A neuronal two P domain K+ channel stimulated by arachidonic acid and polyunsaturated fatty acids.

TWIK-1, TREK-1 and TASK K+ channels comprise a class of pore-forming subunits with four membrane-spanning segments and two P domains. Here we report the cloning of TRAAK, a 398 amino acid protein which is a new member of this mammalian class of K+ channels. Unlike TWIK-1, TREK-1 and TASK which are widely distributed in many different mouse tissues, TRAAK is present exclusively in brain, spinal cord and retina. Expression of TRAAK in Xenopus oocytes and COS cells induces instantaneous and non-inactivating currents that are not gated by voltage. These currents are only partially inhibited by Ba2+ at high concentrations and are insensitive to the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine and Cs+. A particularly salient feature of TRAAK is that they can be stimulated by arachidonic acid (AA) and other unsaturated fatty acids but not by saturated fatty acids. These channels probably correspond to the functional class of fatty acid-stimulated K+ currents that recently were identified in native neuronal cells but have not yet been cloned. These TRAAK channels might be essential in normal physiological processes in which AA is known to play an important role, such as synaptic transmission, and also in pathophysiological processes such as brain ischemia. TRAAK channels are stimulated by the neuroprotective drug riluzole.     

TWIK-2, an inactivating 2P domain K+ channel

We cloned human and rat TWIK-2 and expressed this novel 2P domain K(+) channel in transiently transfected COS cells. TWIK-2 is highly expressed in the gastrointestinal tract, the vasculature, and the immune system. Rat TWIK-2 currents are about 15 times larger than human TWIK-2 currents, but both exhibit outward rectification in a physiological K(+) gradient and mild inward rectification in symmetrical K(+) conditions. TWIK-2 currents are inactivating at depolarized potentials, and the kinetic of inactivation is highly temperature-sensitive. TWIK-2 shows an extremely low conductance, which prevents the visualization of discrete single channel events. The inactivation and rectification are intrinsic properties of TWIK-2 channels. In a physiological K(+) gradient, TWIK-2 is half inhibited by 0.1 mm Ba(2+), quinine, and quinidine. Finally, cysteine 53 in the M1P1 external loop is required for functional expression of TWIK-2 but is not critical for subunit self-assembly. TWIK-2 is the first reported 2P domain K(+) channel that inactivates. The base-line, transient, and delayed activities of TWIK-2 suggest that this novel 2P domain K(+) channel may play an important functional role in cell electrogenesis.     

G protein betagamma11 complex translocation is induced by Gi, Gq and Gs coupling receptors and is regulated by the alpha subunit type

G protein activation by Gi/Go coupling M2 muscarinic receptors, Gq coupling M3 receptors and Gs coupling beta2 adrenergic receptors causes rapid reversible translocation of the G protein gamma11 subunit from the plasma membrane to the Golgi complex. Co-translocation of the beta1 subunit suggests that gamma11 translocates as a betagamma complex. Pertussis toxin ADP ribosylation of the alphai subunit type or substitution of the C terminal domain of alphao with the corresponding region of alphas inhibits gamma11 translocation demonstrating that alpha subunit interaction with a receptor and its activation are requirements for the translocation. The rate of gamma11 translocation is sensitive to the rate of activation of the G protein alpha subunit. alpha subunit types that show high receptor activated rates of guanine nucleotide exchange in vitro support high rates of gamma11 translocation compared to alpha subunit types that have a relatively lower rate of guanine nucleotide exchange. The results suggest that the receptor induced translocation of gamma11 is controlled by the rate of cycling of the G protein through active and inactive forms. They also demonstrate that imaging of gamma11 translocation can be used as a non-invasive tool to measure the relative activities of wild type or mutant receptor and alpha subunit types in a live cell.     

