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Atrial fibrillation is the commonest cardiac arrhythmia and leads to significant clinical morbidity and mortality. It has a complex pathophysiology but is often initiated by atrial ectopic beats and because of atrial remodelling once it occurs it can become established. Thus therapeutic interventions designed to prevent the initial occurrence of the arrhythmia are particularly needed. At the cellular level, these ectopic beats arise because of abnormal calcium release events from the sarcoplasmic reticulum leading to an inward current mediated by the sodium-calcium exchanger. There has been considerable interest in this over the last few years largely focused on the ryanodine receptor and related signalling pathways. However, atrial myocytes also possess a well-developed inositol trisphosphate (IP 3 ) dependent calcium release system and this has been less studied. In this review we focus on pathways and molecules that couple via the G q\11 family of G-proteins including regulators of G-protein signalling that may influence IP 3 mediated calcium release and atrial fibrillation. [ABSTRACT FROM AUTHOR]

Reciprocal physiological modulation of heart rate is controlled by the sympathetic and parasympathetic systems acting on the sinoatrial (SA) node. However, there is little direct in vivo work examining the role of stimulatory and inhibitory G protein signaling in the SA node. Thus, we designed a study to examine the role of the stimulatory (Gs) and inhibitory G protein (Gi2) in in vivo heart rate regulation in the SA node in the mouse. We studied mice with conditional deletion of Gαs and Gαi2 in the conduction system using cre-loxP technology. We crossed mice in which cre recombinase expression was driven by a tamoxifeninducible conduction system-specific construct with "Gαs floxed" and "Gαi2 floxed" mice. We studied the heart rate responses of adult mice compared with littermate controls by using radiotelemetry before and after administration of tamoxifen. The mice with conditional deletion of Gαs and Gαi2 had a loss of diurnal variation and were bradycardic or tachycardic, respectively, in the daytime. In mice with conditional deletion of Gαs, there was a selective loss of low-frequency power, while with deletion of Gαi2, there was a loss of high-frequency power in power spectral analysis of heart rate variability. There was no evidence of pathological arrhythmia. Pharmacological modulation of heart rate by isoprenaline was impaired in the Gαs mice, but a muscarinic agonist was still able to slow the heart rate in Gαi2 mice. We conclude that Gαs- and Gαi2-mediated signaling in the sinoatrial node is important in the reciprocal regulation of heart rate through the autonomic nervous system. [ABSTRACT FROM AUTHOR]

Multiple isoforms of inhibitory Gα-subunits (Gαi1,2,3, as well as Gα0) are present within the heart, and their role in modulating pacemaker function remains unresolved. Do inhibitory Gα-subunits selectively modulate parasympathetic heart rate responses? Published findings using a variety of experimental approaches have implicated roles for Gαi2, Gαi3, and Gα0 in parasympathetic signal transduction. We have compared in vivo different groups of mice with global genetic deletion of Giα1/Gαi3, Gαi2, and Gα0 against littermate controls using implanted ECG telemetry. Significant resting tachycardia was observed in Gαi2-/- and Gα0-/- mice compared with control and Gαi1-/- /Gαi3-/- mice (P < 0.05). Loss of diurnal heart rate variation was seen exclusively in Gα0-/- mice. Using heart rate variability (HRV) analysis, compared with littermate controls (4.02 ms² ± 1.17; n = 6, Gαi2-/-) mice have a selective attenuation of high-frequency (HF) power (0.73 ms² ± 0.31; n = 5, P < 0.05). Gαi1-/- /Gαi3-/- and Gα0-/- cohorts have nonsignificant changes in HF power. Gα0-/- mice have a different basal HRV signature. The observed HRV phenotype in Gαi2-/- mice was qualitatively similar to atropine (1 mg/kg)-treated controls [and mice treated with the GIRK channel blocker tertiapinQ (0.05 mg/kg)]. Maximal cardioinhibitory response to the M2-receptor agonist carbachol (0.5 mg/kg) compared with basal heart rate was attenuated in Gαi2-/- mice (0.08 ± 0.04; n = 6) compared to control (0.27 ± 0.04; n = 7 P < 0.05). Our data suggest a selective defect of parasympathetic heart rate modulation in mice with Gαi2 deletion. Mice with Gα0 deletion also have a defect in short-term heart rate dynamics, but this is qualitatively different to the effects of atropine, tertiapinQ, and Gαi2 deletion. In contrast, Gαi1 and Gαi3 do not appear to be essential for parasympathetic responses in vivo. [ABSTRACT FROM AUTHOR]

