TASK-3 immunoreactivity shows differential distribution in the human gastrointestinal tract

The presence and distribution of TASK-3 immunopositivity (a channel with potential oncogenic significance) was investigated in the human gastrointestinal system. The immunohistochemical reactions were performed with two commercially available polyclonal antibodies, targeting different epitopes of the channel protein. Experiments conducted on frozen and formalin-fixed samples indicated that the application of a suitable antigen retrieval (AR) technique was essential to produce consistent, strong and reproducible TASK-3-specific immunolabelling of the formalin-fixed tissue. The lack of or inappropriate selection of the AR resulted in false-negative reactions. As for the distribution of the TASK-3 channels, strong immunolabelling was observed in the gastric and large intestinal mucosa, with particularly prominent immunoreactivity of the epithelial cells. In contrast, the smooth-muscle layers demonstrated weak TASK-3 positivity. Intense TASK-3 expression was noted in both the exocrine and endocrine pancreas, but the islets of Langerhans exhibited more powerful reactions. The ductal apparatus of the submandibular gland and lymphocytes situated in pericolonic lymph nodes were also TASK-3 positive. Strong TASK-3 positivity could also be observed in malignant gastrointestinal tumours, with intense nuclear-perinuclear labelling of some of the tumour cells. The present findings suggest that TASK-3 channels may have roles in the gastrointestinal functions, including insular hormone secretion.     

G protein modulation of K2P potassium channel TASK-2

TASK-2 (K_2P5.1) is a background K⁺ channel opened by extra- or intracellular alkalinisation that plays a role in renal bicarbonate handling, central chemoreception and cell volume regulation. Here, we present results that suggest that TASK-2 is also modulated by Gβγ subunits of heterotrimeric G protein. TASK-2 was strongly inhibited when GTP-γ-S was used as a replacement for intracellular GTP. No inhibition was present using GDP-β-S instead. Purified Gβγ introduced intracellularly also inhibited TASK-2 independently of whether GTP or GDP-β-S was present. The effects of GTP-γ-S and Gβγ subunits were abolished by neutralisation of TASK-2 C terminus double lysine residues K257–K258 or K296–K297. Use of membrane yeast two hybrid (MYTH) experiments and immunoprecipitation assays using tagged proteins gave evidence for a physical interaction between Gβ1 and Gβ2 subunits and TASK-2, in agreement with expression of these subunits in proximal tubule cells. Co-immunoprecipitation was impeded by mutating C terminus K257–K258 (but not K296–K297) to alanines. Gating by extra- or intracellular pH was unaltered in GTP-γ-S-insensitive TASK-2-K257A-K258A mutant. Shrinking TASK-2-expressing cells in hypertonic solution decreased the current to 36 % of its initial value. The same manoeuvre had a significantly diminished effect on TASK-2-K257A-K258A- or TASK-2-K296-K297-expressing cells, or in cells containing intracellular GDP-β-S. Our data are compatible with the concept that TASK-2 channels are modulated by Gβγ subunits of heterotrimeric G protein. We propose that this modulation is a novel way in which TASK-2 can be tuned to its physiological functions.     

Effects of divalent cations and spermine on the K+ channel TASK-3 and on the outward current in thalamic neurons

The potassium channels TASK-1 and TASK-3 show high sequence homology but differ in their sensitivity to extracellular divalent cations. Heterologous expression in HEK293 cells showed that the single-channel conductance of TASK-3 increased approximately four-fold after removal of external divalent cations, whereas the conductance of TASK-1 was unaffected. Replacing the glutamate at position 70 of TASK-3 by a lysine or arginine residue abolished the sensitivity to divalent cations. The reverse mutation in TASK-1 (K70E) induced sensitivity to divalent cations. The organic polycations spermine and ruthenium red modulated the conductance of TASK-3 in a similar way as Ca²⁺ or Mg²⁺. Our data suggest that these effects were mediated by shielding of the negative charges in the extracellular loops of TASK-3. Whole-cell currents carried by TASK-3 channels were inhibited by spermine and ruthenium red even in the presence of external divalent cations. These data suggest that, in addition to their effect on single-channel conductance, spermine and ruthenium red decreased the open probability of TASK-3 channels, probably by binding to residue E70. The standing outward current in thalamocortical relay neurons, which is largely carried by TASK channels, was also inhibited by divalent cations and spermine. Using the differential sensitivity of TASK-1 and TASK-3 to divalent cations and spermine we found that about 20% of the standing outward current in thalamocortical relay neurons flows through TASK-3 channels. We conclude from our results that inhibition of TASK-3 channels may contribute to the neuromodulatory effect of spermine released from neurons during repetitive activity or during hypoxia.     

