TASK-1 and TASK-3 may form heterodimers in human atrial cardiomyocytes.

TASK-1 channels have emerged as promising drug targets against atrial fibrillation, the most common arrhythmia in the elderly. While TASK-3, the closest relative of TASK-1, was previously not described in cardiac tissue, we found a very prominent expression of TASK-3 in right human auricles. Immunocytochemistry experiments of human right auricular cardiomyocytes showed that TASK-3 is primarily localized at the plasma membrane. Single-channel recordings of right human auricles in the cell-attached mode, using divalent-cation-free solutions, revealed a TASK-1-like channel with a single-channel conductance of about 30pS. While homomeric TASK-3 channels were not found, we observed an intermediate single-channel conductance of about 55pS, possibly reflecting the heteromeric channel formed by TASK-1 and TASK-3. Subsequent experiments with TASK-1/TASK-3 tandem channels or with co-expressed TASK-1 and TASK-3 channels in HEK293 cells or Xenopus oocytes, supported that the 55pS channels observed in right auricles have electrophysiological characteristics of TASK-1/TASK-3 heteromers. In addition, co-expression experiments and single-channel recordings suggest that heteromeric TASK-1/TASK-3 channels have a predominant surface expression and a reduced affinity for TASK-1 blockers. In summary, the evidence for heteromeric TASK-1/TASK-3 channel complexes together with an altered pharmacologic response to TASK-1 blockers in vitro is likely to have further impact for studies isolating ITASK-1 from cardiomyocytes and for the development of drugs specifically targeting TASK-1 in atrial fibrillation treatment.

Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1.

The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion.

A splice variant of the two-pore domain potassium channel TREK-1 with only one pore domain reduces the surface expression of full-length TREK-1 channels.

We have identified a novel splice variant of the human and rat two-pore domain potassium (K2P) channel TREK-1. The splice variant TREK-1e results from skipping of exon 5, which causes a frame shift in exon 6. The frame shift produces a novel C-terminal amino acid sequence and a premature termination of translation, which leads to a loss of transmembrane domains M3 and M4 and of the second pore domain. RT-PCR experiments revealed a preferential expression of TREK-1e in kidney, adrenal gland, and amygdala. TREK-1e was nonfunctional when expressed in Xenopus oocytes. However, both the surface expression and the current density of full-length TREK-1 were reduced by co-expression of TREK-1e. Live cell imaging in COS-7 cells transfected with GFP-tagged TREK-1e showed that this splice variant was retained in the endoplasmic reticulum (ER). Attachment of the C-terminus of TREK-1e to two different reporter proteins (Kir2.1 and CD8) led to a strong reduction in the surface expression of these fusion proteins. Progressive truncation of the C-terminus of TREK-1e in these reporter constructs revealed a critical region (amino acids 198 to 205) responsible for the intracellular retention. Mutagenesis experiments indicated that amino acids I204 and W205 are key residues mediating the ER retention of TREK-1e. Our results suggest that the TREK-1e splice variant may interfere with the vesicular traffic of full-length TREK-1 channels from the ER to the plasma membrane. Thus, TREK-1e might modulate the copy number of functional TREK-1 channels at the cell surface, providing a novel mechanism for fine tuning of TREK-1 currents.

The HCN4 channel mutation D553N associated with bradycardia has a C-linker mediated gating defect.

BACKGROUND/AIMS: The D553N mutation located in the C-linker of the cardiac pacemaker channel HCN4 is thought to cause sino-atrial dysfunction via a pronounced dominant-negative trafficking defect. Since HCN4 mutations usually have a minor defect in channel gating, it was our aim to further characterize the disease causing mechanism of D553N. METHODS: Fluorescence microscopy, FACS, TEVC and patch-clamp recordings were performed to characterize D553N. RESULTS: Surprisingly, we found that D553N channels reach the plasma membrane and have no apparent trafficking defect. Co-expression of D553N with HCN4 also revealed no dominant-negative effect on wild-type channels. Consistent with the normal cell surface expression of D553N, it was possible to extensively characterize D553N mutants in Xenopus oocytes and mammalian cells. D553N channels generate currents with reduced amplitude, while the kinetics of activation and deactivation are not altered. While the regulation of D553N by tyrosine kinases is normal, we observed a change in the cAMP regulation which however cannot account for the strong loss-of-function of the mutant. CONCLUSION: The pronounced current reduction and the regular surface expression indicate a major gating defect of the C-linker gate. We hypothesize that the D553N mutation stabilizes a previously reported salt bridge important for the gating of the channel.

