Salvinorin A: Allosteric Interactions at the μ-Opioid Receptor

Abstract

Salvinorin A [(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(acetyloxy)-2-(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2h-naphtho[2,1-c]pyran-7-carboxylic acid methyl ester] is a hallucinogenic κ-opioid receptor agonist that lacks the usual basic nitrogen atom present in other known opioid ligands. Our first published studies indicated that Salvinorin A weakly inhibited μ-receptor binding, and subsequent experiments revealed that Salvinorin A partially inhibited μ-receptor binding. Therefore, we hypothesized that Salvinorin A allosterically modulates μ-receptor binding. To test this hypothesis, we used Chinese hamster ovary cells expressing the cloned human opioid receptor. Salvinorin A partially inhibited [³H]Tyr-d-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) (0.5, 2.0, and 8.0 nM) binding with E_MAX values of 78.6, 72.1, and 45.7%, respectively, and EC₅₀ values of 955, 1124, and 4527 nM, respectively. Salvinorin A also partially inhibited [³H]diprenorphine (0.02, 0.1, and 0.5 nM) binding with E_MAX values of 86.2, 64, and 33.6%, respectively, and EC₅₀ values of 1231, 866, and 3078 nM, respectively. Saturation binding studies with [³H]DAMGO showed that Salvinorin A (10 and 30 μM) decreased the μ-receptor B_max and increased the K_d in a dose-dependent nonlinear manner. Saturation binding studies with [³H]diprenorphine showed that Salvinorin A (10 and 40 μM) decreased the μ-receptor B_max and increased the K_d in a dose-dependent nonlinear manner. Similar findings were observed in rat brain with [³H]DAMGO. Kinetic experiments demonstrated that Salvinorin A altered the dissociation kinetics of both [³H]DAMGO and [³H]diprenorphine binding to μ receptors. Furthermore, Salvinorin A acted as an uncompetitive inhibitor of DAMGO-stimulated guanosine 5′-O-(3-[³⁵S]thio)-triphosphate binding. Viewed collectively, these data support the hypothesis that Salvinorin A allosterically modulates the μ-opioid receptor.