TASK-1, a two-pore domain K+ channel, is modulated by multiple neurotransmitters in motoneurons

Inhibition of "leak" potassium (K+) channels is a widespread CNS mechanism by which transmitters induce slow excitation. We show that TASK-1, a two pore domain K+ channel, provides a prominent leak K+ current and target for neurotransmitter modulation in hypoglossal motoneurons (HMs). TASK-1 mRNA is present at high levels in motoneurons, including HMs, which express a K+ current with pH- and voltage-dependent properties virtually identical to those of the cloned channel. This pH-sensitive K+ channel was fully inhibited by serotonin, norepinephrine, substance P, thyrotropin-releasing hormone, and 3,5-dihydroxyphenylglycine, a group I metabotropic glutamate receptor agonist. The neurotransmitter effect was entirely reconstituted in HEK 293 cells coexpressing TASK-1 and the TRH-R1 receptor. Given its expression patterns and the widespread prevalence of this neuromodulatory mechanism, TASK-1 also likely supports this action in other CNS neurons.     

Developmental expression of a functional TASK-1 2P domain K+ channel in embryonic chick heart

Background

Background K⁺ channels are the principal determinants of the resting membrane potential (RMP) in cardiac myocytes and thus, influence the magnitude and time course of the action potential (AP).

Methods

RT-PCR and in situ hybridization are used to study the distribution of TASK-1 and whole-cell patch clamp technique is employed to determine the functional expression of TASK-1 in embryonic chick heart.

Results

Chicken TASK-1 was expressed in the early tubular heart, then substantially decreased in the ventricles by embryonic day 5 (ED5), but remained relatively high in ED5 and ED11 atria. Unlike TASK-1, TASK-3 was uniformly expressed in heart at all developmental stages. In situ hybridization studies further revealed that TASK-1 was expressed throughout myocardium at Hamilton-Hamburger stages 11 and 18 (S11 &; S18) heart. In ED11 heart, TASK-1 expression was more restricted to atria. Consistent with TASK-1 expression data, patch clamp studies indicated that there was little TASK-1 current, as measured by the difference currents between pH 8.4 and pH 7.4, in ED5 and ED11 ventricular myocytes. However, TASK-1 current was present in the early embryonic heart and ED11 atrial myocytes. TASK-1 currents were also identified as 3 μM anandamide-sensitive currents. 3 μM anandamide reduced TASK-1 currents by about 58% in ED11 atrial myocytes. Zn²⁺ (100 μM) which selectively inhibits TASK-3 channel at this concentration had no effect on TASK currents. In ED11 ventricle where TASK-1 expression was down-regulated, I_K1 was about 5 times greater than in ED11 atrial myocytes.

Conclusion

Functional TASK-1 channels are differentially expressed in the developing chick heart and TASK-1 channels contribute to background K⁺ conductance in the early tubular embryonic heart and in atria. TASK-1 channels act as a contributor to background K⁺ current to modulate the cardiac excitability in the embryonic heart that expresses little I_K1.     

ATP-dependent activation of the intermediate conductance, Ca2+-activated K+ channel, hIK1, is conferred by a C-terminal domain

We previously demonstrated that hIK1 is activated directly by ATP in excised, inside-out patches in a protein kinase A inhibitor 5-24 dependent manner, suggesting a role for phosphorylation in the regulation of this Ca(2+)-dependent channel. However, mutation of the single consensus cAMP-dependent protein kinase phosphorylation site (S334A) failed to modify the response of hIK1 to ATP (Gerlach, A. C., Gangopadhyay, N. N., and Devor, D. C. (2000) J. Biol. Chem. 275, 585-598). Here we demonstrate that ATP does not similarly activate the highly homologous Ca(2+)-dependent K(+) channels, hSK1, rSK2, and rSK3. To define the region of hIK1 responsible for the ATP-dependent regulation, we generated a series of hIK1 truncations and hIK1/rSK2 chimeras. ATP did not activate a chimera containing the N terminus plus S1-S4 from hIK1. In contrast, ATP activated a chimera containing the hIK1 C-terminal amino acids His(299)-Lys(427). Furthermore, truncation of hIK1 at Leu(414) resulted in an ATP-dependent channel, whereas larger truncations of hIK1 failed to express. Additional hIK1/rSK2 chimeras defined the minimal region of hIK1 required to confer complete ATP sensitivity as including amino acids Arg(355)-Ala(413). An alanine scan of all non-conserved serines and threonines within this region failed to alter the response of hIK1 to ATP, suggesting that hIK1 itself is not directly phosphorylated. Additionally, substitution of amino acids Arg(355)-Met(368) of hIK1 into the corresponding region of rSK2 resulted in an ATP-dependent activation, which was approximately 50% of that of hIK1. These results demonstrate that amino acids Arg(355)-Ala(413) within the C terminus of hIK1 confer sensitivity to ATP. Finally, we demonstrate that the ATP-dependent phosphorylation of hIK1 or an associated protein is independent of Ca(2+).     

