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.     

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.     

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 pK _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.     

K+ channel modulation in arterial smooth muscle

Potassium channels play an essential role in the membrane potential of arterial smooth muscle, and also in regulating contractile tone. Four types of K⁺ channel have been described in vascular smooth muscle: Voltage-activated K⁺ channels (K_V) are encoded by the Kv gene family, Ca²⁺-activated K⁺ channels (BK_Ca) are encoded by the slogene, inward rectifiers (K_IR) by Kir2.0, and ATP-sensitive K⁺ channels (K_ATP) by Kir6.0 and sulphonylurea receptor genes. In smooth muscle, the channel subunit genes reported to be expressed are: Kv1.0, Kv1.2, Kv1.4–1.6, Kv2.1, Kv9.3, Kvβ1–β4, slo α and β, Kir2.1, Kir6.2, and SUR1 and SUR2. Arterial K⁺ channels are modulated by physiological vasodilators, which increase K⁺ channel activity, and vasoconstrictors, which decrease it. Several vasodilators acting at receptors linked to cAMP-dependent protein kinase activate K_ATP channels. These include adenosine, calcitonin gene-related peptide, and β-adrenoceptor agonists. β-adrenoceptors can also activate BK_Ca and K_V channels. Several vasoconstrictors that activate protein kinase C inhibit K_ATP channels, and inhibition of BK_Ca and K_V channels through PKC has also been described. Activators of cGMP-dependent protein kinase, in particular NO, activate BK_Ca channels, and possibly K_ATP channels. Hypoxia leads to activation of K_ATP channels, and activation of BK_Ca channels has also been reported. Hypoxic pulmonary vasoconstriction involves inhibition of K_V channels. Vasodilation to increased external K⁺ involves K_IR channels. Endothelium-derived hyperpolarizing factor activates K⁺ channels that are not yet clearly defined. Such K⁺ channel modulations, through their effects on membrane potential and contractile tone, make important contributions to the regulation of blood flow.     

AMP-activated protein kinase regulates hERG potassium channel

Besides their role in cardiac repolarization, human ether-a-go-go-related gene potassium (hERG) channels are expressed in several tumor cells including rhabdomyosarcoma cells. The channels foster cell proliferation. Ubiquitously expressed AMP-dependent protein kinase (AMPK) is a serine-/threonine kinase, stimulating energy-generating and inhibiting energy-consuming processes thereby helping cells survive periods of energy depletion. AMPK has previously been shown to regulate Na⁺/K⁺ ATPase, Na⁺/Ca²⁺ exchangers, Ca²⁺ channels and K⁺ channels. The present study tested whether AMPK regulates hERG channel activity. Wild type AMPK (α1β1γ1), constitutively active ^γR70QAMPK (α1β1γ1(R70Q)), or catalytically inactive ^αK45RAMPK (α1(K45R)β1γ1) were expressed in Xenopus oocytes with hERG. Tail currents were determined as a measure of hERG channel activity by two-electrode-voltage clamp. hERG membrane abundance was quantified by chemiluminescence and visualized by immunocytochemistry and confocal microscopy. Moreover, hERG currents were measured in RD rhabdomyosarcoma cells after pharmacological modification of AMPK activity using the patch clamp technique. Coexpression of wild-type AMPK and of constitutively active ^γR70QAMPK significantly downregulated the tail currents in hERG-expressing Xenopus oocytes. Pharmacological activation of AMPK with AICAR or with phenformin inhibited hERG currents in Xenopus oocytes, an effect abrogated by AMPK inhibitor compound C. ^γR70QAMPK enhanced the Nedd4-2-dependent downregulation of hERG currents. Coexpression of constitutively active ^γR70QAMPK decreased membrane expression of hERG in Xenopus oocytes. Compound C significantly enhanced whereas AICAR tended to inhibit hERG currents in RD rhabdomyosarcoma cells. AMPK is a powerful regulator of hERG-mediated currents in both, Xenopus oocytes and RD rhabdomyosarcoma cells. AMPK-dependent regulation of hERG may be particularly relevant in cardiac hypertrophy and tumor growth.     

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.     

