Gating properties of GIRK channels activated by Galpha(o)- and Galpha(i)-coupled muscarinic m2 receptors in Xenopus oocytes: the role of receptor precoupling in RGS modulation.
'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 Galpha(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-Galpha coupling were established by uncoupling endogenous pertussis toxin (PTX)-sensitive Galpha(i/o) subunits with PTX, while expressing varying amounts of a single PTX-insensitive subunit (Galpha(i1(C351G)), Galpha(i2(C352G)), Galpha(i3(C351G)), Galpha(oA(C351G)), or Galpha(oB(C351G))). Co-expression of each of the PTX-insensitive Galpha(i/o) subunits rescued acetylcholine (ACh)-elicited GIRK currents (I(K,ACh)) in a concentration-dependent manner, with Galpha(o) isoforms being more effective than Galpha(i) isoforms. Receptor-independent 'basal' GIRK currents (I(K,basal)) were reduced with increasing expression of PTX-insensitive Galpha subunits and were accompanied by a parallel rise in I(K,ACh). These effects together are indicative of increased Gbetagamma scavenging by the expressed Galpha subunit and the subsequent formation of functionally coupled m2 receptor-G protein heterotrimers (Galpha((GDP))betagamma). Co-expression of RGS4 accelerated all the PTX-insensitive Galpha(i/o)-coupled GIRK currents to a similar extent, yet reduced I(K,ACh) amplitudes 60-90 % under conditions of low Galpha(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 Galpha(o)-coupled GIRK currents. Co-expression of Gbeta5, in addition to enhancing the kinetic effects of RGS7, caused a significant reduction (70-85 %) in steady-state GIRK currents indicating RGS7-Gbeta5 complexes disrupt Galpha(o) coupling. Altogether these results provide further evidence for a GPCR-Galphabetagamma-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.
Zhang
Qingli
Q
Department of Physiology and Biophysics, University of South Florida College of Medicine, Tampa, Florida 33612-4799, USA.
Pacheco
Mary A
MA
Doupnik
Craig A
CA
D000109:Acetylcholine / Q000494:pharmacology; D000465:Algorithms; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D009865:Oocytes / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin / Q000494:pharmacology; D024661:Potassium Channels, Inwardly Rectifying / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D011233:Precipitin Tests; D020710:RGS Proteins / Q000494:pharmacology*; D051381:Rats; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D014981:Xenopus
D018076:DNA, Complementary; D024661:Potassium Channels, Inwardly Rectifying; D020710:RGS Proteins; C492039:RGS7 protein, human; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; C492038:Rgs7 protein, mouse; C115771:Rgs7 protein, rat; C098799:RGS4 protein; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000109:Acetylcholine