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At least 19 recordsLinked to original sources
PubMed1999

TWIK-2, a new weak inward rectifying member of the tandem pore domain potassium channel family.

Potassium channels are found in all mammalian cell types, and they perform many distinct functions in both excitable and non-excitable cells. These functions are subserved by several different families of potassium channels distinguishable by primary sequence features as well as by physiological characteristics. Of these families, the tandem pore domain potassium channels are a new and distinct class, primarily distinguished by the presence of two pore-forming domains within a single polypeptide chain. We have cloned a new member of this family, TWIK-2, from a human brain cDNA library. Primary sequence analysis of TWIK-2 shows that it is most closely related to TWIK-1, especially in the pore-forming domains. Northern blot analysis reveals the expression of TWIK-2 in all human tissues assayed except skeletal muscle. Human TWIK-2 expressed heterologously in Xenopus oocytes is a non-inactivating weak inward rectifier with channel properties similar to TWIK-1. Pharmacologically, TWIK-2 channels are distinct from TWIK-1 channels in their response to quinidine, quinine, and barium. TWIK-2 is inhibited by intracellular, but not extracellular, acidification. This new clone reveals the existence of a subfamily in the tandem pore domain potassium channel family with weak inward rectification properties.

Amino Acid Sequence↗
PubMed2006

Tandem-pore domain potassium channels are functionally expressed in retinal (Müller) glial cells.

Tandem-pore domain (2P-domain) K+-channels regulate neuronal excitability, but their function in glia, particularly, in retinal glial cells, is unclear. We have previously demonstrated the immunocytochemical localization of the 2P-domain K+ channels TASK-1 and TASK-2 in retinal Müller glial cells of amphibians. The purpose of the present study was to determine whether these channels were functional, by employing whole-cell recording from frog and mammalian (guinea pig, rat and mouse) Müller cells and confocal microscopy to monitor swelling in rat Müller cells. TASK-like immunolabel was localized in these cells. The currents mediated by 2P-domain channels were studied in isolation after blocking Kir, K(A), K(D), and BK channels. The remaining cell conductance was mostly outward and was depressed by acid pH, bupivacaine, methanandamide, quinine, and clofilium, and activated by alkaline pH in a manner consistent with that described for TASK channels. Arachidonic acid (an activator of TREK channels) had no effect on this conductance. Blockade of the conductance with bupivacaine depolarized the Müller cell membrane potential by about 50%. In slices of the rat retina, adenosine inhibited osmotic glial cell swelling via activation of A1 receptors and subsequent opening of 2P-domain K+ channels. The swelling was strongly increased by clofilium and quinine (inhibitors of 2P-domain K+ channels). These data suggest that 2P-domain K+ channels are involved in homeostasis of glial cell volume, in activity-dependent spatial K+ buffering and may play a role in maintenance of a hyperpolarized membrane potential especially in conditions where Kir channels are blocked or downregulated.

Animals↗
PubMed2005

Enhanced expressions of arachidonic acid-sensitive tandem-pore domain potassium channels in rat experimental acute cerebral ischemia.

To further explore the pathophysiological significance of arachidonic acid-sensitive potassium channels, RT-PCR and Western blot analysis were used to investigate the expression changes of TREK channels in cortex and hippocampus in rat experimental acute cerebral ischemia in this study. Results showed that TREK-1 and TRAAK mRNA in cortex, TREK-1 and TREK-2 mRNA in hippocampus showed significant increases 2 h after middle cerebral artery occlusion (MCAO). While the mRNA expression levels of the all three channel subtypes increased significantly 24 h after MCAO in cortex and hippocampus. At the same time, the protein expressions of all the three channel proteins showed significant increase 24 h after MCAO in cortex and hippocampus, but only TREK-1 showed increased expression 2 h after MCAO in cortex and hippocampus. Immunohistochemical experiments verified that all the three channel proteins had higher expression levels in cortical and hippocampal neurons 24 h after MCAO. These results suggested a strong correlation between TREK channels and acute cerebral ischemia. TREK channels might provide a neuroprotective mechanism in the pathological process.

Animals↗
PubMed1999

Local anesthetic inhibition of baseline potassium channels with two pore domains in tandem.