Abstract: There is accumulating evidence that regulators of G-protein signalling (RGS) can have roles in signal transduction that are not related to GAP activity. Furthermore, RGSs have much more selective effects in vivo than might be anticipated from their behaviour in in vitro assays. I discuss the molecular mechanisms by which these phenomena might be explained including specific interactions between the RGS and G-protein coupled receptor, G-protein and effector. [Copyright &y& Elsevier]

Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the α2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits two, β1, and γ2 and a number of GPCRs including M4, α2A, the adenosine Al receptor, and the dopamine D2 receptor under resting conditions. The interaction between GPCRs and Gα proteins shows specificity: there is interaction between the α2A receptor and Go, but little interaction between the α2A receptor and Gs. In contrast, the predominantly Gs-coupled prostacyclin receptor interacted with Gs, but there was little interaction between the prostacyclin receptor and Go. Inverse agonists did not change the FRET ratio, whereas the addition of agonist resulted in a modest fall. Our work suggests that GPCR dimers and the G protein heterotrimer are present at the cell membrane in the resting state in a pentameric complex. [ABSTRACT FROM AUTHOR]

G protein-gated inward rectifier (Kit3) channels are inhibited by activation of Gq/11-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PIP2). Their precise roles in mediating the inhibition of this family of channels remain controversial. We examine here their relative roles in causing inhibition of Kir3.1/3.2 channels stably expressed in human embryonic kidney (HEK)-293 ceils after muscarinic M3 receptor activation. In perforated patch mode, staurosporine prevented the Gq/11-mediated, M3 receptor, inhibition of channel activity. Recovery from M3-mediated inhibition was wortmannin sensitive. Whole cell currents, where the patch pipette was supplemented with PIP2, were still irreversibly inhibited by M3 receptor stimulation. When adenosine A1 receptors were co-expressed, inclusion of PIP2 rescued the A1-mediated response. Recordings from inside-out patches showed that catalytically active PKC applied directly to the intracellular membrane face inhibited the channels: a reversible effect modulated by okadaic acid. Generation of mutant heteromeric channel Kir3.1S185A/Kir3.2C-S178A, still left the channel susceptible to receptor, pharmacological, and direct kinase-mediated inhibition. Biochemically, labeled phosphate is incorporated into the channel. We suggest that PKC-δ mediates channel inhibition because recombinant PKC-δ inhibited channel activity, M3-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC-δ constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC-δ to the plasma membrane on M3 receptor stimulation. Thus Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent on the activity of PKC-δ after M3 receptor activation in HEK-293 cells. [ABSTRACT FROM AUTHOR]

Activation of G protein-gated inwardly rectifying K+ (GIRK) channels, found in the brain, heart, and endocrine tissue, leads to membrane hyperpolarization that generates neuronal inhibitory postsynaptic potentials, slows the heart rate, and inhibits hormone release. During stimulation of Gi/o-Coupled receptors and subsequent channel activation, it has been observed that the current desensitizes. In this study we examined mechanisms underlying fast desensitization of cloned heteromeric neuronal Kir3.1+3.2A and atrial Kir3.1+3.4 channels and also homomeric Kir3.0 currents in response to stimulation of several Gi/o G protein-coupled receptors (GPCRs) expressed in HEK-293 cells (adenosine A1, adrenergic α2A, dopamine D2S, M4 muscarinic, and GABAB1b/2 receptors). We found that all agonist-induced currents displayed a similar degree of desensitization except the adenosine A1 receptor, which exhibits an additional desensitizing component. Using the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPγS), we found that this is due to a receptor-dependent, G protein-independent process. Using Ca2+ imaging we showed that desensitization is unlikely to be accounted for solely by phospholipase C activation and phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. We examined the contribution of the G protein cycle and found the following. First, agonist concentration is strongly correlated with degree of desensitization. Second, competitive inhibition of GDP/ GTP exchange by using nonhydrolyzable guanosine 5'-O-(2-thiodiphosphate) (GDPβS) has two effects, a slowing of channel activation and an attenuation of the fast desensitization phenomenon. Finally, using specific Ga subunits we showed that ternary complexes with fast activation rates display more prominent desensitization than those with slower activation kinetics. Together our data suggest that fast desensitization of GIRK currents is accounted for by the fundamental properties of the G protein cycle. [ABSTRACT FROM AUTHOR]

