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‘Regulators of G protein Signalling’ (RGSs) accelerate the activation and deactivation kinetics of G protein‐gated inwardly rectifying K+ (GIRK) channels. In an apparent paradox, RGSs do not reduce steady‐state GIRK current amplitudes as expected from the accelerated rate of deactivation when reconstituted in Xenopus oocytes. We present evidence here that this kinetic anomaly is dependent on the degree of G protein‐coupled receptor (GPCR) precoupling, which varies with different Gαi/o‐RGS complexes. The gating properties of GIRK channels (Kir3.1/Kir3.2a) activated by muscarinic m2 receptors at varying levels of G protein expression were examined with or without the co‐expression of either RGS4 or RGS7 in Xenopus oocytes. Different levels of specific m2 receptor‐Gα coupling were established by uncoupling endogenous pertussis toxin (PTX)‐sensitive Gαi/o subunits with PTX, while expressing varying amounts of a single PTX‐insensitive subunit (Gαi1(C351G), Gαi2(C352G), Gαi3(C351G), GαoA(C351G), or GαoB(C351G)). Co‐expression of each of the PTX‐insensitive Gαi/o subunits rescued acetylcholine (ACh)‐elicited GIRK currents (IK,ACh) in a concentration‐dependent manner, with Gαo isoforms being more effective than Gαi isoforms. Receptor‐independent ‘basal’ GIRK currents (IK,basal) were reduced with increasing expression of PTX‐insensitive Gα subunits and were accompanied by a parallel rise in IK,ACh. These effects together are indicative of increased Gβγ scavenging by the expressed Gα subunit and the subsequent formation of functionally coupled m2 receptor‐G protein heterotrimers (Gα(GDP)βγ). Co‐expression of RGS4 accelerated all the PTX‐insensitive Gαi/o‐coupled GIRK currents to a similar extent, yet reduced IK,ACh amplitudes 60‐90 % under conditions of low Gαi/o coupling. Kinetic analysis indicated the RGS4‐dependent reduction in steady‐state GIRK current was fully explained by the accelerated deactivation rate. Thus kinetic inconsistencies associated with RGS4‐accelerated GIRK currents occur at a critical threshold of G protein coupling. In contrast to RGS4, RGS7 selectively accelerated Gαo‐coupled GIRK currents. Co‐expression of Gβ5, in addition to enhancing the kinetic effects of RGS7, caused a significant reduction (70‐85 %) in steady‐state GIRK currents indicating RGS7‐Gβ5 complexes disrupt Gαo coupling. Altogether these results provide further evidence for a GPCR‐Gαβγ‐GIRK signalling complex that is revealed by the modulatory affects of RGS proteins on GIRK channel gating. Our functional experiments demonstrate that the formation of this signalling complex is markedly dependent on the concentration and composition of G protein‐RGS complexes.

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