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Signaling of G protein-activated inwardly rectifying K+ (GIRK) channels is an important mechanism of the parasympathetic regulation of the heart rate and cardiac excitability. GIRK channels are inhibited during stimulation of Gq-coupled receptors (GqPCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP2) and/or channel phosphorylation by protein kinase C (PKC). The GqPCR-dependent modulation of GIRK currents in terms of specific PKC isoform activation was analyzed in voltage-clamp experiments in rat atrial myocytes and in CHO or HEK 293 cells. By using specific PKC inhibitors, we identified the receptor-activated PKC isoforms that contribute to phenylephrine- and angiotensin-induced GIRK channel inhibition. We demonstrate that the cPKC isoform PKCα significantly contributes to GIRK inhibition during stimulation of wildtype α1B-adrenergic receptors (α1B-ARs). Deletion of the α1B-AR serine residues S396 and S400 results in a preferential regulation of GIRK activity by PKCβ. As a novel finding, we report that the AT1-receptor-induced GIRK inhibition depends on the activation of the nPKC isoform PKCε whereas PKCα and PKCβ do not mainly participate in the angiotensin-mediated GIRK reduction. Expression of the dominant negative (DN) PKCε prolonged the onset of GIRK inhibition and significantly reduced AT1-R desensitization, indicating that PKCε regulates both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism. The serine residue S418 represents an important phosphorylation site for PKCε in the GIRK4 subunit. To analyze the functional impact of this PKC phosphorylation site for receptor-specific GIRK channel modulation, we monitored the activity of a phosphorylation-deficient (GIRK4 (S418A)) GIRK4 channel mutant during stimulation of α1B-ARs or AT1-receptors. Mutation of S418 did not impede α1B-AR-mediated GIRK inhibition, suggesting that S418 within the GIRK4 subunit is not subject to PKCα-induced phosphorylation. Furthermore, activation of angiotensin receptors induced pronounced GIRK4 (S418A) channel inhibition, excluding that this phosphorylation site contributes to the AT1-R-induced GIRK reduction. Instead, phosphorylation of S418 has a facilitative effect on GIRK activity that was abolished in the GIRK4 (S418A) mutant. To summarize, the present study shows that the receptor-dependent regulation of atrial GIRK channels is attributed to the GqPCR-specific activation of different PKC isoforms. Receptor-specific activated PKC isoforms target distinct phosphorylation sites within the GIRK4 subunit, resulting in differential regulation of GIRK channel activity with either facilitative or inhibitory effects on GIRK currents. [ABSTRACT FROM AUTHOR]

Activation of Gq protein-coupled receptors (GqPCRs) might induce divergent cellular responses, related to receptor-specific activation of different branches of the Gq signaling pathway. Receptor-specific desensitization provides a mechanism of effector modulation by restricting the spatiotemporal activation of signaling components downstream of Gq. We quantified signaling events downstream of GqPCR activation with FRET-based biosensors in CHO and HEK 293 cells. KCNQ1/KCNE1 channels (IKs) were measured as a functional readout of receptor- specific activation. Activation of muscarinic M1 receptors (M1-Rs) caused robust and reversible inhibition of IKs. In contrast, activation of α1B-adrenergic receptors (α1B-ARs) induced transient inhibition of IKs, which turned into delayed facilitation after agonist withdrawal. As a novel finding, we demonstrate that GqPCR-specific kinetics of IKs modulation are determined by receptor-specific desensitization, evident at the level of Gαq activation, phosphatidylinositol 4,5-bisphosphate (PIP2) depletion, and diacylglycerol production. Sustained IKs inhibition during M1-R stimulation is attributed to robust membrane PIP2 depletion, whereas the rapid desensitization of α1B-AR delimits PIP2 reduction and augments current activation by protein kinaseC(PKC). Overexpression of Ca2+-independent PKCδ did not affect the time course ofα1B-AR-induced diacylglycerol formation, excluding a contribution of PKCδ to α1B-AR desensitization. Pharmacological inhibition of Ca2+-dependent PKC isoforms abolished fast α1B receptor desensitization and augmented IKs reduction, but did not affect IKs facilitation. These data indicate a contribution of Ca2+-dependent PKCs to α1B-AR desensitization, whereas IKs facilitation is induced by Ca2+-independent PKC isoforms. In contrast, neither inhibition of Ca2+-dependent/Ca2+-independent isoforms nor overexpression of PKCδ induced M1 receptor desensitization, excluding a contribution of PKC to M1-R-induced IKs modulation. [ABSTRACT FROM AUTHOR]

