TASK-3 Expression Research Articles

Downregulation of TASK-3 Channel Induces Senescence in Granulosa Cells of Bovine Cystic Ovarian Follicles.

Ovarian cysts are linked to hormone imbalances and altered gene expressions, but the connection between cysts and ion channel expression is understudied. This study explored the role of TWIK-related acid-sensitive K+ (TASK) channels in bovine ovarian cyst formation. The ovarian follicles were split into small (5 to 10 mm in diameter) and large (>25 mm in diameter) groups. Among the measured K+, Na+, and Cl- concentrations in follicular fluid (FF) obtained from small-sized follicles (SFs) and large-sized follicles (LFs), the K+ concentration was significantly lower in LFFF. Quantitative PCR, Western blot, and immunocytochemistry data revealed that TASK-3 expression levels significantly decreased by approximately 50% in LFs and granulosa cells obtained from LFs (LFGCs) compared to the corresponding controls. The TASK-3 protein was localized to the plasma membranes of GCs. The diameters of LFGCs were larger than those of SFGCs. The cell swelling response to exposure to a hypotonic solution (200 mOsm/L) was highly reduced in TASK-3-overexpressing cells compared to vector-transfected cells. TASK-3-knockdown cells showed arrested growth. Senescence markers were detected in LFGCs and TASK-3-knockdown cells. These findings suggest that reduced TASK-3 expression in LFs is associated with the inhibition of GC growth, leading to senescence and cyst formation.

Epigenetic Dysregulation of KCNK9 Imprinting and Triple-Negative Breast Cancer.

Simple SummaryGenomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. Here, we showed that KCNK9 is imprinted in breast tissue and identified the differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). The high frequency of KCNK9 DMR hypomethylation in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may provide a new target for prevention of TNBC.Genomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes for TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. However, KCNK9 gene amplification accounts for increased expression in <10% of these breast cancers. Here, we showed that KCNK9 is imprinted in breast tissue and identified a differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). KCNK9 hypomethylation was also found in non-cancerous tissue from 77% of women at high-risk of developing breast cancer. Functional studies demonstrated that the KCNK9 gene product, TASK3, regulates mitochondrial membrane potential and apoptosis-sensitivity. In TNBC cells and non-cancerous mammary epithelial cells from high-risk women, hypomethylation of the KCNK9 DMR predicts for increased TASK3 expression and mitochondrial membrane potential (p < 0.001). This is the first identification of the KCNK9 DMR in mammary epithelial cells and demonstration that its hypomethylation in breast cancer is associated with increases in both mitochondrial membrane potential and apoptosis resistance. The high frequency of hypomethylation of the KCNK9 DMR in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may serve as a marker of risk and a target for prevention of TNBC, particularly in African American women.

Synthesis and cellular effects of a mitochondria-targeted inhibitor of the two-pore potassium channel TASK-3

The two-pore potassium channel TASK-3 has been shown to localize to both the plasma membrane and the mitochondrial inner membrane. TASK-3 is highly expressed in melanoma and breast cancer cells and has been proposed to promote tumor formation. Here we investigated whether pharmacological modulation of TASK-3, and specifically of mitochondrial TASK-3 (mitoTASK-3), had any effect on cancer cell survival and mitochondrial physiology. A novel, mitochondriotropic version of the specific TASK-3 inhibitor IN-THPP has been synthesized by addition of a positively charged triphenylphosphonium moiety. While IN-THPP was unable to induce apoptosis, mitoIN-THPP decreased survival of breast cancer cells and efficiently killed melanoma lines, which we show to express mitoTASK-3. Cell death was accompanied by mitochondrial membrane depolarization and fragmentation of the mitochondrial network, suggesting a role of the channel in the maintenance of the correct function of this organelle. In accordance, cells treated with mitoIN-THPP became rapidly depleted of mitochondrial ATP which resulted in activation of the AMP-dependent kinase AMPK. Importantly, cell survival was not affected in mouse embryonic fibroblasts and the effect of mitoIN-THPP was less pronounced in human melanoma cells stably knocked down for TASK-3 expression, indicating a certain degree of selectivity of the drug both for pathological cells and for the channel. In addition, mitoIN-THPP inhibited cancer cell migration to a higher extent than IN-THPP in two melanoma cell lines. In summary, our results point to the importance of mitoTASK-3 for melanoma cell survival and migration.

