GPCR-dependent biasing of GIRK channel signaling dynamics by RGS6 in mouse sinoatrial nodal cells

Abstract

How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M₂R) and adenosine (A₁R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M₂R and A₁R activate a shared pool of cardiac G protein-gated inwardly rectifying K⁺ (GIRK) channels in SAN cells from adult mice, but A₁R-GIRK responses are smaller and slower than M₂R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M₂R-GIRK coupling efficiency and kinetics and A₁R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gα_o over Gα_i as a substrate for its catalytic activity and that M₂R signals preferentially via Gα_o, while A₁R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gα_o or Gα_i2 was consistent with these findings. Gα_i2 ablation had minimal impact on M₂R-GIRK and A₁R-GIRK signaling in SAN cells. In contrast, Gα_o ablation decreased the amplitude and slowed the kinetics of M₂R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A₁R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gα_o substrate preference of RGS6, shape A₁R- and M₂R-GIRK signaling dynamics in mouse SAN cells.

Keywords: G protein; Kir3; adenosine; heart rate; muscarinic.

Fig. 1.

Ado- and CCh-induced GIRK currents in SAN cells. (A and B) Whole-cell currents (V_hold = −70 mV) evoked by Ado (10 μM; A) or CCh (10 μM; B) in SAN cells from wild-type (Left, black) and Rgs6^−/− (Right, red) mice; these currents were not observed in SAN cells from Girk4^−/− mice (Bottom, blue). (Scale bars: 5 s/500 pA.) (C) Peak current density of responses elicited by Ado (Left) and CCh (Right) in SAN cells from wild-type (black) and Rgs6^−/− (red) mice. There was a significant interaction between genotype and agonist (F_1,49 = 14.9, P < 0.001; two-way ANOVA); group sizes ranged from 10 to 20 cells (5–7 mice). **P < 0.01 wild-type vs. Rgs6^−/− (within agonist); ^####P < 0.0001 Ado vs. CCh (wild-type). (D) Acute desensitization of responses elicited by Ado (Left) and CCh (Right) in SAN cells from wild-type (black) and Rgs6^−/− (red) mice. Statistical analysis revealed a main effect of agonist (F_1,49 = 30.4, P < 0.0001; two-way ANOVA), but no main effect of genotype (F_1,49 = 0.13, P = 0.72; two-way ANOVA) or genotype x agonist interaction (F_1,49 = 1.6, P = 0.21; two-way ANOVA); group sizes ranged from 9 to 19 cells (5–7 mice). (E) Activation rates of responses elicited by Ado (Left) and CCh (Right) in SAN cells from wild-type (black) and Rgs6^−/− (red) mice. Statistical analysis revealed a genotype x agonist interaction (F_1,45 = 6.8, P < 0.05; two-way ANOVA); group sizes ranged from 9 to 17 cells (5–7 mice). **P < 0.01 wild-type vs. Rgs6^−/− (within agonist); ^####P < 0.0001 Ado vs. CCh (wild type). (F) Deactivation rates of responses elicited by Ado (Left) and CCh (Right) in SAN cells from wild-type (black) and Rgs6^−/− (red) mice. Statistical analysis revealed a genotype x agonist interaction (F_1,48 = 8.0, P < 0.01; two-way ANOVA); group sizes ranged from 12 to 19 cells (5–7 mice). ***P < 0.001 wild-type vs. Rgs6^−/− (within agonist); ^###P < 0.001 Ado vs. CCh (wild type).

Fig. 2.