Abstract: Opening of G-protein-activated inward-rectifying K+ (GIRK, Kir3) channels is regulated by interaction with βγ-subunits of Pertussis-toxin-sensitive G proteins upon activation of appropriate GPCRs. In atrial and neuronal cells agonist-independent activity (Ibasal) contributes to the background K+ conductance, important for stabilizing resting potential. Data obtained from the Kir3 signaling pathway reconstituted in Xenopus oocytes suggest that Ibasal requires free Gβγ. In cells with intrinsic expression of Kir3 channels this issue has been scarcely addressed experimentally. Two Gβγ-binding proteins (myristoylated phosducin — mPhos — and Gαi1) were expressed in atrial myocytes using adenoviral gene transfer, to interrupt Gβγ-signaling. Agonist-induced and basal currents were recorded using whole cell voltage-clamp. Expression of mPhos and Gαi1 reduced activation of Kir3 current via muscarinic M2 receptors (IK(ACh)). Inhibition of IK(ACh) by mPhos consisted of an irreversible component and an agonist-dependent reversible component. Reduction in density of IK(ACh) by overexpressed Gαi1, in contrast to mPhos, was paralleled by substantial slowing of activation, suggesting a reduction in density of functional M2 receptors, rather than Gβγ-scavenging as underlying mechanism. In line with this notion, current density and activation kinetics were rescued by fusing the αi1-subunit to an Adenosine A1 receptor. Neither mPhos nor Gαi1 had a significant effect on Ibasal, defined by the inhibitory peptide tertiapin-Q. These data demonstrate that basal Kir3 current in a native environment is unrelated to G-protein signaling or agonist-independent free Gβγ. Moreover, our results illustrate the importance of physiological expression levels of the signaling components in shaping key parameters of the response to an agonist. [Copyright &y& Elsevier]

Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen-Tawil syndrome (ATS), a familial disorder leading to stresstriggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time-resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patient's individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy. [ABSTRACT FROM AUTHOR]

The effect of β-adrenergic stimulation on endogenous G-protein-activated K+ (GIRK) current has been investigated in atrial myocytes from hearts of adult rats. β-Adrenergic stimulation (10 μm isoprenaline, Iso) had no effect on activation kinetics, peak current or steady-state current but resulted in slowing of deactivation upon washout of acetylcholine (ACh), the time constant (τd) being increased by a factor of about 2.5. The effect of Iso could be mimicked by inclusion of cAMP (500 μm) in the filling solution of the patch clamp pipette. The Iso-induced increase in τd was blocked by the selective β1 receptor antagonist CGP-20112A (2 μm) and by the PKA inhibitor H9 (100 μm included in the pipette solution). A candidate for mediating these effects is RGS10, one of the regulators of G-protein signalling (RGS) species expressed in cardiac myocytes. Overexpression of RGS10 by adenoviral gene transfer resulted in a reduction in τd of 60%. Sensitivity of τd to Iso remained in cells overexpressing RGS10. Overexpression of RGS4 caused a comparable reduction in τd, which became insensitive to Iso. Expression of an RGS10 carrying a mutation (RGS10-S168A), which deletes a PKA phosphorylation site, caused a decrease in τd comparable to overexpression of wild-type RGS10. Sensitivity of τd to Iso was lost in RGS10-S168A-expressing myocytes. Silencing of RGS10 by means of adenovirus-mediated transcription of a short hairpin RNA did not affect basal τd but removed sensitivity to Iso. These data suggest that endogenous RGS10 has GTPase-activating protein (GAP) activity on the G-protein species that mediates activation of atrial GIRK channels. Moreover, RGS10, via PKA-dependent phosphorylation, enables a crosstalk between β-adrenergic and muscarinic cholinergic signalling. [ABSTRACT FROM AUTHOR]

