Inhibition of TASK-1 potassium channel by phospholipase C

Gábor Czirják, Gábor L. Petheo, András Spät, and Péter Enyedi

Faculty of Medicine, Laboratory of Cellular and Molecular Physiology, Department of Physiology, Semmelweis University, H-1444 Budapest, Hungary


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ABSTRACT
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The two-pore-domain K+ channel, TASK-1, was recently shown to be a target of receptor-mediated regulation in neurons and in adrenal glomerulosa cells. Here, we demonstrate that TASK-1 expressed in Xenopus laevis oocytes is inhibited by different Ca2+-mobilizing agonists. Lysophosphatidic acid, via its endogenous receptor, and ANG II and carbachol, via their heterologously expressed ANG II type 1a and M1 muscarinic receptors, respectively, inhibit TASK-1. This effect can be mimicked by guanosine 5'-O-(3-thiotriphosphate), indicating the involvement of GTP-binding protein(s). The phospholipase C inhibitor U-73122 reduced the receptor-mediated inhibition of TASK-1. Downstream signals of phospholipase C action (inositol 1,4,5-trisphosphate, cytoplasmic Ca2+ concentration, and diacylglycerol) do not mediate the inhibition. Unlike the Gq-coupled receptors, stimulation of the Gi-activating M2 muscarinic receptor coexpressed with TASK-1 results in an only minimal decrease of the TASK-1 current. However, additional coexpression of phospholipase C-beta 2 (which is responsive also to Gi beta gamma -subunits) renders M2 receptor activation effective. This indicates the significance of phospholipase C activity in the receptor-mediated inhibition of TASK-1.

voltage clamp; two-pore channel; phosphatidylinositol bisphosphate; wortmannin


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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K+ CHANNELS are major determinants of the resting membrane potential in many cell types. Most mammalian K+ channels can be classified into three structural classes characterized by two, four, or six transmembrane segments (TMS). K+ channels with six TMS and one K+ channel pore domain constitute the voltage-dependent and the Ca2+-activated K+ channels. These channels are activated predominantly when the cell is stimulated, and they are involved in the repolarization, i.e., recovery from the activated state. In contrast to the voltage-dependent and Ca2+-activated K+ channels, inwardly rectifying channels and members of the recently described new family, the background (leak) two-pore-domain (2P) K+ channels, are permanently active at more negative membrane potentials. Consequently, they drive the resting membrane potential toward more hyperpolarized values. In cell types that lack inward rectifiers and that still possess markedly negative resting membrane potential, 2P K+ channels are the most likely candidates for determining the resting K+ conductance (cf. Ref. 20).

Members of the TASK channel family are defined by the characteristic structure of four TMS and two K+ channel pore domains in one polypeptide chain (4TMS/2P). Until now, 10 mammalian 2P K+ channels were cloned, and eight of them were expressed functionally. The expressed channels were reported to be the homodimers of the 4TMS/2P pore-forming units. They induced voltage-independent and noninactivating (leak) K+ current [with the exception of the inactivating TWIK-2 (tandem of pore domains in a weakly inwardly rectifying K+ channel); see Ref. 29]. Although most of the 2P K+ channels are not influenced by membrane potential changes, they are not passive K+-selective pores in the membrane, but they are regulated in many other ways.

The TWIK-related acid-sensitive K+ (TASK) channel subfamily [consisting of TASK-1 (9, 19), TASK-2 (32), and TASK-3 (16, 31)] is characterized by the inhibition by extracellular (EC) acidification in the physiological range. Members of the TREK subfamily [TREK-1 (12), TREK-2 (21), and TRAAK (13)] are mechanosensitive channels. TREK-1 is regulated also by temperature (25). Beyond these physicochemical conditions, 2P K+ channels might be modulated by signal transduction mechanisms too. Members of the TREK family are stimulated by arachidonic acid, and TREK-1 is inhibited by protein kinase C (PKC) and protein kinase A (13). TREK-2 is inhibited by cAMP elevation; furthermore, it was found to be a target of Gq protein-coupled receptors (21). In this paper, we focused on the receptor-mediated regulation of TASK-1.

TASK-1 was cloned from human kidney (9) and rat cerebellum (19), and expression of the channel was detected in heart, lung, and brain. Recently, TASK-1 was suggested as the channel responsible for the background K+ conductance in cerebellar granule cells (26), motoneurons of the brain stem and spinal cord (35), and also in the chemoreceptor type I cells of the carotid body (5). We demonstrated high TASK-1 mRNA expression in rat adrenal cortex, and TASK-1 was shown to be a significant component of the leak K+ current of the aldosterone-producing glomerulosa cells (7).

