Search

Presents information on a study conducted to evaluate the contribution of cholinergic and adrenergic stimuli to acid secretion in gas gland cells. Identification of second messenger systems involved in the control of acid release; Growth of cells as monolayer with typical epithelial appearance on collagen matrix; Methods and materials used in study; Results of study.

As a major cause of aldosterone producing adenomas, numerous gain-of-function mutations in the KCNJ5 gene (encoding the K + channel subunit GIRK4) have been identified. The human adrenocortical carcinoma cell line NCI-H295R is the most frequently used cellular model for in vitro studies related to regulation of aldosterone-synthesis. Because of the undefined role of KCNJ5 (GIRK4) in regulating synthesis of aldosterone, we aimed at identifying basal and G protein-activated GIRK4 currents in this paradigmatic cell line. The GIRK-specific blocker Tertiapin-Q did not affect basal current. Neither loading of the cells with GTP-γ-S via the patch-clamp pipette nor agonist stimulation of an infected A 1 -adenosine receptor resulted in activation of GIRK current. In cells co-infected with KCNJ5, robust activation of basal and adenosine-activated inward-rectifying current was observed. Although GIRK4 protein can be detected in Western blots of H295R homogenates, we suggest that GIRK4 in aldosterone-producing cells does not form functional G βγ -activated channels. [ABSTRACT FROM AUTHOR]

GIRK (G protein-activated inward-rectifying K+ channel) channels, important regulators of membrane excitability in the heart and in the central nervous, are activated by interaction with βγ subunits from heterotrimeric G proteins upon receptor stimulation. For activation interaction of the channel with phosphatidylinositol 4,5-bisphosphate (PtIns(4,5)P2) is conditional. Previous studies have provided evidence that in myocytes PtIns(4,5)P2 levels relevant to GIRK channel regulation are under regulatory control of receptors activating phospholipase C. In the present study a phosphatidyl-4-phosphate 5-kinase was expressed in atrial myocytes by transient transfection. This did not affect basal properties of GIRK current activated by acetylcholine via M2 receptors but completely abolished inhibition of guanosine triphosphate-γ-S activated current by endothelin-1 or α-adrenergic agonists. We conclude that though PtIns(4,5)P2 is conditional for channel gating, its normal level in the membrane is not limiting basal function of GIRK channels. Moreover, our data provide further evidence for a regulation of GIRK channels by α1A receptors and endothelin-A receptors, endogenously expressed in atrial myocytes, via depletion of PtIns(4,5)P2. [Copyright &y& Elsevier]

Ca2+-sensing receptors (CaSRs) belong to the class C of G protein-coupled receptors and are activated by extracellular Ca2+. CaSRs display biased G protein signaling by coupling to different classes of heterotrimeric G proteins depending on agonist and cell type. In this study we used fluorescent biosensors to directly analyze G protein coupling to CaSRs and downstream signaling in living cells. In HEK 293 cells, CaSRs displayed biased signaling: elevation of extracellular Ca2+ or application of the alternative agonist spermine caused activation of G i - and G q -proteins. Adult cardiac myocytes express endogenous CaSRs, which have been implicated in regulating Ca2+ signaling and contractility. Biased signaling of CaSRs has not been investigated in these cells. To evaluate efficiencies of G i - and G q -signaling via CaSRs in rat atrial myocytes, we measured G protein-activated K+ (GIRK) channels. Activation of GIRK requires binding of Gβγ subunits released from G i proteins, whereas G q -signaling results in inhibition of GIRK channel activity. Stimulation of CaSRs by Ca2+ or spermine failed to directly activate G i and GIRK channels. When GIRK channels were pre-activated via endogenous M 2 receptors, stimulation of CaSRs caused pronounced inhibition of GIRK currents. This effect was specific to CaSR activation: GIRK current inhibition was sensitive to NPS-2143, a negative allosteric modulator of CaSRs, and abrogated by FR900359, a direct inhibitor of G q. GIRK current inhibition was also sensitive to the PKC inhibitor chelerythrine, suggesting that following activation of CaSR and G q , GIRK currents are modulated by PKC phosphorylation. We conclude from this data that cardiac CaSRs do not activate G i and affect GIRK currents preferentially via the G q /PKC pathway. [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]

