Potassium Channel TASK-1 Research Articles

The two-pore domain potassium channel TASK3 functionally impacts glioma cell death

Two-pore domain K(+) channels, a recently discovered family of ion channels with a unique membrane topology, have been shown to be critically involved in cell death. We here address the functional role of TASK3 (TWIK-related acid-sensitive K(+) channel, KCNK9) in human glioblastoma in vitro and in vivo. Human glioma cell lines (n = 5) as well as glioma specimens (n = 5) constitutively express TASK3 mRNA and protein. The functional impact of the potassium channel on cell survival was investigated using a medium with high (25 mM) extracellular potassium over 7 days. Using flow cytometric assessment, we show that under these culture conditions 97 +/- 0.76% of all glioma cells survived. Application of the TASK channel opener isoflurane (1 vol%) resulted in a 30 +/- 4% reduction of cell survival in different glioma cell lines. Simultaneous application of isoflurane and the TASK channel blockers bupivacaine (20 microM) and spermine (500 microM) completely reversed this effect. Our results demonstrate the expression of TASK3 in glioma cells in vitro and in vivo and provide a direct link between the TASK3 channel function and glioma cell survival. This implies that TASK3 channels may possibly represent a novel molecular target for the treatment of this type of cancer.

A Di‐Acidic Sequence Motif Enhances the Surface Expression of the Potassium Channel TASK‐3

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

The selectivity, voltage-dependence and acid sensitivity of the tandem pore potassium channel TASK-1: contributions of the pore domains.

We have investigated the contribution to ionic selectivity of residues in the selectivity filter and pore helices of the P1 and P2 domains in the acid sensitive potassium channel TASK-1. We used site directed mutagenesis and electrophysiological studies, assisted by structural models built through computational methods. We have measured selectivity in channels expressed in Xenopus oocytes, using voltage clamp to measure shifts in reversal potential and current amplitudes when Rb+ or Na+ replaced extracellular K+. Both P1 and P2 contribute to selectivity, and most mutations, including mutation of residues in the triplets GYG and GFG in P1 and P2, made channels non-selective. We interpret the effects of these--and of other mutations--in terms of the way the pore is likely to be stabilised structurally. We show also that residues in the outer pore mouth contribute to selectivity in TASK-1. Mutations resulting in loss of selectivity (e.g. I94S, G95A) were associated with slowing of the response of channels to depolarisation. More important physiologically, pH sensitivity is also lost or altered by such mutations. Mutations that retained selectivity (e.g. I94L, I94V) also retained their response to acidification. It is likely that responses both to voltage and pH changes involve gating at the selectivity filter.

Anandamide and NADA bi‐directionally modulate presynaptic Ca2+ levels and transmitter release in the hippocampus

Inhibitory CB(1) cannabinoid receptors and excitatory TRPV(1) vanilloid receptors are abundant in the hippocampus. We tested if two known hybrid endocannabinoid/endovanilloid substances, N-arachidonoyl-dopamine (NADA) and anandamide (AEA), presynapticaly increased or decreased intracellular calcium level ([Ca(2+)](i)) and GABA and glutamate release in the hippocampus. Resting and K(+)-evoked levels of [Ca(2+)](i) and the release of [(3)H]GABA and [(3)H]glutamate were measured in rat hippocampal nerve terminals. NADA and AEA per se triggered a rise of [Ca(2+)](i) and the release of both transmitters in a concentration- and external Ca(2+)-dependent fashion, but independently of TRPV(1), CB(1), CB(2), or dopamine receptors, arachidonate-regulated Ca(2+)-currents, intracellular Ca(2+) stores, and fatty acid metabolism. AEA was recently reported to block TASK-3 potassium channels thereby depolarizing membranes. Common inhibitors of TASK-3, Zn(2+), Ruthenium Red, and low pH mimicked the excitatory effects of AEA and NADA, suggesting that their effects on [Ca(2+)](i) and transmitter levels may be attributable to membrane depolarization upon TASK-3 blockade. The K(+)-evoked Ca(2+) entry and Ca(2+)-dependent transmitter release were inhibited by nanomolar concentrations of the CB(1) receptor agonist WIN55212-2; this action was sensitive to the selective CB(1) receptor antagonist AM251. However, in the low micromolar range, WIN55212-2, NADA and AEA inhibited the K(+)-evoked Ca(2+) entry and transmitter release independently of CB(1) receptors, possibly through direct Ca(2+) channel blockade. We report here for hybrid endocannabinoid/endovanilloid ligands novel dual functions which were qualitatively similar to activation of CB(1) or TRPV(1) receptors, but were mediated through interactions with different targets.