ATP-dependent activation of the intermediate conductance, Ca2+-activated K+ channel, hIK1, is conferred by a C-terminal domain.

We previously demonstrated that hIK1 is activated directly by ATP in excised, inside-out patches in a protein kinase A inhibitor 5-24 dependent manner, suggesting a role for phosphorylation in the regulation of this Ca(2+)-dependent channel. However, mutation of the single consensus cAMP-dependent protein kinase phosphorylation site (S334A) failed to modify the response of hIK1 to ATP (Gerlach, A. C., Gangopadhyay, N. N., and Devor, D. C. (2000) J. Biol. Chem. 275, 585-598). Here we demonstrate that ATP does not similarly activate the highly homologous Ca(2+)-dependent K(+) channels, hSK1, rSK2, and rSK3. To define the region of hIK1 responsible for the ATP-dependent regulation, we generated a series of hIK1 truncations and hIK1/rSK2 chimeras. ATP did not activate a chimera containing the N terminus plus S1-S4 from hIK1. In contrast, ATP activated a chimera containing the hIK1 C-terminal amino acids His(299)-Lys(427). Furthermore, truncation of hIK1 at Leu(414) resulted in an ATP-dependent channel, whereas larger truncations of hIK1 failed to express. Additional hIK1/rSK2 chimeras defined the minimal region of hIK1 required to confer complete ATP sensitivity as including amino acids Arg(355)-Ala(413). An alanine scan of all non-conserved serines and threonines within this region failed to alter the response of hIK1 to ATP, suggesting that hIK1 itself is not directly phosphorylated. Additionally, substitution of amino acids Arg(355)-Met(368) of hIK1 into the corresponding region of rSK2 resulted in an ATP-dependent activation, which was approximately 50% of that of hIK1. These results demonstrate that amino acids Arg(355)-Ala(413) within the C terminus of hIK1 confer sensitivity to ATP. Finally, we demonstrate that the ATP-dependent phosphorylation of hIK1 or an associated protein is independent of Ca(2+).     

Regulation of the epithelial Na+ channel by the RH domain of G protein-coupled receptor kinase, GRK2, and Galphaq/11

The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with α-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na(+) absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant ((K220R)GRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (ΔRH-GRK2) or a GRK2 mutant that cannot interact with Gαq/11/14 ((D110A)GRK2), increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the α-subunits of Gq/11. We further found that expression of constitutively active Gαq/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against Gαq/11 increases ENaC activity. The effect of Gαq on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 α-subunits regulate the activity ENaC.     

Effect of lysophospholipids, arachidonic acid and other fatty acids on regulation of Ca2+ transport in permeabilized pancreatic islets.

The immediate reaction products of PLA2-mediated hydrolysis of phospholipids were tested for their ability to induce Ca2+ mobilization from internal stores in permeabilized ob/ob mouse pancreatic islets. Lysophospholipids and unsaturated fatty acids increased the free Ca2+ concentration in the incubation medium of permeabilized ob/ob mouse pancreatic islets. The potency of the lysophospholipids decreased in the following order: lysophosphatidylcholine = lysophosphatidylglycerol much greater than lysophosphatidylinositol greater than lysophosphatidylserine much greater than lysophosphatidylethanolamine. Arachidonic acid and palmitoleic acid had a potency comparable to lysophosphatidylinositol, while palmitic acid was ineffective. The Ca(2+)-mobilizing effect of inositol-1,4,5-trisphosphate (IP3) in permeabilized islet cells was additive to the lysophospholipid effect, indicating different sites of action. Both Ca(2+)-mobilizing effects were counteracted by the polyamine spermine, while the presence of Mg2+ shifted the Ca2+ concentrations to higher levels. Since not only an activation of a phospholipase C but also an activation of a phospholipase A2 with subsequent generation of lysophospholipids and free fatty acids is reported to occur in glucose-induced insulin secretion, the interaction of the phospholipase C reaction product IP3 with a lysophospholipid or an unsaturated fatty acid may affect the extent and duration of the rise in the free cytoplasmic Ca2+ concentration responsible for initiation of insulin secretion.     