Alteration of the K+ conductance can influence cellular activity via membrane potential changes. Therefore, inhibition of TASK-1 current by different hormones and neurotransmitters (7, 26, 35) is of particular interest. The pH-sensitive K+ current of hypoglossal motoneurons, regarded as the functional counterpart of TASK-1, could be inhibited by Ca2+-mobilizing hormones and neurotransmitters (35). Inhibition of TASK-1 was also demonstrated in heterologous expression systems. In HEK-293 cells cotransfected with TASK-1 and thyrotropin-releasing hormone (TRH)-R1 receptor, TRH inhibited TASK-1 (35). ANG II inhibited the background K+ conductance in glomerulosa cells and in Xenopus laevis oocytes coexpressing TASK-1 and ANG II type 1a (AT1a) receptor (7). In spite of the growing body of evidence for receptor-mediated regulation of TASK-1, the mechanism of inhibition has not been elucidated. Therefore, in the present study, we investigated the signal transduction pathway between different receptors and TASK-1 by taking advantage of the Xenopus heterologous expression system. We demonstrated that receptor-mediated inhibition of TASK-1 is the consequence of phospholipase C (PLC) activation. The effect is not mediated by the Ca2+ signal, emptying of the intracellular Ca2+ store, generation of inositol phosphates, or PKC; rather, it is related to the breakdown of the membrane polyphosphoinositides.


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Chemicals. Restriction enzymes and DNA processing enzymes were purchased from Amersham (Little Chalfont, UK), Fermentas (Vilnius, Lithuania), and New England Biolabs (Beverly, MA); the mMESSAGE mMACHINE T7 in vitro Transcription Kit was from Ambion (Austin, TX); and the Wizard Plus DNA purification kit was from Promega (Madison, WI). Chemicals of analytical grade were obtained from Fluka (Buchs, Switzerland) and Sigma Chemical (St. Louis, MO). [gamma -32P]ATP was obtained from Izinta (Budapest, Hungary).

Synthesis of cRNA. TASK-1, AT1a ANG II receptor, M1 ACh receptor, M2 ACh receptor, and PLC-beta 2 cRNAs were synthesized in vitro according to the manufacturer's instructions (Ambion mMESSAGE mMACHINE T7 In vitro Transcription Kit). Templates for the TASK-1 ion channel cRNA were the XbaI linearized pEXO-TASK1 (9), which contained the total coding sequence (CDS). Templates for the receptor cRNAs (AT1a, M1, and M2) were the NotI linearized plasmid construct comprising the CDS and 5'-untranslated region of rat AT1a receptor, the BamHI linearized pcDNA3.1 (Invitrogen) containing the CDS for the human M1 ACh receptor (14, 30) in its EcoRI site, and the PstI linearized hm2-pGEMHE construct (2). For obtaining stable PLC-beta 2 cRNA for the Xenopus expression system, the EcoRI-EcoRI fragment of pMT2-PLC-beta 2 (28) was cloned into the EcoRI site of the pBluescript (Stratagene)-derived pEXO containing the 5'- and 3'-untranslated regions of the Xenopus globin gene. The resulting pEXO-PLC-beta 2 construct was linearized with NotI for cRNA synthesis.

Animals and tissue preparation. Mature female X. laevis frogs were obtained from Amrep Reptielen (Breda, Netherlands). Frogs were anesthetized by immersing them in benzocaine solution (0.03%). Ovarian lobes were removed, and the tissue was dissected and treated with collagenase [1.45 mg/ml, 148 U/mg, type I; Worthington Biochemical (Freehold, NJ)] and continuous mechanical agitation in Ca2+-free solution containing (in mM) 82.5 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES (pH 7.5) for 1.5-2 h. Stage V and VI oocytes were defolliculated manually and kept at 18°C in modified Barth's saline containing (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.33 Ca(NO3)2, 0.41 CaCl2, and 20 HEPES buffered to pH 7.5 with NaOH and supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), sodium pyruvate (4.5 mM), and theophylline (0.5 mM). The treatment of animals was conducted in accordance with state laws and institutional regulations. The experiments were approved by the Animal Care and Ethics Committee of the Semmelweis University.

Injection of X. laevis oocytes. Oocytes were injected 1 day after defolliculation. The appropriate cRNA mixture (50 nl) was delivered with a Nanoliter Injector (World Precision Instruments, Sarasota, FL). Currents were measured 3 or 4 days after injection.

