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The acid-sensitive steady-state potassium current. ( A ) Representative steady-state current– voltage relationship measured with slow voltage ramps (see Supplementary material online, Figure S1A ) at pH 8.0 (black trace) and at pH 6.0 (red trace); the difference curve is shown in green. The inset shows the averaged difference curve from 30 cardiomyocytes in the voltage range 2 30 to + 30 mV. The currents from one cell were averaged at 10 mV intervals (for example, from 2 5 to + 5 mV) and the corresponding values for all cardiomyocytes were used to calculate the mean + SEM . ( B ) The time course of the currents measured at + 30 mV during changes of extracellular pH from 8.0 to
Source publication
Aims The two-pore-domain potassium channel TASK-1 is robustly inhibited by the activation of receptors coupled to the Gαq subgroup of G-proteins, but the signal transduction pathway is still unclear. We have studied the mechanisms by which endothelin receptors inhibit the current carried by TASK-1 channels (ITASK) in cardiomyocytes.Methods and resu...
Contexts in source publication
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... current-voltage relationships of isolated cardiomyocytes were determined with slow voltage ramps (215 mV s 21 ; see Supplemen- tary material online, Figure S1). Membrane capacitance was measured with fast voltage ramps (500 mV s 21 ; see Supplementary material online, Figure S2). ...
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... To determine the maximal ampli- tude of I TASK , we measured steady-state current -voltage relationships in rat cardiomyocytes before and after a switch from pH 8.0 to 6.0 in the presence of the blocker cocktail designed to eliminate I to , I KATP , I Kr , I Ks , and I Ca (see Section 2). The change to pH 6.0 caused a reduc- tion in the outward currents in the range 250 to +40 mV ( Figure 1A). The current change was complete in 30 s ( Figure 1B), which corre- sponds to the time required for a complete exchange of the bath so- lution. ...
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... change to pH 6.0 caused a reduc- tion in the outward currents in the range 250 to +40 mV ( Figure 1A). The current change was complete in 30 s ( Figure 1B), which corre- sponds to the time required for a complete exchange of the bath so- lution. When the extracellular solution was switched back to pH 8.0, the decrease in outward current was reversed; and a small transient overshoot was observed ( Figure 1B). ...
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... current change was complete in 30 s ( Figure 1B), which corre- sponds to the time required for a complete exchange of the bath so- lution. When the extracellular solution was switched back to pH 8.0, the decrease in outward current was reversed; and a small transient overshoot was observed ( Figure 1B). The changes in extracellular pH could be repeated several times, indicating that the recording con- figuration was stable enough to record small changes in steady-state currents over 30 min. ...
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... mean current amplitude at +30 mV in the presence of the blocker cocktail was 1.12 + 0.05 pA/pF (n ¼ 30 cells). The mean TASK-1 current, defined as the current component blocked by extracellular acidification, was 0.35 + 0.03 pA/pF at +30 mV (n ¼ 30; Figure 1C). ...
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... was slightly smaller but did not differ significantly (P . 0.05) from the current change produced by acidification ( Figure 1C). ...
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... effects of ET-1 reached a steady state within 40 s but were poorly reversible ( Figure 2B). The mean current change produced by 200 nM ET-1 was 0.30 + 0.02 pA/pF (n ¼ 24; Figure 1C and inset of Figure 2A). The a 1 -adrenergic agonist methoxamine (100 mM), acting through another Gq-coupled receptor, produced a similar current change as 200 nM ET-1 at pH 8.0 ( Figure 1C). ...
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... mean current change produced by 200 nM ET-1 was 0.30 + 0.02 pA/pF (n ¼ 24; Figure 1C and inset of Figure 2A). The a 1 -adrenergic agonist methoxamine (100 mM), acting through another Gq-coupled receptor, produced a similar current change as 200 nM ET-1 at pH 8.0 ( Figure 1C). ...
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... obtain an estimate of the EC 50 of ET-1, we repeated these experiments with lower concentrations of ET-1. The current Figure S1A) at pH 8.0 (black trace) and at pH 6.0 (red trace); the difference curve is shown in green. The inset shows the averaged difference curve from 30 cardiomyocytes in the voltage range 230 to +30 mV. ...