G protein-gated inwardly rectifying K[sup +] (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[sub i/o], in a membrane-delimited fashion by the direct binding of Gβγ dimers to the channel complex. In this study we are concerned with the kinetics of deactivation of the cloned neuronal G protein-gated K[sup +] 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 α 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 α subunit. With some combinations of agonist/GPCR, 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 α subunit. G protein isoform and interaction with G protein-signaling proteins play a significant role with this group of GPCRs. [ABSTRACT FROM AUTHOR]

Examines the role of isoforms of pertussis toxin-sensitive G protein alpha subunits in coupling receptors to the G protein-gated inwardly rectifying potassium channel. Characterization of pertussis toxin-insensitive mutants; Delineating different patterns of receptor stimulation of channel activation.

In atrial and nodal cardiac myocytes, M2 muscarinic receptors activate inhibitory G-proteins (Gi/o), which in turn stimulate G-protein-gated inwardly rectifying K+ channels through direct binding of the G/3y subunit. Despite also releasing Gβγ, Gs-coupled receptors such as the β-adrenergic receptor are not able to prominently activate this current. An appealing hypothesis would be if components were sequestered in membrane domains such as caveolae/rafts. Using biochemical fractionation followed by Western blotting and/or radioligand binding experiments, we examined the distribution of the components in stable HEK293 and HL-I cells, which natively express the transduction cascade. The channel, M2 muscarinic, and A1 adenosine receptors were located in noncaveolar/nonraft fractions. Giα12 was enriched in both caveolar/raft and noncaveolar/ nonraft fractions. In contrast, Gsα was only enriched in caveolar/raft fractions. We constructed YFP-tagged caveolin-2 (YFP-Cav2) and chimeras with the M2 (M2-YFP-Cav2) and A1 (A1-YFP-Cav2) receptors. Analysis of gradient fractions showed that these receptor chimeras were now localized to caveolae-enriched fractions. Microscopy showed that M2-YFP and A1-YFP had a diffuse homogenous membrane signal. YFPCav2, M2-YFP-Cav2, and A1-YFP-Cav2 revealed a more punctuate pattern. Finally, we looked at the consequences for signaling. Activation via M2-YFP-Cav2 or A1-YFP-Cav2 revealed substantially slower kinetics compared with M2-YFP or A1-YFP and was reversed by the addition of methyl-β-cyclodextrin. Thus the localization of the channel signal transduction cascade in non-cholesterol rich domains substantially enhances the speed of signaling. The presence of Gsα solely in caveolae may account for signaling selectivity between Gi/o and Gs-coupled receptors. [ABSTRACT FROM AUTHOR]

Regulators of G-protein signaling (RGS) proteins modulate signaling through heterotrimeric G-proteins. They act to enhance the intrinsic GTPase activity of the Ga subunit but paradoxically have also been shown to enhance receptor-stimulated activation. To study this paradox, we used a G-protein gated K+ channel to report the dynamics of the G-protein cycle and fluorescence resonance energy transfer techniques with cyan and yellow fluorescent protein-tagged proteins to report physical interaction. Our data show that the acceleration of the activation kinetics is dissociated from deactivation kinetics and dependent on receptor and RGS type, G-protein isoform, and RGS expression levels. By using fluorescently tagged proteins, fluorescence resonance energy transfer microscopy showed a stable physical interaction between the G-protein α subunit and RGS (RGS8 and RGS7) that is independent of the functional state of the G-protein. RGS8 does not directly interact with G-protein-coupled receptors. Our data show participation of the RGS in the ternary complex between agonist-receptor and G-protein to form a "quaternary complex." Thus we propose a novel model for the action of RGS proteins in the G-protein cycle in which the RGS protein appears to enhance the "kinetic efficacy" of the ternary complex, by direct association with the G-protein α subunit. [ABSTRACT FROM AUTHOR]

Addresses controversial issues regarding models of receptor activation of G-proteins. Data indicating that the combination of agonist, G-protein-coupled receptors (GPCRs) and G-protein isoform uniquely specify the behavior of these channels; Importance of the whole ternary complex at a kinetic level.