Functional modulation of the ATP-sensitive potassium channel by extracellular signal-regulated kinase-mediated phosphorylation

ATP-sensitive potassium (K(ATP)) channels play an important role in controlling insulin secretion and vascular tone as well as protecting neurons under metabolic stress. We have previously demonstrated that stimulation of the K(ATP) channel by nitric oxide (NO) requires activation of Ras- and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) family. However, the mechanistic link between ERK and the K(atp) channel remained unknown. To investigate how ERK modulates the function of K(ATP) channels, we performed single-channel recordings in combination with site-directed mutagenesis. The Kir6.2/SUR1 channel, a neuronal K(ATP) channel isoform, was expressed in human embryonic kidney (HEK) 293 cells by transient transfection. Direct application of the activated ERK2 to the cytoplasmic surface of excised, inside-out patches markedly enhanced the single-channel activity of Kir6.2/SUR1 channels. The normalized open probability (NPo) and opening frequency were significantly increased, whereas the mean closed duration was reduced. The single-channel conductance level was not affected. The ERK2-induced stimulation of Kir6.2/SUR1 channels was prevented by heat-inactivation of the enzyme. Furthermore, alanine substitutions of T341 and S385 to disrupt the potential ERK phosphorylation sites present in the Kir6.2 subunit significantly abrogated the stimulatory effects of ERK2, while aspartate substitutions of T341 and S385 to mimic the (negative) charge effect of phosphorylation rendered a small yet significant reduction in the ATP sensitivity of the channel. Taken together, here we report for the first time that ERK2/MAPK activates neuronal-type K(ATP) channels, and this stimulation requires ERK phosphorylation of the Kir6.2 subunit at T341 and S385 residues. The ERK2-induced K(ATP) channel stimulation can be accounted for by changes in channel gating that destabilize the closed states and by reduction in the ATP sensitivity. As Kir6.2 is the pore-forming subunit of K(ATP) channels, ERK2-mediated phosphorylation may represent a common mechanism for K(ATP) channel regulation in different tissues.     

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. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users. K. H. Yuill, P. J. Stansfeld and I. Ashmole contributed equally to this paper.     

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.     

Serotonin and protein kinase C modulation of a rat brain inwardly rectifying K+ channel expressed inXenopus oocytes

InXenopus laevis oocytes injected with rat brain poly(A)⁺ RNA, perfusion with a high-K⁺ solution (96 mM KCl) generated an inward current (I _HK) which was absent in water-injected oocytes. Part ofI _HK was blocked by low concentrations of Ba²⁺ (half-maximal inhibitory concentration, IC₅₀: 4.2 ± 0.5 M). When serotonin (5-HT) was applied to these oocytes a transient inward oscillating Cl^– current arising from activation of Ca²⁺ -dependent Cl^– channels,I _Cl(Ca), was observed. When this response decayed, a 30% reduction ofI _HK could be detected. Electrophysiological characterization of the K⁺ channel down-modulated by 5-HT revealed that it is an inward rectifier. Antisense suppression experiments revealed that the 5-HT_2C receptor mediates the down-modulatory effect of 5-HT. The nature of the modulatory pathway was investigated by application of phorbol esters and intracellular injection of protein kinase C (PKC) inhibitors, ethylenebis (oxonitrilo)tetraacetate (EGTA) and inositol 1, 4, 5-trisphosphate. The results demonstrate that PKC is responsible for the down-modulatory effect.     

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.     

Possible role of potassium channel, big K in etiology of schizophrenia

Schizophrenia (SZ), a common severe mental disorder, affecting about 1% of the world population. However, the etiology of SZ is still largely unknown. It is believed that molecules that are in an association with the etiology and pathology of SZ are neurotransmitters including dopamine, 5-HT and gamma-aminobutyric acid (GABA). But several lines of evidences indicate that potassium large conductance calcium-activated channel, known as BK channel, is likely to be included. BK channel belongs to a group of ion channels that plays an important role in regulating neuronal excitability and transmitter releasing. Its involvement in SZ emerges as a great interest. For example, commonly used neuroleptics, in clinical therapeutic concentrations, alter calcium-activated potassium conductance in central neurons. Diazoxide, a potassium channel opener/activator, showed a significant superiority over haloperidol alone in the treatment of positive and general psychopathology symptoms in SZ. Additionally, estrogen, which regulates the activity of BK channel, modulates dopaminergic D2 receptor and has an antipsychotic-like effect. Therefore, we hypothesize that BK channel may play a role in SZ and those agents, which can target either BK channel functions or its expression may contribute to the therapeutic actions of SZ treatment.