Context 10
... control solution was buffered to a pH of 8.0 to maximize I TASK . The current amplitude was related to the cell size (pA/pF) by determining the membrane capacitance of each cardi- omyocyte (see Supplementary material online, Figure S1B). The number of cardiomyocytes from which the data were obtained is indicated in brackets. ...
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... conclusion, extracellular acidification, application of the TASK-1 blocker A293, application of ET-1, and application of methoxamine (100 mM) all inhibited a current component that displayed the charac- teristic outwardly rectifying current-voltage relationship of TASK-1. 22 The reductions in outward current measured after these interven- tions did not differ significantly ( Figure 1C). These findings suggest that application of 20, 50, or 200 nM ET-1 caused complete inhibition of I TASK in rat cardiomyocytes and that the amplitude of I TASK at +30 mV was 0.30 pA/pF at pH 8.0. ...
Citations
... Some studies suggest the involvement of canonical signaling pathways (InsP3, calcium, and protein kinase C), which state that Gαq-mediated pathways are crucial in K2P channel regulation (Chen 2006b). Further many data suggest that the activation of phospholipase C (PLC) is required for normal functioning of these channels (Czirják et al. 2001;Lindner et al. 2011;Schiekel et al. 2013). Another research revealed that PIP2 signaling pathway where breakdown and decreased steady state level of PIP2 lead to the regulation of K2P channel (Czirják et al. 2001), particularly as PIP2 containing liposomes, or its water-soluble analogs, were found to activate TASK-1 in inside-out membrane patches (Lopes 2005). ...
K2P channel is the leaky potassium channel that is critical to keep up the negative resting membrane potential for legitimate electrical conductivity of the excitable tissues. Recently, many substances and medication elements are discovered that could either straightforwardly or in a roundabout way influence the 15 distinctive K⁺ ion channels including TWIK, TREK, TASK, TALK, THIK, and TRESK. Opening and shutting of these channels or any adjustment in their conduct is thought to alter the pathophysiological condition of CNS. There is no document available till now to explain in detail about the molecular mechanism of agents acting on K2P channel. Accordingly, in this review we cover the current research and mechanism of action of these channels, we have also tried to mention the detailed effect of drugs and how the channel behavior changes by focusing on recent advances regarding activation and modulation of ion channels.
Graphic Abstract
... Most of these triggers either activate phospholipase C-β (PLC-β) via Gαq or boost cAMP production; FGF23 acts via FGFR4 to stimulate PLC-γ [1][2][3][4][5][6][7][8][9]. Mechanical stretch of cardiomyocytes opens pannexin-1 channels, allowing ATP to leak to the extracellular space, where it can activate P2Y purinoreceptors coupled to Gαq [10,11]. ...
Although well documented drug therapies are available for the management of ventricular hypertrophy (VH) and heart failure (HF), most patients nonetheless experience a downhill course, and further therapeutic measures are needed. Nutraceutical, dietary, and lifestyle measures may have particular merit in this regard, as they are currently available, relatively safe and inexpensive, and can lend themselves to primary prevention as well. A consideration of the pathogenic mechanisms underlying the VH/HF syndrome suggests that measures which control oxidative and endoplasmic reticulum (ER) stress, that support effective nitric oxide and hydrogen sulfide bioactivity, that prevent a reduction in cardiomyocyte pH, and that boost the production of protective hormones, such as fibroblast growth factor 21 (FGF21), while suppressing fibroblast growth factor 23 (FGF23) and marinobufagenin, may have utility for preventing and controlling this syndrome. Agents considered in this essay include phycocyanobilin, N-acetylcysteine, lipoic acid, ferulic acid, zinc, selenium, ubiquinol, astaxanthin, melatonin, tauroursodeoxycholic acid, berberine, citrulline, high-dose folate, cocoa flavanols, hawthorn extract, dietary nitrate, high-dose biotin, soy isoflavones, taurine, carnitine, magnesium orotate, EPA-rich fish oil, glycine, and copper. The potential advantages of whole-food plant-based diets, moderation in salt intake, avoidance of phosphate additives, and regular exercise training and sauna sessions are also discussed. There should be considerable scope for the development of functional foods and supplements which make it more convenient and affordable for patients to consume complementary combinations of the agents discussed here. Research Strategy: Key word searching of PubMed was employed to locate the research papers whose findings are cited in this essay.