Electrophysiology: Two-electrode voltage clamp. Membrane currents of oocytes were recorded by two-electrode voltage clamp (OC-725-C; Warner Instruments, Hamden, CT) using microelectrodes made of borosilicate glass (Clark Electromedical Instruments, Pangbourne, UK) with resistance of 0.3-3 MOmega when filled with 3 M KCl. Currents were filtered at 1 kHz, digitally sampled at 2.5 kHz with a Digidata Interface (Axon Instruments, Foster City, CA), and stored on a PC/AT computer. Recording and data analysis were performed using pCLAMP software 6.0.4 (Axon Instruments). Experiments were carried out at room temperature. Solutions were applied by a gravity-driven perfusion system. Low K+ concentration ([K+]) solution contained (in mM) 95.4 NaCl, 2 KCl, 1.8 CaCl2, and 5 HEPES. High [K+] solution contained 80 mM K+ (78 mM Na+ of the low [K+] solution was replaced with K+). The pH of every solution was adjusted to 7.5 with NaOH. Concentrated stock solutions of wortmannin (10 mM) and U-73122 (4 mM) were made up in DMSO, and the concentration of the solvent in the final solution was a maximum 0.2%. Control solutions contained DMSO at an identical concentration.

Presentation of the electrophysiological data. K+ conductance of the TASK-1-expressing oocytes was estimated by measuring the inward current at -100 mV in high (80 mM) [K+] medium [TASK current (ITASK)]. At this membrane potential, leak current and the current through endogenous channels conducting ions other than K+ contribute to the actually measured value. However, in oocytes with appropriate TASK-1 channel expression (larger current than 1 µA in 80 mM [K+] at -100 mV), these had only a minor contribution to ITASK (which was verified routinely by measuring the current in 2 mM [K+] at -100 mV). Furthermore, where possible, ITASK was corrected for by subtracting even this small current. In experiments where the time course of the K+ conductance change in response to different stimuli is shown, the presented curves were not corrected, and, where indicated, the curves were normalized to the value at the beginning of the stimulation. When ITASK of the individual oocytes was measured both before and after the experimental manipulation, the ratio of the two values is presented and used in the calculations. Measurement of the K+ current similarly to ITASK in water-injected oocytes resulted in currents <0.1 µA (7), which ensured that at least 90% of ITASK in the oocytes of appropriate expression was indeed the consequence of the TASK-1 current.

Measurement of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in Xenopus oocytes. For radiolabeling, the oocytes were injected with [gamma -32P]ATP (20 kBq in 50-nl volume) and incubated for 2 h at room temperature in modified Barth's solution with or without 3 µM wortmannin. The incubation was terminated by acidic methanol and by freezing the oocytes in liquid nitrogen. The amount of injected total radioactivity was checked, and two oocytes were pooled for each analysis. The oocytes were homogenized in a hand-driven Potter homogenizer before the extraction and separation of phospholipids, which were performed as described previously (10). Radioactivity of the separated lipid fractions was detected and quantified by a GS-525 Phosphor-Imager (Bio-Rad, Hercules, CA).

Statistics. Data are expressed as means ± SE. Statistical significance was estimated by t-test for independent samples [see Fig. 2B; phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) meaurements], one-way ANOVA (see Fig. 7A), and 2-way repeated-measures ANOVA (see Figs. 2C, 3, 4, 5C, and 7B) using the STATISTICA program package (StatSoft, Tulsa, OK).


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Stimulation of Gq protein-coupled receptors inhibits TASK-1. Injection of TASK-1 cRNA in X. laevis oocytes induces the expression of a time- and voltage-independent (background) K+ current (7, 9). In oocytes with appropriate channel expression, this conductance is responsible for >90% of the inward current at -100 mV when measured in high (80 mM) EC [K+]. Challenge of the oocytes expressing TASK-1 with lysophosphatidic acid (LPA, 500 nM), which activates the endogenous LPA receptor (11), inhibited the current by 67 ± 3.5% (n = 10). Similarly, when AT1a ANG II receptor or M1 type muscarinic ACh receptors were coexpressed with TASK-1, activation of these receptors with their appropriate agonist, ANG II (10 nM) or carbachol (1 µM), reduced the K+ current by 77 ± 1% (n = 16) and by 77 ± 3% (n = 9), respectively.