The role of 14-3-3 proteins in the trafficking of the potassium channels TASK-1 and TASK-3

The role of 14-3-3 proteins in the trafficking of the potassium channels TASK-1 and TASK-3

The acid-sensitive potassium channel TASK-1 in rat cardiac muscle

The outward current flowing through the two-pore domain acid-sensitive potassium channel TASK-1 (I(TASK)) and its inhibition via alpha1-adrenergic receptors was studied in rat ventricular cardiomyocytes. Quantitative RT-PCR experiments were carried out with mRNA from rat heart. Patch-clamp recordings were performed in isolated rat cardiomyocytes. TASK-1 and other K+ channels were expressed in Xenopus oocytes to study the pharmacological properties of a new TASK-1 channel blocker, A293. TASK-1 channels were found to be strongly expressed in rat heart. Analysis of the sensitivity of various K+ channels to A293 in Xenopus oocytes showed that at low concentrations A293 was a selective blocker of TASK-1 channels. I(TASK) in rat cardiomyocytes was dissected by application of A293 and by extracellular acidification to pH 6.0; it had an amplitude of approximately 0.30 pA/pF at +30 mV. Application of 200 nM A293 increased action potential duration (APD(50)) by 31+/-3% at a stimulation rate of 4 Hz. The plausibility of the effects of A293 on APD50 was checked with a mathematical action potential model. Application of the alpha1-adrenergic agonist methoxamine inhibited I(TASK) in Xenopus oocytes co-injected with cRNA for TASK-1 and alpha1A-receptors. In cardiomyocytes, methoxamine inhibited an outward current with characteristics similar to I(TASK). This effect was abolished in the presence of the alpha1A-antagonist 5-methyl-urapidil. Our results suggest that in rat cardiomyocytes I(TASK) makes a substantial contribution to the outward current flowing in the plateau range of potentials and that this current component can be inhibited via alpha1A-adrenergic receptors.

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.

The selectivity filter of the tandem pore potassium channel TASK-1 and its pH-sensitivity and ionic selectivity.

We have studied pH sensitivity and ionic selectivity of the tandem pore K(+) channel TASK-1 heterologously expressed in Xenopus oocytes. We fit pH sensitivity assuming that only one of the two residues H98 need be protonated for channels to be shut. The effect of protons was weakly voltage dependent with a p K(a) of 6.02 at +40 mV. Replacement of His (H98D, H98N) reduced pH sensitivity but did not abolish it. Use of a concatameric channel permitted replacement of one His residue only; this concatamer was fully pH-sensitive. Increasing the number of His residues to 4 (mutant D204H) abolished pH sensitivity over the physiological range. The implication that D204 plays a role in pH-sensitivity was confirmed by the finding that pH sensitivity over the physiological range was also abolished in the mutant D204N. Ionic selectivity was also altered in D204H, D204N and H98D mutants. P(Rb)/ P(K) was increased from 0.80+/-0.04 (n=19) in wild type to 1.06+/-0.04 (n=19) in D204H. H98D, D204H and D204N were permeable to Na(+) with P(Na)/ P(K)=0.39+/-0.03 (n=14) in H98D, 0.64+/-0.04 (n=18) in D204H and 0.33+/-0.07 (n=3) in D204N. Thus, the arrangement of ring of residues HDHD appears to optimise both pH sensitivity and ionic selectivity.

Cardiorespiratory neurons of the rat ventrolateral medulla contain TASK-1 and TASK-3 channel mRNA.