... Several lines of evidence support the involvement of phospholipase C (PLC) in GPCR-mediated inhibition of TASK channel activity. Firstly, the PLC inhibitor, U73122, abrogated TASK1 and TASK3 channel inhibition by endothelin in rat cardiac myocytes and Chinese hamster ovary (CHO) cells expressing TASK1 and endothelin type A (ET A ) receptor, whereas its inactive derivative U73343, which has no action on PLC, failed to abrogate the inhibition [88]. Secondly, the exogenous expression of PLC resulted in an enhancement of the inhibition of TASK channel activity in oocytes expressing M 2 receptors and TASK1 in response to muscarinic receptor stimulation [18]. ...
TWIK-related acid-sensitive K+ (TASK) channels contribute to the resting membrane potential in various kinds of cells, such as brain neurons, smooth muscle cells, and endocrine cells. Loss-of-function mutations at multiple sites in the KCNK3 gene encoding for TASK1 channels are one of the causes of pulmonary arterial hypertension in humans, whereas a mutation at only one site is reported for TASK3 channels, resulting in a syndrome of mental retardation, hypotonia, and facial dysmorphism. TASK channels are subject to regulation by G protein-coupled receptors (GPCRs). Two mechanisms have been proposed for the GPCR-mediated inhibition of TASK channels: a change in gating and channel endocytosis. The most feasible mechanism for altered gating is diacylglycerol binding to a site in the C-terminus, which is shared by TASK1 and TASK3. The inhibition of channel function by endocytosis requires the presence of a tyrosine residue subjected to phosphorylation by the non-receptor tyrosine kinase Src and a dileucine motif in the C-terminus of TASK1. Therefore, homomeric TASK1 and heteromeric TASK1-TASK3 channels, but not homomeric TASK3, are internalized by GPCR stimulation. Tyrosine phosphorylation by Src is expected to result in a conformational change in the C-terminus, allowing for AP-2, an adaptor protein for clathrin, to bind to the dileucine motif. It is likely that a raft membrane domain is a platform where TASK1 is located and the signaling molecules protein kinase C, Pyk2, and Src are recruited in sequence in response to GPCR stimulation.
... Direct experimental support for this hypothesis was provided with the demonstration that the pathophysiological effects of platelet-activating factor on the myocardium are directly linked to inhibition of KCNK3 (TASK-1)-encoded (or closely related) K + channels in ventricular myocytes [439]. The effects of both platelet-activating factor and TASK-1 are dependent on protein kinase C [439,440]. Although it has been demonstrated that different KCNKencoded K2P subunits can coassemble to form heterodimer channels [441,442], it is presently unclear whether heterodimeric K2P subunit assembly is physiologically relevant in the myocardium. ...
The physiology of the heart is controlled by an indigenous electrical system that regulates heart rhythm and contractile activity. The timing of cardiac electrical events is critical to proper heart function, and key to this activity is duration of the excitation of tissues in ventricular chambers. The duration of these events must be critically controlled: depolarization that is too brief presents vulnerability of the heart to premature excitation and a congenital disorder – the short QT syndrome. Prolonged depolarization underlies both congenital and drug-induced long QT syndrome that can lead to arrhythmias and sudden death. The critical electrical event in determining the duration of ventricular depolarization is the timing of repolarization of ventricular muscle cells. The timing is due to a critical balance of ion channel and electrogenic exchange currents such that a balance that tips in favor of the outward flow of positive ions begins the repolarization process. The molecular determinants of these currents are ions passing through a network of ion channels. In this chapter we review the key ion channels that underlie cardiac repolarization and focus on critical potassium ion channels. Multiple potassium channels contribute to this electrical activity, and together they underlie repolarization reserve of the heart. When this reserve is compromised by drug- or mutation-induced decreases in individual ion channels, risk of arrhythmia is increased. Here we focus on cardiac ionic currents, their molecular determinants, and the roles they play in cardiac repolarization.