Ionotropic glutamate receptors are the most important excitatory receptors in the central nervous system, and their impairment can lead to multiple neuronal diseases. Here, we show that glutamate-induced currents in oocytes expressing GluA1 are increased by coexpression of the schizophrenia-associated phosphoinositide kinase PIP5K2A. This effect was due to enhanced membrane abundance and was blunted by a point mutation (N251S) in PIP5K2A. An increase in GluA1 currents was also observed upon acute injection of PI(4,5)P, the main product of PIP5K2A. By expression of wild-type and mutant PIP5K2A in human embryonic kidney cells, we were able to provide evidence of impaired kinase activity of the mutant PIP5K2A. We defined the region K813-K823 of GluA1 as critical for the PI(4,5)P effect by performing an alanine scan that suggested PI(4,5)P binding to this area. A PIP strip assay revealed PI(4,5)P binding to the C-terminal GluA1 peptide. The present observations disclose a novel mechanism in the regulation of GluA1. [ABSTRACT FROM AUTHOR]

Kv7.1 to Kv7.5 α-subunits belong to the family of voltage-gated potassium channels (Kv). Assembled with the β-subunit KCNE1, Kv7.1 conducts the slowly activating potassium current IKs, which is one of the major currents underlying repolarization of the cardiac action potential. A known regulator of Kv7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 increases the macroscopic current amplitude by stabilizing the open conformation of 7.1/KCNE1 channels. However, knowledge about the exact nature of the interaction is incomplete. The aim of this study was the identification of the amino acids responsible for the interaction between Kv7.1 and PIP2. We generated 13 charge neutralizing point mutations at the intracellular membrane border and characterized them electrophysiologically in complex with KCNE1 under the influence of diC8-PIP2. Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP2 regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP2 binding, molecular dynamics simulations of Kv7.1/KCNE1 complexes containing two PIP2 molecules in each subunit at specific sites were performed. Here, we identified a subset of nine residues participating in the interaction of PIP2 and Kv7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP2 binding. [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]

Infections with coxsackieviruses of type B (CVBs), which are known to induce severe forms of acute and chronic myocarditis, are often accompanied by ventricular arrhythmias and sudden cardiac death. The mechanisms underlying the development of virus-induced, life-threatening arrhythmias, which are phenotypically similar to those observed in patients having functionally impaired cardiac ion channels, remain, however, enigmatic. In the present study, we show, for the first time, modulating time-dependent effects of CVB3 on the cardiac ion channels KCNQ1, hERG1, and Cav1.2 in heterologous expression. Channel protein abundance in cellular plasma membrane and patterns of their subcellular distribution were altered in infected murine hearts. The antiviral compound AG7088 did not prevent these effects on channels. In silico analyses of infected human myocytes suggest pronounced alterations of electrical and calcium signaling and increased risk of arrhythmogenesis. These modifications are attenuated by the common Asian polymorphism KCNQ1 P448R, a genetic determinant preventing coxsackievirus-induced effects in vitro. This study provides a previously unknown explanation for the development of arrhythmias in enteroviral myocarditis, which will help to develop therapeutic strategies for arrhythmia treatment. [ABSTRACT FROM AUTHOR]

Abstract: Shortened action-potential duration (APD) and blunted APD rate adaptation are hallmarks of chronic atrial fibrillation (cAF). Basal and muscarinic (M)-receptor-activated inward-rectifier K+ currents (IK1 and IK,ACh, respectively) contribute to regulation of human atrial APD and are subject to cAF-dependent remodeling. Intracellular Na+ ([Na+]i) enhances IK,ACh in experimental models but the effect of [Na+]i-dependent regulation of inward-rectifier K+ currents on APD in human atrial myocytes is currently unknown. Here, we report a [Na+]i-dependent inhibition of outward IK1 in atrial myocytes from sinus rhythm (SR) or cAF patients. In contrast, IK,ACh activated by carbachol, a non-selective M-receptor agonist, increased with elevation of [Na+]i in SR. This [Na+]i-dependent IK,ACh regulation was absent in cAF. Including [Na+]i dependence of IK1 and IK,ACh in a recent computational model of the human atrial myocyte revealed that [Na+]i accumulation at fast rates inhibits IK1 and blunts physiological APD rate dependence in both groups. [Na+]i-dependent IK,ACh augmentation at fast rates increased APD rate dependence in SR, but not in cAF. These results identify impaired Na+-sensitivity of IK,ACh as one potential mechanism contributing to the blunted APD rate dependence in patients with cAF. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”. [Copyright &y& Elsevier]