The potassium channels TASK-1 and TASK-3 are neuronal 'leak' channels that are inhibited by extracellular acidification and numerous neurotransmitters. Here we tested whether in the rat TASK-1 and TASK-3 mRNAs are present in ventrolateral medulla neurons that regulate respiration or circulation (C1 adrenergic neurons). The mRNAs were identified by in situ hybridization. Respiratory neurons were identified by anatomical markers (neurokinin-1 receptors, NK1R, or somatostatin) or directly by juxtacellular labeling of bulbospinal neurons. C1 neurons were identified by the presence of tyrosine-hydroxylase. TASK-1 and TASK-3 transcripts were present in all hypoglossal and facial motor neurons, in most C1 cells (85-95%), in most small and large NK1R-ir neurons (>90%) of the ventral respiratory group (VRG) and in all the inspiratory-augmenting bulbospinal neurons of the rostral ventral respiratory group. In conclusion, TASK channels are expressed by respiratory cells with putative rhythmogenic function and by premotor and motor neurons. TASK channels presumably mediate excitatory effects of numerous transmitters on these neurons and may contribute to their pH-sensitivity.

Oncogenic potential of TASK3 (Kcnk9) depends on K+ channel function.

TASK3 gene (Kcnk9) is amplified and overexpressed in several types of human carcinomas. In this report, we demonstrate that a point mutation (G95E) within the consensus K+ filter of TASK3 not only abolished TASK3 potassium channel activity but also abrogated its oncogenic functions, including proliferation in low serum, resistance to apoptosis, and promotion of tumor growth. Furthermore, we provide evidence that TASK3G95E is a dominant-negative mutation, because coexpression of the wild-type and the mutant TASK3 resulted in inhibition of K+ current of wild-type TASK3 and its tumorigenicity in nude mice. These results establish a direct link between the potassium channel activity of TASK3 and its oncogenic functions and imply that blockers for this potassium channel may have therapeutic potential for the treatment of cancers.

Ruthenium red inhibits TASK-3 potassium channel by interconnecting glutamate 70 of the two subunits.

TASK channels are highly pH-sensitive two-pore-domain background potassium channels expressed in the central nervous system and in some peripheral tissues. Their current can be regulated by receptor-mediated activation of phospholipase C and also by pharmacological means. We have reported previously that the cationic dye, ruthenium red (RR), inhibited homodimeric TASK-3 (kcnk9), whereas TASK-1 (kcnk3) homodimer and TASK-1/TASK-3 heterodimer were not affected by this compound. In the present study, we identify the molecular determinant of the RR-mediated TASK-3 inhibition. Mutation of the negatively charged Glu 70 of TASK-3 to Arg (E70R) or Cys (E70C) abolished the inhibitory action of RR. When two TASK-3 coding sequences were concatenated, and the entire homodimer was expressed as a single polypeptide chain, the resulting tandem channel was also sensitive to RR. Mutation of Glu 70 in either the first (E70R) or the second (E465R) linked subunit prevented the action of the inhibitor. Together with the Hill coefficient of 1.0 for TASK-3 inhibition, these data indicate that simultaneous binding of one polycationic RR molecule to Glu 70 of both subunits is required for the inhibitory action. The pivotal role of this residue in the inhibitory mechanism of RR is confirmed by the gained RR sensitivity of the mutant TASK-1 in which Lys 70 was changed to Glu. Our results indicate that RR inhibits TASK-3 by tethering its two subunits and identify amino acid 70 as a possible target for designing selective inhibitors against the different TASK channels.

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 14-3-3zeta cRNA increased TASK-1 current in Xenopus oocytes by about 70 %. After co-transfection in HEK293 cells, TASK-1 and 14-3-3zeta (but not TASK-1DeltaC5 and 14-3-3zeta) 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 role of Ca2+ stores in the muscarinic inhibition of the K+ current IK(SO) in neonatal rat cerebellar granule cells.