A 5' UTR GGN repeat controls localisation and translation of a potassium leak channel mRNA through G-quadruplex formation.

RNA G-quadruplexes (G4s) are secondary structures proposed to function as regulators of post-transcriptional mRNA localisation and translation. G4s within some neuronal mRNAs are known to control distal localisation and local translation, contributing to distinct local proteomes that facilitate the synaptic remodelling attributed to normal cellular function. In this study, we characterise the G4 formation of a (GGN)13 repeat found within the 5′ UTR of the potassium 2-pore domain leak channel Task3 mRNA. Biophysical analyses show that this (GGN)13 repeat forms a parallel G4 in vitro exhibiting the stereotypical potassium specificity of G4s, remaining thermostable under physiological ionic conditions. Through mouse brain tissue G4-RNA immunoprecipitation, we further confirm that Task3 mRNA forms a G4 structure in vivo. The G4 is inhibitory to translation of Task3 in vitro and is overcome through activity of a G4-specific helicase DHX36, increasing K+ leak currents and membrane hyperpolarisation in HEK293 cells. Further, we observe that this G4 is fundamental to ensuring delivery of Task3 mRNA to distal primary cortical neurites. It has been shown that aberrant Task3 expression correlates with neuronal dysfunction, we therefore posit that this G4 is important in regulated local expression of Task3 leak channels that maintain K+ leak within neurons.

Withaferin A suppresses breast cancer cell proliferation by inhibition of the two-pore domain potassium (K2P9) channel TASK-3

Withaferin A (WFA), a C5,C6-epoxy steroidal lactone isolated from the medicinal plant Withania somnifera (L.) Dunal, inhibits growth of tumor cells in different cancer types. However, the mechanisms underlying the effect of WFA on tumor cells are not fully understood. In the present study, we evaluated the blockade of TASK-3 channels by WFA in TASK-3-expressing HEK-293 cells. Explore if the WFA-mediated TASK-3 blockade can be used as a pharmacological tool to decrease the cell viability in cancer cells. A combination of functional experiments (patch-clamp, gene downregulation, overexpression and pharmacological inhibition) and molecular docking analysis were used to get insights into the mechanism by which the inhibition of TASK-3 by WFA affects the growth and viability of cancer cells. Withaferin A was found to inhibit the activity of TASK-3 channels. The inhibitory effect of Withaferin A on TASK-3 potassium currents was dose-dependent and independent of voltage. Molecular modeling studies identified putative WFA-binding sites in TASK-3 channel involved the channel blockade. In agreements with the molecular modeling predictions, mutation of residues F125 to A (F125A), L197 to V (L197 V) and the double mutant F125A-L197 V markedly decreased the WFA-induced inhibition of TASK-3. Finally, the cytotoxic effect of WFA was tested in MDA-MB-231 human breast cancer cells transfected with TASK-3 or shRNA that decreases TASK-3 expression. Together, our results show that the cytotoxic effect of WFA on fully transformed MDA-MB-231 cells depends on the expression of TASK-3. Herein, we also provide insights into the mechanism of TASK-3 inhibition by WFA.

Spinal TASK-1 and TASK-3 modulate inflammatory and neuropathic pain

This study assessed the participation of spinal TWIK-related acid-sensitive K+ channels 1 and 3 (TASK-1 and TASK-3) in inflammatory (formalin test) and neuropathic (spinal nerve ligation, SNL) pain in rats. Intrathecal pre-treatment (−10 min) with the TASK-1 blocker ML365 or TASK-3 blocker PK-THPP, but not vehicle, enhanced in a dose-dependent manner 1% formalin-induced acute and long-lasting secondary mechanical allodynia and mechanical hyperalgesia in rats. In contrast, intrathecal pre-treatment with terbinafine, an activator of TASK-3, reduced formalin-induced flinching and allodynia/hyperalgesia. Both blockers and terbinafine had similar effects on female and male rats. In addition, intrathecal injection of ML365 or PK-THPP blocked the terbinafine-induced antiallodynic effect in neuropathic rats, but they did not modify baseline withdrawal threshold in naïve or sham-operated rats. TASK-1 and TASK-3 mRNA and protein were expressed in L4 and L5 dorsal root ganglia (DRG) and dorsal and ventral spinal cord of naïve animals. Interestingly, formalin injection increased TASK-1 expression in ipsilateral L5 DRG, but not in the spinal cord. Moreover, formalin injection transiently enhanced TASK-3 expression in ipsilateral L5 DRG and dorsal spinal cord. In contrast, SNL down-regulated TASK-3 expression in the ipsilateral L4 and L5 DRG but not in dorsal or ventral spinal cord, while SNL did not modify TASK-1 expression at any tissue. The pharmacological and molecular results suggest that TASK-1 and TASK-3 have a relevant antinociceptive role in inflammatory and neuropathic pain.