Cyclic AMP-dependent modulation of cardiac L-type Ca2+ and transient outward K+ channel activities by epoxyeicosatrienoic acids

The three major enzyme systems, cyclo-oxygenase, lipoxygenase, and cytochrome P450 (P450/CYP), metabolize arachidonic acid (AA) to biologically active compounds. P450 and its associated monooxygenase activities have been identified in mammalian cardiac tissue, including humans. The four regioisomeric eicosanoids, 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) of AA metabolites derived by P450 epoxygenases have shown to possess potent biological effects in numerous tissues. In the coronary circulation the EETs are leading candidates for endothelial-derived hyperpolarizing factors that hyperpolarize vascular smooth muscle cells by opening Ca2+-activated K+ channels. Recently, the effects of the CYP pathways and their metabolites on cardiac ischemia-reperfusion injury have been evaluated in animal models. Some of these AA metabolites are cardioprotective and some are detrimental. However, EETs appear to be cardioprotective in CYP2J2 transgenic mice and in a canine ischemic model. Multiple effects of EETs on cardiac ion channels have been observed, such as activation of ATP-sensitive K+ channels and L-type Ca2+ channels in cardiomyocytes and inhibition of cardiac Na+ channels and L-type Ca2+ channels reconstructed in planar lipid bilayers. This brief review summarizes EET-induced modulation of cardiac ion channels.     

Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids

The ability of the Src family kinases Fyn and Lck to participate in signaling through the T cell receptor is critically dependent on their dual fatty acylation with myristate and palmitate. Here we identify a palmitate analog, 2-bromopalmitate, that effectively blocks Fyn fatty acylation in general and palmitoylation in particular. Treatment of COS-1 cells with 2-bromopalmitate blocked myristoylation and palmitoylation of Fyn and inhibited membrane binding and localization of Fyn to detergent-resistant membranes (DRMs). In Jurkat T cells, 2-bromopalmitate blocked localization of the endogenous palmitoylated proteins Fyn, Lck, and LAT to DRMs. This resulted in impaired signaling through the T cell receptor as evidenced by reductions in tyrosine phosphorylation, calcium release, and activation of mitogen-activated protein kinase. We also examined the ability of long chain polyunsaturated fatty acids (PUFAs) to inhibit protein fatty acylation. PUFAs have been reported to inhibit T cell signaling by excluding Src family kinases from DRMs. Here we show that the PUFAs arachidonic acid and eicosapentaenoic acid inhibit Fyn palmitoylation and consequently block Fyn localization to DRMs. We propose that inhibition of protein palmitoylation represents a novel mechanism by which PUFAs exert their immunosuppressive effects.     

Tissue polyunsaturated fatty acids and a digestive phospholipase A2 in the primary screwworm,Cochliomyia hominivorax

We report on the presence of arachidonic acid in larval and adult tissues of the primary screwworm, Cochliomyia hominivorax and of the secondary screwworm, C. macellaria. Arachidonic acid is present in the phospholipids of whole animal extracts of both species. This fatty acid appears to be accumulated during the larval stages, because proportions of arachidonic acid were higher in adults than in larvae. These insects probably obtain the arachidonic acid from dietary phospholipids. We also report on a phospholipase A₂ activity in midgut preparations from third instars of the primary screwworm. Phospholipase A₂ is responsible for hydrolyzing fatty acids from the sn-2 position of dietary phospholipids to release essential fatty acids. The screwworm enzyme is similar to mammalian digestive phospholipase A₂s because it depends on calcium for high catalytic activity, it is sensitive to the site-specific inhibitor oleyloxyethylphosphorylcholine, and it interacts with heparin. We further characterized the screwworm midgut phospholipase A₂ by altering the reaction conditions, including reaction time, radioactive substrate concentration, protein concentration, pH and temperature. We speculate that the biological significance of this enzyme relates to acquiring essential fatty acids, including arachidonic acid, from dietary phospholipids.     