Aims Apoptosis of cardiomyocytes significantly contributes to the development of post-ischaemic cardiomyopathy. Although mitochondria have been suggested to play a crucial role in this process, the precise mechanisms controlling the mitochondria-dependent apoptosis in cardiomyocytes under ischaemia/reperfusion are still poorly understood. Here we aimed to analyse the role of the soluble adenylyl cyclase (sAC). Methods and results Adult rat cardiomyocytes were subjected to simulated in vitro ischaemia (SI) consisting of glucose-free anoxia at pH 6.4. Apoptosis was detected by DNA laddering, chromatin condensation, and caspases cleavage. SI led to the translocation of sAC to the mitochondria and mitochondrial depolarization followed by cytochrome c release, caspase-9/-3 cleavage and apoptosis during simulated reperfusion (SR). Pharmacological inhibition of sAC during SI, but not during SR, significantly reduced the SI/SR-induced mitochondrial injury and apoptosis. Similarly, sAC knock-down mediated by an adenovirus coding for shRNA targeting sAC prevented the activation of the mitochondrial pathway of apoptosis. Analysis of the link between sAC and apoptosis revealed a sAC and protein kinase A-dependent Bax phosphorylation at Thr(167) and its translocation to mitochondria during SI, which subsequently caused mitochondrial oxygen radical formation followed by cytochrome c release and caspase-9 cleavage during SR. Conclusion These results suggest a key role of sAC in SI-induced mitochondrial Bax translocation and activation of the mitochondrial pathway of apoptosis in adult cardiomyocytes. [ABSTRACT FROM PUBLISHER]

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]

Background: Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P2 in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Fö rster resonance energy transfer (FRET), to monitor changes in plasma membrane. For controlled and reversible depletion of PtdIns(4,5)P2, voltagesensing phosphoinositide phosphatases (VSD) have been demonstrated as a superior tool, since they are independent of cellular signaling pathways. Combining these methods in intact cells requires multiple transfections. We used self-cleaving viral 2A-peptide sequences for adenovirus driven expression of the PH-domain of phospholipase-C&dgr;1 (PLC&dgr;1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes. Methods and Results: Expression and correct targeting of ECFP-PH-PLC&dgr;1, EYFP-PH-PLC&dgr;1, and Ci-VSP from a single tricistronic vector containing 2A-peptide sequences first was demonstrated in HEK293 cells by voltage-controlled FRET measurements and Western blotting. Adult rat cardiac myocytes expressed Ci-VSP and the two fluorescent PH-domains within 4 days after gene transfer using the vector integrated into an adenoviral construct. Activation of Ci-VSP by depolarization resulted in rapid changes in FRET ratio indicating depletion of PtdIns(4,5)P2 in the plasma membrane. This was paralleled by inhibition of endogenous G protein activated K<+ (GIRK) current. By comparing changes in FRET and current, a component of GIRK inhibition by adrenergic receptors unrelated to depletion of PtdIns(4,5)P2 was identified. Conclusions: Expression of a FRET sensor pair and Ci-VSP from a single ORF provides a useful approach to study regulation of ion channels by phosphoinositides in cell lines and transfection-resistant postmitotic cells. Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes. [ABSTRACT FROM AUTHOR]

A large conductance (~300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca2+ release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca2+ release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities. [ABSTRACT FROM AUTHOR]

G protein-activated K channels composed of Kir3 (GIRK) subunits contribute to regulation of heart rate and excitability. Opening of these channels in myocytes is increased by binding of G upon activation of muscarinic M receptors (M-R) or A adenosine receptors (A-R). It has been shown that saturating activation of A-R resulted in a smaller GIRK current than activation of M-R. Adenovirus-driven overexpression of the A-R caused an increase in current induced by adenosine (I), whereas the M-R-activated current (I) was reduced. Here, we sought to get deeper insight into the mechanism causing this negative crosstalk. GIRK current in cultured rat atrial myocytes was recorded in whole cell mode. Adenovirus-driven RNA interference targeting the M-R resulted in a reduction in I without affecting I, arguing against a competition of the two receptors for common signaling complexes. The negative effect of A-R overexpression on I was reduced by the A-R antagonist DPCPX and augmented by the agonist chloro-N6-cyclopentyladenosin (CCPA). In native myocytes incubation with either CCPA or the muscarinic agonist carbachol resulted in reduction in I and I, suggesting common pathways of A-R and M-R downregulation. In the absence of agonist, inhibition of adenosine deaminase by EHNA or exposure to AMP, less to ADP, but not ATP resulted in reduction of I and I. Our data indicate that atrial myocytes generate adenosine from extracellular AMP, which activates A-R in an autocrine fashion. Chronic activation of A-R causes parallel downregulation of both A-R and M-R. [ABSTRACT FROM AUTHOR]