Silencing the KCNK9 potassium channel (TASK-3) gene disturbs mitochondrial function, causes mitochondrial depolarization, and induces apoptosis of human melanoma cells.

TASK-3 (KCNK9 or K2P9.1) channels are thought to promote proliferation and/or survival of malignantly transformed cells, most likely by increasing their hypoxia tolerance. Based on our previous results that suggested mitochondrial expression of TASK-3 channels, we hypothesized that TASK-3 channels have roles in maintaining mitochondrial activity. In the present work we studied the effect of reduced TASK-3 expression on the mitochondrial function and survival of WM35 and A2058 melanoma cells. TASK-3 knockdown cells had depolarized mitochondrial membrane potential and contained a reduced amount of mitochondrial DNA. Compared to their scrambled shRNA-transfected counterparts, they demonstrated diminished responsiveness to the application of the mitochondrial uncoupler [(3-chlorophenyl)hydrazono]malononitrile (CCCP). These observations indicate impaired mitochondrial function. Further, TASK-3 knockdown cells presented reduced viability, decreased total DNA content, altered cell morphology, and reduced surface area. In contrast to non- and scrambled shRNA-transfected melanoma cell lines, which did not present noteworthy apoptotic activity, almost 50% of the TASK-3 knockdown cells exhibited strong Annexin-V-specific immunofluorescence signal. Sequestration of cytochrome c from the mitochondria to the cytosol, increased caspase 3 activity, and translocation of the apoptosis-inducing factor from mitochondria to cell nuclei were also demonstrated in TASK-3 knockdown cells. Interference with TASK-3 channel expression, therefore, induces caspase-dependent and -independent apoptosis of melanoma cells, most likely via causing mitochondrial depolarization. Consequently, TASK-3 channels may be legitimate targets of future melanoma therapies.

Stable Cell Line of Human SH-SY5Y Uniformly Expressing TWIK-Related Acid-Sensitive Potassium Channel and eGFP Fusion

TWIK-related acid-sensitive potassium channels (TASK3) are pharmacological targets of CNS inflammation induced by acidification. They function as molecular switches between survival and death of neurons. In this report, TASK3 cloned from human brain cDNA was tagged with enhanced green fluorescent protein (eGFP), and the fusion gene was transiently expressed in human neuroblastoma SH-SY5Y cells. A cell line stably expressing TASK-eGFP fusion proteins was generated from transient expression cells by using fluorescence-activated cell sorting followed by antibiotic selection. The uniform expression of TASK3 fusion proteins was further confirmed by flow cytometry. Moreover, the localization of TASK3 tagged with eGFP was checked by confocal microcopy. TASK3-eGFP fusion proteins are observed on the SH-SY5Y cell membrane. The strategies using eGFP as a fusion tag facilitate the monitoring of the TASK3 expression and enable the successful employment of FACS for screening and construction of cell lines stably expressing TASK3. The TASK3 overexpression cell line will lay a fundamental for the in vitro evaluation of TASK3 function during hypoxic/ischemic injury.

Immunocytochemical localization of TASK-3 protein (K2P9.1) in the rat brain.

