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

Significance

Mechanisms promoting the compartmentalization of G-protein signaling are poorly understood in native cells. This study examined muscarinic (M₂R) and adenosine (A₁R) receptor signaling to a common effector—the G protein-gated inwardly rectifying K⁺ (GIRK) channel—in mouse sino-atrial nodal (SAN) cells. Regulator of G protein Signaling 6 (RGS6) exerts a GPCR-dependent influence on the efficacy, kinetics, and sensitivity of GIRK-dependent signaling in SAN cells, suppressing M₂R-GIRK signaling kinetics and sensitivity and A₁R-GIRK signaling amplitude. The distinctions can be explained by the differential utilization of G protein isoforms by M₂R and A₁R and a Gα_o substrate preference of RGS6. Thus, GPCR-G protein signaling and RGS-G protein substrate biases shape GIRK-dependent signaling dynamics in the mouse heart.

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.

G protein-coupled receptors (GPCRs) constitute the largest family of cell-surface receptors in the mammalian genome, and they mediate the influence of a diverse array of ligands on cell signaling, organ physiology, and behavior (1). GPCRs evoke biological responses by coupling to a limited array of heterotrimeric G proteins, which then regulate the activity of a relatively small pool of effectors. Mechanisms underlying the compartmentalization of G protein-dependent signaling pathways in native cell types are not well understood.

Regulation of heart rate (HR) offers an opportunity to investigate how different GPCRs engage intracellular signaling networks to shape a critical physiological response. Activation of the parasympathetic branch of the autonomic nervous system elicits bradycardia (2), an effect mediated by the acetylcholine (ACh)-induced activation of M₂ muscarinic receptors (M₂R) on sinoatrial nodal (SAN) cells (3). M₂R activation stimulates inhibitory G proteins, which then modulate the activity of downstream effectors, including the G protein-gated inwardly rectifying K⁺ (GIRK) channel (4–6). The cardiac GIRK channel is a heterotetramer consisting of GIRK1 and GIRK4 subunits (7–9). Studies involving Girk1^−/− and Girk4^−/− mice have shown that activation of cardiac GIRK channels mediates most of the parasympathetic impact on HR, as well as atrial and atrio-ventricular (AV) impulse conduction (10–13). Cardiac GIRK channels are also activated by adenosine (Ado), an autacoid generated under ischemic conditions that activates the A₁ adenosine receptor (A₁R), resulting in GIRK-dependent bradycardia (10, 14–16). The ability of Ado to suppress HR and AV impulse conduction is exploited clinically to treat and diagnose supraventricular tachycardias (14).

GPCR-dependent signaling dynamics are influenced by Regulator of G protein Signaling (RGS) proteins (17). RGS proteins possess GTPase-activating protein (GAP) catalytic activity that accelerates the intrinsic GTP hydrolysis rate of G protein alpha (Gα) subunits. Previous work from our group and others has identified RGS6 as a critical negative regulator of M₂R-GIRK signaling in mouse SAN cells and atrial myocytes (18–20). RGS6 is a member of the R7 RGS subfamily of RGS proteins, members of which form obligate dimers with the atypical Gβ subunit Gβ5 (21). Genetic ablation of Rgs6 in mice increased GIRK channel sensitivity to muscarinic receptor activation, prolonged the deactivation rate of currents elicited by the nonselective cholinergic agonist carbachol (CCh), and enhanced CCh-induced bradycardia (18–20). The extent to which RGS6 regulates A₁R-GIRK signaling in the heart is currently unknown.

Here, we probed the functional compartmentalization of GPCR-dependent signaling by investigating signaling engaged by M₂R and A₁R activation in SAN cells. Despite converging on a common effector, M₂R-GIRK and A₁R-GIRK signaling pathways display notable differences in amplitude and kinetics. Using an array of knockout mouse lines, as well as electrophysiological, molecular, and optical imaging approaches, we found that the differences in M₂R-GIRK and A₁R-GIRK signaling dynamics in mouse SAN cells can be explained by GPCR-specific coupling biases for inhibitory G protein isoforms and the Gα_o substrate specificity of RGS6.

Results

A₁R-GIRK and M₂R-GIRK Signaling Distinctions in Mouse SAN Cells.

