KCNK13 potassium channels in the ventral tegmental area of rats are important for excitation of ventral tegmental area neurons by ethanol.

BACKGROUND: Alcohol excites neurons of the ventral tegmental area (VTA) and the release of dopamine from these neurons is a key event in ethanol (EtOH)-induced reward and reinforcement. Many mechanisms have been proposed to explain EtOH's actions on neurons of the VTA, but antagonists generally do not eliminate the EtOH-induced excitation of VTA neurons. We have previously demonstrated that the ion channel KCNK13 plays an important role in the EtOH-related excitation of mouse VTA neurons. Here, we elaborate on that finding and further assess the importance of KCNK13 in rats. METHODS: Rats (Sprague-Dawley and Fisher 344) were used in these studies. In addition to single-unit electrophysiology in brain slices, we used quantitative PCR and immunohistochemistry to discern the effects of EtOH and the brain slice preparation method on the expression levels of the Kcnk13 gene and KCNK13 protein. RESULTS: Immunohistochemistry demonstrated that the levels of KCNK13 were significantly reduced during procedures normally used to prepare brain slices for electrophysiology, with a reduction of about 75% in KCNK13 protein at the time that electrophysiological recordings would normally be made. Extracellular recordings demonstrated that EtOH-induced excitation of VTA neurons was reduced after knockdown of Kcnk13 using a small interfering RNA (siRNA) delivered via the recording micropipette. Real-time PCR demonstrated that the expression of Kcnk13 was altered in a time-dependent manner after alcohol withdrawal. CONCLUSIONS: KCNK13 plays an important role in EtOH-induced stimulation of rat VTA neurons and is dynamically regulated by cell damage and EtOH exposure, and during withdrawal. KCNK13 is a novel alcohol-sensitive protein, and further investigation of this channel may offer new avenues for the development of agents useful in altering the rewarding effect of alcohol.

The glycine hinge of transmembrane segment 2 modulates the subcellular localization and gating properties in TREK channels.

TWIK-Related K+ channels (TREK), including TREK-1 and TREK-2, belong to the TREK/TRAAK subclass of two-pore domain K+ (K2P) family. The important functions of transmembrane segment 4 (M4)-glycine hinge in TREK channel gating have been characterized, but the roles of M2-hinge (the equivalent residue of M4-hinge) remain unclear. Here, by characterizing the macroscopic currents, subcellular localization and gating properties of their M2-hinge mutants (G166A for TREK-1 and G196A for TREK-2), we investigated the functions of M2-hinge. G166A displayed decreased whole-cell currents, whereas no current was produced by G196A. Subcellular analysis indicated that both mutants were aggregated near the perinuclear region, and most of them were retented within the endoplasmic reticulum (ER). Next, to explore the roles of M2-hinge in the gating mechanism, we tested the responses of the related M2-hinge mutants to 2-Aminoethoxydiphenyl borate (2-APB) and extracellular pH alteration (ΔpHo). TREK-1mut7-G166A displayed reduced sensitivity to 2-APB activation, but similar sensitivity to ΔpHo, when compared with TREK-1mut7. WT-ΔpCt, a TREK-2 tandom dimer, was used to assess the function of M2-hinge in the cis-type gating of TREK-2. The sensitivities of G196A-ΔpCt to both 2-APB and ΔpHo decreased compared with WT-ΔpCt. Taken together, our results reveal that the M2-hinge of TREK channels control their macroscopic current, subcellular localization and gating process.

POPDC1(S201F) causes muscular dystrophy and arrhythmia by affecting protein trafficking.

The Popeye domain-containing 1 (POPDC1) gene encodes a plasma membrane-localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1(S201F) displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1(S201F) and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1(S201F) in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1(S191F)) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases.

Gain-of-function mutation in TASK-4 channels and severe cardiac conduction disorder.

Analyzing a patient with progressive and severe cardiac conduction disorder combined with idiopathic ventricular fibrillation (IVF), we identified a splice site mutation in the sodium channel gene SCN5A. Due to the severe phenotype, we performed whole-exome sequencing (WES) and identified an additional mutation in the KCNK17 gene encoding the K2P potassium channel TASK-4. The heterozygous change (c.262G>A) resulted in the p.Gly88Arg mutation in the first extracellular pore loop. Mutant TASK-4 channels generated threefold increased currents, while surface expression was unchanged, indicating enhanced conductivity. When co-expressed with wild-type channels, the gain-of-function by G88R was conferred in a dominant-active manner. We demonstrate that KCNK17 is strongly expressed in human Purkinje cells and that overexpression of G88R leads to a hyperpolarization and strong slowing of the upstroke velocity of spontaneously beating HL-1 cells. Thus, we propose that a gain-of-function by TASK-4 in the conduction system might aggravate slowed conductivity by the loss of sodium channel function. Moreover, WES supports a second hit-hypothesis in severe arrhythmia cases and identified KCNK17 as a novel arrhythmia gene.

