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 Gαoover Gαias a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαoor Gαi2was consistent with these findings. Gαi2ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαoablation 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 Gαosubstrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

Common coupling map advances GPCR-G protein selectivity

“…Furthermore, the finding that on average only 27% of GPCRs activate only a subset of the G proteins in each family ( Figure 4 ) opens the possibility that signaling bias on the level of specific subtypes may be much more frequent than currently appreciated. G proteins that belong to the same family can have diverse functional outcomes pertaining to effector engagement selectivity and kinetic profiles ( Jiang and Bajpayee, 2009 ; Ho and Wong, 2001 ), suggesting that bias within a G protein family may have physiological and/or therapeutic implications ( Anderson et al, 2020 ). The observation that many GPCRs can couple to more than one family of G proteins but still can show selectivity between members of a same family, opens important questions concerning the structural determinants of such multifaceted selectivity profiles.…”

Characterization of VU0468554, a New Selective Inhibitor of Cardiac G Protein–Gated Inwardly Rectifying K+ Channels

“…2A). Previously, we demonstrated that CCh-induced currents in wild-type SAN cells were reversed by application of tertiapin-Q (Anderson et al, 2020). VU0468554 reversed CCh-induced GIRK currents in SAN cells in a dose-dependent manner, inhibiting approximately 73% of the CCh-induced response at the highest dose tested (10 M; Fig.…”

“…Previous work has explored the potential of GIRK channel inhibitors for the treatment of cardiac arrhythmias. Tertiapin, a bee venom peptide that is relatively selective for GIRK channels , completely reversed CCh-induced currents in SAN cells from wildtype mice (Anderson et al, 2020), and it suppressed atrial tachyarrhythmias in canines following pacing-induced cardiac remodeling (Cha et al, 2006). NIP-142, which inhibits GIRK channels at low concentrations (EC 50 =0.64 μM) and hERG channels at substantially higher concentrations (EC 50 =44 μM), reversed CCh-and adenosine-induced shortening of APD (Matsuda et al, 2006), and was shown to terminate and prevent re-initiation of AF and atrial flutter in atrial canine preparations (Nagasawa et al, 2002).…”

“…SAN cell culture. SAN cells were isolated from young adult (2-3 months) mice (male and female) as described (Anderson et al, 2018;Anderson et al, 2020). In brief, mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and hearts were excised into Tyrode's solution (in mM): 140 NaCl, 5.4 KCl, 1.2 KH 2 PO 4 , 1.0 MgCl 2 , 1.8 CaCl 2 , 5.55 glucose, 5 HEPES (pH 7.4 with NaOH).…”

Common coupling map advances GPCR-G protein selectivity

“…The copyright holder for this preprint this version posted September 8, 2021. ; https://doi.org/10.1101/2021.09.07.459250 doi: bioRxiv preprint subtypes may be much more frequent than currently appreciated. G proteins that belong to the same family can have diverse functional outcomes pertaining to effector engagement selectivity and kinetic profiles (26,27), suggesting that bias within a G protein family may have physiological and/or therapeutic implications (28). The observation that many GPCRs can couple to more than one family of G proteins but yet can show selectivity between members of a same family, opens important questions concerning the structural determinants of such multifaceted selectivity profiles.…”