We began by measuring whole-cell currents evoked by a saturating concentration of either Ado (10 μM) or CCh (10 μM) in isolated SAN cells from C57BL/6J (wild-type) mice (Fig. 1). Both agonists reliably evoked currents in SAN cells from wild-type mice, but not in SAN cells from congenic Girk4^−/− mice (Fig. 1 A and B), indicating that the evoked responses were mediated by GIRK channel activation. Currents elicited by Ado in wild-type SAN cells were smaller than CCh-induced currents (Fig. 1C) and did not exhibit acute desensitization (Fig. 1D). In addition, activation and deactivation rates of Ado-induced GIRK currents were slower than those of CCh-induced responses (Fig. 1 E and F).

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).

RGS6 Exerts a GPCR-Dependent Influence on GIRK-Dependent Signaling.

In parallel, we explored the impact of Rgs6 ablation on Ado- and CCh-induced currents in SAN cells. While Ado-induced currents were significantly larger in SAN cells from Rgs6^−/− mice relative to wild-type controls (Fig. 1 A and C), there was no impact of Rgs6 ablation on CCh-induced current amplitudes (Fig. 1 B and C). Rgs6 ablation also exerted a GPCR-dependent impact on response kinetics. Loss of RGS6 correlated with prolonged activation rate of Ado-induced currents (Fig. 1E), but there was no impact on deactivation rate (Fig. 1F). In contrast, and consistent with published reports (18, 19), there was no impact of Rgs6 ablation on the activation rate of CCh-induced currents (Fig. 1F), but deactivation rate was prolonged (Fig. 1F). Acute desensitization of CCh- and Ado-induced currents was not impacted by Rgs6 ablation (Fig. 1D). Thus, RGS6 normally suppresses the amplitude and accelerates the activation rate of A₁R-GIRK signaling in mouse SAN cells, while accelerating the deactivation rate of M₂R-GIRK signaling.

Ado-induced currents in SAN cells from Rgs6^−/− mice were completely reversed by the GIRK channel inhibitor rTertiapin-Q (TTQ, 300 nM; SI Appendix, Fig. S1) (22, 23), indicating that the enhanced Ado-induced responses in these cells involves an increase in GIRK-dependent signaling. There was no difference in GIRK1, GIRK4, or A₁R mRNA levels in atrial tissue samples containing the SAN region from wild-type and Rgs6^−/− mice (SI Appendix, Fig. S2). Additionally, GIRK channel currents evoked by ML297, a direct and selective activator of GIRK1-containing GIRK channels (24, 25), were modestly but significantly smaller in SAN cells from Rgs6^−/− mice (SI Appendix, Fig. S3). Thus, the increase in Ado-induced GIRK currents in SAN cells from Rgs6^−/− mice does not appear to be linked to an increase in the expression of A₁R or GIRK channels.

To address the impact of Rgs6 ablation on the sensitivity of M₂R-GIRK and A₁R-GIRK signaling pathways, we measured GIRK currents evoked by increasing concentrations of Ado and CCh in SAN cells from wild-type and Rgs6^−/− mice. While there was no impact of Rgs6 ablation on the sensitivity of GIRK channels to Ado (Fig. 2 A–C), GIRK channels in SAN cells from Rgs6^−/− mice were more sensitive to CCh than GIRK channels in SAN cells from wild-type controls (Fig. 2 D–F) (19). Thus, RGS6 decreases GIRK channel sensitivity to CCh, but not Ado, in SAN cells.

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).

RGS6 Impacts GIRK-Dependent Bradycardia in a GPCR-Dependent Manner.

We also evaluated the impact of Rgs6 ablation on the bradycardic effect of A₁R and M₂R activation using an isolated heart model (Fig. 3). Both the stable A₁R agonist N6-cyclopentyl-adenosine (CPA) (Fig. 3 A and B) and CCh (Fig. 3 C and D) evoked dose-dependent decreases in HR in wild-type hearts. The bradycardic effect of CPA and CCh was strongly diminished in hearts from Girk4^−/− mice (Fig. 3 B and D), suggesting that M₂R- and A₁R-induced bradycardia is largely dependent on GIRK channel activation. The magnitude of CPA-induced bradycardia was larger in Rgs6^−/− as compared to wild-type hearts (Fig. 3B), but there was no detectable shift in CPA potency. While CCh-induced bradycardia was also more pronounced in Rgs6^−/− hearts (18, 19, 26), there was a clear leftward shift in the dose–response curve (Fig. 3D). Thus, the results from the isolated heart model mirror the results obtained from isolated SAN cells, indicating that RGS6 exerts a prominent negative influence on A₁R-GIRK signaling amplitude and M₂R-GIRK signaling sensitivity.