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. 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.

TASK-1 channels may modulate action potential duration of human atrial cardiomyocytes.

BACKGROUND/AIMS: Atrial fibrillation is the most common arrhythmia in the elderly, and potassium channels with atrium-specific expression have been discussed as targets to treat atrial fibrillation. Our aim was to characterize TASK-1 channels in human heart and to functionally describe the role of the atrial whole cell current I(TASK-1). METHODS AND RESULTS: Using quantitative PCR, we show that TASK-1 is predominantly expressed in the atria, auricles and atrio-ventricular node of the human heart. Single channel recordings show the functional expression of TASK-1 in right human auricles. In addition, we describe for the first time the whole cell current carried by TASK-1 channels (I(TASK-1)) in human atrial tissue. We show that I(TASK-1) contributes to the sustained outward current I(Ksus) and that I(TASK-1) is a major component of the background conductance in human atrial cardiomyocytes. Using patch clamp recordings and mathematical modeling of action potentials, we demonstrate that modulation of I(TASK-1) can alter human atrial action potential duration. CONCLUSION: Due to the lack of ventricular expression and the ability to alter human atrial action potential duration, TASK-1 might be a drug target for the treatment of atrial fibrillation.

Knock-out of the potassium channel TASK-1 leads to a prolonged QT interval and a disturbed QRS complex.

BACKGROUND/AIMS: The aim of the study was to characterize the whole cell current of the two-pore domain potassium channel TASK-1 (K2P3) in mouse ventricular cardiomyocytes (I(TASK-1)) and to analyze the cardiac phenotype of the TASK-1(-/-) mice. METHODS AND RESULTS: We have quantified the ventricular I(TASK-1) current using the blocker A293 and TASK-1(-/-) mice. Surface electrocardiogram recordings of TASK-1(-/-) mice showed a prolonged QTc interval and a broadened QRS complex. The differences in electrocardiograms between wild type and TASK-1(-/-) mice disappeared during sympathetic stimulation of the animals. Quantitative RT-PCR, patch clamp recordings and measurements of hemodynamic performance of TASK-1(-/-) mice revealed no major compensatory changes in ion channel transcription. Action potential recordings of TASK-1(-/-) mouse cardiomyocytes indicated that I(TASK-1) modulates action potential duration. Our in vivo electrophysiological studies showed that isoflurane, which activates TASK-1, slowed heart rate and atrioventricular conduction of wild-type but not of TASK-1(-/-) mice. CONCLUSION: The results of an invasive electrophysiological catheter protocol in combination with the observed QRS time prolongation in the surface electrocardiogram point towards a regulatory role of TASK-1 in the cardiac conduction system.

Calcium-activated K(+) channel (K(Ca)3.1) activity during Ca(2+) store depletion and store-operated Ca(2+) entry in human macrophages.

