol effects on agonist binding in the presence and absence of the cations suggested that those cation effects involve the exchange of –SH/‐SS– bonds between receptor subunits. All of the behaviour is interpreted in terms of a model involving association‐dissociation equilibria of homologous and/or heterologous receptor subunits of an oligomeric opioid receptor structure.