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^−/−.

RGS6 Limits the Access of A₁R to GIRK Channels.

The differential impact of Rgs6 ablation on M₂R-GIRK and A₁R-GIRK signaling in mouse SAN cells could reflect the coupling of the GPCRs to separate pools of GIRK channels. To test this possibility, we examined whether Ado-induced currents were occluded by a maximal CCh-induced GIRK channel response. In both wild-type and Rgs6^−/− SAN cells, Ado failed to evoke an additive current during application of a saturating concentration of CCh (Fig. 4 A and B). When the order of agonist application was reversed, CCh evoked an additive current during the Ado-induced response (Fig. 4C). The size of the CCh-induced additive response was substantially smaller, however, in SAN cells from Rgs6^−/− mice (Fig. 4D). Indeed, the Ado-induced GIRK current occluded nearly all of the CCh-induced response. Collectively, these findings show that M₂R and A₁R couple to a shared pool of GIRK channels in SAN cells and that RGS6 effectively limits the access of A₁R to GIRK channels in these cells.

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).

M₂R and A₁R Display Overlapping but Distinct G Protein Coupling Preferences.

We next investigated whether the utilization of different Gα isoforms by M₂R and A₁R could explain the differences in GIRK channel-dependent signaling and modulation by RGS6. Treatment with pertussis toxin abolished CCh- and Ado-induced currents in wild-type SAN cells (SI Appendix, Fig. S4), indicating that M₂R– and A₁R-GIRK signaling pathways are mediated by inhibitory (G_i/o) G proteins. To probe the inhibitory G protein coupling preferences for M₂R and A₁R, we employed a Bioluminescence Resonance Energy Transfer (BRET) assay to measure G protein activation in response to GPCR stimulation in living cells (Fig. 5A) (27).

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.

We quantified maximal BRET response amplitudes and G protein activation rates for M₂R and A₁R in the presence of specific G protein isoforms (Fig. 5B). Analysis of BRET amplitudes showed that both M₂R and A₁R can activate all members of the G_i/o subfamily, as well as G₁₅ (Fig. 5C). M₂R displayed faster activation rates with G⍺_o relative to G⍺_i isoforms, however, while A₁R exhibited similar activation rates for G⍺_o and G⍺_i isoforms (Fig. 5D). Since G protein activation rates closely reflect the catalytic activity of GPCRs (28), whereas amplitudes in the BRET assay could be influenced by intrinsic differences in the extent of heterotrimer dissociation (29), we relied on activation rate as a proxy for G⍺ coupling preferences for M₂R and A₁R. Accordingly, M₂R exhibits a coupling preference for G⍺_o over G⍺_i, whereas A₁R does not discriminate between the inhibitory G protein isoforms.

Gα_o Is the Preferred Substrate for RGS6 GAP Activity.

Given the distinct G protein isoform coupling preferences displayed by M₂R and A₁R, and the differential impact of RGS6 on G protein-mediated regulation of GIRK channels, we next tested whether the RGS6 exhibits a G protein isoform preference for its catalytic activity. The G protein deactivation rates of all Gα_i/o members were measured in the presence of RGS6/Gβ5 using a modified version of the BRET assay (Fig. 6A). The D₂ dopamine receptor (D₂R), dopamine, and haloperidol were chosen for this study due to previous work showing that D₂R can efficiently activate all Gα_i/o G proteins and that GTPase activity is the rate-limiting step in response deactivation for this combination of receptor and ligands (28). Consistent with results from a study involving purified RGS6/Gβ5 and G proteins (30), RGS6/Gβ5 strongly accelerated the deactivation of Gα_oA/B, while showing only weak activity toward Gα_i1-3 isoforms and no activity toward Gα_z (Fig. 6 B and C). Thus, Gα_o is the preferred substrate for RGS6 and its GAP activity.

