Abstract

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 Gαo 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-Ala², N-MePhe⁴, 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 Gα peptide inhibitors for Gαo₁ and Gαi_1-3 that block receptor–G protein interactions were used to test the preference of agonists for MOR-Gα complexes. The Gαo₁ inhibitor reduced DAMGO inhibition of eIPSCs, but Gαi_1-3 inhibitors had no effect. Both Gαo₁ and Gαi_1-3 inhibitors separately reduced fentanyl inhibition of eIPSCs but had no effects on ME inhibition. Gαi_1-3 inhibitors blocked the inhibitory effects of ME and fentanyl on miniature postsynaptic current (mIPSC) frequency, but both Gαo₁ and Gαi_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 Gαo₁ and Gαi_1-3 inhibitor peptides, suggesting that GABA_B receptor coupling to G proteins in vlPAG presynaptic terminals is different than MOR coupling.