Impact of Rgs6 ablation on GIRK channel sensitivity to CCh and Ado. (A) Concentration-response experiments for Ado in SAN cells from wild-type (Scale bars: Top, 10 s/200 pA.) and Rgs6^−/− (Scale bars: Bottom, 10 s/500 pA.) mice. (B and C) Summary of concentration-response experiments for Ado-induced currents in SAN cells from wild-type and Rgs6^−/− mice. There was no difference in EC₅₀ values for Ado-induced currents in SAN cells from wild-type (n = 24 cells/7 mice) and Rgs6^−/− (n = 13 cells/5 mice) mice (t₃₅ = 1.8, P = 0.08; unpaired t test). (D) Concentration-response experiments for CCh-induced currents in SAN cells from wild-type (Scale bars: Top, 10 s/200 pA.) and Rgs6^−/− (Scale bars: Bottom, 10 s/500 pA.) mice. (E and F) Summary of concentration-response experiments of CCh-induced currents in SAN cells from wild-type and Rgs6^−/− mice. The EC₅₀ values for CCh-induced currents in SAN cells from Rgs6^−/− (n = 12 cells/4 mice) mice were lower than the EC₅₀ values measured in wild-type counterparts (n = 10 cells/4 mice) (t₂₀ = 4.8, ***P < 0.001; unpaired t test).

Fig. 3.

CPA- and CCh-induced bradycardia in isolated hearts from wild-type and Rgs6^−/− mice. (A) Segments of ECG traces from isolated wild-type (Top) and Rgs6^−/− (Bottom) hearts perfused with the A₁R-selective agonist CPA (30 nM). (Scale bar: 5 s.) (B) Percentage decrease in HR (relative to baseline) following perfusion of increasing concentrations of CPA in hearts from wild-type (n = 6), Rgs6^−/− (n = 7), and Girk4^−/− (n = 4) mice; there was a genotype x CPA concentration interaction for wild-type and Rgs6^−/− hearts (F_4,44 = 2.81; P < 0.05; two-way ANOVA with repeated measures). **P < 0.01 wild type vs. Rgs6^−/−. (C) Segments of ECG traces of isolated wild-type (Top) and Rgs6^−/− (Bottom) hearts perfused with CCh (1 μM). (Scale bar: 5 s.) (D) Percentage decrease in HR (relative to baseline) following perfusion of increasing concentrations of CCh in hearts from wild-type (n = 3), Rgs6^−/− (n = 5), and Girk4^−/− (n = 6) mice; there was a genotype x CCh concentration interaction for wild-type and Rgs6^−/− hearts (F_3,18 = 5.1, P < 0.05; two-way ANOVA with repeated measures). *P < 0.05 and ***0.001, respectively, wild type vs. Rgs6^−/−.

Fig. 4.

Occlusion of CCh- and Ado-induced GIRK currents. (A) Occlusion experiments showing whole-cell currents elicited by a maximal concentration of CCh (10 μM), followed by Ado application (10 μM) in SAN cells from wild-type (Left) and Rgs6^−/− (Right) mice. (Scale bars: 10 s/500 pA.) (B) There was no difference in the Ado-induced additive response in SAN cells isolated from wild-type (n = 9 cells/4 mice) and Rgs6^−/− (n = 6 cells/5 mice) mice (t₁₃ = 0.11, P = 0.91; unpaired t test). (C) Occlusion experiments showing whole-cell currents elicited by a maximal concentration of Ado (10 μM), followed by CCh application (10 μM) in SAN cells from wild-type (Left) and Rgs6^−/− (Right) mice. (Scale bars: 10 s/500 pA.) (D) There was a significant difference in the CCh-induced additive response in SAN cells from wild-type (n = 7 cells/4 mice) and Rgs6^−/− (n = 6 cells/4 mice) mice (t₁₁ = 6.0; ****P < 0.0001; unpaired t test).

Fig. 5.

