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Gating properties of GIRK channels activated by G_o- and G_i-coupled muscarinic m2 receptors in Xenopus oocytes: the role of receptor precoupling in RGS modulation

Qingli Zhang, Mary A. Pacheco* and Craig A. Doupnik

'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 G_i/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-G coupling were established by uncoupling endogenous pertussis toxin (PTX)-sensitive G_i/o subunits with PTX, while expressing varying amounts of a single PTX-insensitive subunit (G_i1(C351G), G_i2(C352G), G_i3(C351G), G_oA(C351G), or G_oB(C351G)). Co-expression of each of the PTX-insensitive G_i/o subunits rescued acetylcholine (ACh)-elicited GIRK currents (I_K,ACh) in a concentration-dependent manner, with G_o isoforms being more effective than G_i isoforms. Receptor-independent 'basal' GIRK currents (I_K,basal) were reduced with increasing expression of PTX-insensitive G subunits and were accompanied by a parallel rise in I_K,ACh. These effects together are indicative of increased G scavenging by the expressed G subunit and the subsequent formation of functionally coupled m2 receptor-G protein heterotrimers (G_(GDP)). Co-expression of RGS4 accelerated all the PTX-insensitive G_i/o-coupled GIRK currents to a similar extent, yet reduced I_K,ACh amplitudes 60-90 % under conditions of low G_i/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 G_o-coupled GIRK currents. Co-expression of G5, in addition to enhancing the kinetic effects of RGS7, caused a significant reduction (70-85 %) in steady-state GIRK currents indicating RGS7-G5 complexes disrupt G_o coupling. Altogether these results provide further evidence for a GPCR-G-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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