Cerebellar granule neurons (CGNs) possess a standing outward potassium current (IK(SO)) which shares many similarities with current through the two-pore domain potassium channel TASK-1 and which is inhibited following activation of muscarinic acetylcholine receptors. The action of muscarine on IK(SO) was unaffected by the M2 receptor antagonist methoctramine (100 nM) but was blocked by the M3 antagonist zamifenacin, which, at a concentration of 100 nM, shifted the muscarine concentration-response curve to the right by around 50-fold. Surprisingly, M3 receptor activation rarely produced a detectable increase in [Ca2+]i unless preceded by depolarization of the cells with 25 mM K+. Experiments with thapsigargin and ionomycin suggested that the endoplasmic reticulum Ca2+ stores in CGNs were depleted at rest. In contrast, cerebellar glial cells in the same fields of cells possessed substantial endoplasmic reticulum Ca2+ stores at rest. Pretreatment of the cells with BAPTA AM, thapsigargin or the phospholipase C (PLC) inhibitor U-73122 all blocked the muscarine-induced Ca2+ signal but had little or no effect on muscarinic inhibition of IK(SO). Raising [Ca2+]i directly with ionomycin caused a small but significant inhibition of IK(SO). It is concluded that muscarine acts on M3 muscarinic acetylcholine receptors both to inhibit IK(SO) and to mobilize Ca2+ from intracellular stores in CGNs. While the mobilization of Ca2+ occurs through activation of PLC, this does not seem to be the primary mechanism underlying muscarinic inhibition of IK(SO).

A functional role for the two-pore domain potassium channel TASK-1 in cerebellar granule neurons

Cerebellar granule neurons (CGNs) are one of the most populous cells in the mammalian brain. They express an outwardly rectifying potassium current, termed a “standing-outward” K + current, or IK SO , which does not inactivate. It is active at the resting potential of CGNs, and blocking IK SO leads to cell depolarization. IK SO is blocked by Ba 2+ ions and is regulated by activation of muscarinic M 3 receptors, but it is insensitive to the classical broad-spectrum potassium channel blocking drugs 4-aminopyridine and tetraethylammonium ions. The molecular nature of this important current has yet to be established, but in this study, we provide strong evidence to suggest that IK SO is the functional correlate of the recently identified two-pore domain potassium channel TASK-1. We show that IK SO has no threshold for activation by voltage and that it is blocked by small extracellular acidifications. Both of these are properties that are diagnostic of TASK-1 channels. In addition, we show that TASK-1 currents expressed in Xenopus oocytes are inhibited after activation of endogenous M 3 muscarinic receptors. Finally, we demonstrate that mRNA for TASK-1 is found in CGNs and that TASK-1 protein is expressed in CGN membranes. This description of a functional two-pore domain potassium channel in the mammalian central nervous system indicates its physiological importance in controlling cell excitability and how agents that modify its activity, such as agonists at G protein-coupled receptors and hydrogen ions, can profoundly alter both the neuron's resting potential and its excitability.

A functional role for the two-pore domain potassium channel TASK-1 in cerebellar granule neurons

Cerebellar granule neurons (CGNs) are one of the most populous cells in the mammalian brain. They express an outwardly rectifying potassium current, termed a "standing-outward" K(+) current, or IK(SO), which does not inactivate. It is active at the resting potential of CGNs, and blocking IK(SO) leads to cell depolarization. IK(SO) is blocked by Ba(2+) ions and is regulated by activation of muscarinic M(3) receptors, but it is insensitive to the classical broad-spectrum potassium channel blocking drugs 4-aminopyridine and tetraethylammonium ions. The molecular nature of this important current has yet to be established, but in this study, we provide strong evidence to suggest that IK(SO) is the functional correlate of the recently identified two-pore domain potassium channel TASK-1. We show that IK(SO) has no threshold for activation by voltage and that it is blocked by small extracellular acidifications. Both of these are properties that are diagnostic of TASK-1 channels. In addition, we show that TASK-1 currents expressed in Xenopus oocytes are inhibited after activation of endogenous M(3) muscarinic receptors. Finally, we demonstrate that mRNA for TASK-1 is found in CGNs and that TASK-1 protein is expressed in CGN membranes. This description of a functional two-pore domain potassium channel in the mammalian central nervous system indicates its physiological importance in controlling cell excitability and how agents that modify its activity, such as agonists at G protein-coupled receptors and hydrogen ions, can profoundly alter both the neuron's resting potential and its excitability.