G protein coupling preferences of M₂R and A₁R. (A) Schematic representation of the BRET assay for real-time optical imaging of G protein activity. Agonist-induced activation of a GPCR leads to the dissociation of Gα-GTP and Venus-Gβγ subunits. The released Venus-Gβγ then interacts with the Gβγ effector mimetic masGRK3ct-Nluc-HA to produce the BRET signal. (B) Representative real-time monitoring of G protein activation by M₂R (Top) or A₁R (Bottom). HEK293T/17 cells were transfected with the BRET sensor pair (A) and GPCR, together with either Gα_oA (black) or Gα_i1 (green). Acetylcholine (100 μM) or Ado (100 μM) was applied at the 5-s time point, and the BRET signal was followed across time. (C and D) G protein coupling summary diagrams for M₂R and A₁R. Maximum amplitudes (red) and activation rate constants (blue) for 15 different G proteins were normalized to the largest value and plotted in the wheel diagrams. Line thickness represents the SEM of three technical replicates performed independently.

Fig. 6.

Gα substrate specificity of RGS6/Gβ5. (A) Schematic representation of the BRET assay for measuring RGS GAP activity. Application of antagonist after G protein activation by a GPCR agonist initiates the deactivation of G proteins, decreasing the BRET signal. (B) Real-time monitoring of G protein deactivation. HEK293T/17 cells were transfected with D₂R, Gα, Venus-Gβγ, and masGRK3ct-Nluc-HA with (red) or without (black) RGS6/Gβ5. Dopamine (100 μM) and haloperidol (100 μM) were applied to activate D₂R and initiate G protein deactivation, respectively. Representative data from three independent experiments are shown. (C) Comparison of kGAP activity of RGS6/Gβ5 on the G_i/o isoforms (F_5,12 = 372.6, P < 0.0001; one-way ANOVA). ****P < 0.0001 vs. Gα_o isoforms.

Fig. 7.

Impact of Gα_o and Gα_i2 ablation on CCh- and Ado-induced GIRK currents. (A) Whole-cell currents evoked by CCh (10 μM) in SAN cells from SLNCre(−):Gα_o^fl/fl (Left, gray) and SLNCre(+):Gα_o^fl/fl (Right, green) mice. (Scale bars: 5 s/500 pA.) (B and C) Peak CCh-induced current density (t₂₀ = 4.3, ***P < 0.001; unpaired t test) and current desensitization (t₁₉ = 2.6, *P < 0.05; unpaired t test) were smaller in SAN cells from SLNCre(+):Gα_o^fl/fl mice as compared to their SLNCre(−):Gα_o^fl/fl counterparts; group sizes ranged from 9 to 12 cells (5 mice) per genotype. There was no difference in CCh-induced current density (t₁₃ = 0.4, P = 0.69; unpaired t test) or desensitization (t₁₃ = 0.5, P = 0.62; unpaired t test) in SAN cells from SLNCre(−):Gα_i2^fl/fl mice and SLNCre(+):Gα_i2^fl/fl mice; group sizes ranged from 7 to 8 cells (3 mice) per genotype. (D and E) Activation (t₁₉ = 3.0, **P < 0.01; unpaired t test) and deactivation (t₁₈ = 2.5, *P < 0.05; unpaired t test) rates of CCh-induced currents were prolonged in SAN cells from SLNCre(+):Gα_o^fl/fl mice, relative to SLNCre(−):Gα_o^fl/fl counterparts; group sizes ranged from 8 to 12 cells (5 mice) per genotype. There was no difference in activation (t₁₁ = 0.9, P = 0.38; unpaired t test) or deactivation (t₁₃ = 1.0, P = 0.36; unpaired t test) rates of CCh-induced currents in SAN cells from SLNCre(−):Gα_i2^fl/fl and SLNCre(+):Gα_i2^fl/fl mice; group sizes ranged from 6 to 8 cells (3 mice) per genotype. (F) Whole-cell currents evoked by Ado (10 μM) in SAN cells from SLNCre(−):Gα_o^fl/fl (Left, gray) and SLNCre(+):Gα_o^fl/fl (Right, green) mice. (Scale bars: 5 s/500 pA.) (G and H) There was no difference in peak current density (t₂₀ = 1.8, P = 0.08; unpaired t test) or desensitization (t₂₁ = 0.9, P = 0.38; unpaired t test) of Ado-induced currents in SAN cells from SLNCre(−):Gα_o^fl/fl and SLNCre(+):Gα_o^fl/fl mice; group sizes ranged from 11 to 12 cells (4 mice) per genotype. There were no differences in Ado-induced current density (t₁₃ = 0.1, P = 0.91; unpaired t test) or desensitization (t₁₃ = 0.7, P = 0.51; unpaired t test) in SAN cells from SLNCre(−):Gα_i2^fl/fl and SLNCre(+):Gα_i2^fl/fl mice; group sizes ranged from 7 to 8 cells (3 mice) per genotype. (I and J) There was no difference in peak current density (t₂₀ = 1.8, P = 0.08; unpaired t test) or desensitization (t₂₁ = 0.9, P = 0.38; unpaired t test) of Ado-induced currents in SAN cells from SLNCre(−):Gα_o^fl/fl and SLNCre(+):Gα_o^fl/fl mice; group sizes ranged from 11 to 12 cells (4 mice) per genotype. There was an increase in activation rate (t₁₃ = 2.2, *P < 0.05; unpaired t test) of Ado-induced currents in SAN cells from SLNCre(+):Gα_i2^fl/fl mice as compared to SLNCre(−):Gα_i2^fl/fl littermates. There was no difference in deactivation rate (t₁₂ = 0.08, P = 0.94; unpaired t test) of Ado-induced currents in SAN cells from SLNCre(−):Gα_i2^fl/fl and SLNCre(+):Gα_i2^fl/fl mice; group sizes ranged from 7 to 8 cells (3 mice) per genotype.

