Abstract
IntroductionM2 muscarinic (M2R) activation of G proteinâgated inwardly rectifying K+ (GIRK) channels in sinoatrial nodal (SAN) cells is a key mediator of vagal/parasympathetic slowing of heart rate. The strength of M2RâGIRK signaling is negatively regulated by Regulator of G protein Signaling 6 (RGS6). Adenosine, used clinically to diagnose and treat certain arrhythmias, exerts these actions through A1 adenosine receptor (A1R) activation of GIRK channels. The extent to which RGS6 regulates A1RâGIRK signaling, however, is unclear. Here, we investigated the impact of RGS6 on A1RâGIRK and M2RâGIRK signaling in mouse SAN cells.Methods and ResultsWe measured the efficacy and potency of M2Râ and A1RâGIRK signaling in adult SAN cells from wildâtype and RGS6â/â mice using wholeâcell patchâclamp electrophysiology. As previously reported, SAN cells from RGS6â/â mice displayed prolonged channel deactivation kinetics and increased channel sensitivity to the nonâselective cholinergic agonist carbachol (CCh), relative to wildâtype counterparts. In contrast, we observed a striking increase in the amplitude of the GIRK response evoked by adenosine (Ado) in RGS6â/â SAN cells compared to wildâtype controls, with no impact on channel deactivation kinetics or sensitivity. Further recordings in wildâtype SAN cells revealed that M2R activation completely occludes the Adoâinduced response, whereas Ado application activates only a smaller fraction of GIRK channels. Both CChâ and Adoâ induced currents were abolished in SAN cells treated by pertussis toxin (PTX), indicating these responses are mediated by Gi/o G proteins. Utilizing a bioluminescence resonance energy transfer (BRET) assay in transfected HEK cells, we found that RGS6 prefers Gαo as a substrate for over Gαi. Additionally, we found that while A1R does not discriminate between Gαo and Gαi, M2R displays a clear preference for Gαo. We next employed a Cre/flox strategy to remove Gαo from atrial tissue, including SAN cells. Loss of Gαo yielded significantly blunted CChâinduced responses, which also displayed prolonged activation and deactivation kinetics. While Adoâinduced responses had prolonged deactivation kinetics, activation kinetics and response amplitude were unaffected.ConclusionsThese results suggest that RGS6 negatively regulates A1RâGIRK signaling distinct from its influence on M2RâGIRK signaling in mouse SAN cells likely due to differing utilization of Gao by M2R and A1R.Support or Funding InformationThis work was supported by NIH grants R01 HL105550 and F31 HL139090.
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