... In this regard, it is of interest that other regulatory pathways contributing to cell proliferation and vasoconstriction in the pulmonary arteries alter the function of TASK-1 channels in PASMCs. For example, endothelin-1, a potent vasoconstrictor that stimulates vascular remodelling, inhibits TASK-1 channels via both a protein kinase C-dependent pathway (Tang et al. 2009, Schiekel et al. 2013 and Rho kinase (Seyler et al. 2012). Similarly, treprostinil, a stable analogue of prostacyclin, acts via PKA to up-regulate TASK-1 current in human PASMCs (Olschewski et al. 2006). ...
Key points
The TASK‐1 channel gene (KCNK3) has been identified as a possible disease‐causing gene in heritable pulmonary arterial hypertension (PAH).
In the present study, we show that novel mutated TASK‐1 channels, seen in PAH patients, have a substantially reduced current compared to wild‐type TASK‐1 channels.
These mutated TASK‐1 channels are located at the plasma membrane to the same degree as wild‐type TASK‐1 channels.
ONO‐RS‐082 and alkaline pH 8.4 both activate TASK‐1 channels but do not recover current through mutant TASK‐1 channels.
We show that the guanylate cyclase activator, riociguat, a novel treatment for PAH, enhances current through TASK‐1 channels but does not recover current through mutant TASK‐1 channels.
Abstract
Pulmonary arterial hypertension (PAH) affects ∼15–50 people per million. KCNK3, the gene that encodes the two pore domain potassium channel TASK‐1 (K2P3.1), has been identified as a possible disease‐causing gene in heritable PAH. Recently, two new mutations have been identified in KCNK3 in PAH patients: G106R and L214R. The present study aimed to characterize the functional properties and regulation of wild‐type (WT) and mutated TASK‐1 channels and determine how these might contribute to PAH and its treatment. Currents through WT and mutated human TASK‐1 channels transiently expressed in tsA201 cells were measured using whole‐cell patch clamp electrophysiology. Localization of fluorescence‐tagged channels was visualized using confocal microscopy and quantified with in‐cell and on‐cell westerns. G106R or L214R mutated channels were located at the plasma membrane to the same degree as WT channels; however, their current was markedly reduced compared to WT TASK‐1 channels. Functional current through these mutated channels could not be restored using activators of WT TASK‐1 channels (pH 8.4, ONO‐RS‐082). The guanylate cyclase activator, riociguat, enhanced current through WT TASK‐1 channels; however, similar to the other activators investigated, riociguat did not have any effect on current through mutated TASK‐1 channels. Thus, novel mutations in TASK‐1 seen in PAH substantially alter the functional properties of these channels. Current through these channels could not be restored by activators of TASK‐1 channels. Riociguat enhancement of current through TASK‐1 channels could contribute to its therapeutic benefit in the treatment of PAH.
... The principal modifier of K 2p 3.1 channel functionality is considered to be external pH with the channel showing a dynamic functional response within the normal physiological range, with maximal activation at pH >7.8 but complete inhibition at pH values <6.4. [1][2][3] The pK of the channel is ~7.3 with a Hill coefficient of ~1.6, thus showing a steep sensitivity to pH across this key physiological and pathophysiological range. 1 Channel functionality of K 2p 3.1 can also be modulated by several other factors: inhibition occurs during hypoxia; in response to alpha adrenergic, 4 and endothelin-1 receptor stimulation, 5 and in the presence of the endocannabinoid anandamide (or its stable analogue methanandamide). 6,7 It also shows phosphorylation-dependent inhibition by protein kinase C, 7 and inhibition by platelet activating factor. ...
Introduction
K2p3.1, also known as TASK‐1, is a twin‐pore acid‐sensitive repolarizing K⁺ channel, responsible for a background potassium current that significantly contributes to setting the resting membrane potential of cardiac myocytes. Inhibition of IK2p3.1 alters cardiac repolarization and is pro‐arrhythmogenic. In this study, we have examined the expression of K2p3.1 and function of this channel in tissue and myocytes from across the left ventricular free wall.