Simultaneously with the TASK-1 inhibition, all three agonists induced an outward current at +20 mV. At this membrane potential (being close to the K+ equilibrium potential in 80 mM EC [K+]), the outward current is dominated by the Cl- conductance. Considering the high abundance of Ca2+-activated Cl- channels of the oocytes, the observed outward current is the reflection of the elevation of the cytoplasmic Ca2+ concentration ([Ca2+]) in response to the Ca2+-mobilizing agonists. The increased Cl- conductance causes a minor increase in the inward current also at -100 mV, but this increase is negligible if it is compared with the decrease of the robust K+ current of TASK-1. This allowed the simultaneous and fairly separate measurement of the K+ current and the Ca2+-activated Cl- current (ICl,Ca) with a voltage protocol comprising repeated steps to -100 and +20 mV (Fig. 1).


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Fig. 1.   Ca2+-mobilizing hormones inhibit the two-pore-domain K+ channel TASK-1 expressed in Xenopus oocytes. A: membrane currents of an oocyte expressing TASK-1 and M1 muscarinic ACh receptor were measured in high-K+ (80 mM) extracellular (EC) solution. Every 3 s, the voltage protocol (see inset) was applied from a holding potential of 6 mV. Currents before stimulation (15 s), at the peak of the Ca2+-activated Cl- current (39 s), and at maximal inhibition of TASK-1 (60 s) are shown from the same oocyte. B: currents of the same oocyte measured at the end of the steps to -100 and +20 mV (as indicated by arrows in A) were plotted as a function of time. C: membrane currents of an oocyte expressing TASK-1 and ANG II type 1a (AT1a) receptor. D: stimulation of the endogenous lysophosphatidic acid (LPA) receptor in an oocyte expressing TASK-1. (The voltage protocol for C and D was the same as for B.)

Inhibition of TASK-1 is G protein dependent. ITASK was measured before and 1-3 h after injection of the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) or control solution. GTPgamma S (2 mM) was injected in 50 nl. This resulted in an estimated 10-fold dilution within the oocyte. Because GTPgamma S was a tetralithium salt, the control solution contained 8 mM LiCl. The ratio of ITASK measured after and before the injection is depicted in Fig. 2B. ITASK was inhibited significantly by GTPgamma S (by 55 ± 4%, n = 6, P < 0.0001) and was not altered by injection of the control solution. GTPgamma S also modified the kinetics of TASK-1 inhibition upon M1 receptor stimulation. When oocytes coexpressing TASK-1 and M1 receptor were injected with GTPgamma S (2 mM, 50 nl) 13-15 min before the application of carbachol (this short pretreatment with the nonhydrolyzable analog reduced the current by 23 ± 10%, n = 7), the recovery of the current after the withdrawal of carbachol from the perifusing solution became incomplete (Fig. 2C; P < 0.03).


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Fig. 2.   TASK-1 is inhibited by guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) injection. A: oocytes expressing TASK-1 were kept at -30 mV holding potential. Every 3 s a 300-ms voltage step to -100 mV was applied. Currents at the end of these steps were plotted as the function of time. The K+ current ([K+]) of the EC solution was changed from the control 2 mM to 80 mM as indicated by the bar. Calculation of TASK current (ITASK; the difference of inward currents in 80 and 2 mM EC [K+] at -100 mV) is shown by the arrow. B: effect of injection of GTPgamma S (2 mM, 50 nl) or solvent (control) on ITASK in oocytes. The current measured 1-3 h after injection is normalized to that measured before injection (nos. in the bars represent no. of oocytes). C: oocytes expressing TASK-1 and M1 muscarinic ACh receptor were injected with GTPgamma S (2 mM, 50 nl, n = 7) or solvent (n = 8). They were stimulated with carbachol (1 µM) 13-15 min after injection as indicated by the bar.

Activation of PLC mediates TASK-1 inhibition. The effect of different Gq-coupled receptors and involvement of G protein(s) suggested that TASK-1 inhibition could be related to PLC action. Therefore, a PLC inhibitor was used to prevent receptor-induced PLC activation in oocytes coexpressing TASK-1 and M1 receptor. The oocytes were preincubated with U-73122 (2.5 µM) for 45-75 min, and the drug was present also in the perifusing solutions. U-73122 by itself did not influence the TASK-1 current (data not shown). The efficiency of U-73122 in the oocytes is indicated by the extinction of ICl,Ca in response to carbachol (300 nM). The inhibitory effect of the stimulus on the TASK-1 current measured simultaneously was also significantly reduced (Fig. 3; P < 0.0001).