Fig. 8.

Impact of Gα_o or Gα_i2 ablation on GIRK channel sensitivity to CCh and Ado. (A) Concentration-response experiments of CCh-induced currents in SAN cells from SLNCre(−):Gα_o^fl/fl (Top) and SLNCre(+):Gα_o^fl/fl (Bottom) mice. (B) Summary of CCh sensitivity experiments in SAN cells from SLNCre(−):Gα_o^fl/fl and SLNCre(+):Gα_o^fl/fl mice. (C) There was no difference in EC₅₀ values of CCh-induced GIRK currents in SAN cells from SLNCre(−):Gα_o^fl/fl (n = 12 cells/3 mice) and SLNCre(+):Gα_o^fl/fl (n = 8 cells/3 mice) mice (t₁₈ = 0.8, P = 0.44; unpaired t test), but there was a decrease in the EC₅₀ value in SAN cells from SLNCre(+):Gα_i2^fl/fl (n = 5 cells/3 mice) compared to SLNCre(−):Gα_i2^fl/fl (n = 8 cells/3 mice) mice (t₁₁ = 2.5, *P < 0.05; unpaired t test). (D) Concentration-response experiments of Ado-induced GIRK currents in SAN cells from SLNCre(−):Gα_o^fl/fl (Top) and SLNCre(+):Gα_o^fl/fl (Bottom) mice. (E) Summary of concentration-response experiments of Ado-induced GIRK currents in SAN cells from SLNCre(−):Gα_o^fl/fl and SLNCre(+):Gα_o^fl/fl mice. (F) The EC₅₀ value for Ado-induced signaling in SAN cells from SLNCre(+):Gα_o^fl/fl (n = 15 cells/3 mice) mice was lower than that measured in SAN cells from SLNCre(−):Gα_o^fl/fl (n = 12 cells/3 mice) mice (t₂₅ = 3.3, **P < 0.01; unpaired t test), but there was no difference in EC₅₀ value in SAN cells from SLNCre(−):Gα_i2^fl/fl (n = 7 cells/3 mice) and SLNCre(+):Gα_i2^fl/fl (n = 7 cells/3 mice) mice (t₁₂ = 1.0, P = 0.35; unpaired t test).