Ligand binding and activation of UTP-activated G protein-coupled P2Y2 and P2Y4 receptors elucidated by mutagenesis,pharmacological and computational studies

The nucleotide receptors P2Y₂ and P2Y₄ are the most closely related G protein-coupled receptors (GPCRs) of the P2Y receptor (P2YR) family. Both subtypes couple to G_q proteins and are activated by the pyrimidine nucleotide UTP, but only P2Y₂R is also activated by the purine nucleotide ATP. Agonists and antagonists of both receptor subtypes have potential as drugs e.g. for neurodegenerative and inflammatory diseases. So far, potent and selective, “drug-like” ligands for both receptors are scarce, but would be required for target validation and as lead structures for drug development. Structural information on the receptors is lacking since no X-ray structures or cryo-electron microscopy images are available. Thus, we performed receptor homology modeling and docking studies combined with mutagenesis experiments on both receptors to address the question how ligand binding selectivity for these closely related P2YR subtypes can be achieved. The orthosteric binding site of P2Y₂R appeared to be more spacious than that of P2Y₄R. Mutation of Y197 to alanine in P2Y₄R resulted in a gain of ATP sensitivity. Anthraquinone-derived antagonists are likely to bind to the orthosteric or an allosteric site depending on their substitution pattern and the nature of the orthosteric binding site of the respective P2YR subtype. These insights into the architecture of P2Y₂- and P2Y₄Rs and their interactions with structurally diverse agonists and antagonist provide a solid basis for the future design of potent and selective ligands.     

Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs.

Coupling of the three alpha 2-adrenergic receptor (alpha 2AR) subtypes to Gi and Gs was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the alpha 2AR agonist UK-14304, alpha 2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, alpha 2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of alpha 2C10 to both Gi and Gs. To isolate functional Gs and Gi coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only alpha 2C10-mediated inhibition (approximately 50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only alpha 2C10-mediated stimulation (approximately 60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl-terminal portion of Gs alpha blocked this functional alpha 2AR.Gs coupling to the same extent as that found for beta 2AR.Gs coupling. In addition to functional Gs coupling, we also verified direct, agonist-dependent, physical coupling of alpha 2AR to Gs alpha. In agonist-treated membranes, an agonist-receptor-Gs alpha complex was immunoprecipitated with a specific alpha 2C10 antibody, and the Gs component identified by both western blots using Gs alpha antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the alpha 2AR subtypes, we investigated whether G-protein coupling was subtype-selective, using UK-14304 and cells with the same alpha 2AR expression levels (approximately 5 pmol/mg). Coupling to Gi was equivalent for alpha 2C10, alpha 2C4, and alpha 2C2: 53.4 +/- 8.8% versus 54.9 +/- 1.0% versus 47.6 +/- 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to Gs were found between the three alpha 2AR subtypes: stimulation of adenylyl cyclase was 57.9 +/- 6.3% versus 30.7 +/- 1.1% versus 21.8 +/- 1.7% for alpha 2C10, alpha 2C4, and alpha 2C2, respectively. Thus, alpha 2AR have the potential to couple physically and functionally to both Gi and Gs; for Gi coupling we found a rank order of alpha 2C10 = alpha 2C4 = alpha 2C2, while for Gs coupling, alpha 2C10 greater than alpha 2C4 greater than alpha 2C2.     

Identification of a protein component of the Ca2+-dependent K+ channel by affinity labelling with apamin

The red blood cell membrane proteins and plasma proteins of normal and spontaneously hypertensive rats were studied by uni- and bidimensional polyacrylamide gel electrophoresis. The amount of band 3, the major intrinsic protein of the erythrocyte membrane, was observed to be significantly reduced in spontaneously hypertensive rats. Plasma from these rats contained two additional heat stable proteins, characterized by a molecular weight of 16,000 daltons and isoelectric points of 4.7 and 5.1, respectively. These proteins were not detected in normotensive control Wistar Kyoto rats, in normal Wistar rats, or in Wistar rats with experimentally induced hypertension.     