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.

Gα_o and Gα_i2 Ablation Impact GIRK-Dependent Signaling in a GPCR-Dependent Manner.

Previous work utilizing constitutive or conditional knockout mice has implicated G⍺_o and Gα_i2 isoforms in HR regulation (31–33). Accordingly, we crossed an atrial-specific Cre driver line (SLNCre) (34) with conditional G⍺_o (G⍺_o^fl/fl) (35) or Gα_i2 (G⍺_i2^fl/fl) (32) knockout mice to generate mice lacking G⍺_o (SLNCre:G⍺_o^fl/fl) or Gα_i2 (SLNCre:Gα_i2^fl/fl) in atrial (including SAN) tissue. We then recorded currents evoked by CCh or Ado in SAN cells from Cre(+) and Cre(−) littermates (Fig. 7). While maximal CCh-induced currents and desensitization were smaller in SLNCre(+):G⍺_o^fl/fl SAN cells compared to Cre(−) littermates (Fig. 7 A–C), Ado-induced currents and desensitization were not impacted by G⍺_o ablation (Fig. 7 F–H). Gα_i2 ablation had no impact on CCh- or Ado-induced response amplitudes or desensitization (Fig. 7 B, C, G, and H). Loss of G⍺_o prolonged activation and deactivation rates of CCh-induced GIRK currents, whereas Gα_i2 ablation was without effect (Fig. 7 D and E). The deactivation rate of Ado-induced GIRK currents was also prolonged by G⍺_o ablation (Fig. 7J), but interestingly, the activation rate of Ado-induced GIRK currents was accelerated by Gα_i2 ablation (Fig. 7I). Collectively, these results align with observations that M₂R signals preferentially through G⍺_o, whereas A₁R does not discriminate between G⍺_o and G⍺_i. Moreover, the lack of impact of Gα_i2 ablation on the CCh- and Ado-induced GIRK current deactivation rates is consistent with the Gα_o substrate preference of RGS6.

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.

Finally, we evaluated the impact of G⍺_o or Gα_i2 ablation on GIRK channel sensitivity to CCh and Ado. While the loss of Gα_o did not impact GIRK channel sensitivity to CCh (Fig. 8 A–C), Gα_i2 ablation increased the sensitivity of GIRK channels to CCh. Conversely, Gα_o ablation increased GIRK channel sensitivity to Ado, whereas Gα_i2 ablation was without effect (Fig. 8 D–F). Thus, loss of Gα_o increases the sensitivity of A₁R-GIRK channel signaling, whereas loss of Gα_i2 increases the sensitivity of M₂R-GIRK channel signaling.

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).

Discussion

While M₂R and A₁R signal to a shared pool of GIRK channels in mouse SAN cells, the amplitude and kinetics of these signaling pathways are distinct. RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in mouse SAN cells, modulating the sensitivity and kinetics of M₂R-GIRK signaling and the amplitude of A₁R-GIRK signaling. Innate GPCR-G protein coupling preferences appear to contribute to the distinctions in M₂R- and A₁R-GIRK signaling pathways in SAN cells. Our data suggest that Gα_o plays a more important role in mediating M₂R-GIRK than A₁R-GIRK signaling in mouse SAN cells. This, together with the strong Gα_o substrate preference of RGS6, provides a plausible explanation for the GPCR-dependent influence of RGS6 on GIRK-dependent signaling in mouse SAN cells.

A₁R-GIRK responses are smaller than M₂R-GIRK responses in mouse SAN cells; parallel observations have been noted in mouse, rat, and guinea pig atrial myocytes (36–38). A maximal M₂R-induced GIRK response completely occludes the A₁R-GIRK response in SAN cells, suggesting that M₂R activation can elicit a maximal GIRK-dependent response, while A₁R activation can engage only a fraction of available GIRK channels. Different levels of M₂R and A₁R expression likely contribute to the differences in response amplitudes and activation kinetics. Indeed, overexpression of A₁R in rat atrial myocytes increased Ado-induced GIRK currents and accelerated the current activation rate (39). Thus, the level of A₁R and/or G protein intermediate appears to limit A₁R-GIRK signaling amplitude in mouse SAN cells.