Among all K2P channels, TASK-3 shows the most widespread expression in rat brain, regulating neuronal excitability and transmitter release. Using a recently purified and characterized polyclonal monospecific antibody against TASK-3, the entire rat brain was immunocytochemically analyzed for expression of TASK-3 protein. Besides its well-known strong expression in motoneurons and monoaminergic and cholinergic neurons, TASK-3 expression was found in most neurons throughout the brain. However, it was not detected in certain neuronal populations, and neuropil staining was restricted to few areas. Also, it was absent in adult glial cells. In hypothalamic areas, TASK-3 was particularly strongly expressed in the supraoptic and suprachiasmatic nuclei, whereas other hypothalamic nuclei showed lower protein levels. Immunostaining of hippocampal CA1 and CA3 pyramidal neurons showed strongest expression, together with clear staining of CA3 mossy fibers and marked staining also in the dentate gyrus granule cells. In neocortical areas, most neurons expressed TASK-3 with a somatodendritic localization, most obvious in layer V pyramidal neurons. In the cerebellum, TASK-3 protein was found mainly in neurons and neuropil of the granular cell layer, whereas Purkinje cells were only faintly positive. Particularly weak expression was demonstrated in the forebrain. This report provides a comprehensive overview of TASK-3 protein expression in the rat brain.

Expression of background potassium channels in rat DRG is cell-specific and down-regulated in a neuropathic pain model

Neuropathic pain is associated with hyperexcitability of DRG neurons. Despite the importance of leakage potassium channels for neuronal excitability, little is known about their cell-specific expression in DRGs and possible modulation in neuropathic pain. Multiple leakage channels are expressed in DRG neurons, including TASK1, TASK3, TRESK, TRAAK, TWIK1, TREK1 and TREK2 but little is known about their distribution among different cell types. Our immunohistochemical studies show robust TWIK1 expression in large and medium size neurons, without overlap with TRPV1 or IB4 staining. TASK1 and TASK3, on the contrary, are selectively expressed in small cells; TASK1 expression closely overlaps TRPV1-positive cells, while TASK3 is expressed in TRPV1- and IB4-negative cells. We also studied mRNA expression of these channels in L4-L5 DRGs in control conditions and up to 4 weeks after spared nerve injury lesion. We found that TWIK1 expression is much higher than TASK1 and TASK3 and is strongly decreased 1, 2 and 4 weeks after neuropathic injury. TASK3 expression, on the other hand, decreases 1 week after surgery but reverts to baseline by 2weeks; TASK1 shows no significant change at any time point. These data suggest an involvement of TWIK1 in the maintenance of the pain condition.

Immunocytochemical localization of TASK-3 channels in rat motor neurons.

Motor neurons are large cholinergic neurons located in the brain stem and spinal cord. In recent years, a functional role for TASK channels in cellular excitability and vulnerability to anesthetics of motor neurons has been described. Using a polyclonal monospecific antibody against the tandem pore domain K(+) channel (K2P channel) TWIK-related acid-sensitive K(+) channel (TASK-3), we analyzed the expression of the TASK-3 protein in motor systems of the rat CNS. Immunocytochemical staining showed strong TASK-3 expression in motor neurons of the facial, trigeminal, ambiguus, and hypoglossal nuclei. Oculomotor nuclei (including trochlear and abducens nucleus) were also strongly positive for TASK-3. The parasympathetic Edinger-Westphal nucleus and dorsal vagal nucleus showed significant, but weaker expression compared with somato- and branchiomotoric neurons. In addition, motor neurons in the anterior horn of the spinal cord were also strongly labeled for TASK-3 immunoreactivity. Based on morphological criteria, TASK-3 was found in the somatodendritic compartment of motor neurons. Cellular staining using methyl green and immunofluorescence double-labeling with anti-vesicular acetylcholine transporter (anti-vAChT) indicated ubiquitous TASK-3 expression in motor neurons, whereas in other brain regions TASK-3 showed a widespread but not ubiquitous expression. In situ hybridization using a TASK-3 specific riboprobe verified the expression of TASK-3 in motor neurons at the mRNA level.

Reduction of breast cancer cell migration via up‐regulation of TASK‐3 two‐pore domain K+ channel

