Sagit Peleg

Gαi Controls the Gating of the G Protein-Activated K+ Channel, GIRK

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.

Gαi3 primes the G protein‐activated K+ channels for activation by coexpressed Gβγ in intact Xenopus oocytes

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 Gαi3–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 Gαi3 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 Gαi3 and membrane‐attached Gβγ scavenger protein, m‐phosducin, reduce the basal channel activity. However, Gαi3 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, Gαi3 also does not impair the Gβγ‐dependent attenuation of the channel rectification, in contrast to m‐phosducin, which prevents this Gβγ‐induced modulation. The Gαi3‐induced enhancement of direct activation of GIRK by Gβγ, demonstrated here for the first time in intact cells, strongly supports the hypothesis that Gαi regulates GIRK gating under physiological conditions.

Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by GαiGDP and Gβγ

G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of Gi/o proteins via G protein‐coupled receptors (GPCRs). Although Gαi 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 Gαi3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the ‘inactive’ GDP‐bound Gαi3GDP and involves the formation of Gαi3βγ 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 Gαi3, Gαi3G203A, which strongly binds Gβγ, but not by the ‘constitutively active’ mutant, Gαi3Q204L, or by Gβγ‐scavenging proteins. Furthermore, regulation by Gαi3G203A 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 Gαi3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a ‘classical’ Gβγ effector, showing low Ibasal and strong Gβγ‐dependent activation. Expression of Gαi3G203A did not affect either Ibasal or Gβγ‐induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by Gβγ. Expression of Gαi3GDP reduced Ibasal 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 Gαi3GDP and Gβγ, while GIRK2 is a Gβγ‐effector insensitive to Gαi3GDP.

Expression levels of RGS7 and RGS4 proteins determine the mode of regulation of the G protein-activated K+ channel and control regulation of RGS7 by Gβ5

Regulators of G protein signaling RGS4 and RGS7 accelerate the kinetics of K+ channels (GIRKs) in the Xenopus oocyte system. Here, via quantitative analysis of RGS expression, we reveal biphasic effects of RGSs on GIRK regulation. At low concentrations, RGS4 inhibited basal GIRK activity, but stimulated it at high concentrations. RGS7, which is associated with the G protein subunit Gβ5, is regulated by Gβ5 by two distinct mechanisms. First, Gβ5 augments RGS7 activity, and second, it increases its expression. These dual effects resolve previous controversies regarding RGS4 and RGS7 function and indicate that they modulate signaling by mechanisms supplementary to their GTPase‐activating protein activity.

Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by GalphaiGDP and Gbetagamma.

G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of Gi/o proteins via G protein‐coupled receptors (GPCRs). Although Gαi 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 Gαi3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the ‘inactive’ GDP‐bound Gαi3GDP and involves the formation of Gαi3βγ 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 Gαi3, Gαi3G203A, which strongly binds Gβγ, but not by the ‘constitutively active’ mutant, Gαi3Q204L, or by Gβγ‐scavenging proteins. Furthermore, regulation by Gαi3G203A 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 Gαi3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a ‘classical’ Gβγ effector, showing low Ibasal and strong Gβγ‐dependent activation. Expression of Gαi3G203A did not affect either Ibasal or Gβγ‐induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by Gβγ. Expression of Gαi3GDP reduced Ibasal 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 Gαi3GDP and Gβγ, while GIRK2 is a Gβγ‐effector insensitive to Gαi3GDP.

Recruitment of Gβγ controls the basal activity of G‐protein coupled inwardly rectifying potassium (GIRK) channels: crucial role of distal C terminus of GIRK1

The G‐protein coupled inwardly rectifying potassium (GIRK) channel is an important mediator of neurotransmission via Gβγ subunit of the heterotrimeric Gi/o protein released by G‐protein coupled receptor (GPCR) activation. Channels containing the GIRK1 subunit exhibit high basal currents, whereas channels that are formed by the GIRK2 subunit have very low basal currents. GIRK1‐containing channels, but not channels consisting of GIRK2 only, recruit Gβγ to the plasma membrane. The Gα subunit of the G protein is not recruited by either GIRK1/2 or GIRK2. The unique distal C terminus of GIRK1 (G1‐dCT) endows the channel with strong interaction with Gβγ, and deletion of G1‐dCT abolishes the Gβγ recruitment and reduces the basal currents. These findings suggest that the basal activity of GIRK channels depends on channel‐induced recruitment of Gβγ. The unique C terminus of GIRK1 subunit plays an important role in Gβγ recruitment.

G protein‐activated K+ channels: a reporter for rapid activation of G proteins by lysophosphatidic acid in Xenopus oocytes

Threshold concentrations of lysophosphatidic acid (LPA) or acetylcholine (ACh) induce pertussis toxin (PTX)‐sensitive rapid desensitization of responses to LPA in Xenopus oocytes. To demonstrate that threshold [LPA] rapidly activates Gi/o proteins, we used the G protein‐activated K+ channel (GIRK) as a reporter. Low [LPA] induced I K + in <3 s of the agonist addition with little or no activation of chloride current. Depletion of Gαo/Gαo1 each decreased the LPA‐induced I K + by approximately 40–50%, while PTX completely abolished it. This is the first direct evidence showing the activation of GIRK by LPA, and the involvement of G proteins of the Go family in rapid desensitization of LPA responses.

Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+channel by GαiGDPand Gβγ: Divergent regulation of GIRK1 and GIRK2 subunits by Gαiand Gβγ

G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of Gi/o proteins via G protein‐coupled receptors (GPCRs). Although Gαi 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 Gαi3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the ‘inactive’ GDP‐bound Gαi3GDP and involves the formation of Gαi3βγ 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 Gαi3, Gαi3G203A, which strongly binds Gβγ, but not by the ‘constitutively active’ mutant, Gαi3Q204L, or by Gβγ‐scavenging proteins. Furthermore, regulation by Gαi3G203A 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 Gαi3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a ‘classical’ Gβγ effector, showing low Ibasal and strong Gβγ‐dependent activation. Expression of Gαi3G203A did not affect either Ibasal or Gβγ‐induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by Gβγ. Expression of Gαi3GDP reduced Ibasal 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 Gαi3GDP and Gβγ, while GIRK2 is a Gβγ‐effector insensitive to Gαi3GDP.