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

Peleg, Sagit; Varon, Dalia; Ivanina, Tatiana; Dessauer, Carmen W.; Dascal, Nathan

doi:10.1016/s0896-6273(01)00567-0

Cited by 150 publications

(248 citation statements)

References 40 publications

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“…6, b and d), as suggested by functional data (11). Correspondingly, DL-G1/G2 and G1/DL-G2 expressed alone at high levels yield high I basal (10) and an intermediate i-FRET signal. A substantial basal GIRK activity has been reported in hippocampal and cortical neurons (31,32); therefore, the preactivated states are physiologically relevant.…”

Section: G␤␥ Is Crucial For Strong Interaction Between Girk1mentioning

confidence: 56%

Gαi and Gβγ Jointly Regulate the Conformations of a Gβγ Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

Berlin

Keren-Raifman

Castel

et al. 2010

Journal of Biological Chemistry

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Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical G␤␥ effector G protein-activated K ؉ channel (GIRK; Kir3) physically interacts with G␤␥ but also with G␣ i/o . Whether and how G␣ i/o subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, G␣ i3 and G␤␥. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that G␤␥ and G␣ i3 distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that G␤␥ greatly enhances the binding of GIRK1 subunit to G␣ i3 GDP and, unexpectedly, to G␣ i3 GTP . i-FRET showed that both G␣ i3 and G␤␥ induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a "constitutively active" G␣ i3 mutant and G␤␥ together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either G␣ i3 or G␤␥. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-G␣-G␤␥ signaling complex and mediates allosteric interactions between G␣ i GTP and G␤␥. Our findings imply that G␣ i/o and the G␣ i ␤␥ heterotrimer can regulate a G␤␥ effector both before and after activation by neurotransmitters.It is believed that signaling via G protein-coupled receptors (GPCRs) 5 occurs within multiprotein complexes that include GPCRs, G proteins, and effectors (1-3). The G protein-activated K ϩ channel (GIRK, Kir3), an important mediator of neuronal inhibition (4), is activated by the binding of G␤␥ to the cytosolic N and C termini (NT and CT, respectively) of GIRK. G␤␥ associates with the channel before and after receptor activation (5, 6). GIRK NT and CT segments also bind G␣ i/o (see Ref. 7), but it is not clear whether and how G␣ i/o regulates GIRK in vivo. G␣ clearly plays a role in determining specificity of signaling from GPCR to GIRK (8, 9). In heterologous systems, expression of G␣ i reduces GIRK basal activity (I basal ) and increases the relative extent of activation by added or coexpressed G␤␥ (10 -12). Recently, we have demonstrated that this regulation is exerted by the GDP-bound form of G␣ i , G␣ i GDP (12); no role for G␣ i GTP in GIRK gating could be assigned so far. We proposed that regulation of GIRK by G␣ i relies upon the formation of the G␣ i GDP -G␤␥ heterotrimer, which forms a persistent, dynamic signaling complex with GIRK to ensure proper gating with low I basal and high signal-to-background ratio upon G␤␥ activation (11,12).We hypothesized that within a multiprotein signaling complex, the partner proteins alter each other's conformation and activity at various stages of the signaling process. Thus, G␣ i and G␤␥ may regulate the conformation of the channel both before and after activation. ...

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Section: G␤␥ Is Crucial For Strong Interaction Between Girk1mentioning

confidence: 56%

Gαi and Gβγ Jointly Regulate the Conformations of a Gβγ Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

Berlin

Keren-Raifman

Castel

et al. 2010

Journal of Biological Chemistry

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“…Binding of the G␤␥ subunit activates Kir3, whereas G␣ i binding suppresses basal Kir3 activity and enhances G protein activation (9). This action of G␣ i binding resembles the effect of BDNF-induced tyrosine phosphorylation on Kir3.…”

mentioning

confidence: 85%

“…Recent evidence shows that both arms of the heterotrimeric G protein complex, G␣ i and G␤␥, interact with Kir3 (9). Binding of the G␤␥ subunit activates Kir3, whereas G␣ i binding suppresses basal Kir3 activity and enhances G protein activation (9).…”

mentioning

confidence: 99%

N-terminal Tyrosine Residues within the Potassium Channel Kir3 Modulate GTPase Activity of Gαi

