High potency μ-opioid receptor agonists engage sodium-bound receptor states

The simple 2-state conformational selection model of G protein-coupled receptor activation suggests that, by binding to a high-affinity state, an agonist shifts receptor equilibrium in favor of active state (R∗) conformations that recruit heterotrimeric G proteins over inactive (R) conformations. Agonist binding affinity at the μ-opioid receptor is reduced in the presence of Na⁺ ions, which stabilize R conformations. The binding of higher efficacy opioid agonists, such as [D-Ala², NMe-Phe⁴, Gly-ol⁵]-enkephalin and fentanyl, is more sensitive to Na⁺ ions than lower efficacy ligands. However, the binding of the highly potent agonists etorphine and dihydroetorphine is less sensitive to Na⁺ ions than expected, such that the prevailing models fail to explain their pharmacology. To understand this discrepancy, experiments were performed to evaluate the binding properties and G protein activation of several high-affinity, high-potency agonists, including carfentanil, etonitazene, and several peptidomimetics in comparison to [D-Ala², NMe-Phe⁴, Gly-ol⁵]-enkephalin, Met-enkephalin, fentanyl, and morphine in the presence of Na⁺ or K⁺ ions. Several very potent agonists retained high-affinity and potency in both ionic conditions, whereas the standard agonists displayed enhanced binding and signaling only in the presence of K⁺ ions. Comparison of the difference in binding affinity with degree of agonism afforded a negative correlation in which several ligands with a very high degree of agonism were least sensitive to Na⁺ ions. These data suggest that select highly potent μ-opioid receptor agonists have high-affinity for Na⁺ bound receptor states and shift the receptors into active conformations that efficiently couple to G protein. SIGNIFICANCE STATEMENT: The theory of conformational selection suggests that agonists bind to active states (R∗) of G protein-coupled receptors, including the μ-opioid receptor, to shift the receptor equilibrium in favor of R∗ over inactive states (R). This study demonstrates that ligands with very high-affinity and potency at the μ-opioid receptor can engage low-affinity Na⁺-bound receptor states (R) and convert these to active R∗ states that efficiently couple to G protein, suggesting a conformational induction model.