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.

Presence of task-1 channel in the laryngeal mucosa in the newborn lamb.

Nearly 40 potassium channels have been described in respiratory epithelial cells. Of these are found several members of the 4-transmembrane domain, 2-pore K(+) channel family (K2P family), namely Twik-1 and -2, Trek-1 and -2, Task-2, -3, and -4, Thik-1, and KCNK7. The aim of this study was to verify whether the Twik-related acid-sensitive K(+) channel, subtype 1 (Task-1) (also known as KCNK3), is present in the laryngeal mucosa in the newborn lamb. Through the use of immunohistochemistry and nested polymerase chain reaction (PCR) amplification, results indicate that Task-1 protein and mRNA are present in the laryngeal mucosa, in both the ciliated, pseudostratified columnar (respiratory) epithelium and the nonkeratinized, stratified squamous epithelium. The complete ovine Task-1 protein sequence showed high homology levels with previously reported mouse, bovine, and human Task-1 sequences. This includes a complete homology for the C-terminal amino acid sequence, which is mandatory for protein trafficking to the cell membrane. These results represent the first demonstration that Task-1, a pH-sensitive channel responsible for setting membrane potential, is present in the laryngeal mucosa of a newborn mammal.

Expression and localization of two-pore domain K(+) channels in bovine germ cells.

Two-pore domain K(+) (K(2P)) channels that help set the resting membrane potential of excitable and nonexcitable cells are expressed in many kinds of cells and tissues. However, the expression of K(2P) channels has not yet been reported in bovine germ cells. In this study, we demonstrate for the first time that K(2P) channels are expressed in the reproductive organs and germ cells of Korean cattle. RT-PCR data showed that members of the K(2P) channel family, specifically KCNK3, KCNK9, KCNK2, KCNK10, and KCNK4, were expressed in the ovary, testis, oocytes, embryo, and sperm. Out of these channels, KCNK2 and KCNK4 mRNAs were abundantly expressed in the mature oocytes, eight-cell stage embryos, and blastocysts compared with immature oocytes. KCNK4 and KCNK3 were significantly increased in eight-cell stage embryos. Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed at the membrane of oocytes and blastocysts. KCNK10 and KCNK4 were strongly expressed and distributed in oocyte membranes. These channel proteins were also localized to the acrosome sperm cap. In particular, KCNK3 and KCNK4 were strongly localized to the post-acrosomal region of the sperm head and the equatorial band within the sperm head respectively. These results suggest that K(2P) channels might contribute to the background K(+) conductance of germ cells and regulate various physiological processes, such as maturation, fertilization, and development.

Potassium channels involved in human sperm volume regulation--quantitative studies at the protein and mRNA levels.

KCNE1, KCNA5 and KCNK5 have been identified, by using specific blockers, as K(+)-channels involved in sperm volume regulation under physiological conditions. All three channels were localised on the cytoplasmic droplets and tail of human ejaculated spermatozoa by fluorescence microscopy. Using flow cytometric quantification, KCNE1 was found to be present in 80% or more spermatozoa and KCNK5 in only about 20%, with KCNA5 expressed by 20-90% of cells. Whereas the extents of such protein expression did not differ statistically between semen donors and subfertile patients, the former group exhibited higher capacities for sperm volume regulation which were correlated with other sperm qualities including normal morphology and motile sperm number in the ejaculate. Channel identification was further confirmed at the protein level using Western blotting. RT-PCR analysis of testicular and sperm RNA of proven quality indicated the presence of Kcne1, Kcna5 and Kcnk5 transcripts. Subsequent sequencing of PCR products demonstrated that the nucleotide sequences of the entire encoding regions of Kcne1 and Kcnk5 were identical to those published in the database, whereas that of Kcna5 mRNA showed a single nucleotide synonymous deviation that agrees with the published genomic sequence. Quantitative real-time PCR analysis of sperm RNA revealed the amounts of Kcne1 > Kcna5 > Kcnk5, in the same order as for protein expression. Thus, KCNE1 is probably the major K(+)-channel involved in regulatory volume decrease in human spermatozoa, and channel activity is regulated beyond the extent of protein expression.

Altered acetylcholine, bradykinin and cutaneous pressure-induced vasodilation in mice lacking the TREK1 potassium channel: the endothelial link.