Rationale - The plateau phase of the ventricular action potential is the result of balanced Ca2+ influx and K+ efflux. The action potential is terminated by repolarizing K+ currents. Under β-adrenergic stimulation, both the Ca2+-influx and the delayed rectifier K+ currents IK are stimulated to adjust the cardiac action potential duration to the enhanced heart rate and to ascertain adequate increase in net Ca2+ influx. Intracellularly, a Calsequestrin2 (CASQ2)-ryanodine receptor complex serves as the most effective Ca2+ reservoir/release system to aid the control of intracellular Ca2+ levels. Currently, it is unclear if disease-associated CASQ2 gene variants alter intracellular free Ca2+ concentrations and if cardiac ion channels are affected by it. Objective The goal of this study is to test if CASQ2 determines intracellular free Ca2+ concentrations and to identify cardiac ion channels that are affected by it. Further, we aim to study disease-associated CASQ2 gene variants in this context. Methods and Results - Here, we study the effects of the CASQ2 mutations R33Q, F189L, and D307H, located in highly conserved regions, on the functions of cardiac potassium channels in Xenopus oocytes using two electrode voltage clamp. As a result, CASQ2 wild type and CASQ2-mutants modulated hERG functions differently. Free Ca2+ measurements and molecular dynamics simulations imply alterations in Ca2+ buffer capacity paralled by changes in the dynamic behavior of the CASQ2-mutants compared to CASQ2 wild type. Conclusions - These in vitro and in silico data suggest a regulatory role of CASQ2 on cytosolic Ca2+ and hERG channels which may contribute to the etiology of CPVT. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The R145G amino acid exchange in the inhibitory subunit (cTnI) of cardiac troponin, which regulates muscle contraction, is related to familial hypertrophic cardiomyopathy. Information on its impact on contractility of adult cardiomyocytes is scarce. We studied shortening of adult rat cardiomyocytes before and during ß-adrenergic stimulation using adenovirus-driven expression of human cTnI-wild type (wt) and cTnI-R145G. Baseline sarcomere shortening was significantly decreased by cTnI-R145G expression. Upon ß-adrenergic stimulation using isoproterenol (ISO), nearly identical amplitudes of shortening were obtained with cells expressing cTnI-R145G and control cardiomyocytes (native and cTnI-wt). However, rates of shortening and relengthening were depressed in cTnI-R145G-expressing cells but were comparable to those of control cells upon addition of forskolin or ISO and ICI118,551. This indicates that cTnI-R145G expression influences the response to ß-adrenergic stimulation dependent on the receptor subtype. [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]

Parasympathetic slowing of the heart rate is predominantly mediated by acetylcholine-dependent activation of the G protein-gated potassium (K+) channel (IK,ACh). This channel is composed of 2 inward-rectifier K+ (Kir) channel subunits, Kir3.1 and Kir3.4, that display distinct functional properties. Here we show that subunit composition of IK,ACh changes during embryonic development. At early stages, IK,ACh is primarily formed by Kir3.1, while in late embryonic and adult cells, Kir3.4 is the predominant subunit. This change in subunit composition results in reduced rectification of IK,ACh, allowing for marked K+ currents over the whole physiological voltage range. As a consequence, IK,ACh is able to generate the membrane hyperpolarization that underlies the strong negative chronotropy occurring in late- but not early-stage atrial cardiomyocytes upon application of muscarinic agonists. Both strong negative chronotropy and membrane hyperpolarization can be induced in early-stage cardiomyocytes by viral overexpression of the mildly rectifying Kir3.4 subunit. Thus, a switch in subunit composition is used to adopt IK,ACh to its functional role in adult cardiomyocytes. [ABSTRACT FROM AUTHOR]

