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

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 GPCRdependent 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_oover Gαi 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_oor Gai2 was consistent with these findings. Ga_i2ablation hadminimal impact onM2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gα_oablation 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_osubstrate preference of RGS6, shape A₁R- and M₂R-GIRK signaling dynamics in mouse SAN cells.