Expression of TWIK-related acid sensitive K+ channels in capsaicin sensitive and insensitive cells of rat dorsal root ganglia

Previous reports have demonstrated that small- to medium-diameter dorsal root ganglia (DRG) cells in rats can be subgrouped into individual cell types by patterns of voltage-activated currents. These cell types have consistent responses to algesic compounds and maintain characteristic histochemical phenotypes. Using immunocytochemical methods, we have now examined expression of TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)-related acid sensitive K+ (TASK) channels, TASK-1, TASK-2 and TASK-3, in nine electrophysiologically identified small- to medium-diameter DRG cell types. The immunoreactivity in DRG cells was diverse, with all nine cell types expressing one to all three TASK channels. Some cells expressed TASK-1 (types 1, 4, 6 and 9), some TASK-2 (types 2, 4, 5, 6, 7 and 9), and some TASK-3 (types 1, 2, 3, 4, 5, 6 and 8). The co-expression of TASK-1 and TASK-3 in cell types 1, 4 and 6 suggests that these sensory afferents might contain functional heterodimeric channels. In peripheral sensory afferents, TASK channels have been implicated in the pain sensory transduction pathway, and can be modulated by anesthetics and neuroprotective agents. This study seeks to identify TASK channel populations in electrophysiologically characterized populations of putative nociceptive afferents.     

Selective block of the human 2-P domain potassium channel, TASK-3, and the native leak potassium current, IKSO, by zinc

Background potassium channels control the resting membrane potential of neurones and regulate their excitability. Two-pore-domain potassium (2-PK) channels have been shown to underlie a number of such neuronal background currents. Currents through human TASK-1, TASK-2 and TASK-3 channels expressed in Xenopus oocytes were inhibited by extracellular acidification. For TASK-3, mutation of histidine 98 to aspartate or alanine considerably reduced this effect of pH. Zinc was found to be a selective blocker of TASK-3 with virtually no effect on TASK-1 or TASK-2. Zinc had an IC₅₀ of 19.8 μ m for TASK-3, at +80 mV, with little voltage dependence associated with this inhibition. TASK-3 H98A had a much reduced sensitivity to zinc suggesting this site is important for zinc block. Surprisingly, TASK-1 also has histidine in position 98 but is insensitive to zinc block. TASK-3 and TASK-1 differ at position 70 with glutamate for TASK-3 and lysine for TASK-1. TASK-3 E70K also had a much reduced sensitivity to zinc while the corresponding reverse mutation in TASK-1, K70E, induced zinc sensitivity. A TASK-3–TASK-1 concatamer channel was comparatively zinc insensitive. For TASK-3, it is concluded that positions E70 and H98 are both critical for zinc block. The native cerebellar granule neurone (CGN) leak current, IK_SO, is sensitive to block by zinc, with current reduced to 0.58 of control values in the presence of 100 μ m zinc. This suggests that TASK-3 channels underlie a major component of IK_SO. It has recently been suggested that zinc is released from inhibitory synapses onto CGNs. Therefore it is possible that inhibition of IK_SO in cerebellar granule cells by synaptically released zinc may have important physiological consequences.     

Acid-sensitive two-pore domain potassium (K2P) channels in mouse taste buds

Sour (acid) taste is postulated to result from intracellular acidification that modulates one or more acid-sensitive ion channels in taste receptor cells. The identity of such channel(s) remains uncertain. Potassium channels, by regulating the excitability of taste cells, are candidates for acid transducers. Several 2-pore domain potassium leak conductance channels (K(2)P family) are sensitive to intracellular acidification. We examined their expression in mouse vallate and foliate taste buds using RT-PCR, and detected TWIK-1 and -2, TREK-1 and -2, and TASK-1. Of these, TWIK-1 and TASK-1 were preferentially expressed in taste cells relative to surrounding nonsensory epithelium. The related TRESK channel was not detected, whereas the acid-insensitive TASK-2 was. Using confocal imaging with pH-, Ca(2+)-, and voltage-sensitive dyes, we tested pharmacological agents that are diagnostic for these channels. Riluzole (500 microM), selective for TREK-1 and -2 channels, enhanced acid taste responses. In contrast, halothane (< or = approximately 17 mM), which acts on TREK-1 and TASK-1 channels, blocked acid taste responses. Agents diagnostic for other 2-pore domain and voltage-gated potassium channels (anandamide, 10 microM; Gd(3+), 1 mM; arachidonic acid, 100 microM; quinidine, 200 microM; quinine, 100 mM; 4-AP, 10 mM; and TEA, 1 mM) did not affect acid responses. The expression of 2-pore domain channels and our pharmacological characterization suggest that a matrix of ion channels, including one or more acid-sensitive 2-pore domain K channels, could play a role in sour taste transduction. However, our results do not unambiguously identify any one channel as the acid taste transducer.     