Considering the GAP activity of RGS6, Rgs6 ablation should result in increased steady-state levels of activated Gα_o (and Gβγ) following M₂R and A₁R activation. Indeed, A₁R-GIRK response amplitudes were enhanced by Rgs6 ablation without up-regulation of A₁R or GIRK channels. Thus, the amplitude of A₁R-GIRK signaling in mouse SAN cells is likely limited by the amount of activated G protein released by A₁R activation. Interestingly, loss of RGS6 had no impact on CCh-induced GIRK current amplitude, suggesting that the GIRK channel limits the M₂R-GIRK responses in these cells, a contention supported by our occlusion data. The enhanced sensitivity of GIRK channels to CCh in SAN cells from Rgs6^−/− mice further suggests that the loss of RGS6 effectively increases the fraction of spare M₂R receptors in these cells. Interestingly, we recently reported an increase in M₂R-GIRK current amplitude in ventricular myocytes from Rgs6^−/− mice, where expression M₂R is lower than in the atria (40). Thus, RGS6 may impact GPCR-effector response amplitudes when the level of G protein, rather than effector, is limiting.

RGS proteins may also facilitate GIRK-dependent signaling in a GAP-independent manner. Mice heterozygous for an RGS-insensitive Gα_o, for example, exhibit blunted GIRK channel responses to mu opioid receptor (MOR) activation in some neurons (41), suggesting that RGS proteins promote MOR-GIRK coupling. Notably, we observed a modest decrease in GIRK currents elicited by the GIRK channel activator ML297 in SAN cells from Rgs6^−/− mice. Thus, RGS6 may facilitate efficient GIRK channel signaling in SAN cells, perhaps via physical interaction (42). The increase in A₁R-GIRK channel signaling seen with Rgs6 ablation, however, suggests that this is not the main influence of RGS6 in SAN cells. Other work has called attention to the role of adaptor proteins, including R7BP and GPR158, in fine-tuning the influence of RGS proteins (21, 43–45). While R7BP or GPR158 expression has not been observed in the heart (46, 47), an unknown adaptor protein(s) may influence the impact of RGS6/Gβ5 on GIRK-dependent signaling in cardiomyocytes.

Previous work has implicated Gα_o in the bradycardic effects of A₁R and M₂R activation. For example, isolated hearts from mice with a global knockout of Gα_o, but not of Gα_i2 or Gα_i3, exhibited diminished CCh-induced bradycardia (33). Additionally, work with embryonic stem cell-derived cardiomyocytes (ESDCs) showed that the reduction in isoproterenol-stimulated beating rate elicited by M₂R and A₁R activation was exaggerated in cells containing an RGS-insensitive version of Gα_o (48). Our findings that Gα_o is a critical mediator of M₂R-GIRK signaling in mouse SAN cells, and that Rgs6 ablation enhances both M₂R- and A₁R-mediated bradycardia, are in line with these reports. Interestingly, the enhanced A₁R-induced suppression of isoproterenol-stimulated beating rate seen in ESDCs harboring a RGS-insensitive Gα_o mutant was not blunted by TTQ, suggesting that this effect of A₁R activation may not be GIRK-dependent (48). This contrasts with our finding that A₁R-mediated bradycardia is absent in Girk4^−/− mouse hearts. These discrepancies likely arise from the different cell types and model systems employed, where the relative complements of GPCRs, G proteins, effectors, and RGS proteins may differ.