BACKGROUND: Recently, a new structural family of potassium channels characterized by two pore domains in tandem within their primary amino acid sequence was identified. These tandem pore domain potassium channels are not gated by voltage and appear to be involved in the control of baseline membrane conductances. The goal of this study was to identify mechanisms of local anesthetic action on these channels. METHODS: Oocytes of Xenopus laevis were injected with cRNA from five cloned tandem pore domain baseline potassium channels (TASK, TREK-1, TOK1, ORK1, and TWIK-1), and the effects of several local anesthetics on the heterologously expressed channels were assayed using two-electrode voltage-clamp and current-clamp techniques. RESULTS: Bupivacaine (1 mM) inhibited all studied tandem pore potassium channels, with TASK inhibited most potently. The potency of inhibition was directly correlated with the octanol: buffer distribution coefficient of the local anesthetic, with the exception of tetracaine, to which TASK is relatively insensitive. The approximate 50% inhibitory concentrations of TASK were 709 microM mepivacaine, 222 microM lidocaine, 51 microM R(+)-ropivacaine, 53 microM S(-)-ropivacaine, 668 microM tetracaine, 41 microM bupivacaine, and 39 microM etidocaine. Local anesthetics (1 mM) significantly depolarized the resting membrane potential of TASK cRNA-injected oocytes compared with saline-injected control oocytes (tetracaine 22+/-6 mV rs. 7+/-1 mV, respectively, and bupivacaine 31+/-7 mV vs. 6+/-4 mV). CONCLUSIONS: Local anesthetics inhibit tandem pore domain baseline potassium channels, and they could depolarize the resting membrane potential of cells expressing these channels. Whether inhibition of these channels contributes to conduction blockade or to the adverse effects of local anesthetics remains to be determined.

Anesthetics, Local↗
PubMed2000

Volatile anesthetics activate the human tandem pore domain baseline K+ channel KCNK5.

BACKGROUND: Previous studies have identified a volatile anesthetic-induced increase in baseline potassium permeability and concomitant neuronal inhibition. The emerging family of tandem pore domain potassium channels seems to function as baseline potassium channels in vivo. Therefore, we studied the effects of clinically used volatile anesthetics on a recently described member of this family. METHODS: A cDNA clone containing the coding sequence of KCNK5 was isolated from a human brain library. Expression of KCNK5 in the central nervous system was determined by Northern blot analysis and reverse-transcription polymerase chain reaction. Functional expression of the channel was achieved by injection of cRNA into Xenopus laevis oocytes. RESULTS: Expression of KCNK5 was detected in cerebral cortex, medulla, and spinal cord. When heterologously expressed in Xenopus oocytes, KCNK5 currents exhibited delayed activation, outward rectification, proton sensitivity, and modulation by protein kinase C. Clinical concentrations of volatile general anesthetics potentiated KCNK5 currents by 8-30%. CONCLUSION: Human KCNK5 is a tandem pore domain potassium channel exhibiting delayed activation and sensitivity to volatile anesthetics and may therefore have a role in suppressing cellular excitability during general anesthesia.

Anesthetics, Inhalation↗
PubMed1995

A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem.

Potassium channels catalyse the permeation of K+ ions across cellular membranes and are identified by a common structural motif, a highly conserved signature sequence of eight amino acids in the P domain of each channel's pore-forming alpha-subunit. Here we describe a novel K+ channel (TOK1) from Saccharomyces cerevisiae that contains two P domains within one continuous polypeptide. Xenopus laevis oocytes expressing the channel exhibit a unique, outwardly rectifying, K(+)-selective current. The channel is permeable to outward flow of ions at membrane potentials above the K+ equilibrium potential; its conduction-voltage relationship is thus sensitive to extracellular K+ ion concentration. In excised membrane patches, external divalent cations block the channel in a voltage-dependent manner, and their removal in this configuration allows inward channel current. These attributes are similar to those described for inwardly rectifying K+ channels, but in the opposite direction, a previously unrecognized channel behaviour. Our results identify a new class of K+ channel which is distinctive in both its primary structure and functional properties. Structural homologues of the channel are present in the genome of Caenorhabditis elegans.

Amino Acid Sequence↗
PubMed2000

TASK-3, a novel tandem pore domain acid-sensitive K+ channel. An extracellular histiding as pH sensor.