TASK-5, a novel member of the tandem pore K+ channel family

We have cloned a novel member of the tandem pore K+ channel family from human brain cDNA. The novel cDNA encodes a 330-residue polypeptide of predicted molecular mass 36 kDa. We have named the channel TASK-5 owing to its sequence homology with TASK-1 and TASK-3. TASK-5 mRNA is expressed in pancreas, liver, kidney, lung, ovary, testis and heart. However, expression of TASK-5 in heterologous systems failed to elicit ionic currents. Removal of a putative endoplasmic reticulum retention sequence did not alter this finding and the distribution of channel proteins in HEK293 cells was similar for both TASK-1 and TASK-5. We tested whether TASK-5 could form heteromers with TASK-1. We show a mutant form of TASK-1 (H98N) to have a radically reduced sensitivity to acidification. Proton sensitivity could be rescued by injecting equimolar amounts of wild-type and mutant TASK-1 cRNA into Xenopus oocytes; the effect was that expected if half the channels formed are heteromers. Co-expression of TASK-5 with TASK-1 H98N does not affect the proton sensitivity of mutant TASK-1; thus TASK-5 appears not to form heteromers with TASK-1. Nonetheless, TASK-5 may require some other, unidentified partner subunit to form functional channels in the plasma membrane or it may form a channel in an intracellular organelle.     

TASK-3钾离子通道的研究现状

双孔钾离子通道是一个膜蛋白家族,广泛分布于可兴奋和不可兴奋细胞中。TASK-3钾离子通道是新发现的一个双孔钾离子通道家族成员,编码374个氨基酸,对细胞凋亡和增殖有重要的作用。在多种肿瘤组织中TASK-3钾离子通道基因呈高表达,并表现出与钾离子通道功能相关的原癌基因特性。     

Taste receptor cells express pH-sensitive leak K+ channels

Two-pore domain K+ channels encoded by genes KCNK1-17 (K2p1-17) play important roles in regulating cell excitability. We report here that rat taste receptor cells (TRCs) highly express TASK-2 (KCNK5; K2p5.1), and to a much lesser extent TALK-1 (KCNK16; K2p16.1) and TASK-1 (KCNK3; K2p3.1), and suggest potentially important roles for these channels in setting resting membrane potentials and in sour taste transduction. Whole cell recordings of isolated TRCs show that a leak K+ (Kleak) current in a subset of TRCs exhibited high sensitivity to acidic extracellular pH similar to reported properties of TASK-2 and TALK-1 channels. A drop in bath pH from 7.4 to 6 suppressed 90% of the current, resulting in membrane depolarization. K+ channel blockers, BaCl2, but not tetraethylammonium (TEA), inhibited the current. Interestingly, resting potentials of these TRCs averaged -70 mV, which closely correlated with the amplitude of the pH-sensitive Kleak, suggesting a dominant role of this conductance in setting resting potentials. RT-PCR assays followed by sequencing of PCR products showed that TASK-1, TASK-2, and a functionally similar channel, TALK-1, were expressed in all three types of lingual taste buds. To verify expression of TASK channels, we labeled taste tissue with antibodies against TASK-1, TASK-2, and TASK-3. Strong labeling was seen in some TRCs with antibody against TASK-2 but not TASK-1 and TASK-3. Consistent with the immunocytochemical staining, quantitative real-time PCR assays showed that the message for TASK-2 was expressed at significantly higher levels (10-100 times greater) than was TASK-1, TALK-1, or TASK-3. Thus several K2P channels, and in particular TASK-2, are expressed in rat TRCs, where they may contribute to the establishment of resting potentials and sour reception.     