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Fig. 3.   U-73122 reduces the inhibition of TASK-1. Oocytes coexpressing TASK-1 and M1 muscarinic ACh receptor were incubated in 2.5 µM U-73122 (n = 5) or solvent (control, n = 6) for 45-75 min. After pretreatment, they were stimulated with carbachol (300 nM) as indicated by the bar. ITASK was normalized to the value at the beginning of the stimulation. (The voltage protocol was the same as in Fig. 1.)

The role of PLC action in the inhibition of TASK-1 was also tested by taking advantage of the regulatory characteristics of PLC-beta 2 (28). This isoform of the enzyme is known to also be activated by the beta gamma -subunit; accordingly, receptors acting via Gi proteins can effectively turn it on (6). To enable release of beta gamma -subunits of Gi-type G proteins, M2 muscarinic ACh receptor (3) was coexpressed with TASK-1. When the oocytes coexpressing TASK-1 and M2 receptor were stimulated with carbachol (1 µM), the activation of the endogenous PLC activity was moderate. This was indicated by the absence of marked stimulation of ICl,Ca, although in some oocytes small current oscillations (amplitude in the 20- to 200-nA range) appeared at +20 mV (data not shown). [This effect is not detectable on the average plot (Fig. 4, top).] In these oocytes, the simultaneously observed inhibition of TASK-1 was also moderate, only 24 ± 3% (n = 10, Fig. 4, bottom).


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Fig. 4.   Phospholipase C (PLC) activation inhibits TASK-1. Oocytes coexpressing TASK-1 and M2 muscarinic receptor (control group, n = 10) or PLC-beta 2, TASK-1, and M2 ACh receptor (PLC-beta 2 group, n = 10) were stimulated with carbachol (1 µM) at 0 min. ITASK was normalized to the value at the beginning of the stimulation with carbachol. (The voltage protocol was the same as in Fig. 1.)

However, when PLC-beta 2 was coexpressed with M2 ACh receptor and TASK-1 (Fig. 4, PLC-beta 2 curves), carbachol evoked more considerable activation of ICl,Ca (verifying the success of PLC-beta 2 expression), and the TASK-1 inhibition was significantly stronger (48 ± 3%, n = 10, P < 0.0001) than that in the control group. Because the only difference between the two groups was the expression of PLC-beta 2, the stronger TASK-1 inhibition must have been the consequence of PLC activation.

TASK-1 inhibition is not mediated by Ca2+, by inositol 1,4,5-trisphosphate, or its metabolites, PKC or tyrosine kinase. Although in almost every oocyte stimulated by ANG II the onset of the TASK-1 inhibition slightly preceded the beginning of ICl,Ca, theoretically the Ca2+ signal and emptying of the intracellular Ca2+ store were possible candidates for inhibiting TASK-1. Thus oocytes expressing ANG II receptor and TASK-1 were pretreated with thapsigargin (1 µM, 5-6.5 h), an inhibitor of the Ca2+-ATPase of intracellular Ca2+ stores of oocytes. Subsequent ANG II (10 nM) stimulation failed to evoke ICl,Ca, but the mechanism responsible for TASK-1 inhibition remained effective (63 ± 4% inhibition, n = 7, Fig. 5A). The Ca2+ ionophore ionomycin induces rapid elevation of cytoplasmic [Ca2+] by releasing the ion predominantly from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular stores of the oocytes, without inducing Ca2+ influx (37). Accordingly, ionomycin (1 µM) generated large ICl,Ca; however, it induced only a slight reduction of ITASK. ICl,Ca became refractory to repeated application of ionomycin, but the subsequent addition of ANG II strongly inhibited TASK-1 (by 80 ± 1%, n = 4) in spite of the apparent depletion of the Ca2+ stores (Fig. 5B). The ANG II-induced ICl,Ca was also abolished by injection of the Ca2+ chelator EGTA (50 nl, 100 mM; data not shown). Although the ensuing approx 10 mM intracellular concentration of EGTA slowed down the TASK-1 inhibition evoked by ANG II (P < 0.0001, ANOVA), the final extent of inhibition was not altered (Fig. 5C). Downstream of the PLC activation but upstream of the Ca2+ signal, there is InsP3. Theoretically, InsP3 or one of its numerous metabolites might have transmitted the signal from PLC activation to TASK-1 inhibition. To examine this possibility, InsP3 (10 ng/injection) was injected in oocytes expressing TASK-1. This concentration of InsP3 evoked maximal ICl,Ca, but it did not cause any apparent TASK-1 inhibition (Fig. 6). Therefore, PLC activation reduces TASK-1 current, but the inhibitory mechanism does not involve InsP3.