STIM1 'senses' decreases in endoplasmic reticular (ER) luminal Ca(2+) and induces store-operated Ca(2+) (SOC) entry through plasma membrane Orai channels. The Ca(2+)/calmodulin-activated K(+) channel K(Ca)3.1 (previously known as SK4) has been implicated as an 'amplifier' of the Ca(2+)-release activated Ca(2+) (CRAC) current, especially in T lymphocytes. We have previously shown that human macrophages express K(Ca)3.1, and here we used the whole-cell patch-clamp technique to investigate the activity of these channels during Ca(2+) store depletion and store-operated Ca(2+) influx. Using RT-PCR, we found that macrophages express the elementary CRAC channel components Orai1 and STIM1, as well as Orai2, Orai3 and STIM2, but not the putatively STIM1-activated channels TRPC1, TRPC3-7 or TRPV6. In whole-cell configuration, a robust Ca(2+)-induced outwardly rectifying K(+) current inhibited by clotrimazole and augmented by DC-EBIO could be detected, consistent with K(Ca)3.1 channel current (also known as intermediate-conductance IK1). Introduction of extracellular Ca(2+) following Ca(2+) store depletion via P2Y(2) receptors induced a robust charybdotoxin (CTX)- and 2-APB-sensitive outward K(+) current and hyperpolarization. We also found that SOC entry induced by thapsigargin treatment induced CTX-sensitive K(+) current in HEK293 cells transiently expressing K(Ca)3.1. Our data suggest that SOC and K(Ca)3.1 channels are tightly coupled, such that a small Ca(2+) influx current induces a much large K(Ca)3.1 channel current and hyperpolarization, providing the necessary electrochemical driving force for prolonged Ca(2+) signaling and store repletion.

Intracellular traffic of the K+ channels TASK-1 and TASK-3: role of N- and C-terminal sorting signals and interaction with 14-3-3 proteins.

The two-pore-domain potassium channels TASK-1 (KCNK3) and TASK-3 (KCNK9) modulate the electrical activity of neurons and many other cell types. We expressed TASK-1, TASK-3 and related reporter constructs in Xenopus oocytes, mammalian cell lines and various yeast strains to study the mechanisms controlling their transport to the surface membrane and the role of 14-3-3 proteins. We measured potassium currents with the voltage-clamp technique and fused N- and C-terminal fragments of the channels to various reporter proteins to study changes in subcellular localisation and surface expression. Mutational analysis showed that binding of 14-3-3 proteins to the extreme C-terminus of TASK-1 and TASK-3 masks a tri-basic motif, KRR, which differs in several important aspects from canonical arginine-based (RxR) or lysine-based (KKxx) retention signals. Pulldown experiments with GST fusion proteins showed that the KRR motif in the C-terminus of TASK-3 channels was able to bind to COPI coatomer. Disabling the binding of 14-3-3, which exposes the KRR motif, caused localisation of the GFP-tagged channel protein mainly to the Golgi complex. TASK-1 and TASK-3 also possess a di-basic N-terminal retention signal, KR, whose function was found to be independent of the binding of 14-3-3. Suppression of channel surface expression with dominant-negative channel mutants revealed that interaction with 14-3-3 has no significant effect on the dimeric assembly of the channels. Our results give a comprehensive description of the mechanisms by which 14-3-3 proteins, together with N- and C-terminal sorting signals, control the intracellular traffic of TASK-1 and TASK-3.

Impaired interaction between the slide helix and the C-terminus of Kir2.1: a novel mechanism of Andersen syndrome.

OBJECTIVE: Andersen syndrome (AS) is a rare genetic disease caused by mutations of the potassium channel Kir2.1 (KCNJ2). We identified two unrelated patients with mutations in the slide helix of Kir2.1 leading to AS. The functional consequences of these two mutations, Y68D and D78Y, were studied and compared with previously reported slide helix mutations. METHODS: Channel function and surface expression were studied by voltage clamp recordings and a chemiluminescence assay in Xenopus laevis oocytes and by patch clamp recordings and fluorescence microscopy in HEK293 cells. In addition, a phosphatidylinositol bisphosphate (PIP(2)) binding assay and a yeast-two-hybrid assay were used to characterize the molecular mechanisms by which slide helix mutations cause AS. RESULTS: Neither mutant channel produced any current, but both had dominant negative effects on Kir2.2, Kir2.3, and Kir2.4 channels. We show that Y68D, D78Y, and previously reported AS mutations are clustered on the hydrophilic, cytosolic side of the slide helix and traffic normally to the plasma membrane. The in vitro lipid binding assay indicated that Y68D or D78Y N-terminal peptides bind PIP(2) similar to wild-type peptides. Yeast-two-hybrid assays showed that AS-associated mutations disturb the interaction between the slide helix and the C-terminal domain of the channel protein. CONCLUSION: Our experiments indicate a new disease-causing mechanism independent of trafficking and PIP(2) binding defects. Our findings suggest that the hydrophilic side of the slide helix interacts with a specific domain of the C-terminus facing the membrane. This interaction, which may be required for normal gating both in homomeric and heteromeric Kir2 channels, is disturbed by several mutations causing AS.