The TWIK related K+ channel TREK1 is an important member of the class of two-pore-domain K+ channels. It is a background K+ channel and is regulated by hormones, neurotransmitters, intracellular pH and mechanical stretch. This work shows that TREK1 is present both in mesenteric resistance arteries and in skin microvessels. It is particularly well expressed in endothelial cells. Deletion of TREK1 in mice leads to an important alteration in vasodilation of mesenteric arteries induced by acetylcholine and bradykinin. Iontophoretic delivery of acetylcholine and bradykinin in the skin of TREK1+/+ and TREK1-/- mice also shows the important role of TREK1 in cutaneous endothelium-dependent vasodilation. The vasodilator response to local pressure application is also markedly decreased in TREK1-/- mice, mimicking the decreased response to pressure observed in diabetes. Deletion of TREK1 is associated with a marked alteration in the efficacy of the G-protein-coupled receptor-associated cascade producing NO that leads to major endothelial dysfunction.

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.

TASK channel expression in human placenta and cytotrophoblast cells.

OBJECTIVE: The multinucleate syncytiotrophoblast is the transporting epithelium of the human placental villus, formed throughout pregnancy by fusion and differentiation of underlying mononucleate cytotrophoblast cells. Similar to other epithelia, K+ channels will impact on syncytiotrophoblast transport properties during its development and differentiation. Therefore we investigated expression and activity of the two-pore domain K+ channels TASK1 and 2 in relation to gestation and differentiation, using villous tissue from first and third trimester and cultured cytotrophoblast cells at mononucleate and multinucleate stages of culture. METHODS: Quantitative real-time polymerase chain reaction (PCR), immunofluorescence, and 86Rb+ (K) efflux were used to investigate TASK channel expression and function. RESULTS: TASK2 mRNA expression was higher in first trimester than term (10 to 13 vs 38 to 40 weeks, P < .05). Other K+ alpha-subunit mRNAs, including TASK1, remained unaltered but the regulatory BKCa beta-subunit, like TASK2, was higher in first trimester than term (P < .001). Immunofluorescence showed that TASK2 had an intracellular localization within the trophoblast of first trimester villi but was less abundant and restricted to stem villi at term. TASK2 also showed intracellular localization in mononucleate cytotrophoblast cells in culture and expression was lost with multinucleation. By contrast, TASK1 was localised, independently of cell nucleation, to cytotrophoblast cell plasma membranes. 86Rb+ (K) efflux was measured from multinucleated cytotrophoblast cells. Both basal and pH 8.0-stimulated efflux was inhibited by the TASK1 antagonist anandamide (n = 5 for both conditions; P < .01 and P < .001, respectively). CONCLUSION: TASK1 and 2 are expressed in placental trophoblast cells and TASK1 activity may have a role in regulating syncytiotrophoblast homeostasis and/or solute transport functions.

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.

Cross-talk between the mechano-gated K2P channel TREK-1 and the actin cytoskeleton.

TREK-1 (KCNK2) is a K(2P) channel that is highly expressed in fetal neurons. This K(+) channel is opened by a variety of stimuli, including membrane stretch and cellular lipids. Here, we show that the expression of TREK-1 markedly alters the cytoskeletal network and induces the formation of actin- and ezrin-rich membrane protrusions. The genetic inactivation of TREK-1 significantly alters the growth cone morphology of cultured embryonic striatal neurons. Cytoskeleton remodelling is crucially dependent on the protein kinase A phosphorylation site S333 and the interactive proton sensor E306, but is independent of channel permeation. Conversely, the actin cytoskeleton tonically represses TREK-1 mechano-sensitivity. Thus, the dialogue between TREK-1 and the actin cytoskeleton might influence both synaptogenesis and neuronal electrogenesis.

Expression of TASK and TREK, two-pore domain K+ channels, in human myometrium.

Two-pore domain K+ channels are an emerging family of K+ channels that may contribute to setting membrane potential in both electrically excitable and non-excitable cells and, as such, influence cellular function. The human uteroplacental unit contains both excitable (e.g. myometrial) and non-excitable cells, whose function depends upon the activity of K+ channels. We have therefore investigated the expression of two members of this family, TWIK (two-pore domain weak inward rectifying K+ channel)-related acid-sensitive K+ channel (TASK) and TWIK-related K+ channel (TREK) in human myometrium. Using RT-PCR the mRNA expression of TASK and TREK isoforms was examined in myometrial tissue from pregnant women. mRNAs encoding TASK1, 4 and 5 and TREK1 were detected whereas weak or no signals were observed for TASK2, TASK3 and TREK2. Western blotting for TASK1 gave two bands of approximately 44 and 65 kDa, whereas TREK1 gave bands of approximately 59 and 90 kDa in myometrium from pregnant women. TASK1 and TREK1 immunofluorescence was prominent in intracellular and plasmalemmal locations within myometrial cells. Therefore, we conclude that the human myometrium is a site of expression for the two-pore domain K+ channel proteins TASK1 and TREK1.