Interaction with 14-3-3 proteins promotes functional expression of the potassium channels TASK-1 and TASK-3

The two-pore-domain potassium channels TASK-1, TASK-3 and TASK-5 possess a conserved C-terminal motif of five amino acids. Truncation of the C-terminus of TASK-1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP-tagged channel proteins. Two-hybrid analysis showed that the C-terminal domain of TASK-1, TASK-3 and TASK-5, but not TASK-4, interacts with isoforms of the adapter protein 14-3-3. A pentapeptide motif at the extreme C-terminus of TASK-1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14-3-3, whereas the last 40 amino acids of TASK-1 were required for strong binding. Deletion of a single amino acid at the C-terminal end of TASK-1 or TASK-3 abolished binding of 14-3-3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK-1 mutants that failed to interact with 14-3-3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK-1 S409A, which interacts with 14-3-3-like wild-type channels, displayed normal macroscopic currents. Co-injection of 14-3-3ζ cRNA increased TASK-1 current in Xenopus oocytes by about 70 %. After co-transfection in HEK293 cells, TASK-1 and 14-3-3ζ (but not TASK-1ΔC5 and 14-3-3ζ) could be co-immunoprecipitated. Furthermore, TASK-1 and 14-3-3 could be co-immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14-3-3 with TASK-1 or TASK-3 may promote the trafficking of the channels to the surface membrane.     

The 2P-domain K+ channels: role in apoptosis and tumorigenesis

Two-pore (2P)-domain K⁺ channels have been shown recently to play a critical role in both cell apoptosis and tumorigenesis. The activity of two-pore, (TWIK)-related acid-sensitive-3 (TASK-3) K⁺ channels, is responsible for K⁺-dependent apoptosis of cultured cerebellar granule neurons. Neuron death can be prevented by conditions that specifically reduce K⁺ efflux through the TASK-3 channels. Moreover, genetic transfer of TASK subunits into hippocampal neurons that lack TASK-3, induces apoptosis. These results indicate a direct link between TASK K⁺ channel activity and the physiological process of programmed cell death. The TASK-3 K⁺ channel gene has also been shown to be amplified genomically and over-expressed in a significant number of breast tumours. TASK-3 has a potent oncogenic potential that appears to be related directly to its K⁺ channel function. In the present review, we will examine the pro-apoptotic and oncogenic properties of TASK-3. We will discuss: (1) the molecular and functional properties of the novel family of mammalian 2P domain K⁺ channels; (2) the role of TASK-3 in cerebellar granule neuron apoptosis and (3) the role of TASK-3 in breast tumorigenesis.     

Modulation of K2P3.1 (TASK-1), K2P9.1 (TASK-3), and TASK-1/3 heteromer by reactive oxygen species

Reactive oxygen species (ROS) generated by mitochondria or NADPH oxidase have been implicated in the inhibition of K⁺ current by hypoxia in chemoreceptor cells. As TASKs are highly active background K⁺ channels in these cells, we studied the role of ROS in hypoxia-induced inhibition of TASKs. In HeLa cells expressing TASKs, H₂O₂ applied to inside-out patches activated TASK-1, TASK-3, and TASK-1/3 heteromer starting at ~16?mM. When applied to cell-attached or outside-out patches, 326?mM H₂O₂ did not affect TASK activity. Other K_2P channels (TREK-1, TREK-2, TASK-2, TALK-1, TRESK) were not affected by H₂O₂ (tested up to 326?mM). A reducing agent (dithiothreitol) and a cysteine-modifying agent (2-aminoethyl methanethiosulfonate hydrobromide) had no effect on basal TASK activity and did not block the H₂O₂-induced increase in channel activity. A TASK mutant in which the C-terminus of TASK-3 was replaced with that of TREK-2 showed a normal sensitivity to H₂O₂. Xanthine/xanthine oxidase mixture used to generate superoxide radical showed no effect on TASK-1, TASK-3, and TASK-1/3 heteromer from either side of the membrane, but it strongly activated TASK-2 from the extracellular side. Acute H₂O₂ (32–326?mM) exposure did not affect hSlo1/b1(BK) expressed in HeLa cells and BK in carotid body glomus cells. In carotid body glomus cells, adrenal cortical cells, and cerebellar granule neurons that show abundant hypoxia-sensitive TASK activity, H₂O₂ (>16?mM) activated the channels only when applied intracellularly, similar to that observed with cloned TASKs. These findings show that ROS do not support or inhibit TASK and BK activity and therefore are unlikely to be the hypoxic signal that causes cell excitation via inhibition of these K⁺ channels.     