Inwardly-rectifying K+ channel subunits are not homogenously expressed in different cardiac tissues. In ventricular myocytes (VM) the background current-voltage relation is dominated by IK1, carried by channels composed of Kir2.x subunits, which is less important in atrial myocytes (AM). On the other hand in AM a large G protein gated current carried by Kir3.1/3.4 complexes can be activated by stimulation of muscarinic M2 receptors (IK(ACh)), which is assumed to be marginal in VM. Recent evidence suggests that total current carried by cardiac inward-rectifiers (IK(ATP), IK(ACh), IK1) in both, AM and VM is limited, due to K+ accumulation/depletion. This lead us to hypothesize that in conventional whole celI recordings IK(ACh) in VM is underestimated as a consequence of constitutive IK1. In that case a reduction in density of IK1 should be paralleled by an increase in density of IK(ACh). Three different experimental strategies have been used to test for this hypothesis: (i) Adenovirus-driven expression of a dominant-negative mutant of Kir2.1, one of the subunits supposed to form IK1 channels, in VM caused a reduction in IK1-density by about 80 %. In those cells IK(ACh) was increased about 4 fold. (ii) A comparable increase in IK(ACh) was observed upon reduction of IK1 caused by adenovirus-mediated RNA interference.(iii) Ba2+ in a concentration of 2 μM blocks IK1 in VM by about 60 % without affecting atrial IK(ACh). The reduction in IK1 by 2 μM Ba2+ is paralleled by a reversible increase in IK(ACh) by about 100%. These data demonstrate that the increase in K+ conductance underlying ventricular IK(ACh) is largely underestimated, suggesting that it might be of greater physiological relevance than previously thought. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Apart from gating by interaction with βγ subunits from heterotrimeric G proteins upon stimulation of appropriate receptors, Kir.3 channels have been shown to be gated by intracellular Na+. However, no information is available on how Na+-dependent gating affects endogenous Kir3.1/Kir3.4 channels in mammalian atrial myocytes. We therefore studied how loading of adult atrial myocytes from rat hearts via the patch pipette filling solution with different concentrations of Na+ ([Na+]pip) affects Kir3 current. Surprisingly, in a range between 0 and 60 mm, Na+ neither had an effect on basal inward-rectifier current nor on the current activated by acetylcholine. Overexpression of Kir3.4 in adult atrial myocytes forced by adenoviral gene transfer results in formation of functional homomeric channels that interact with βγ subunits upon activation of endogenous muscarinic receptors. These channels are activated at [Na+]pip≥ 15 mm, resulting in a receptor-independent basal inward rectifier current ( Ibir). Ibir was neither affected by pertussis toxin nor by GDP-β-S, suggesting G-protein-independent activation. PIP2 depletion via endogenous PLC-coupled α1 adrenergic receptors causes inhibition of endogenous Kir3.1/3.4 channel currents by about 75%. In contrast, inhibition of Na+-activated Ibir amounts to < 20%. The effect of the Kir3 channel blocker tertiapin-Q can be described using an IC 50 of 12 nm (endogenous IK(ACh)) and 0.61 nm ( Ibir). These data clearly identify Ibir as a homotetrameric Kir3.4 channel current with novel properties of regulation and pharmacology. Ibir shares some properties with a basal current recently described in atrial myocytes from an animal model of atrial fibrillation (AF) and AF patients. [ABSTRACT FROM AUTHOR]

In this study we have investigated the voltage dependence of ATP-dependent K+ current (IK(ATP)) in atrial and ventricular myocytes from hearts of adult rats and in CHO cells expressing Kir6.2 and SUR2A. The current–voltage relation of 2,4-dinitrophenole (DNP) -inducedIK(ATP) in atrial myocytes and expressed current in CHO cells was linear in a voltage range between 0 and−100 mV. In ventricular myocytes, the background current–voltage relation of which is dominated by a large constitutive inward rectifier (IK1), the slope conductance ofIK(ATP) was reduced at membrane potentials negative toEK (around−50 mV), resulting in an outwardly rectifyingI–Vrelation. Overexpression of Kir2.1 by adenoviral gene transfer, a subunit contributing toIK1 channels, in atrial myocytes resulted in a largeIK1-like background current. TheI–Vrelation ofIK(ATP) in these cells showed a reduced slope conductance negative toEK similar to ventricular myocytes. In atrial myocytes with an increased background inward-rectifier current through Kir3.1/Kir3.4 channels (IK(ACh)), irreversibly activated by intracellular loading with GTP-γ-S, theI–Vrelation ofIK(ATP) showed a reduced slope negative toEK, as in ventricular myocytes and atrial myocytes overexpressing Kir2.1. It is concluded that the voltage dependencies of membrane currents are not only dependent on the molecular composition of the charge-carrying channel complexes but can be affected by the activity of other ion channel species. We suggest that the interference between inwardIK(ATP) and other inward rectifier currents in cardiac myocytes reflects steady-state changes in K+ driving force due to inward K+ current. [ABSTRACT FROM AUTHOR]

