RGS8 accelerates G-protein-mediated modulation of K+currents

107

141

In mammals, basal currents through G protein-coupled inwardly rectifying K ؉ (GIRK) channels are repressed by G␣i/oGDP, and the channels are activated by direct binding of free G␤␥ subunits released upon stimulation of G␣ i/o-coupled receptors. However, essentially all information on G protein regulation of GIRK electrophysiology has been gained on the basis of coexpression studies in heterologous systems. A major advantage of the model organism, Arabidopsis thaliana, is the ease with which knockout mutants can be obtained. We evaluated plants harboring mutations in the sole Arabidopsis G␣ (AtGPA1), G␤ (AGB1), and Regulator of G protein Signaling (AtRGS1) genes for impacts on ion channel regulation. In guard cells, where K ؉ fluxes are integral to cellular regulation of stomatal apertures, inhibition of inward K ؉ (Kin) currents and stomatal opening by the phytohormone abscisic acid (ABA) was equally impaired in Atgpa1 and agb1 single mutants and the Atgpa1 agb1 double mutant. AGB1 overexpressing lines maintained a wild-type phenotype. The Atrgs1 mutation did not affect K in current magnitude or ABA sensitivity, but Kin voltage-activation kinetics were altered. Thus, Arabidopsis cells differ from mammalian cells in that they uniquely use the G␣ subunit or regulation of the heterotrimer to mediate K in channel modulation after ligand perception. In contrast, outwardly rectifying (K out) currents were unaltered in the mutants, and ABA activation of slow anion currents was conditionally disrupted in conjunction with cytosolic pH clamp. Our studies highlight unique aspects of ion channel regulation by heterotrimeric G proteins and relate these aspects to stomatal aperture control, a key determinant of plant biomass acquisition and drought tolerance.G protein-coupled inwardly rectifying potassium or ''GIRK'' channels (also known as Kir3 channels) comprise important targets of heterotrimeric G protein regulation in mammals (1, 2). GIRK channels mediate signals from muscarinic, adrenergic, opioid, dopaminergic, and GABA B receptors (3). Basal activity of GIRK channels is repressed by their direct binding of G␣ i/o GDP (4, 5) within a macromolecular complex that includes G␤␥ (4-7). Upon activation of G i/o -coupled G protein-coupled receptors (GPCRs), formation of the GTP-bound form of G␣ i/o both alleviates G␣-mediated repression and releases ␤␥ dimers that independently interact with the channel (7,8). G␤ 1-4 ␥ binding strengthens GIRK interaction with phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ), thereby promoting conformational changes that increase channel open time (3, 9-11). Conversely, G␣ q -based activation of phospholipase C opposes GIRK activity via both depletion of PtdInsP 2 and activation of PKC-based phosphorylation events (12).Numerous studies analyzing GIRK activity in Xenopus oocytes and cultured mammalian cells have led to the beautifully intricate model described above. However, studies analyzing GIRK activity in the appropriate native cell context upon genetic depletion of G␤ subunits are lack...

Section: Regulation Of Plant Kin Channels By G Protein Complex Componsupporting

confidence: 62%

Abscisic acid regulation of guard-cell K ⁺ and anion channels in Gβ- and RGS-deficient Arabidopsis lines

Fan

Zhang

Chen³

et al. 2008

107

141

“…Ion-channel data come from the m2 MAchR, Gi, RGS4, and G protein-gated inward rectifier K ϩ ion channel in oocytes (23), which is closely related to the m1 MAchR-Gq-RGS4 system we have modeled. As mentioned in the Introduction, these data appear paradoxical because RGS proteins do not necessarily alter G proteinstimulated ion-channel current amplitude but do accelerate the onset and desensitization of current (23)(24)(25)(26). In our simulations, we cannot directly assess the activation of ion channels; however, we predict that Z decreases from 0.98 (path RG) to 0.096 (path RGA) in the presence of RGS4 and speculate that Z ϭ 0.096 is sufficient to saturate ion channels.…”

Section: Table 2 Ratio Of G:r (And G:gap) Required For Activation (Omentioning

confidence: 82%

“…GAPs are expected to decrease amplitude and hasten G protein deactivation upon removal of active receptor. However, RGS proteins do not affect the amplitude of G protein-induced changes in K ϩ channel current, but do accelerate the timing of both current onset upon agonist addition and current desensitization upon agonist removal (23)(24)(25)(26).…”

mentioning

confidence: 99%

Computational modeling reveals how interplay between components of a GTPase-cycle module regulates signal transduction