Striatal cholinergic interneurons express a receptor-insensitive homomeric TASK-3-like background K+ current

Large aspiny cholinergic interneurons provide the sole source of striatal acetylcholine, a neurotransmitter essential for normal basal ganglia function. Cholinergic interneurons engage in multiple firing patterns that depend on interactions among various voltage-dependent ion channels active at different membrane potentials. Leak conductances, particularly leak K(+) channels, are of primary importance in establishing the prevailing membrane potential. We have combined molecular neuroanatomy with whole cell electrophysiology to demonstrate that TASK-3 (K(2P)9.1, Kcnk9) subunits contribute to leak K(+) currents in striatal cholinergic interneurons. Immunostaining for choline acetyltransferase was combined with TASK-3 labeling, using nonradioactive cRNA probes or antisera selective for TASK-3, to demonstrate that striatal cholinergic neurons universally express TASK-3. Consistent with this, we isolated a pH-, anesthetic-, and Zn(2+)-sensitive current with properties expected of TASK-3 homodimeric channels. Surprisingly, activation of Galphaq-linked receptors (metabotropic glutamate mGluR1/5 or histamine H1) did not appear to modulate native interneuron TASK-3-like currents. Together, our data indicate that homomeric TASK-3-like background K(+) currents contribute to establishing membrane potential in striatal cholinergic interneurons and they suggest that receptor modulation of TASK channels is dependent on cell context.     

MMC-E cells--origin and changes in karyotype accompanying malignant transformation

Karyotype analysis of an established nontumorigenic cell line (MMC-E) indicated that the cells are of rat origin instead of mouse, as interpreted earlier. This cell line, now termed RE (rat epithelial) had a karyotype of 39,X,-5,-15,-?16,+t(3q11q) in the stem line. There was also one subline that had a karyotype of 40,X,-5,-15,-?16,+t(3q11q), +?t(5;?). Chromosome changes and frequency of sister chromatid exchanges (SCEs) were studied from seven malignantly transformed RE cell lines. These included cells transformed by Moloney murine sarcoma virus, the acute transforming retrovirus 3611-MSV, murine leukemia virus, or ethylnitrosourea. Changes in chromosomes #3 and #5 seemed to be associated with malignant transformation of RE cells. In addition to the t(3q11q) and monosomy for chromosome #5 seen in the parent cell line, monosomy for chromosome #3 and the t(5;?) were observed in all malignant cell lines. The latter changes either were absent (monosomy for chromosome #3) or present only occasionally [t(5;?)] in the parental cells. The results of SCEs showed that the malignantly transformed cell lines do not have increased frequency of SCEs as compared with that of the parental cell line.     

Changes in oxygen sensitivity of TASK in carotid body glomus cells during early postnatal development

A post-natal increase in carotid body (CB) hypoxia responsiveness occurs at the level of carotid sinus nerve activity, intracellular calcium, cell membrane depolarization and hypoxic inhibition of O(2)-sensitive background K(+) conductance. TASK-1, TASK-1/3 and TASK-3 are functionally expressed in CB glomus cells, with TASK-1/3 providing the major part of the O(2)-sensitive TASK-like background K(+) conductance. Here we report the effects of graded hypoxia on TASK-like channel activity in CB glomus cells from rats aged 0 to 1, 6 to 7 and 16 to 18 days; the time frame of postnatal CB functional maturation. TASK was active in nearly all cell-attached patches and TASK activity during normoxia did not differ across ages. Hypoxia produced a progressive decrease in channel opening frequency with graded decreases in O(2) level and also produced glomus cell depolarization, as assessed by the shift in reversal potential of TASK single channel current. Hypoxic inhibition of TASK activity was least at P0-P1 and increased with age mainly between 6-7 and 16-18 days. The O(2)-sensitive TASK activity was significantly greater in glomus cells from P16 to P18 when compared to cells from P0 to P1 day old rats. These results support the hypothesis that postnatal carotid body functional maturation is due, at least in part, to changes in the sensitivity of TASK to the hypoxic signals generated in glomus cells.     

Associated expression of HLA class I and class II antigens on melanoma cells in surgically removed metastases

Malignant transformation of melanocytes and further neoplastic progression may be associated with qualitative and/or quantitative changes in expression of HLA class I and class II antigens. Since previous immunohistochemical studies of surgically removed melanoma lesions have suggested a relationship in the expression of HLA class I and class II antigens, we have investigated the expression of these antigens at the single cell level. Double immunofluorescence staining of frozen sections of melanoma metastases and immunoelectron microscopic double labelling of melanoma cell suspensions prepared from three of these lesions has detected three HLA phenotypes on the large majority of melanoma cells: either both HLA class I and class II antigens, neither HLA antigen or only HLA class I antigens. In four out of the 11 lesions a few melanoma cells were found to express HLA class II antigens and to lack HLA class I antigens. A relationship was also found in the level of expression of HLA class I and class II antigens, as estimated by the intensity of staining with monoclonal antibodies. The level of expression of HLA class II antigens appeared to be similar to or lower than that of HLA class I antigens on the large majority of melanoma cells. This coordinated heterogeneity in the expression of HLA class I and class II antigens by melanoma cells may have implications in the interactions of tumour cells with the host's immune system.