Many kinds of K(+) channels are expressed in a variety of cells, including cancer cells. However, only a small amount of research has explored the relationship between voltage-independent K(+) channels and breast cancer. This study was performed to investigate whether changes in two-pore domain K(+) (K(2P) ) channel expression levels are related to the migration of human breast cancer cells. K(2P) channel gene/protein expression levels were compared between MCF-7 (a non-invasive cell) and MDA-MB-231 (an invasive cell) using reverse transcriptase (RT)-polymerase chain reaction (PCR), real-time PCR, Western blotting and immunocytochemistry. The relationship between K(2P) channel expression level and cell migration was analysed using gene overexpression and knock-down techniques. Functional expression of TASK-3 in MCF-7 and MDA-MB-231 cells was recorded using patch-clamp technique. Of K(2P) channels, TASK-3 mRNA and protein were highly expressed in MCF-7 cells compared with those in MDA-MB-231 cells. Overexpression of TASK-3 in breast cancer cells reduced migration and invasion, whereas silencing of TASK-3 increased the migration and invasion. The TASK-3 expression level was decreased by phorbol myristate acetate (PMA), a PKC activator. PMA also enhanced the cell migration in MDA-MB-231 cells. These results show that an increase in TASK-3 expression levels, which could be modulated by PKC activation, reduces cell migration/invasion in breast cancer cells and suggest that modulation of TASK-3 expression may regulate metastasis of breast cancer cells.

Inhibition of TASK-3 (KCNK9) channel biosynthesis changes cell morphology and decreases both DNA content and mitochondrial function of melanoma cells maintained in cell culture

TASK-3 channel overexpression was shown to facilitate the survival of malignantly transformed cells, possibly by providing greater hypoxia tolerance through a still unknown mechanism. Although it has been suggested previously that TASK-3 channels are expressed in the mitochondrial membranes, their role here remains elusive. In this study, a transient transfection of TASK-3 knockdown melanoma cell cultures was produced to show the significance of TASK-3 expression. Reduction of the TASK-3 protein biosynthesis induced characteristic changes in cell morphology, reduced the amount of DNA and decreased metabolic activity and mitochondrial function of melanoma cells when compared with control. These findings indicate that TASK-3 channel expression and function is indispensable for the proliferation and/or survival of the melanoma cells, as they seem to contribute to their mitochondrial functions. The significance is that, in this study, we have shown that TASK-3 channels are expressed in the mitochondria of melanoma malignum cells, and they are essential for maintaining cellular integrity and viability. The TASK-3 knockdown melanoma cell line had altered morphology, reduced DNA content, decreased metabolic activity and impaired mitochondrial function. These data indicate that TASK-3 channels are functionally present in the mitochondria of the melanoma cells, and their function is essential for the survival of these cells, thus TASK-3 channels may be the possible targets of future anticancer therapy.

Immunocytochemical localization of TASK-3 (K(2P)9.1) channels in monoaminergic and cholinergic neurons.

Monoaminergic and cholinergic systems are important regulators of cortical and subcortical systems, and a variety of vegetative functions are controlled by the respective neurotransmitters. Neuronal excitability and transmitter release of these neurons are strongly regulated by their potassium conductances carried by Kir and K(2P) channels. Here we describe the generation and characterization of a polyclonal monospecific antibody against rat TASK-3, a major brain K(2P) channel. After removal of cross-reactivities and affinity purification the antibody was characterized by ELISA, immunocytochemistry of TASK-3 transfected cells, and Western blots indicating that the antibody only detects TASK-3 protein, but not its paralogs TASK-1 and TASK-5. Western blot analysis of brain membrane fractions showed a single band around 45 kD, close to the predicted molecular weight of the TASK-3 protein. In addition, specific immunolabeling using the anti-TASK-3 antibody in Western blot analysis and immunocytochemistry was blocked in a concentration dependent manner by its cognate antigen only. Immunocytochemical analysis of rat brain revealed strong expression of TASK-3 channels in serotoninergic neurons of the dorsal and median raphe, noradrenergic neurons of the locus coeruleus, histaminergic neurons of the tuberomammillary nucleus and in the cholinergic neurons of the basal nucleus of Meynert. Immunofluorescence double-labeling experiments with appropriate marker enzymes confirmed the expression of TASK-3 in cholinergic, serotoninergic, and noradrenergic neurons. In the dopaminergic system strong TASK-3 expression was found in the ventral tegmental area, whereas TASK-3 immunoreactivity in the substantia nigra compacta was only weak. All immunocytochemical results were supported by in situ hybridization using TASK-3 specific riboprobes.