Ippolito

Temkin

Rogalski

et al. 2002

Journal of Biological Chemistry

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trkB activation results in tyrosine phosphorylation of N-terminal Kir3 residues, decreasing channel activation. To determine the mechanism of this effect, we reconstituted Kir3, trkB, and the mu opioid receptor in Xenopus oocytes. Activation of trkB by BDNF (brainderived neurotrophic factor) accelerated Kir3 deactivation following termination of mu opioid receptor signaling. Similarly, overexpression of RGS4, a GTPaseactivating protein (GAP), accelerated Kir3 deactivation. Blocking GTPase activity with GTP␥S also prevented Kir3 deactivation, and the GTP␥S effect was not reversed by BDNF treatment. These results suggest that BDNF treatment did not reduce Kir3 affinity for G␤␥ but rather acted to accelerate GTPase activity, like RGS4. Tyrosine phosphatase inhibition by peroxyvanadate pretreatment reversibly mimicked the BDNF/trkB effect, indicating that tyrosine phosphorylation of Kir3 may have caused the GTPase acceleration. Tyrosine to phenylalanine substitution in the N-terminal domain of Kir3.4 blocked the BDNF effect, supporting the hypothesis that phosphorylation of these tyrosines was responsible. Like other GAPs, Kir3.4 contains a tyrosine-arginine-glutamine motif that is thought to function by interacting with G protein catalytic domains to facilitate GTP hydrolysis. These data suggest that the N-terminal tyrosine hydroxyls in Kir3 normally mask the GAP activity and that modification by phosphorylation or phenylalanine substitution reveals the GAP domain. Thus, BDNF activation of trkB could inhibit Kir3 by facilitating channel deactivation.The family of G protein-activated potassium channels (Kir3 or GIRK) 1 is a principal effector mediating the actions of a wide range of pertussis toxin-sensitive, G protein-coupled receptors (GPCR) (1). Channel activation provides key regulation of both cardiac and neuronal excitability. The activity of this channel is controlled by a large number of modulators including phosphatidylinositol bisphosphate, Na ϩ , eicosanoids, ATP, Mg 2ϩ , and phosphorylation (1-6). For example, in a previous study (7), we found that tyrosine phosphorylation of Kir3 resulted in channel inhibition. Brain-derived neurotrophic factor (BDNF) activation of trkB receptors caused the phosphorylation of specific tyrosine residues in the N-terminal domain of Kir3.1 and Kir3.4, reducing basal channel conductance. G␤␥, released by opioid receptor activation, overcame the inhibition and evoked the original maximal effect. The results suggest that channel phosphorylation regulates the specific interaction with G␤␥, but the mechanism of this effect is unclear. However, the finding may be physiologically significant because tyrosine kinase cascades initiated by neurotrophic factors such as BDNF and nerve growth factor are up-regulated under conditions such as inflammation (8). It is possible that tyrosine phosphorylation of Kir3 may mediate neuronal excitability in conjunction with GPCRs under conditions of inflammation. The goal of the present study was to define the mechanism responsible for BDNF inhib...

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“…G ␤␥ Microinjection. G␤␥ (purified recombinant G␤1␥2) was kindly provided by Carmen W. Dessauer (University of Texas, Houston) and stored as described (52,53) in a stock solution (40 M). Denatured G␤␥ was obtained by heating to 70°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Molecular mechanisms that control initiation and termination of physiological depolarization-evoked transmitter release

Kupchik

Rashkovan

Ohana

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Ca 2؉ is essential for physiological depolarization-evoked synchronous neurotransmitter release. But, whether Ca 2؉ influx or another factor controls release initiation is still under debate. The time course of ACh release is controlled by a presynaptic inhibitory G protein-coupled autoreceptor (GPCR), whose agonist-binding affinity is voltage-sensitive. However, the relevance of this property for release control is not known. To resolve this question, we used pertussis toxin (PTX), which uncouples GPCR from its Gi/o and in turn reduces the affinity of GPCR toward its agonist. We show that PTX enhances ACh and glutamate release (in mice and crayfish, respectively) and, most importantly, alters the time course of release without affecting Ca 2؉ currents. These effects are not mediated by G␤␥ because its microinjection into the presynaptic terminal did not alter the time course of release. Also, PTX reduces the association of the GPCR with the exocytotic machinery, and this association is restored by the addition of agonist. We offer the following mechanism for control of initiation and termination of physiological depolarization-evoked transmitter release. At rest, release is under tonic block achieved by the transmitter-bound high-affinity presynaptic GPCR interacting with the exocytotic machinery. Upon depolarization, the GPCR uncouples from its G protein and consequently shifts to a low-affinity state toward the transmitter. The transmitter dissociates, the unbound GPCR detaches from the exocytotic machinery, and the tonic block is alleviated. The free machinery, together with Ca 2؉ that had already entered, initiates release. Release terminates when the reverse occurs upon repolarization.G protein-coupled receptor ͉ neurotransmitter release ͉ pertussis toxin ͉ presynaptic receptors C a 2ϩ influx is essential for physiological depolarizationinduced neurotransmitter (NT) release (1, 2). A broader, Ca 2ϩ voltage, hypothesis suggests that two factors control release: Ca 2ϩ and G protein-coupled receptors (GPCRs), whose agonist-binding affinity is voltage-dependent (3). The mechanism suggested for this control is as follows. (i) At resting potential and rest concentration (nMs) of transmitter, the release machinery (SNARE proteins and synaptotagmin) is under tonic block imposed by the transmitter-bound high-affinity (nMs) GPCR. (ii) Depolarization shifts the GPCR to a lowaffinity state ( Ms), resulting in rapid transmitter dissociation (it should be emphasized that at this stage release of NT did not occur yet, and the concentration of NT in the synapse is still in the nM range). (iii) The unbound GPCR detaches from the release machinery to relieve the tonic block. The free-release machinery together with Ca 2ϩ , which had already entered, initiates release. (iv) Upon repolarization, release terminates because the receptor returns to its high-affinity state and the tonic block is reinstated.Much of this suggested mechanism was supported experimentally (3-8) by using mainly the cholinergic neuromuscular junction (N...

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

Cited by 150 publications

References 40 publications

Gαi and Gβγ Jointly Regulate the Conformations of a Gβγ Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

Gαi and Gβγ Jointly Regulate the Conformations of a Gβγ Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

N-terminal Tyrosine Residues within the Potassium Channel Kir3 Modulate GTPase Activity of Gαi

Molecular mechanisms that control initiation and termination of physiological depolarization-evoked transmitter release

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