Mice Expressing Regulators of G protein Signaling–insensitive Gαo Define Roles of μ Opioid Receptor Gαo and Gαi Subunit Coupling in Inhibition of Presynaptic GABA Release

Regulators of G protein signaling (RGS) proteins modulate signaling by G protein-coupled receptors. Using a knock-in transgenic mouse model with a mutation in G α o that does not bind RGS proteins (RGS-insensitive), we determined the effect of RGS proteins on presynaptic μ opioid receptor (MOR)-mediated inhibition of GABA release in the ventrolateral periaqueductal gray (vlPAG). The MOR agonists [d-Ala 2 , N -MePhe 4 , Gly-ol]-enkephalin (DAMGO) and met-enkephalin (ME) inhibited evoked inhibitory postsynaptic currents (eIPSCs) in the RGS-insensitive mice compared with wild-type (WT) littermates, respectively. Fentanyl inhibited eIPSCs similarly in both WT and RGS-insensitive mice. There were no differences in opioid agonist inhibition of spontaneous GABA release between the genotypes. To further probe the mechanism underlying these differences between opioid inhibition of evoked and spontaneous GABA release, specific myristoylated G α peptide inhibitors for G α o 1 and G α i 1-3 that block receptor-G protein interactions were used to test the preference of agonists for MOR-G α complexes. The G α o 1 inhibitor reduced DAMGO inhibition of eIPSCs, but G α i 1-3 inhibitors had no effect. Both G α o 1 and G α i 1-3 inhibitors separately reduced fentanyl inhibition of eIPSCs but had no effects on ME inhibition. G α i 1-3 inhibitors blocked the inhibitory effects of ME and fentanyl on miniature postsynaptic current (mIPSC) frequency, but both G α o 1 and G α i 1-3 inhibitors were needed to block the effects of DAMGO. Finally, baclofen-mediated inhibition of GABA release is unaffected in the RGS-insensitive mice and in the presence of G α o 1 and G α i 1-3 inhibitor peptides, suggesting that GABA B receptor coupling to G proteins in vlPAG presynaptic terminals is different than MOR coupling. SIGNIFICANCE STATEMENT: Presynaptic μ opioid receptors (MORs) in the ventrolateral periaqueductal gray are critical for opioid analgesia and are negatively regulated by RGS proteins. These data in RGS-insensitive mice provide evidence that MOR agonists differ in preference for Gαo versus Gαi and regulation by RGS proteins in presynaptic terminals, providing a mechanism for functional selectivity between agonists. The results further define important differences in MOR and GABA B receptor coupling to G proteins that could be exploited for new pain therapies.