Bornheimer

Maurya

Farquhar

et al. 2004

Heterotrimeric G protein signaling is regulated by signaling modules composed of heterotrimeric G proteins, active G proteincoupled receptors (Rs), which activate G proteins, and GTPaseactivating proteins (GAPs), which deactivate G proteins. We term these modules GTPase-cycle modules. The local concentrations of these proteins are spatially regulated between plasma membrane microdomains and between the plasma membrane and cytosol, but no data or models are available that quantitatively explain the effect of such regulation on signaling. We present a computational model of the GTPase-cycle module that predicts that the interplay of local G protein, R, and GAP concentrations gives rise to 16 distinct signaling regimes and numerous intermediate signaling phenomena. The regimes suggest alternative modes of the GTPasecycle module that occur based on defined local concentrations of the component proteins. In one mode, signaling occurs while G protein and receptor are unclustered and GAP eliminates signaling; in another, G protein and receptor are clustered and GAP can rapidly modulate signaling but does not eliminate it. Experimental data from multiple GTPase-cycle modules is interpreted in light of these predictions. The latter mode explains previously paradoxical data in which GAP does not alter maximal current amplitude of G protein-activated ion channels, but hastens signaling. The predictions indicate how variations in local concentrations of the component proteins create GTPase-cycle modules with distinctive phenotypes. They provide a quantitative framework for investigating how regulation of local concentrations of components of the GTPase-cycle module affects signaling.T he timing and amplitude of signaling in cellular signaling networks depends on the local concentrations and kinetics of component proteins. The complexity of these networks has precluded comprehensive quantitative analysis of signaling. However, the networks can be divided into discrete signaling modules that can be quantitatively modeled and analyzed independently (1-5). Later, such models of modules can be experimentally verified and then reconnected to build larger signaling networks.G protein-mediated signaling networks (6) are ideal for such quantitative analysis. A key signaling module in these networks is the GTPase-cycle module, which controls signaling by regulating G protein activity. This module consists of a heterotrimeric G protein, a guanine nucleotide exchange factor (GEF) such as agonist-bound and active G protein-coupled receptor, a GTPase-activating protein (GAP) such as regulator of G protein signaling (RGS) protein or phospholipase C-␤, GTP, GDP, and P i . The extent of G protein activation reflects the balance between the rates of GDP͞GTP exchange, which activates the G protein, and GTP hydrolysis, which deactivates it (7-9). GEFs facilitate GDP͞GTP exchange (9), and GAPs accelerate GTP hydrolysis (10, 11). Additional roles of GEFs (12) and GAPs (10,11,13,14) are not included in this module. At present, we lack quantitativ...

“…To date, these issues have largely been addressed by using the cloned atrial channel, Kir3.1 ϩ 3.4, and the muscarinic M 2 receptor expressed in Xenopus laevis oocytes (17,18). In particular, a number of studies have sought to explain why these channels when expressed in X. laevis oocytes deactivate more slowly than the native atrial current following stimulation of M 2 receptors.…”

mentioning

confidence: 99%

“…RGS proteins interact with G i/o and G q/11 ␣ subunits to increase the intrinsic GTPase rate of the G␣ subunit (19-23). Overexpression of RGS4, for example, accelerates the channel-deactivation kinetics and changes other kinetic parameters such that the measured time constants are more consistent with those occurring after stimulation of native channels in atrial cells (17,18,(24)(25)(26). …”

mentioning

confidence: 99%

Agonist unbinding from receptor dictates the nature of deactivation kinetics of G protein-gated K ⁺ channels

Benians

Leaney

Tinker

2003

G protein-gated inwardly rectifying K ؉ (Kir) channels are found in neurones, atrial myocytes, and endocrine cells and are involved in generating late inhibitory postsynaptic potentials, slowing the heart rate and inhibiting hormone release. They are activated by G proteincoupled receptors (GPCRs) via the inhibitory family of G protein, G i/o, in a membrane-delimited fashion by the direct binding of G␤␥ dimers to the channel complex. In this study we are concerned with the kinetics of deactivation of the cloned neuronal G protein-gated K ؉ channel, Kir3.1 ؉ 3.2A, after stimulation of a number of GPCRs. Termination of the channel activity on agonist removal is thought to solely depend on the intrinsic hydrolysis rate of the G protein ␣ subunit. In this study we present data that illustrate a more complex behavior. We hypothesize that there are two processes that account for channel deactivation: agonist unbinding from the GPCR and GTP hydrolysis by the G protein ␣ subunit. With some combinations of agonist͞GPCR, the rate of agonist unbinding is slow and rate-limiting, and deactivation kinetics are not modulated by regulators of G protein-signaling proteins. In another group, channel deactivation is generally faster and limited by the hydrolysis rate of the G protein ␣ subunit. G protein isoform and interaction with G protein-signaling proteins play a significant role with this group of GPCRs.M embers of the family of inwardly rectifying K ϩ (Kir) channels gated by G proteins were first identified in atrial myocytes, where they are activated through stimulation of M 2 muscarinic receptors by acetylcholine (1). Physiologically, activation of this current is partly responsible for slowing of the heart rate in response to vagal-nerve stimulation (2, 3). It is now known that channel activation is membrane-delimited (4), mimicked by nonhydrolyzable GTP analogues (5), and sensitive to pertussis toxin (PTx), implicating the inhibitory family of G proteins (G i/o ) (6). Channel activation occurs because of direct binding of G␤␥ dimers, released from G i/o ␣-containing heterotrimers, to domains on the channel (7-9). G protein-gated Kir channels are also expressed in many central neurones, where they can be activated by a large variety of neurotransmitters acting at G i/o -coupled receptors (10) including ␥-aminobutyric acid (GABA) at the GABA type B (GABA B ) receptor complex and adenosine at A 1 receptors, and they mediate postsynaptic inhibitory events (9,11,12). The molecular counterparts of these currents have now been identified by cloning techniques (13-16): the channel is a heterotetramer of members of the Kir3.0 family of K ϩ channels. Coexpression of Kir3.1 with Kir3.2 or Kir3.4 in heterologous expression systems results in currents that show many of the basic characteristics of the native channels in neurones and atria, respectively.The kinetic behavior of these channels after agonist application and withdrawal has been a subject of intense investigation. To date, these issues have largely been addressed by using...