Comparison of the relative efficacy and potency of μ-opioid agonists to activate Gαi/o proteins containing a pertussis toxin-insensitive mutation

Pertussis toxin (PTX)-insensitive mutants of G alpha(i/o) proteins expressed in C6 mu cells were used to examine the hypothesis that there are agonist-specific conformational states of the mu-opioid receptor with coupling preferences to different G alpha(i/o) subtypes, as measured by the degree of stimulation of [S-35] guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) binding. Binding of [S-35]GTP gamma S to endogenous G alpha(i/o) proteins stimulated by the full mu-opioid agonist [D-Ala(2), MePhe(4), Gly(5)-ol]enkephalin (DAMGO) was completely blocked by overnight treatment with 100 ng/ml PTX. Treatment for 4 h with lower concentrations led to a PTX-dependent reduction in the maximal effect of DAMGO but no alteration in the potency of DAMGO or morphine nor in the relative maximal effect (relative efficacy) of the partial agonists morphine and buprenorphine compared with the full agonist DAMGO. Using PTX-insensitive G alpha mutants in which the PTX-sensitive cysteine was replaced with isoleucine, the potency for a series of mu-opioid agonists was highest in cells expressing G alpha(i3) and G alpha(o) and lowest with G alpha(i1) and G alpha(i2), with no significant change in the order of potency, namely, etorphine >> endomorphin-1 = DAMGO = endomorphin-2 = fentanyl = morphine >> meperidine. The order of agonist relative efficacy, etorphine = DAMGO = endomorphin-1 = endomorphin-2 = fentanyl >= morphine >= meperidine > buprenorphine >= nalbuphine, was also the same across all of the PTX-insensitive G alpha(i/o) subtypes. Highest relative efficacy to stimulate [S-35]GTP gamma S binding was seen with G alpha(i3). Consequently, reported observations of agonist-directed trafficking at mu-opioid receptors most likely involve non-PTX-sensitive G alpha protein mechanisms.