Angiotensin receptors and α-adrenergic receptors regulate native IK and phosphorylation-deficient GIRK4 (S418A) channels through different PKC isoforms.

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 G-coupled receptors (GPCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP) and/or channel phosphorylation by protein kinase C (PKC). The GPCR-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.

Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors.

In vitro culture systems that structurally model human myogenesis and promote PAX7 myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis toward limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation.

PKC-isoform specific regulation of receptor desensitization and KCNQ1/KCNE1 K channel activity by mutant α-adrenergic receptors.

Activation of a specific protein kinase C (PKC) isoform during stimulation of G protein-coupled receptors (GPCRs) is determined by homologous receptor desensitization that controls the spatiotemporal formation of downstream G signalling molecules. Furthermore, GPCR-activated PKC isoforms specifically regulate receptor activity via a negative feedback mechanism. In the present study, we investigated the contribution of several phosphorylation sites in the α-adrenergic receptor (α-AR) for PKC and G protein coupled receptor kinase 2 (GRK2) to homologous receptor desensitization and effector modulation.

Receptor-specific regulation of atrial GIRK channel activity by different Ca-dependent PKC isoforms.

G Protein-activated K channels (GIRK) channels are inhibited by depletion of PtdIns(4,5)P(PIP), and/or channel phosphorylation by proteinkinase C (PKC). By using FRET-based biosensors, expressed in HEK293 cells or in atrial myocytes, we quantified receptor-specific G-coupled receptor (GPCR) signalling on the level of phospholipase C (PLC) activation by monitoring PIP-depletion and diacylglycerol (DAG) formation. Simultaneous voltage-clamp experiments on GIRK channel activity were performed as a functional readout for G-coupled α- and ET-receptor-induced signalling.

Biased signaling of Ca-sensing receptors in cardiac myocytes regulates GIRK channel activity.

Ca-sensing receptors (CaSRs) belong to the class C of G protein-coupled receptors and are activated by extracellular Ca. 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.

Familial Sinus Node Disease Caused by a Gain of GIRK (G-Protein Activated Inwardly Rectifying K Channel) Channel Function.

Background: Inherited forms of sinus node dysfunction (SND) clinically include bradycardia, sinus arrest, and chronotropic incompetence and may serve as disease models to understand sinus node physiology and impulse generation. Recently, a gain-of-function mutation in the G-protein gene GNB2 led to enhanced activation of the GIRK (G-protein activated inwardly rectifying K channel). Thus, human cardiac GIRK channels are important for heart rate regulation and subsequently, genes encoding their subunits Kir3.

Receptor Species-dependent Desensitization Controls KCNQ1/KCNE1 K+ Channels as Downstream Effectors of Gq Protein-coupled Receptors.

Activation of G protein-coupled receptors (GPCRs) might induce divergent cellular responses, related to receptor-specific activation of different branches of the G signaling pathway. Receptor-specific desensitization provides a mechanism of effector modulation by restricting the spatiotemporal activation of signaling components downstream of G We quantified signaling events downstream of GPCR activation with FRET-based biosensors in CHO and HEK 293 cells. KCNQ1/KCNE1 channels (I) were measured as a functional readout of receptor-specific activation.

NCI-H295R cell line as in vitro model of hyperaldosteronism lacks functional KCNJ5 (GIRK4; Kir3.4) channels.

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.

Synergistic modulation of KCNQ1/KCNE1 K(+) channels (IKs) by phosphatidylinositol 4,5-bisphosphate (PIP2) and [ATP]i.

Cardiac KCNQ1/KCNE1 channels (IKs) are dependent on the concentration of membrane phosphatidylinositol-4,5-bisphosphate (PIP2) and on cytosolic ATP by two distinct mechanisms. In this study we measured IKs and FRET between PH-PLCδ-based fluorescent PIP2 sensors in a stable KCNQ1/KCNE1 CHO cell line. Effects of activating either a muscarinic M3 receptor or the switchable phosphatase Ci-VSP on IKs were analyzed.

Early subcellular Ca2+ remodelling and increased propensity for Ca2+ alternans in left atrial myocytes from hypertensive rats.