M2 muscarinic receptors induce airway smooth muscle activation via a dual, Gbetagamma-mediated inhibition of large conductance Ca2+-activated K+ channel activity

Airway smooth muscle is richly endowed with muscarinic receptors of the M(2) and M(3) subtype. Stimulation of these receptors inhibits large conductance calcium-activated K(+) (BK) channels, a negative feed back regulator, in a pertussis toxin-sensitive manner and thus facilitates contraction. The underlying mechanism, however, is unknown. We therefore studied the activity of bovine trachea BK channels in HEK293 cells expressing the M(2) or M(3) receptor (M(2)R or M(3)R). In M(2)R- but not M(3)R-expressing cells, maximal effective concentrations of carbamoylcholine (CCh) inhibited whole cell BK currents by 53%. This M(2)R-induced inhibition was abolished by pertussis toxin treatment or overexpression of the Gbetagamma scavenger transducin-alpha. In inside-out patches, direct application of 300 nm purified Gbetagamma decreased channel open probability by 55%. The physical interaction of Gbetagamma with BK channels was confirmed by co-immunoprecipitation. Interestingly, inhibition of phospholipase C as well as protein kinase C activities also reversed the CCh effect but to a smaller (approximately 20%) extent. Mouse tracheal cells responded similarly to CCh, purified Gbetagamma and phospholipase C/protein kinase C inhibition as M(2)R-expressing HEK293 cells. Our results demonstrate that airway M(2)Rs inhibit BK channels by a dual, Gbetagamma-mediated mechanism, a direct membrane-delimited interaction, and the activation of the phospholipase C/protein kinase C pathway.     

Biologic activity and tolerance of a preparation of polyunsaturated fatty acids, obtained by a microbiological route ["biopolyene"]]

Biopolyene is a mixture of ethyl ethers of polyunsaturated fatty acids isolated from biomass of Entomophthora virulenta, a mycelial fungus. Its acute and chronic toxicity was studied on rats and guinea pigs. After oral administration of the preparation in single doses exceeding 50 g/kg there were no disorders in the general state of the rats. In chronic experiments with oral biopolyene in doses of 100 and 500 mg/kg and its local application to the intact skin of the animals in a dose of 1 g/kg there were no significant changes in the functional state of the liver and kidneys as well as the peripheral blood count. Insignificant changes in the serum levels of liver enzymes and coagulation were transient. The preparation showed no allergenic or immunomodulating effects. It had neither embryotoxic, teratogenic nor mutagenic action.     

Dexamethasone-induced up-regulation of two-pore domain K+ channel genes, TASK-1 and TWIK-2, in cultured human periodontal ligament fibroblasts

Two-pore domain K(+) channels are widely expressed in many types of cells, and have various important functions, especially maintaining the resting membrane potential. In the previous report, we have confirmed the presence of several kinds of two-pore domain K(+) channels in the periodontal ligament (PDL) fibroblasts. It is well known that dexamethasone (Dex) regulates the functions of various kinds of ion channels. In this work, we investigate if Dex affects the gene expressions of the two-pore domain K(+) channels in the PDL fibroblasts. We also examined the effects of other steroid hormones on the K(+) channels gene expression. The mRNA levels of two-pore domain K(+) channels in human PDL fibroblasts were examined in the presence or absence of Dex by RT-PCR. The effects of other steroid hormones (aldosterone, estrogen, 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], and retinoic acid) were also examined. Dex significantly induced the expression of TASK-1 and TWIK-2 in mRNA levels in both a dose- and a time-dependent manner. The stimulatory effects of Dex were completely abolished by a glucocorticoid receptor antagonist. 1,25-(OH)(2)D(3) also increased the TASK-1 mRNA levels but had no effect on TWIK-2 expression. Dex, one of the potent glucocorticoid, probably have a protective role against external stimuli by maintaining the membrane potential of PDL fibroblasts through the up-regulation of TASK-1 and TWIK-2 K(+) channels.