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Fig. 5.   TASK-1 inhibition may develop without Ca2+ signal, and large Ca2+ signal exerts only limited inhibition of ITASK. TASK-1 and AT1 ANG II receptor were coexpressed. A: oocytes were pretreated with thapsigargin (1 µM, 5-6.5 h). Effect of ANG II (10 nM) on the TASK-1 current (at -100 mV) and on the Ca2+-activated Cl- current (at +20 mV) was measured as shown in Fig. 1. At the end of the measurement, [K+] in the EC solution was changed from 80 to 2 mM (representative of 7 similar experiments). B: effect of repeated application of ionomycin (1 µM). After the third ionomycin challenge, which already failed to elicit detectable Ca2+-activated Cl- current, the oocyte was stimulated with ANG II. (Methodical details are the same as in A; representative of 4 similar experiments.) C: effect of EGTA (100 mM, 50 nl) or distilled water (control) injection on ITASK inhibition evoked by ANG II. ITASK is normalized to the value at the beginning of the stimulation with ANG II (10 nM, at 0 min). Means ± SE of 11 (EGTA treated) and 8 (control) experiments are shown.



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Fig. 6.   Inositol 1,4,5-trisphosphate (InsP3) injection fails to inhibit TASK-1. Arrow 1: insertion of the injection capillary in the oocyte (increase of the nonspecific leak); arrow 2: injection of 10 ng InsP3 (Ca2+-activated Cl- current, no TASK-1 inhibition); arrow 3: injection of 10 ng InsP3 again (no further response). Currents at +20 and -100 mV were measured as described in Fig. 1. EC 80 mM [K+] was periodically replaced by 2 mM EC [K+] (as indicated by the bars) for estimating TASK-1 current. (Representative of 4 similar experiments.)

The other main consequence of Gq-PLC signaling is the activation of PKC. The possible contribution of this branch of the signal transduction to the receptor-mediated TASK-1 inhibition was investigated by two approaches. First, before receptor stimulation, the oocytes (expressing TASK-1 and AT1 receptor) were treated with staurosporine (3 µM). The treatment with the PKC inhibitor failed to affect the inhibition of ITASK induced by ANG II (data not shown). The effect of pharmacological activation of PKC by phorbol 12-myristate 13-acetate (PMA) was also tested. ITASK were measured before and 12 min after treating the cell with PMA (100 nM). The inactive analog 4alpha -phorbol 12,13-didecanoate (4alpha -PDD, 100 nM) was used as a control. Only a small identical decrease of ITASK was observed both in the PMA-treated and in the control group during the 12-min incubation time [18 ± 3 and 23 ± 2% (n = 6), respectively], indicating that PKC activation failed to inhibit TASK-1. It was verified that PMA activated PKC, since PMA treatment desensitized the cells to subsequent ANG II stimulation (data not shown).

In addition to the Gq-PLC pathway stimulation, the AT1 receptor activates tyrosine kinases (1, 15). Because the COOH-terminal cytoplasmic tail of TASK-1 contains a tyrosine phosphorylation consensus sequence, it was tempting to test the idea of regulation via tyrosine phosphorylation. Injection of the oocytes with the nonspecific tyrosine kinase inhibitor genistein (1 mM, 50 nl, n = 4) 1-2 h before the current measurement or preincubation with the Src kinase-specific 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine (1 µM, n = 5) failed to affect the degree of inhibition of ITASK by 10 nM ANG II (72 ± 2 and 80 ± 4%, respectively). In vitro site-directed mutagenesis of the tyrosine in the consensus sequence (in position 323) of TASK-1 to phenylalanine also did not alter the inhibition (data not shown). Thus the inhibition by ANG II is not related to tyrosine phosphorylation.

Effect of wortmannin on TASK-1 current. To examine the possible role of polyphosphoinositide changes in the modulation of the TASK-1 current, we used wortmannin, an inhibitor of phosphatidylinositol 3-kinase that also affects phosphatidylinositol 4-kinase at higher concentration. Preincubation of [32P]ATP-injected oocytes with wortmannin (3 µM) for 2 h reduced the labeling of PIP and PIP2 lipid fractions by 26% (P < 0.05) and 28% (P < 0.01), respectively (n = 9, t-test). Similar pretreatment of oocytes expressing TASK-1 with wortmannin resulted in a concentration-dependent inhibition of ITASK (P < 0.001, ANOVA, n = 4-5 at each concentration, Fig. 7A).


