μ-Opioid Receptor Superagonists Engage a Sodium-Bound Active State

The simple two-state conformational selection model of G-protein coupled receptor (GPCR) activation suggests that, by binding to a high affinity site, an agonist will shift receptor equilibrium in favor of active state (R*) conformations that recruit heterotrimeric G proteins over inactive state (R) conformations. Agonist binding to the -opioid receptor is highly sensitive to Na+ ions which stabilize an inactive receptor state. Higher efficacy opioid agonists, such as DAMGO and fentanyl, are sensitive to Na+ compared to lower efficacy ligands at the -opioid receptor. However, the binding of the highly potent oripavine agonists etorphine and dihydroetorphine are less sensitive to Na+ than expected such that the prevailing models fail to explain their pharmacology. To explain this discrepancy, experiments were performed to evaluate the binding properties and G protein activation of the highly potent agonists carfentanil, BU72, etorphine, etonitazene and similarly potent opioid peptidomimetics in comparison to the standard agonists DAMGO, fentanyl, and morphine in the presence or absence of Na+ or K+ ions. Several of the superagonists retained high affinity and potency in both ionic conditions, whereas DAMGO, fentanyl and morphine displayed enhanced binding and signaling in K+, compared to Na+ ions. These functional parameters were used to determine an intrinsic efficacy value, determined as [Formula]. Comparison of affinity shifts with intrinsic efficacy afforded a negative correlation in which superagonists with the highest intrinsic efficacy are least sensitive to Na+. These data suggest that select -opioid receptor superagonists have high affinity for the Na+ bound receptor states (R) and shift these species into active receptor conformations (R*) that efficiently couple to G proteins. Significance StatementThe simple theory of conformational selection suggests the binding affinities of high efficacy -opioid receptor ligands, such as fentanyl and DAMGO, are more sensitive to Na+ and guanine nucleotide which stabilize inactive receptor states than lower efficacy agonists and antagonists. Here, we show that ligands with high intrinsic efficacy (superagonists) are much less sensitive to Na+ and guanine nucleotide. This work demonstrates that highly potent ligands can engage a low affinity Na+-bound receptor state that may then convert to a receptor species that efficiently couples to G protein - i.e. a conformational induction.