The predominant histamine receptor subtype in the supraventricular and ventricular tissue of various mammalian species is the H2 receptor (H2-R) subtype, which is known to couple to stimulatory G proteins (Gs), i.e. the major effects of this autacoid are an increase in sinus rate and in force of contraction. To investigate histamine effects in H2-R-transfected rat atrial myocytes, endogenous GIRK currents and L-type Ca2+ currents were used as functional assays. In H2-R-transfected myocytes, exposure to His resulted in a reversible augmentation of L-type Ca2+ currents, consistent with the established coupling of this receptor to the Gs–cAMP–PKA signalling pathway. Mammalian K+ channels composed of GIRK (Kir3.x) subunits are directly controlled by interaction with βγ subunits released from G proteins, which couple to seven-helix receptors. In mock-transfected atrial cardiomyocytes, activation of muscarinic K+ channels (IK(ACh)) was limited to Gi-coupled receptors (M2R, A1R). In H2-R-overexpressing cells, histamine activated IK(ACh) via Gs-derived βγ subunits since the histamine-induced current was insensitive to pertussis toxin. These data indicate that overexpression of Gs-coupled H2-R results in a loss of target specificity due to an increased agonist-induced release of Gs-derived βγ subunits. When IK(ACh) was maximally activated by GTP-γ–S, histamine induced an irreversible inhibition of the inward current in a fraction of H2-R-transfected cells. This inhibition is supposed to be mediated via a Gq/11-PLC-mediated depletion of PIP2, suggesting a partial coupling of overexpressed H2-R to Gq/11. Dual coupling of H2-Rs to Gs and Gq is demonstrated for the first time in cardiac myocytes. It represents a novel mechanism to augment positive inotropic effects by activating two different signalling pathways via one type of histamine receptor. Activation of the Gs–cAMP–PKA pathway promotes Ca2+ influx through phosphorylation of L-type Ca2+ channels. Simultaneous activation of Gq-signalling pathways might result in phosphoinositide turnover and Ca2+ release from intracellular stores, thereby augmenting H2-induced increases in [Ca2+]i. [Copyright &y& Elsevier]

A recently described reduction in sensitivity of G protein-activated inward-rectifying K+ (GIRK) channels to stimulation of muscarinic M2 receptors (M2AChR) in atrial myocytes overexpressing purinergic A1 receptors (A1AdoR) was further investigated by heterologous expression of a 5-HT1A receptor (5-HT1AR) and by reducing the expression level of endogenous M2AChR receptors using antisense. In 5-HT1AR-expressing myocytes, in line with previous studies, sizable GIRK currents could be activated by 5-HT. In these cells, the mean current density and activation rate of M2AChR-activated current were significantly reduced, supporting the notion that signalling via this receptor is negatively regulated by other G protein-coupled receptors (GPCR) coupling to the same class (Gi/o) of G proteins. To study if reducing M2AChR expression affects sensitivity of GIRK current to stimulation of A1AdoR, antisense oligodinucleotides (AsODN) against the M2AChR were used. Incubation of myocytes with M2AChR-specific AsODN resulted in a significant reduction in mean amplitude and activation rate of ACh-induced currents. This was paralleled by an increase in mean amplitude and activation rate of current activated by stimulation of A1AdoR. Plotting amplitudes of 5-HT- or Ado-induced currents from individual manipulated cells against the amplitude of ACh-induced current yielded a positive correlation between these data. Although difficult to interpret in mechanistic terms, this argues against a competition of receptors for a common pool of Gi/o. The mutual interaction between Gi/o-coupled receptors depends on manipulation of the expression level, since long-term desensitization or down regulation of M2AChR by treatment with carbachol did not affect sensitivity of GIRK current to A1AdoR stimulation, despite a substantial reduction in amplitude and activation rate of M2AChR-activated currents. These data suggest a novel crosstalk between parallel receptors converging on the same class of G proteins. [Copyright &y& Elsevier]

Potassium (K+) channels are highly selective for K+ ions but their unitary conductances are quite divergent. Although Kir6.1 and Kir6.2 are highly homologous and both form functional K+ channels with sulfonylurea receptors, their unitary conductances measured with 150 mM extracellular K+ are ~35 and 80 pS, respectively. We found that a chain of three amino acid residues N123-V124-R125 of Kir6.1 and S113-I114-H115 of Kir6.2 in the M1-H5 extracellular link and single residues M148 of Kir6.1 and V138 of Kir6.2 in the H5-M2 link accounted for the difference. By using a 3D structure model of Kir6.2, we were able to recognize two independent plausible mechanisms involved in the determination of single channel conductance of the Kir6.0 subunits: (i) steric effects at Kir6.2V138 or Kir6.1M148 in the H5-M2 link influence directly the diffusion of K+ ions; and (ii) structural constraints between Kir6.2S113 or Kir6.1N123 in the M1-H5 link and Kir6.2R136 or Kir6.1R146 near the H5 region control the conformation of the permeation pathway. These mechanisms represent a novel and possibly general aspect of the control of ion channel permeability. [ABSTRACT FROM AUTHOR]