Tandem pore domain acid-sensitive K(+) channel 3 (TASK-3) is a new member of the tandem pore domain potassium channel family. A cDNA encoding a 365- amino acid polypeptide with four putative transmembrane segments and two pore regions was isolated from guinea pig brain. An orthologous sequence was cloned from a human genomic library. Although TASK-3 is 62% identical to TASK-1, the cytosolic C-terminal sequence is only weakly conserved. Analysis of the gene structure identified an intron within the conserved GYG motif of the first pore region. Reverse transcriptase-polymerase chain reaction analysis showed strong expression in brain but very weak mRNA levels in other tissues. Cell-attached patch-clamp recordings of TASK-3 expressed in HEK293 cells showed that the single channel current-voltage relation was inwardly rectifying, and open probability increased markedly with depolarization. Removal of external divalent cations increased the mean single channel current measured at -100 mV from -2.3 to -5.8 pA. Expression of TASK-3 in Xenopus oocytes revealed an outwardly rectifying K(+) current that was strongly decreased in the presence of lower extracellular pH. Substitution of the histidine residue His-98 by asparagine or tyrosine abolished pH sensitivity. This histidine, which is located at the outer part of the pore adjacent to the selectivity filter, may be an essential component of the extracellular pH sensor.

Amino Acid Sequence↗
PubMed2006

VOCCs and TREK-1 ion channel expression in human tenocytes.

Mechanosensitive and voltage-gated ion channels are known to perform important roles in mechanotransduction in a number of connective tissues, including bone and muscle. It is hypothesized that voltage-gated and mechanosensitive ion channels also may play a key role in some or all initial responses of human tenocytes to mechanical stimulation. However, to date there has been no direct investigation of ion channel expression by human tenocytes. Human tenocytes were cultured from patellar tendon samples harvested from five patients undergoing routine total knee replacement surgery (mean age: 66 yr; range: 63-73 yr). RT-PCR, Western blotting, and whole cell electrophysiological studies were performed to investigate the expression of different classes of ion channels within tenocytes. Human tenocytes expressed mRNA and protein encoding voltage-operated calcium channel (VOCC) subunits (Ca alpha(1A), Ca alpha(1C), Ca alpha(1D), Ca alpha(2)delta(1)) and the mechanosensitive tandem pore domain potassium channel (2PK(+)) TREK-1. They exhibit whole cell currents consistent with the functional expression of these channels. In addition, other ionic currents were detected within tenocytes consistent with the expression of a diverse array of other ion channels. VOCCs and TREK channels have been implicated in mechanotransduction signaling pathways in numerous connective tissue cell types. These mechanisms may be present in human tenocytes. In addition, human tenocytes may express other channel currents. Ion channels may represent potential targets for the pharmacological management of chronic tendinopathies.

Aged↗
PubMed2000

Cloning, localisation and functional expression of a novel human, cerebellum specific, two pore domain potassium channel.

We have isolated, by degenerate PCR, a complementary DNA encoding a novel two pore domain potassium channel. This is the 7th functional member of the human tandem pore domain potassium channel family to be reported. It has an open reading frame of 1.125 kb and encodes a 374 amino acid protein which shows 62% identity to the human TASK-1 gene: identity to other human members of the family is 31-35% at the amino acid level. We believe this gene to be human TASK-3, the ortholog of the recently reported rat TASK-3 gene: amino acid identity between the two is 74%. 'Taqman' mRNA analysis demonstrated a very specific tissue distribution pattern, showing human TASK-3 mRNA to be localised largely in the cerebellum, in contrast rat TASK-3 was reported to be widely distributed. We have shown by radiation hybrid mapping that human TASK-3 can be assigned to chromosome 8q24.3. Human TASK-3 was demonstrated to endow Xenopus oocytes with a negative resting membrane potential through the presence of a large K(+) selective conductance. TASK-3 is inhibited by extracellular acidosis with a mid-point of inhibition around pH 6. 5, supporting the predictions from the sequence data that this is a third human TASK (TWIK-related acid sensitive K(+) channel) gene.

Amino Acid Sequence↗
PubMed2001

Molecular physiology of oxygen-sensitive potassium channels.

