Differential effects of the Gβ5-RGS7 complex on muscarinic M3 receptor-induced Ca²⁺ influx and release

The G protein β subunit Gβ5 uniquely forms heterodimers with R7 family regulators of G protein signaling (RGS) proteins (RGS6, RGS7, RGS9, and RGS11) instead of Gγ. Although the Gβ5-RGS7 complex attenuates Ca²⁺ signaling mediated by the muscarinic M3 receptor (M3R), the route of Ca²⁺entry (i.e., release from intracellular stores and/or influx across the plasma membrane) is unknown. Here, we show that, in addition to suppressing carbachol-stimulated Ca²⁺ release, Gβ5-RGS7 enhanced Ca²⁺ influx. This novel effect of Gβ5-RGS7 was blocked by nifedipine and 2-aminoethoxydiphenyl borate. Experiments with pertussis toxin, an RGS domain-deficient mutant of RGS7, and UBO-QIC {L-threonine, (3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl- 2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl- (3R)-3-[[(2S,3R)-3- hydroxy-4- methyl-1-oxo-2-[(1-oxopropyl)amino] pentyl]oxy]-L-leucyl-N,O- dimethyl-,(7→1)-lactone (9CI)}, a novel inhibitor of Gq, showed that Gβ5-RGS7 modulated a Gq-mediated pathway. These studies indicate that Gβ5-RGS7, independent of RGS7 GTPase-accelerating protein activity, couples M3R to a nifedipine-sensitive Ca²⁺ channel. We also compared the action of Gβ5-RGS7 on M3R-induced Ca²⁺ influx and release elicited by different muscarinic agonists. Responses to Oxo-M [oxotremorine methiodide N,N,N,-trimethyl-4-(2-oxo-1-pyrrolidinyl)-2-butyn-1- ammonium iodide] were insensitive to Gβ5-RGS7. Pilocarpine responses consisted of a large release and modest influx components, of which the former was strongly inhibited whereas the latter was insensitive to Gβ5-RGS7. McN-A-343 [(4-hydroxy-2- butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride] was the only compound whose total Ca²⁺ response was enhanced by Gβ5- RGS7, attributed to, in part, by the relatively small Ca²⁺ release this partial agonist stimulated. Together, these results show that distinct agonists not only have differential M3R functional selectivity, but also confer specific sensitivity to the Gβ5-RGS7 complex.