Methods and Results
Using fluorescence immunocytochemistry, the expression of K2p3.1 protein in myocytes from the sub‐endocardial region was found to be twice (205 ± 13.5 %) that found in myocytes from the sub‐epicardial region of the left ventricle (100 ± 5.3 %). The left ventricular free‐wall exhibited a marked transmural gradient of K2p3.1 protein expression. Western blot analysis confirmed significantly higher K2p3.1 protein expression in sub‐endocardial tissue (156 ± 2.5 %) than sub‐epicardial tissue (100 ± 5.0 %); however, there was no difference in K2p3.1 mRNA expression. Whole‐cell patch clamp identified IK2p3.1 current density to be significantly greater in myocytes isolated from the sub‐endocardium (7.66 ± 0.53 pA/pF) compared with those from the sub‐epicardium (3.47 ± 0.74 pA/pF).
Conclusions
This is the first study to identify a transmural gradient of K2p3.1 in the left ventricle. This gradient has implications for understanding ventricular arrhythmogenesis under conditions of ischemia but also in response to other modulatory factors such as adrenergic stimulation and the presence of anesthetics which inhibit or activate this channel.
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... Activation of the receptor coupled with a PTX-insensitive G protein has been shown to inhibit TASK channel activity in various types of cells including brain neurons [4], endocrine cells [13], and cardiac myocytes [63]. The proposed inhibition mechanism involves binding to TASK channel of G protein αq subunits [7] or of diacylglycerol [77]. ...
Adrenal medullary chromaffin cells in mammals are innervated by sympathetic preganglionic nerve fibers, as are sympathetic ganglion neurons. Acetylcholine in the ganglion neurons is well established as mediating fast and slow excitatory postsynaptic potentials through nicotinic and muscarinic acetylcholine receptors (AChRs), respectively. The role of muscarinic AChRs during neuronal transmission in chromaffin cells varies among different mammals. Furthermore, the ion channel mechanisms associated with the muscarinic AChR-mediated increase in excitability of chromaffin cells are complicated and different from the excitation of ganglion neurons, which has been ascribed to the inhibition of M-type K⁺ channels. In this review, we focus on muscarinic receptor-mediated excitation in rodent and guinea pig chromaffin cells, in particular, on the role of muscarinic receptors in neuronal transmission, the muscarinic receptor subtypes involved in excitation and secretion, and the muscarinic regulation of ion channels including TWIK-related acid-sensitive K⁺ channels. Finally, we discuss prospectively the future of muscarinic receptor research in adrenal chromaffin cells.
... It has been reported that Gαq can directly bind to and inhibit TASK-1 [67]. However, a more commonly held view, currently, is that the activation of phospholipase C (PLC) is required for inhibition of the channels [68][69][70]. Originally it has been suggested that the breakdown and reduced steady state level of PIP2 was responsible for the inhibition [68], particularly as PIP2 containing liposomes, or its water soluble analogues, were found to activate TASK-1 in inside-out membrane patches [71]. TASK-1 activity, and the lipid end product diacylglycerol (DAG) of PLC enzyme mediates the effect [72]. ...
TWIK-related acid-sensitive potassium channel 1 (TASK-1 encoded by KCNK3) belongs to the family of two-pore domain potassium channels. This gene subfamily is constitutively active at physiological resting membrane potentials in excitable cells, including smooth muscle cells, and has been particularly linked to the human pulmonary circulation. TASK-1 channels are sensitive to a wide array of physiological and pharmacological mediators that affect their activity such as unsaturated fatty acids, extracellular pH, hypoxia, anaesthetics and intracellular signalling pathways. Recent studies show that modulation of TASK-1 channels, either directly or indirectly by targeting their regulatory mechanisms, has the potential to control pulmonary arterial tone in humans. Furthermore, mutations in KCNK3 have been identified as a rare cause of both familial and idiopathic pulmonary arterial hypertension. This review summarises our current state of knowledge of the functional role of TASK-1 channels in the pulmonary circulation in health and disease, with special emphasis on current advancements in the field.