TASK3 and its role in neuro and systemic oncogenesis

To the editor: I read with interest the recent article by Meuth et al. [1] about the impact of TASK3 functionality on glioma cell death. The past few years have seen a rapid increase in the understanding of TASK3 and their potential role in not just neuro-oncogenesis but systemic oncogenesis as well. For instance, over-expression of TASK3 has been reported in a number of systemic malignancies. Kim et al. [2] in a recent study of TASK3 expression in individuals with colorectal cancer reported that half of the patients with stage D cancers were immuno-positive for TASK3 while nearly 30% of those with stage A cancer were immunopositive for TASK3. Similarly, Mu et al. [3] in another recent study have reported that TASK3 is over expressed in nearly 44% of breast cancers. TASK3 may also have a role in the pathogenesis of malignant melanomas [4]. Pei et al. [5] in a recent study have even reported a point mutation G95E that decreases the oncogenic potential secondary to TASK3. Recent studies suggest that TASK3 may also be involved in the development of cerebellar neurons [6]. Holter et al. [7] in a recent study have even reported that TASK3 mutations may be involved in the evolution of absence seizures. Clearly, TASK3 has a major role to play in neuro-oncogenesis as well as in the pathogenesis of systemic cancers. Further studies are needed to identify agents that might regulate TASK3 function and thus may be used as potential anti carcinogenic agents in the management of malignancies such as gliomas.

Changes in expression of some two-pore domain potassium channel genes ( KCNK) in selected brain regions of developing mice

Two P loop domain potassium (K2P or KCNK) channels produce transmitter-modulated K + currents that could influence brain development. We mapped by in situ hybridization the expression of the K2P gene family in the developing mouse brain. All the K2P genes had different expression patterns, and it is likely that many neuronal types change their K2P channel subunit composition during development. Fitting with a possible role in the control of cell division, three K2P genes ( tandem of P domains in a weak inwardly-rectifying K + channel– related K + channel (TREK) -1, TREK-2 and weak inwardly-rectifying K + channel–related acid- sensitive K + channel (TASK) -2) had high expression in the embryonic subventricular and ventricular zones, and the tandem of P domains in a weak inwardly-rectifying K + channel (TWIK) -1, TREK-1, TREK-2 and TASK-3 genes were significantly expressed in the external cerebellar granule cell layer. There were also some clear changes in developmental expression of the K2P genes: for example, TREK-1 goes from high to low expression in post-migratory cerebellar granule cells; TREK-2 has one of the highest expressions in the embryonic and early postnatal brain of any K2P gene, but transcript levels fall strongly in the postnatal periods, except for cerebellar granule cells. TASK-1 and tandem pore domain halothane- inhibited K + channel (THIK) -2 genes both turn on specifically in post-migratory cerebellar granule cells, whereas the TASK-3 gene, for example, is strongly expressed in pre-migratory cells as well as post-migratory cells. On the other hand, young postnatal dentate granule cells express TWIK-1, TREK-1 and TREK-2 before P7, but TASK-3 expression only begins to become clear in these cells in the second postnatal week. THIK-2 mRNA was up-regulated with TASK-1 and TASK-3 transcripts in cerebella of GABA A receptor α6 subunit knockout mice, possibly implying a functional association of THIK-2, TASK-1 and TASK-3.

Mitochondrial expression of the two-pore domain TASK-3 channels in malignantly transformed and non-malignant human cells

The presence of TASK-3 channels has been described in a number of healthy and malignantly transformed cells, showing mainly intracellular distribution with relatively insignificant labelling of the cell surface membrane. In this work, immunochemical and molecular biology methods were utilised to establish the intracellular organelle whose TASK-3 expression accounts for this strong intracellular labelling using cultured melanoma and HaCaT cells. Before the immunocytochemical experiments, the presence of TASK-3 mRNA was also confirmed in melanoma cells. Comparison of the results of the TASK-3- and mitochondrion-specific labelling indicated that the TASK-3 channel subunits were strongly expressed by mitochondria in both investigated cell types. Moreover, prominent TASK-3 expression of keratinocytes could also be demonstrated in histological sections excised from the human skin. These results indicate that TASK-3 channels are present in the mitochondria in both malignantly transformed and healthy cells, suggesting that they might have roles in ensuring mitochondrial functions.