Physiological adaptation to acute hypoxia involves oxygen-sensing by a variety of specialized cells including carotid body type I cells, pulmonary neuroepithelial body cells, pulmonary artery myocytes and foetal adrenomedullary chromaffin cells. Hypoxia induces depolarization by closing a specific set of potassium channels and triggers cellular responses. Molecular biology strategies have recently allowed the identification of the K+ channel subunits expressed in these specialized cells. Several voltage-gated K+ channel subunits comprising six transmembrane segments and a single pore domain (Kv1.2, Kv1.5, Kv2.1, Kv3.1, Kv3.3, Kv4.2 and Kv9.3) are reversibly blocked by hypoxia when expressed in heterologous expression systems. Additionally, the background K+ channel subunit TASK-1, which comprises four transmembrane segments and two pore domains, is also involved in both oxygen- and acid-sensing in peripheral chemoreceptors. Progress is currently being made to identify the oxygen sensors. Regulatory beta subunits may play an important role in the modulation of Kv channel subunits by oxygen.

Adaptation, Physiological↗
PubMed2003

Cellular localization of TWIK-1, a two-pore-domain potassium channel in the rodent inner ear.

K(+) channels in the inner ear regulate the secretion and homeostasis of K(+), i.e. the flux of K(+) ions required to ensure good mechanosensory transduction. We studied the expression and cellular localization of TWIK-1 and TWIK-2, two-pore-domain K(+) channels responsible for background K(+) currents. Reverse transcription-polymerase chain reaction showed that TWIK-1 mRNA is present in the vestibular end organs, vestibular ganglion and cochlea. In contrast, the TWIK-2 mRNA was not detected in the inner ear. Immunocytochemical experiments using confocal microscopy showed that TWIK-1 is specifically localized in 'non-sensory' cells of the inner ear, in the dark cells of the vestibule and in the strial marginal cells of the cochlea. All of these cell types secrete and regulate the K(+) endolymph production and homeostasis. The labeling was strictly limited to the apical membranes of these cells. TWIK-1 was also detected in the cytoplasm of the large neurons of vestibular ganglion and their fibers. The finding that TWIK-1 is specifically distributed in certain areas of the inner ear suggests that this type of K(+) channel plays a role in the regulation of K(+) homeostasis in dark cells and in strial marginal cells. This role has yet to be identified.

Aging↗
PubMed2006

Voltage-gated and two-pore-domain potassium channels in murine spiral ganglion neurons.

The systematically varied firing features of spiral ganglion neurons provide an excellent model system for the exploration of how graded ion channel distributions can be used to organize neuronal firing across a population of neurons. Elucidating the underlying mechanisms that determine neuronal response properties requires a complete understanding of the combination of ion channels, auxiliary proteins, modulators, and second messengers that form this highly organized system in the auditory periphery. Toward this goal, we built upon previous studies of voltage-gated K+-selective ion channels (Kv), and expanded our analysis to K+-selective leak channels (KCNK), which can play a major role in setting the basic firing characteristics of spiral ganglion neurons. To begin a more comprehensive analysis of Kv and KCNK channels, a screening approach was employed. RT-PCR was utilized to examine gene expression, the major results of which were confirmed with immunocytochemistry. Initial studies validated this approach by accurately detecting voltage-dependent K+ channels that were documented previously in the spiral ganglion. Furthermore, an additional channel type within the Kv3 family, Kv3.3, was identified and further characterized. The major focus of the study, however, was to systematically examine gene expression levels of the KCNK family of K+-selective leak channels. These channel types determine the resting membrane potential which has a major impact on setting the level of neuronal excitation. TWIK-1, TASK-3, TASK-1, and TREK-1 were expressed in the spiral ganglion; TWIK-1 was specifically localized with immunocytochemistry to the neuronal somata and initial processes of spiral ganglion neurons in vitro.

Animals↗
PubMed2004

Genomic structure, cochlear expression, and mutation screening of KCNK6, a candidate gene for DFNA4.

KCNK6 encodes a tandem pore domain potassium channel, TWIK-2, that maps to chromosome 19. Both STS and linkage maps established KCNK6 as a positional candidate gene for DFNA4, a form of autosomal dominant nonsyndromic hereditary hearing loss. Identification and characterization of Kcnk6 expression within the mammalian cochlea established the gene as a functional candidate for DFNA4. Identification of Twik-2 expression in the mouse cochlea was initially established via RT-PCR assay of cochlear RNA. Subsequent immunoblot analysis of cochlear homogenate yielded a distinct 35-kDa band corresponding to the calculated molecular weight of the mouse Twik-2. Immunohistochemical studies localized Twik-2 expression in the cochlea predominantly within the stria vascularis. This vascular tissue borders the cochlear duct and is a critical regulator of potassium concentration in the endolymph. Genomic structure of TWIK-2 was subsequently determined and shown to consist of three coding exons with splice acceptor and donor sites in accordance with the consensus GT-AG rule. Two separate DFNA4 families were screened for KCNK6 sequence alterations. No mutations were found, thus excluding TWIK-2 as the DFNA4 candidate disease gene. Nevertheless, expression of Twik-2 within the stria vascularis suggests a potential role for this protein as one of the terminal components of the potassium ion-recycling pathway that contributes toward its reabsorption into the endolymph.