... 23 Previous studies have used anandamide and A293, inhibitors of KCNK3 channel activity, to analyze the functional contributions of KCNK3 on hPASMC excitability. 8,9,38,39 We confirmed loss of native KCNK3 channel activity in cultured hPASMCs using a recently developed, more selective KCNK3 inhibitor, ML365 ( Figure 2E through 2H and S1B). 21 We exploited the properties of cultured hPASMCs to compare the relative impact of expressed mutant versus WT KCNK3 channels in a more physiological environment. ...
... More likely, ONO acts on membrane phospholipase pathways to alter KCNK3 phosphorylation. It has been shown that phospholipase C activation leads to KCNK3 inhibition, 39 and endothelin-1, a vasoconstrictor and mediator of PAH pathogenesis, inhibits KCNK3 in hPASMCs leading to depolarization, the sensitivity of which requires endothelin-A receptors, phospholipase C, phosphatidylinositol 4,5-bisphosphate, diacylglycerol, and protein kinase C. 40 Diacylglycerol was recently identified as a direct regulator of KCNK3 downstream of activated G protein-coupled receptors. 41 Ultimately, elucidating the specific pathways involved in KCNK3 activation by ONO will further cultivate KCNK3 activation as a PAH treatment paradigm. ...
Background
Heterozygous loss of function mutations in the KCNK3 gene cause hereditary pulmonary arterial hypertension (PAH). KCNK3 encodes an acid‐sensitive potassium channel, which contributes to the resting potential of human pulmonary artery smooth muscle cells. KCNK3 is widely expressed in the body, and dimerizes with other KCNK3 subunits, or the closely related, acid‐sensitive KCNK9 channel.
Methods and Results
We engineered homomeric and heterodimeric mutant and nonmutant KCNK3 channels associated with PAH. Using whole‐cell patch‐clamp electrophysiology in human pulmonary artery smooth muscle and COS7 cell lines, we determined that homomeric and heterodimeric mutant channels in heterozygous KCNK3 conditions lead to mutation‐specific severity of channel dysfunction. Both wildtype and mutant KCNK3 channels were activated by ONO‐RS‐082 (10 μmol/L), causing cell hyperpolarization. We observed robust gene expression of KCNK3 in healthy and familial PAH patient lungs, but no quantifiable expression of KCNK9, and demonstrated in functional studies that KCNK9 minimizes the impact of select KCNK3 mutations when the 2 channel subunits co‐assemble.
Conclusions
Heterozygous KCNK3 mutations in PAH lead to variable loss of channel function via distinct mechanisms. Homomeric and heterodimeric mutant KCNK3 channels represent novel therapeutic substrates in PAH. Pharmacological and pH‐dependent activation of wildtype and mutant KCNK3 channels in pulmonary artery smooth muscle cells leads to membrane hyperpolarization. Co‐assembly of KCNK3 with KCNK9 subunits may provide protection against KCNK3 loss of function in tissues where both KCNK9 and KCNK3 are expressed, contributing to the lung‐specific phenotype observed clinically in patients with PAH because of KCNK3 mutations.
... U73122 has been utilized extensively to probe PLC signalling in cells of the immune system (Heissmeyer et al., 2004), in neurons (e.g. Xiang et al., 2002) and in cardiomyocytes (Kobrinsky et al., 2000;Schiekel et al., 2013), among many other cell types. U73122 has also been used to analyse the PI(4,5)P 2 -dependence of ion channels during activation of the PLC pathway in native tissues and in heterologous expression systems (Horowitz et al., 2005;Schiekel et al., 2013;Richter et al., 2014). ...
... Xiang et al., 2002) and in cardiomyocytes (Kobrinsky et al., 2000;Schiekel et al., 2013), among many other cell types. U73122 has also been used to analyse the PI(4,5)P 2 -dependence of ion channels during activation of the PLC pathway in native tissues and in heterologous expression systems (Horowitz et al., 2005;Schiekel et al., 2013;Richter et al., 2014). ...