Invalidation of TASK1 potassium channels disrupts adrenal gland zonation and mineralocorticoid homeostasis

TASK1 (KCNK3) and TASK3 (KCNK9) are two-pore domain potassium channels highly expressed in adrenal glands. TASK1/TASK3 heterodimers are believed to contribute to the background conductance whose inhibition by angiotensin II stimulates aldosterone secretion. We used task1-/- mice to analyze the role of this channel in adrenal gland function. Task1-/- exhibited severe hyperaldosteronism independent of salt intake, hypokalemia, and arterial 'low-renin' hypertension. The hyperaldosteronism was fully remediable by glucocorticoids. The aldosterone phenotype was caused by an adrenocortical zonation defect. Aldosterone synthase was absent in the outer cortex normally corresponding to the zona glomerulosa, but abundant in the reticulo-fasciculata zone. The impaired mineralocorticoid homeostasis and zonation were independent of the sex in young mice, but were restricted to females in adults. Patch-clamp experiments on adrenal cells suggest that task3 and other K+ channels compensate for the task1 absence. Adrenal zonation appears as a dynamic process that even can take place in adulthood. The striking changes in the adrenocortical architecture in task1-/- mice are the first demonstration of the causative role of a potassium channel in development/differentiation.

Relationship between TWIK-related acid-sensitive K+ channel-1 and TWIK-related acid-sensitive K+ channel-3 expression and the occurrence of central sleep apnea: experiment with rats

To investigate the role of the TWIK-related acid-sensitive K(+) channel-1 (TASK-1) in the occurrence of sleep apnea. Brain and muscle electrodes were put on 27 SD rats to monitor the apneic episodes. Then the rats were killed with their brains taken out. Western blotting was used to examine the expression of TASK-1 as well as its family member TASK-3 in the brainstem. Another 32 rats were randomly divided into 3 groups: intermittent hypercapnic hypoxia (IHH) group (n = 14, undergoing IHH 2 hours a day for 14 days), continuous hypoxia (CH) group (n = 6, undergoing CH for 2 hours everyday for 14 days), and normal control group (n = 10). On the day 15 the rats were killed with venous blood samples collected to undergo blood routine examination and brainstem samples collected to undergo Western blotting to measure the expression of TASK-1 and TASK-3. The relationship between TASK-1 and TASK-3 expression and the occurrence of central sleep apnea was analyzed. In the normal rats the spontaneous apnea index (SPAI) was positively correlated with the protein expression of TASK-in the brainstem and the ratio of the TASK-1 protein expression to the TASK-3 protein expression (r = 0.547 and 0.406 respectively, both P < 0.05). However, the post-sign apnea index (PSAI) was not correlated with the TASK-1 protein expression (P = 0.986), and both the SPAI and PSAI were not correlated with the TASK-3 protein expression (P = 0.986 and 0.916 respectively). Only the SPAI in the non-rapid eye movement. stage was positively correlated with the TASK-1 protein expression and the ratio of TASK-1 protein expression to TASK-3 protein expression (r = 0.601, 0.523 respectively, both P < 0.01). The TASK-1 protein expression of the IHH group was significantly higher than those of the CH group and normal control group (P = 0.032 and 0.004 respectively). The TASK-3 protein expression of the IHH group was significantly higher than that of the CH group (P = 0.005). There were no significant differences in the TASK-1 protein expression and that of TASK-3 between the CH group and the normal control group (both P > 0.05). TASK-1 may play an important role in spontaneous sleep apnea.

Melanoma cells exhibit strong intracellular TASK-3-specific immunopositivity in both tissue sections and cell culture.

Amplification of the kcnk9 gene and overexpression of the encoded channel protein (TASK-3) seems to be involved in carcinogenesis. In the present work, TASK-3 expression of melanoma cells has been studied. For the investigation of TASK-3-specific immunolabelling, a monoclonal antibody has been developed and applied along with two, commercially available polyclonal antibodies targeting different epitopes of the channel protein. Both primary and metastatic melanoma cells proved to be TASK-3 positive, showing prominent intracellular TASK-3-specific labelling; mostly concentrating around or in the proximity of the nuclei. The immunoreaction was associated with the nuclear envelope, and with the processes of the cells and it was also present in the cell surface membrane. Specificity of the immunolabelling was confirmed by Western blot and transfection experiments. As TASK-3 immunopositivity of benign melanocytes could also be demonstrated, the presence or absence of TASK-3 channels cannot differentiate between malignant and non-malignant melanocytic tumours.