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Fig. 7.   Wortmannin inhibits TASK-1 current. A: oocytes expressing TASK-1 were treated with wortmannin for 2-3 h. Ratio of the TASK-1 currents (ITASK, see Fig. 2A) measured before and after the wortmannin treatment was calculated for each oocyte. The control group was incubated with 0.03% DMSO (the same concentration as in the group treated with 3 µM wortmannin). B: oocytes coexpressing TASK-1 and M1 muscarinic ACh receptor were pretreated with wortmannin (20 µM, n = 8) or only DMSO (0.2%, n = 8) for 25-40 min. Afterward they were stimulated with carbachol (1 µM) as indicated by the bar. [Perifusing solutions of the oocytes (wortmannin or control group) contained 3 µM wortmannin or DMSO (0.03%) only.]

Wortmannin also dramatically affected the effect of M1 receptor activation on TASK-1 current. Oocytes coexpressing TASK-1 and M1 receptor were preincubated in a high (20 µM) concentration of wortmannin for a short (25-40 min) period of time to reach an efficient intracellular wortmannin concentration at a relatively high steady-state PIP2 level. TASK-1 was inhibited by this pretreatment by ~50% [control group: 4,134 ± 954 nA (n = 8); wortmannin group: 2,072 ± 384 nA (n = 8)]. After the preincubation, oocytes were stimulated with carbachol (1 µM) for 2 min. Rapid recovery of the current after the inhibition evoked by carbachol was slowed down significantly in oocytes treated with wortmannin (Fig. 7B, P < 0.0001). [Wortmannin (3 µM) was included in the perifusing solutions to avoid washout of the drug.]


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REFERENCES

It was previously found by us (7) and others (26, 35) that TASK-1, the 2P-type acid-sensitive K+ channel, is inhibited by different Ca2+-mobilizing agonists in adrenal glomerulosa cells and in neurons. In the present experiments, ITASK, expressed in Xenopus oocytes, was inhibited 70-80% by LPA acting via endogenous receptor and by ANG II or by carbachol acting via the heterologously expressed AT1a ANG II or M1 ACh receptor, respectively. The inhibition could be mimicked by injection of the nonhydrolyzable GTP analog GTPgamma S, suggesting the role of G protein(s) in the inhibitory process. GTPgamma S injection slowed down and prevented the complete recovery of ITASK after withdrawal of the stimulus, thereby confirming the functional involvement of G protein(s) in the receptor-mediated inhibition of TASK-1.

With the exception of a few "variant donor" frogs (23), having high endogenous muscarinic receptor expression (and therefore excluded from the present study), the effect of carbachol on ICl,Ca and ITASK was negligible in oocytes expressing only TASK-1. In oocytes coexpressing TASK-1 and the M2 muscarinic receptor (which interacts predominantly with Gi-type G proteins), stimulation with carbachol often resulted in a moderate inhibition of TASK-1 current. This inhibition may be attributed either to a slight activation of PLC by a low number of endogenous M3 receptors (liberating alpha q; see Ref. 8) or to beta gamma -subunit-induced activation of endogenous PLC-Xbeta (24). (The involvement of PLC in this phenomenon is supported by the simultaneous occurrence of an also small-amplitude, oscillatory Ca2+ signal.) The inhibitory effect of carbachol, however, was significantly augmented when PLC-beta 2 was also coexpressed with TASK-1 and the M2 receptor, clearly indicating that the channel inhibition is mediated by event(s) induced by PLC action.

If Ca2+-mobilizing agonists inhibit TASK-1 via the activation of PLC, the inhibition of the enzyme should prevent or at least reduce the inhibition of the current. In a previous report on cerebellar granule neurons, the PLC inhibitor U-73122 failed to inhibit a current identical to TASK-1 (4). In contrast to this observation, in the present study, U-73122 abolished ICl,Ca and diminished the TASK-1 inhibition by the M1 receptor. Although we have no explanation for this discrepancy, the efficiency of the enzyme inhibitor in antagonizing the current-reducing effect of the Ca2+-mobilizing agonist in our system is in accordance with the results obtained in oocytes coexpressing PLC-beta 2 and M2 receptor, indicating the significance of PLC in the control of channel function.

An immediate effect of PLC enzyme activation is the generation of the diffusible second messengers InsP3 and diacylglycerol (DAG). In addition to its Ca2+-mobilizing effect, InsP3 may serve as a precursor for a large number of inositol phosphate isomers. Some of these metabolites may also act as intracellular regulators of channel functions, inositol 3,4,5,6-tetrakisphosphate for Cl- channels (36) and inositol pentakisphosphate and inositol hexakisphosphate for Ca2+ channels (17). However, neither the Ca2+ signal nor inositol phosphate isomers appear to be the major inducer of TASK-1 inhibition.

