GIRK (Kir3) channels are activated by neurotransmitters coupled to G proteins, via a direct binding of G(beta)(gamma). The role of G(alpha) subunits in GIRK gating is elusive. Here we demonstrate that G(alpha)(i) is not only a donor of G(beta)(gamma) but also regulates GIRK gating. When overexpressed in Xenopus oocytes, GIRK channels show excessive basal activity and poor activation by agonist or G(beta)(gamma). Coexpression of G(alpha)(i3) or G(alpha)(i1) restores the correct gating parameters. G(alpha)(i) acts neither as a pure G(beta)(gamma) scavenger nor as an allosteric cofactor for G(beta)(gamma). It inhibits only the basal activity without interfering with G(beta)(gamma)-induced response. Thus, GIRK is regulated, in distinct ways, by both arms of the G protein. G(alpha)(i) probably acts in its GDP bound form, alone or as a part of G(alpha)(beta)(gamma) heterotrimer.

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G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of G i/o proteins via G protein-coupled receptors (GPCRs). Although Gα i subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed Gα i3 decreases the basal activity (I basal ) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the 'inactive' GDP-bound Gα i3 GDP and involves the formation of Gα i3 βγ heterotrimers, by a mechanism distinct from mere sequestration of Gβγ 'away' from the channel. The regulation of basal and Gβγ-evoked current was produced by the 'constitutively inactive' mutant of Gα i3 , Gα i3 G203A, which strongly binds Gβγ, but not by the 'constitutively active' mutant, Gα i3 Q204L, or by Gβγ-scavenging proteins. Furthermore, regulation by Gα i3 G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gβγ enhanced the binding of Gα i3 GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a 'classical' Gβγ effector, showing low I basal and strong Gβγ-dependent activation. Expression of Gα i3 G203A did not affect either I basal or Gβγ-induced activation. In contrast, homomeric GIRK1 * (a pore mutant able to form functional homomeric channels) exhibited large I basal and was poorly activated by Gβγ. Expression of Gα i3 GDP reduced I basal and restored the ability of Gβγ to activate GIRK1 * , like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1 * . These results demonstrate that GIRK1 containing channels are regulated by both Gα i3 GDP and Gβγ, while GIRK2 is a Gβγ-effector insensitive to Gα i3 GDP .

G protein-activated K؉ channels (GIRKs; Kir3) are activated by direct binding of G␤␥ subunits released from heterotrimeric G proteins. In native tissues, only pertussis toxin-sensitive G proteins of the G i/o family, preferably G␣ i3 and G␣ i2 , are donors of G␤␥ for GIRK. How this specificity is achieved is not known. Here, using a pulldown method, we confirmed the presence of G␣ i3-GDP binding site in the N terminus of GIRK1 and identified novel binding sites in the N terminus of GIRK2 and in the C termini of GIRK1 and GIRK2. The non-hydrolyzable GTP analog, guanosine 5-3-O-(thio)triphosphate, reduced the binding of G␣ i3 by a factor of 2-4. G␣ i1-GDP bound to GIRK1 and GIRK2 much weaker than G␣ i3-GDP . Titrated expression of components of signaling pathway in Xenopus oocytes and their activation by m2 muscarinic receptors revealed that G i3 activates GIRK more efficiently than G i1 , as indicated by larger and faster agonist-evoked currents. Activation of GIRK by purified G␤␥ in excised membrane patches was strongly augmented by coexpression of G␣ i3 and less by G␣ i1 . Differences in physical interactions of GIRK with GDP-bound G␣ subunits, or G␣␤␥ heterotrimers, may dictate different extents of G␣␤␥ anchoring, influence the efficiency of GIRK activation by G␤␥, and play a role in determining signaling specificity.

G protein-activated K + channels (GIRK) mediate postsynaptic inhibitory effects of neurotransmitters in the atrium and in the brain by coupling to G protein-coupled receptors (GPCRs). In neurotransmitter-dependent GIRK signalling, Gβγ is released from the heterotrimeric Gαβγ complex upon GPCR activation, activating the channel and attenuating its rectification. Now it becomes clear that Gα is more than a mere Gβγ donor. We have proposed that Gα i3 -GDP regulates GIRK gating, keeping its basal activity low but priming (predisposing) the channel for activation by agonist in intact cells, and by Gβγ in excised patches. Here we have further investigated GIRK priming by Gα i3 using a model in which the channel was activated by coexpression of Gβγ, and the currents were measured in intact Xenopus oocytes using the two-electrode voltage clamp technique. This method enables the bypass of GPCR activation during examination of the regulation of the channel in intact cells. Using this method, we further characterize the priming phenomenon. We tested and excluded the possibility that our estimates of priming are affected by artifacts caused by series resistance or large K + fluxes. We demonstrate that both Gα i3 and membrane-attached Gβγ scavenger protein, m-phosducin, reduce the basal channel activity. However, Gα i3 allows robust channel activation by coexpressed Gβγ, in sharp contrast to m-phosducin, which causes a substantial reduction in the total Gβγ-induced current. Furthermore, Gα i3 also does not impair the Gβγ-dependent attenuation of the channel rectification, in contrast to m-phosducin, which prevents this Gβγ-induced modulation. The Gα i3 -induced enhancement of direct activation of GIRK by Gβγ, demonstrated here for the first time in intact cells, strongly supports the hypothesis that Gα i regulates GIRK gating under physiological conditions.