Animals↗
PubMed1997

Alterations in Ito1, IKr and Ik1 density in the BIO TO-2 strain of syrian myopathic hamsters.

The BIO TO-2 strain of cardiomyopathic hamster provides a model of dilated low output heart failure. The goal of the study was to determine whether changes in potassium currents occur in this model of heart failure. The densities of Ito1, IKr and IK1 in 8-month-old myopathic hamsters were not significantly different from their age-matched controls. The half-maximum activation voltage (V1/2) of IKr and Ito1, as well as the voltage-dependence of Ito1 inactivation were also similar in both groups at 8 months. Ito1 inactivation exhibited a double exponential time-course; the slow component (tau2), but not the rapid component (tau1), was larger in the myopathic animals. The densities of Ito1, IKr and IK1 were not significantly different in the 8- and 10-month-old control animals. However, the densities of Ito1, IKr and IK1 were all significantly lower in the 10-month-old myopathic hamsters relative to the 10-month-old controls. The V1/2 for IKr and Ito1 activation was the same in myopathic and control animals. tau2, but not tau1, of Ito1 inactivation was again larger in the myopathic animals. The voltage-dependence of Ito1 inactivation was shifted slightly, but significantly, positive in the myopathic animals. Lastly, a sustained outwardly rectifying current that activated upon depolarization was found to be larger in the myopathic animals at both 8 and 10 months of age. In conclusion, many of the alterations in potassium current densities in the 10-month-old cardiomyopathic hamsters are qualitatively similar to the changes observed in the failing human heart.

Animals↗
PubMed2003

Modulation of native TREK-1 and Kv1.4 K+ channels by polyunsaturated fatty acids and lysophospholipids.

The modulation of TREK-1 leak and Kv1.4 voltage-gated K+ channels by fatty acids and lysophospholipids was studied in bovine adrenal zona fasciculata (AZF) cells. In whole-cell patch-clamp recordings, arachidonic acid (AA) (1-20 microM) dramatically and reversibly increased the activity of bTREK-1, while inhibiting bKv1.4 current by mechanisms that occurred with distinctly different kinetics. bTREK-1 was also activated by the polyunsaturated cis fatty acid linoleic acid but not by the trans polyunsaturated fatty acid linolelaidic acid or saturated fatty acids. Eicosatetraynoic acid (ETYA), which blocks formation of active AA metabolites, failed to inhibit AA activation of bTREK-1, indicating that AA acts directly. Compared to activation of bTREK-1, inhibition of bKv1.4 by AA was rapid and accompanied by a pronounced acceleration of inactivation kinetics. Cis polyunsaturated fatty acids were much more effective than trans or saturated fatty acids at inhibiting bKv1.4. ETYA also effectively inhibited bKv1.4, but less potently than AA. bTREK-1 current was markedly increased by lysophospholipids including lysophosphatidyl choline (LPC) and lysophosphatidyl inositol (LPI). At concentrations from 1-5 microM, LPC produced a rapid, transient increase in bTREK-1 that peaked within one minute and then rapidly desensitized. The transient lysophospholipid-induced increases in bTREK-1 did not require the presence of ATP or GTP in the pipette solution. These results indicate that the activity of native leak and voltage-gated K+ channels are directly modulated in reciprocal fashion by AA and other cis unsaturated fatty acids. They also show that lysophospholipids enhance bTREK-1, but with a strikingly different temporal pattern. The modulation of native K+ channels by these agents differs from their effects on the same channels expressed in heterologous cells, highlighting the critical importance of auxiliary subunits and signaling. Finally, these results reveal that AZF cells express thousands of bTREK-1 K+ channels that lie dormant until activated by metabolites including phospholipase A2 (PLA2)-generated fatty acids and lysophospholipids. These metabolites may alter the electrical and secretory properties of AZF cells by modulating bTREK-1 and bKv1.4 K+ channels.

Animals↗