Mice Expressing Regulators of G protein Signaling-insensitive Gαo Define Roles of μ Opioid Receptor G α o and G α i Subunit Coupling in Inhibition of Presynaptic GABA Release

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. 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.

Fig. 1.

Opioid agonist inhibition of evoked IPSCs is differentially affected in RGS-insensitive mice. (A) Representative traces depicting inhibition of eIPSCs by DAMGO (5 µM) in WT and RGS-insensitive (Het) mice. The inhibition is reversed by naloxone. (B) Combined experiments of % inhibition (±S.D.) by a maximal DAMGO concentration (20 µM; gray bar) and a submaximal concentration (5 µM) in WT compared with Het mice (one-way ANOVA; F_{(2, 25)} = 4.9, P = 0.02; Dunnett’s, *P < 0.05). (C) Combined experiments of % inhibition (±S.D.) by a maximal ME concentration (30 µM; gray bar) and a submaximal concentration (10 µM) in WT compared with Het mice (one-way ANOVA; F_{(2, 15)}= 17.7, P = 0.0001; Dunnett’s, *P < 0.05; ****P < 0.0001). (D) Combined experiments of % inhibition (±S.D.) by a maximal fentanyl concentration (10 µM; gray bar) and a submaximal concentration (1 µM) in WT compared with Het mice (one-way ANOVA; F_{(2, 18)}= 4.1, P = 0.03; Dunnett’s, *P < 0.05). Symbols in bars denote recordings, and numbers denote number of animals used in each group.

Fig. 2.

MOR agonists differentially activate Gα subunits to inhibit evoked GABA release. (A) DAMGO (5 µM)-mediated inhibition of eIPSCs in the absence (control) and presence of Gαo peptide inhibitor and Gαi peptide inhibitors (one-way ANOVA, F_{(3, 22)} = 19.1, P = 0.0001; Dunnett’s, **P < 0.001, ****P < 0.0001). (B) ME (10 µM)-mediated inhibition of eIPSCs in absence and presence of inhibitors (one-way ANOVA, F_{(3, 19)} = 4.2; P = 0.02; Dunnett’s, **P < 0.001). (C) Fentanyl (1 µM)-mediated inhibition of eIPSCs in absence and presence of inhibitors (one-way ANOVA, F_{(2, 14)} = 7.3, P = 0.007, Dunnett’s, *P < 0.05, ** P < 0.001). Symbols in bars denote recordings, and numbers denote number of animals used in each bar. inhib, inhibitor; ns, not significant.

Fig. 3.

Opioid inhibition of GABAergic mIPSCs is not altered in RGS-insensitive mice. (A) Combined experiments of % inhibition (±S.D.) by a maximal DAMGO concentration (20 µM; gray bar) and a submaximal concentration (5 µM) in WT compared with Het mice (one-way ANOVA; F_{(2, 21)} = 0.3, P = 0.8). (B) Combined experiments of % inhibition (±S.D.) by a maximal ME concentration (ME 30 µM; gray bar) and a submaximal concentration (10 µM) in WT compared with Het mice (one-way ANOVA, F_{(2, 24)} = 0.5, P = 0.6). (C) Combined experiments of % inhibition (±S.D.) by a maximal fentanyl concentration (10 µM; gray bar) and a submaximal concentration (1 µM) in WT compared with Het mice (one-way ANOVA; F_{(2, 25)}= 1.2, P = 0.3). Symbols in bars denote recordings, and numbers denote number of animals used in each bar. freq., frequency.

Fig. 4.

MOR-Gαi coupling is more important for inhibition of spontaneous GABA release. (A) Inhibition of mIPSCs by DAMGO (5 µM) is unaffected by Gαo and Gαi inhibitors alone (one-way ANOVA; F_{(3, 32)} = 12.6, P = 0.0001; Dunnett’s, ****P = 0.0001). (B) Inhibition by ME is reduced in the presence of Gαi inhibitors but not by the Gαo inhibitor (F_{(2, 19)} = 11.8, P = 0.001, Dunnett’s, **P < 0.01). (C) Inhibition by fentanyl is reduced in the presence of Gαi inhibitors but not by the Gαo inhibitor (F_{(2, 19)} = 6.2, P = 0.01, Dunnett’s, **P < 0.01). Symbols in bars denote recordings, and numbers denote number of animals used in each bar. inhib, inhibitor.

Fig. 5.

Baclofen-mediated inhibition of evoked GABA release is not affected in slices from RGS-insensitive mice or by Gαo/i peptide inhibitors. (A) Baclofen (5 µM) inhibition is similar in WT and RGS-insensitive (Het) mice (t₍₁₂₎ = 1.3, P = 0.2). (B) Baclofen (5 µM)-mediated inhibition is not altered in the presence of peptide inhibitors (F_{(3, 20)} = 0.2, P = 0.9). (C) Baclofen (20 µM)-mediated inhibition is not altered by the peptide inhibitors (F_{(2, 24)} = 0.3, P = 0.7). Symbols in bars denote recordings, and numbers denote number of animals used in each bar. inhib, inhibitor.

Fig. 6.

Baclofen-mediated inhibition of spontaneous GABA release in slices is not affected in RGS-insensitive mice or in the presence of Gαo/i peptide inhibitors. (A) Baclofen (5 µM) inhibition is similar in WT and RGS-insensitive (Het) mice (t₍₁₁₎ = 1.8, P = 0.1). (B) Baclofen (5 µM)-mediated inhibition is not altered in the peptide inhibitors (F_{(2, 14)} = 0.4, P = 0.7). (C) Baclofen (20 µM) inhibition is similar in WT and RGS-insensitive (Het) mice (t₍₁₇₎ = 0.005, P = 1.0). (D) Baclofen (20 µM)-mediated inhibition is not altered in the peptide inhibitors (F_{(2, 46)} = 0.03, P = 1.0). Symbols in bars denote recordings, and numbers denote number of animals used in each bar. inhib, inhibitor.