There is little consensus on relevance of Gα_i2 to HR regulation. Mice with a global deletion of Gα_i2 exhibited diminished CCh-induced bradycardia and resting tachycardia (31). Mice harboring an RGS-insensitive Gα_i2 displayed enhanced M₂R-induced bradycardia, and the enhanced effect of CCh on beating rate of ESDCs harboring this mutation was reversed by TTQ (48). These findings are consistent with a prominent role for Gα_i2 in mediating M₂R-GIRK signaling in SAN cells. However, atrial myocytes from mice lacking Gα_i2 exhibited increased CCh-induced GIRK responses, which was attributed to increased GIRK subunit expression (49). Moreover, mice lacking Gα_i2 selectively in the cardiac conduction system exhibited resting tachycardia but normal CCh-induced bradycardia (32). Thus, the impact of Gα_i2 ablation and RGS-insensitive Gα_i2 on CCh-induced bradycardia is model-dependent and may reflect an indirect (noncardiac) influence of Gα_i2 on HR regulation. Additionally, the impact of Gα_i2 on resting HR may be exerted through GIRK-independent mechanisms (50). For example, M₂R activation inhibits the hyperpolarization-activated channel (I_f) by suppressing adenylyl cyclase activity and cAMP production (3). Interestingly, M₂R-I_f coupling is more sensitive than M₂R-GIRK signaling in SAN cells (51). Our data show that Gα_o plays a critical role in M₂R-GIRK signaling. This pathway is subject to negative regulation by RGS6 and therefore may be engaged only by strong parasympathetic activation. Gα_i2, which is less susceptible to the GAP activity of RGS6, may mediate M₂R-I_f coupling during periods of weaker parasympathetic activity.

It is important to recognize that genetic ablation of an individual Gα isoform redirects signaling to residual G protein isoforms. Ablation of Gα_o, for example, routes M₂R-GIRK signaling through Gα_i. In our hands, this yielded small CCh-induced GIRK currents with slow activation rates, reminiscent of Ado-induced GIRK currents in wild-type SAN cells. In contrast, routing A₁R-GIRK signaling through Gα_i had no impact on Ado-induced current amplitude or activation kinetics. Gα_o ablation did increase the sensitivity of GIRK channels to Ado, suggesting that (residual) Gα_i is more efficient than Gα_o at mediating A₁R-GIRK signaling in mouse SAN cells. Similarly, Gα_i2 ablation increased the sensitivity of GIRK channels to CCh, suggesting that (residual) Gα_o is a more efficient mediator than Gα_i2 of M₂R-GIRK signaling in mouse SAN cells. The modest overall impact of Gα_i2 ablation, particularly on A₁R-GIRK signaling, is likely attributable to the presence of residual Gα_i1 and Gα_i3 isoforms (49). Indeed, this likely explains why there was no impact of global ablation of any single G-protein isoform (Gα_o, Gα_i2, Gα_i1/3) on GIRK-dependent bradycardia elicited by the A₁R agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) (32).

Ado can provoke atrial fibrillation through shortening of the effective refractory period, an effect thought to be mediated by GIRK channel activation in atrial myocytes (14). Enhanced A₁R and GIRK channel expression are associated with increased bradycardia in a tachypacing-induced chronic heart failure model in dogs (52). Moreover, atrial fibrillation induced by A₁R activation or M₂R activation (via vagal nerve stimulation) is thought to be mediated in part by GIRK channel activity (53, 54). While loss of GIRK4 in mice conferred resistance to pacing-induced atrial fibrillation (55), Rgs6 ablation resulted in a higher incidence of atrial fibrillation induction (26). Similar to the impact of Girk4 ablation, peptides targeting specific Gα_i isoforms reduced atrial fibrillation susceptibility during vagal nerve stimulation in dogs (56). These and related observations, together with our results, show that interventions that suppress the influence of inhibitory G-protein signaling in atria by targeting signaling mediators and regulators or effectors could prove beneficial for treatment of certain arrhythmias and heart failure.

Insights gained in this study are likely to be relevant to the compartmentalization of GPCR-dependent signaling in other cell types, including neurons. GIRK channel regulation by inhibitory GPCRs has been implicated in neurological disorders including epilepsy, pain and analgesia, anxiety and depression, and addiction (57, 58). Neuronal GIRK-dependent signaling pathways regulated by GABA_BR, 5-HT_1AR, and A₁R exhibit distinct amplitude and kinetic profiles and are differentially susceptible to plasticity (59–62). Our previous work has highlighted the integral role of RGS7, another member of the R7 RGS family, in modulating the kinetics and sensitivity of GABA_BR-GIRK signaling in hippocampal neurons (42, 43, 63, 64). Furthermore, RGS7 prefers Gα_o over Gα_i as a substrate for its GAP activity (28). Thus, GPCR-G protein and RGS-G protein substrate preferences likely shape neuronal GPCR-GIRK signaling dynamics in neurons and may help the functional compartmentalization of these signaling pathways.