A di-acidic sequence motif enhances the surface expression of the potassium channel TASK-3.

We have characterized a sequence motif, EDE, in the proximal C-terminus of the acid-sensitive potassium channel TASK-3. Human TASK-3 channels were expressed in Xenopus oocytes, and the density of the channels at the surface membrane was studied with two complementary techniques: a luminometric surface expression assay of hemagglutinin epitope-tagged TASK-3 channels and voltage-clamp measurements of the acid-sensitive potassium current. Both approaches showed that mutation of the two glutamate residues of the EDE motif to alanine (ADA mutant) markedly reduced the transport of TASK-3 channels to the cell surface. Mutation of the central aspartate of the EDE motif had no effect on surface expression. The functional role of the EDE motif was further characterized in chimaeric constructs consisting of truncated Kir2.1 channels to which the C-terminus of TASK-3 was attached. In these constructs, too, replacement of the EDE motif by ADA strongly reduced surface expression. Live-cell imaging of enhanced green fluorescent protein-tagged channels expressed in COS-7 cells showed that 24 h after transfection wild-type TASK-3 was mainly localized to the cell surface whereas the ADA mutant was largely retained in the endoplasmic reticulum (ER). Mutation of a second di-acidic motif in the C-terminus of TASK-3 (DAE) had no effect on surface expression. Coexpression of TASK-3 with a GTP-restricted mutant of the coat recruitment GTPase Sar1 (Sar1H79G) resulted in ER retention of the channel. Our data suggest that the di-acidic motif, EDE, in human TASK-3 is a major determinant of the rate of ER export and is required for efficient surface expression of the channel.

Knock-out mice reveal the contributions of P2Y and P2X receptors to nucleotide-induced Ca2+ signaling in macrophages.

Immune cell function is modulated by changes in extracellular nucleotide levels. Here we used reverse transcription-PCR analyses, single cell Ca2+ imaging, and knock-out mice to define the receptors mediating nucleotide-induced Ca2+ signaling in resident peritoneal macrophages. In Ca2+-free buffer, the potent (K0.5<1 microm) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y2/P2Y4 double knock-out mice. Moreover, P2Y4(0/-), but not P2Y2-/-, macrophages responded to UTP. In P2Y2-/- macrophages, we could elicit Ca2+ responses to "pure" P2X receptor activation by applying ATP in buffer containing Ca2+. Purified UDP and ADP were ineffective agonists, although modest UDP-induced Ca2+ responses could be elicited in macrophages after "activation" with lipopolysaccharide and interferon-gamma. Notably, in Ca2+-free buffer, UTP-induced Ca2+ transients decayed within 1 min, and there was no response to repeated agonist challenge. Measurements of ER [Ca2+] with mag-fluo-4 showed that ER Ca2+ stores were depleted under these conditions. When extracellular Ca2+ was available, ER Ca2+ stores refilled, but Ca2+ increased to only approximately 40% of the initial value upon repeated UTP challenge. This apparent receptor desensitization persisted in GRK2+/- and GRK6-/- macrophages and after inhibition of candidate kinases protein kinase C and calmodulin-dependent kinase II. Initial challenge with UTP also reduced Ca2+ mobilization by complement component C5a (and vice versa). In conclusion, homologous receptor desensitization is not the major mechanism that rapidly dampens Ca2+ signaling mediated by P2Y2, the sole Gq-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y6 receptor expression) increases following macrophage activation.

The retention factor p11 confers an endoplasmic reticulum-localization signal to the potassium channel TASK-1.