We have investigated the acute desensitization of acetylcholine-activated GIRK current (IK(ACh)) in cultured adult rat atrial myocytes. Acute desensitization ofIK(ACh) is observed as a partial relaxation of current with a half-time of<5 s when muscarinic M2 receptors are stimulated by a high concentration (>2μmol l−1) of ACh. Under this condition experimental manoeuvres that cause a decrease in the amplitude ofIK(ACh), such as partial block of M2 receptors by atropine, intracellular loading with GDP-β-S, or exposure to Ba2+, caused a reduction in desensitization. Acute desensitization was also identified as a decrease in current amplitude and a blunting of the response to saturating[ACh] (20μmol l−1) when the current had been partially activated by a low concentration of ACh or by stimulation of adenosine A1 receptors. A reduction in current analogous to acute desensitization was observed when ATP-dependent K+ current (IK(ATP)) was activated either by mitochondrial uncoupling using 2,4-dinitrophenole (DNP) or by the channel opener rilmakalim. Adenovirus-driven overexpression of Kir2.1, a subunit of constitutively active inwardly rectifying K+ channels, resulted in a large Ba2+-sensitive background K+ current and a dramatic reduction of ACh-activated current. Adenovirus-driven overexpression of GIRK4 (Kir3.4) subunits resulted in an increased agonist-independent GIRK current paralleled by a reduction inIK(ACh) and removal of the desensitizing component. These data indicate that acute desensitization depends on K+ current flow, independent of the K+ channel species, suggesting that it reflects a reduction in electrochemical driving force rather than abona fidesignalling mechanism. This is supported by the observation that desensitization is paralleled by a significant negative shift in reversal potential ofIK(ACh). Since the ACh-induced hyperpolarization shows comparable desensitization properties asIK(ACh), this novel current-dependent desensitization is a physiologically relevant process, shaping the time course of parasympathetic bradycardia. [ABSTRACT FROM AUTHOR]

In this study we have investigated the voltage dependence of ATP-dependent K+ current (IK(ATP)) in atrial and ventricular myocytes from hearts of adult rats and in CHO cells expressing Kir6.2 and SUR2A. The current–voltage relation of 2,4-dinitrophenole (DNP) -inducedIK(ATP) in atrial myocytes and expressed current in CHO cells was linear in a voltage range between 0 and−100 mV. In ventricular myocytes, the background current–voltage relation of which is dominated by a large constitutive inward rectifier (IK1), the slope conductance ofIK(ATP) was reduced at membrane potentials negative toEK (around−50 mV), resulting in an outwardly rectifyingI–Vrelation. Overexpression of Kir2.1 by adenoviral gene transfer, a subunit contributing toIK1 channels, in atrial myocytes resulted in a largeIK1-like background current. TheI–Vrelation ofIK(ATP) in these cells showed a reduced slope conductance negative toEK similar to ventricular myocytes. In atrial myocytes with an increased background inward-rectifier current through Kir3.1/Kir3.4 channels (IK(ACh)), irreversibly activated by intracellular loading with GTP-γ-S, theI–Vrelation ofIK(ATP) showed a reduced slope negative toEK, as in ventricular myocytes and atrial myocytes overexpressing Kir2.1. It is concluded that the voltage dependencies of membrane currents are not only dependent on the molecular composition of the charge-carrying channel complexes but can be affected by the activity of other ion channel species. We suggest that the interference between inwardIK(ATP) and other inward rectifier currents in cardiac myocytes reflects steady-state changes in K+ driving force due to inward K+ current. [ABSTRACT FROM AUTHOR]