Materials and Methods

Animals.

All procedures involving mice were approved by the Institutional Animal Care and Use Committee of the University of Minnesota, and experiments were conducted in accordance with guidelines set by the NIH. The generation of Girk4^−/− and Rgs6^−/− mice was described previously (10, 19). C57BL/6J mice, bred on site or purchased from The Jackson Laboratory, were used as wild-type controls for these studies. Mice lacking either Gα_o or Gα_i2 selectively in atrial/SAN tissue were generated by crossing the SLNCre driver mouse line (34) with floxed versions of either Gα_o (35) or Gα_i2 (32), resulting in SLNCre:Gα_o^fl/fl and SLNCre:Gα_i2^fl/fl mouse lines, respectively. Male and female mice were group-housed on a 12-h light/dark cycle, given free access to food and water, and used for experiments at ages 8 to 12 wk.

SAN Cell Culture and Recordings.

SAN cells were prepared for electrophysiological analysis as described (40). See SI Appendix for further details.

Isolated Heart Recordings.

Hearts were excised and placed into ice-cold, oxygenated Tyrode’s solution, and the aorta was quickly cannulated. Cannulated hearts were then placed into a warm (37 ± 1 °C) Tyrode’s bath, and iWorx platinum recording electrodes were placed near/on the surface of the heart. Oxygenated Tyrode’s solution was then perfused at 2 to 3 mL/min, and a baseline HR was recorded. Increasing concentrations of CCh or CPA (Tocris Bioscience) were then perfused via peristaltic pump for at least 15 min per dose. The electrocardiogram (ECG) signal was acquired with LabScribe v.3 software (iWorx) and filtered as appropriate. The derivative of that channel was computed to account for movements in baseline and to amplify the signal for subsequent analysis. A 30-s segment from the last minute of exposure to each agonist dose was then exported to Kubios HRV v.2 (65) for HR analysis, utilizing artifact correction as appropriate.

Quantitative RT-PCR.

Total RNA was isolated from freshly isolated atrial tissue samples from adult wild-type mice and Rgs6^−/− mice using the RNeasy fibrous tissue kit (Qiagen), according to manufacturer recommendations. Reverse transcription of 1.2 μg of total RNA per sample was performed using Maxima H Minus First Strand cDNA Synthesis Kit (ThermoFisher Scientific). Quantitative PCR was performed in a QuantStudio3 Real Time PCR System (Applied Biosystems) with the Fast SYBR Green Master Mix (ThermoFisher Scientific). See SI Appendix for further details, including target-specific primer sequences, reaction conditions, and analysis information.

Fast Kinetic BRET Assay.

HEK293T/17 cells were grown in Dulbecco’s Modified Eagle Media (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Millipore Sigma), minimum Eagle’s medium nonessential amino acids, 1 mM sodium pyruvate, and antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin) at 37 °C in a humidified incubator containing 5% CO₂. Culture dishes (3.5 cm) were incubated for 10 min at 37 °C with 1 mL of 10 mg/mL growth-factor–reduced Matrigel (BD Biosciences) in culture medium. For transfection, cells were seeded into 3.5-cm dishes at a density of 2 × 10⁶ cells/dish. After 2 h, expression constructs (total 5 μg/dish) were added to the cells using PLUS (5 μg/dish) and Lipofectamine LTX (6 μL/dish) reagents (Thermo Fisher Scientific). BRET measurements of Venus-Gβ1γ2 and masGRK3ct-Nluc-HA were performed to measure G protein activation by M₂R and A₁R, or the GAP activity of RGS6/Gβ5 in living cells, as described (27, 66). See SI Appendix for further details, including information about DNA constructs and transfection as well as BRET assay procedures and analysis.

Statistical Analysis.

All data were analyzed using Prism v.8.2.1 software (GraphPad Software) and are presented as mean ± SEM. Statistical outliers were identified and excluded with Grubb’s outlier test. The level of statistical significance was set at P < 0.05. Specific statistical analyses are denoted within the figure legends.

Data Availability.

All experimental procedures and data supporting the findings of this paper are included in the article and SI Appendix. Requests for reagents used in this study should be directed to K.W.