The interaction of the adaptor protein p11, also denoted S100A10, with the C-terminus of the two-pore-domain K+ channel TASK-1 was studied using yeast two-hybrid analysis, glutathione S-transferase pull-down, and co-immunoprecipitation. We found that p11 interacts with a 40 amino-acid region in the proximal C-terminus of the channel. In heterologous expression systems, deletion of the p11-interacting domain enhanced surface expression of TASK-1. Attachment of the p11-interacting domain to the cytosolic tail of the reporter protein CD8 caused retention/retrieval of the construct in the endoplasmic reticulum (ER). Attachment of the last 36 amino acids of p11 to CD8 also caused ER localization, which was abolished by removal or mutation of a putative retention motif (H/K)xKxxx, at the C-terminal end of p11. Imaging of EGFP-tagged TASK-1 channels in COS cells suggested that wild-type TASK-1 was largely retained in the ER. Knockdown of p11 with siRNA enhanced trafficking of TASK-1 to the surface membrane. Our results suggest that binding of p11 to TASK-1 retards the surface expression of the channel, most likely by virtue of a di-lysine retention signal at the C-terminus of p11. Thus, the cytosolic protein p11 may represent a 'retention factor' that causes localization of the channel to the ER.

The stretch-activated potassium channel TREK-1 in rat cardiac ventricular muscle.

OBJECTIVE: The biophysical properties and the regulation of the two-pore-domain potassium channel TREK-1 were studied in rat cardiomyocytes. METHODS: RT-PCR, immunohistochemistry and patch-clamp recording were performed in isolated rat ventricular cardiomyocytes. In some whole-cell-clamp experiments the myocytes were mechanically stretched using a glass stylus. RESULTS: We found strong expression of a splice variant of TREK-1 in rat heart. Immunohistochemistry with antibodies against TREK-1 showed localization of the channel in longitudinal stripes at the external surface membrane of cardiomyocytes. When the cardiomyocytes were mechanically stretched, an outwardly rectifying K+ current component could be detected in whole-cell recordings. In single-channel recordings with symmetrical high K+ solution, two TREK-like channels with 'flickery-burst' kinetics were found: a 'large conductance' K+ channel (132+/-5 pS at positive potentials) and a novel 'low-conductance' channel (41+/-5 pS at positive potentials). The low-conductance channel could be activated by negative pressure in inside-out patches, positive pressure in outside-out patches, intracellular acidification and application of arachidonic acid. Its open probability was strongly increased by depolarization, due to decreased duration of gaps between bursts. The biophysical properties of the two cardiac TREK-like channels were similar to those of TREK-1 channels expressed in HEK293 cells, which both displayed low- and high-conductance modes. CONCLUSIONS: Our results suggest that the two TREK-like channels found in rat cardiomyocytes may reflect two different operating modes of TREK-1. The novel low-conductance channels described here may represent the major operating mode of TREK-1. The current flowing through mechanogated TREK-1 channels may serve to counterbalance the inward current flowing through stretch-activated non-selective cation channels during the filling phase of the cardiac cycle and thus to prevent the occurrence of ventricular extrasystoles.

Cytotoxicity of the novel anti-cancer drug rViscumin depends on HER-2 levels in SKOV-3 cells.

rViscumin is a recombinant mistletoe lectin under clinical investigation as new anti-cancer drug. The relationship between oncogene, e.g., HER-2/neu (c-erbB2) receptor activation and tumor cell chemosensitivity, is of considerable importance to better predict the response to chemotherapy. Here, we analyze the cellular and molecular effects of HER-2 expression on rViscumin chemotoxicity in SKOV-3 cells. We show that selective depletion of HER-2 by ribozyme-targeting markedly decreases cellular sensitivity towards rViscumin. These findings are confirmed by treatment with the well-established inhibitory HER-2 antibody trastuzumab (Herceptin). Using clonal ribozyme-transfected cell lines, we establish a 'HER-2 gene dose' dependence of rViscumin cytotoxicity, which is due to differential induction of apoptosis and is not mediated by cell cycle alterations or altered cellular rViscumin binding/internalization. We further demonstrate an rViscumin-mediated, HER-2-dependent down-regulation of bcl-2 and the dose-dependent activation of members of the MAPK family, p42/44, SAPK/JNK, and p38, but not of caspases-3 and -7.