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 Gao 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 Ga peptide inhibitors for Gao1 and Gai1-3 that block receptor-G protein interactions were used to test the preference of agonists for MOR-Ga complexes. The Gao1 inhibitor reduced DAMGO inhibition of eIPSCs, but Gai1-3 inhibitors had no effect. Both Gao1 and Gai1-3 inhibitors separately reduced fentanyl inhibition of eIPSCs but had no effects on ME inhibition. Gai1-3 inhibitors blocked the inhibitory effects of ME and fentanyl on miniature postsynaptic current (mIPSC) frequency, but both Gao1 and Gai1-3 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 Gao1 and Gai1-3 inhibitor peptides, suggesting that GABAB receptor coupling to G proteins in vlPAG presynaptic terminals is different than MOR coupling. SIGNIFICANCE STATEMENT Presynaptic l 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 Gao versus Gai 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 GABAB receptor coupling to G proteins that could be exploited for new pain therapies.
ASJC Scopus subject areas
- Molecular Medicine