When the receptor-mediated Ca2+ signal was prevented by preincubation of the oocytes with thapsigargin, a treatment that depleted the intracellular Ca2+ stores, the channel was still inhibited by subsequent exposure to ANG II. Even after the application of EGTA, known to reduce cytoplasmic [Ca2+] to subnormal levels, full inhibition of TASK-1 by ANG II was attained. (The slower development of the inhibition may be attributed to the Ca2+ demand of PLC.) These observations are in good agreement with previous results (9, 19) in which manipulation of the intracellular [Ca2+] in both directions failed to influence the expressed channel activity.

Because InsP3 injection, which caused a robust Ca2+ signal, apparently failed to inhibit the channel activity, the possible significance of inositol phosphate isomers can also be ruled out. In addition, the rapid onset of inhibition, which even precedes the activation of ICl,Ca, argues against the possible role of biologically active inositol phosphate isomers, which accumulate more slowly (36). The other messenger molecule generated during the action of PLC is DAG, which activates PKC. However, neither inhibition nor activation of PKC influenced ITASK under conditions in which changes in enzyme activity were clearly verified. This confirms previous observations (19) that, although PKC consensus phosphorylation sites can be detected in TASK-1, PKC activation does not affect the TASK-1 current.

In addition to the PKC consensus phosphorylation sites, TASK-1 also possesses a consensus site for tyrosine kinase phosphorylation. Because ANG II is known to activate the tyrosine kinase pathway as well, this possibility was also tested. The contribution of a tyrosine kinase to the inhibitory process was excluded by two different approaches. ANG II inhibited ITASK both after the application of the tyrosine kinase inhibitor genistein and after mutation of TASK-1 at the tyrosine phosphorylation site.

Given that products of PLC do not mediate TASK-1 inhibition, it is feasible that the depletion of its substrate may be responsible for the effect. Actually, the steady-state membrane level of PIP and PIP2 may decrease far below the control level as a consequence of PLC activation (18). Polyphosphoinositides represent only a minor fraction of the plasma membrane phospholipids; yet, several proteins bind to, and can be regulated by, these negatively charged phospholipids. Specifically, inwardly rectifying K+ channels are activated by PIP2 (22, 33). High concentrations of the phosphatidylinositol 3-kinase inhibitor wortmannin have several nonspecific effects (34), including the inhibition of phosphatidylinositol 4-kinase (27). Taking advantage of this effect, we revealed that wortmannin, while lowering the PIP and PIP2 pools in the oocytes, also reduced the steady-state TASK-1 current. Moreover, wortmannin slowed down the recovery of ITASK after M1 receptor stimulation in accordance with the decelerated resynthesis of the polyphosphoinositides. Irrespective of other side effects of the drug, these results are consistent with the hypothesis that the breakdown of polyphosphoinositides is responsible for the inhibition of TASK-1.

Summarizing our observations, we provided evidence that inhibition of TASK-1 by Ca2+-mobilizing agonists is mediated by activation of PLC. Because none of the generated second messengers seems to be responsible for the inhibition, it is reasonable to assume that it is the membrane level of polyphosphoinositides that regulates the function of the channel. According to this idea, polyphosphoinositides may be necessary for constitutive activity of the TASK-1 channels, and PLC-mediated reduction in those phospholipids could be responsible for ITASK inhibition after receptor activation.


    ACKNOWLEDGEMENTS

We thank M. Lazdunski and Dr. F. Lesage for the pEXO and pEXO-TASK plasmids, Dr. László Hunyady for the ANG II (AT1a) receptor plasmid construct, Dr. Xin-Yun Huang for the M1 and Dr. T. I. Bonner for the M2 muscarinic receptor plasmid construct, and Dr. Sue Goo Rhee for pMT2-PLC-beta 2. The skillful technical assistance of Erika Kovács and Irén Veres is highly appreciated.


    FOOTNOTES

This work was supported by the Hungarian National Research Fund (OTKA 032159) and by the Hungarian Medical Research Council (ETT-244/2000).

Address for reprint requests and other correspondence: P. Enyedi, Dept. of. Physiology, Semmelweis Univ., Faculty of Medicine, P.O. Box 259, H-1444 Budapest, Hungary (E-mail: enyedi{at}puskin.sote.hu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 November 2000; accepted in final form 23 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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