Aims: Hypertension is a major risk factor for atrial fibrillation. We hypothesized that arterial hypertension would alter atrial myocyte calcium (Ca2+) handling and that these alterations would serve to trigger atrial tachyarrhythmias.

Methods And Results: Left atria or left atrial (LA) myocytes were isolated from spontaneously hypertensive rats (SHR) or normotensive Wistar-Kyoto (WKY) controls.

Differential effects of genetically-encoded Gβγ scavengers on receptor-activated and basal Kir3.1/Kir3.4 channel current in rat atrial myocytes.

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 (I(basal)) contributes to the background K(+) conductance, important for stabilizing resting potential. Data obtained from the Kir3 signaling pathway reconstituted in Xenopus oocytes suggest that I(basal) requires free G(βγ).

Voltage-dependent open-channel block of G protein-gated inward-rectifying K(+) (GIRK) current in rat atrial myocytes by tamoxifen.

Tamoxifen (Tmx) is a nonsteroidal selective estrogen receptor antagonist and is frequently used in the treatment and prevention of breast cancer. The compound and its metabolites have been reported to inhibit functions of different classes of membrane proteins, including various ion channels. For members of the inward-rectifying K(+) (Kir) channel family, interference of Tmx with binding of phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been suggested as the mechanism underlying such inhibition.

Altered stress stimulation of inward rectifier potassium channels in Andersen-Tawil syndrome.

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 stress-triggered periodic paralysis and ventricular arrhythmia.

A genetically encoded tool kit for manipulating and monitoring membrane phosphatidylinositol 4,5-bisphosphate in intact cells.

Background: Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P(2) in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Förster resonance energy transfer (FRET), to monitor changes in plasma membrane(.

Autocrine signaling via A(1) adenosine receptors causes downregulation of M(2) receptors in adult rat atrial myocytes in vitro.

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(βγ) upon activation of muscarinic M(2) receptors (M(2)-R) or A(1) adenosine receptors (A(1)-R). It has been shown that saturating activation of A(1)-R resulted in a smaller GIRK current than activation of M(2)-R.

Pannexin 1 constitutes the large conductance cation channel of cardiac myocytes.

A large conductance (∼300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca(2+) 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.

Adenovirus-mediated delivery of short hairpin RNA (shRNA) mediates efficient gene silencing in terminally differentiated cardiac myocytes.

RNA interference (RNAi) represents the most frequently utilized technique to analyze proteins by loss of function assays. Protein synthesis is impaired by sequence-specific degradation of mRNA, which is triggered by short (19-28 nt) silencing RNAs (siRNA). Efficient gene silencing using RNAi has been demonstrated in numerous cell lines and primary cultured cells.

G protein-activated (GIRK) current in rat ventricular myocytes is masked by constitutive inward rectifier current (I(K1)).

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 I(K1), carried by channels composed of Kir2.x subunits, which is less important in atrial myocytes (AM).

A role for RGS10 in beta-adrenergic modulation of G-protein-activated K+ (GIRK) channel current in rat atrial myocytes.

The effect of beta-adrenergic stimulation on endogenous G-protein-activated K(+) (GIRK) current has been investigated in atrial myocytes from hearts of adult rats. Beta-adrenergic stimulation (10 microm 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 (tau(d)) being increased by a factor of about 2.5.

Generation of a constitutive Na+-dependent inward-rectifier current in rat adult atrial myocytes by overexpression of Kir3.4.

Apart from gating by interaction with betagamma 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.

Gene silencing in adult rat cardiac myocytes in vitro by adenovirus-mediated RNA interference.

RNA interference (RNAi) by short double stranded RNA (siRNA) represents an efficient and frequently used tool for gene silencing to study gene function. Whereas efficient ablation of genes has been demonstrated in neonatal cardiac myocytes, thus far information on successful application of this technique in adult cardiac myocytes (ACM), a standard experimental model in cardiac physiology and pathophysiology, is sparse. Here we demonstrate efficient ablation of a transgene encoding for enhanced green fluorescent protein (EGFP) and a cell specific endogenous gene encoding for an inward-rectifier channel subunit (Kir2.