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-2 (which is responsive also to Gi
-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
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INTRODUCTION |
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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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MATERIALS AND METHODS |
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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). [-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-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-
2 cRNA for the Xenopus
expression system, the EcoRI-EcoRI fragment of
pMT2-PLC-
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-
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 M 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
[-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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RESULTS |
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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.
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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) (GTPS) or control
solution. GTP
S (2 mM) was injected in 50 nl. This resulted in an
estimated 10-fold dilution within the oocyte. Because GTP
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 GTP
S (by
55 ± 4%, n = 6, P < 0.0001) and
was not altered by injection of the control solution. GTP
S also
modified the kinetics of TASK-1 inhibition upon M1 receptor
stimulation. When oocytes coexpressing TASK-1 and M1
receptor were injected with GTP
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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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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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 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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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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DISCUSSION |
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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 GTPS, suggesting the role of G protein(s) in the inhibitory process. GTP
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 q;
see Ref. 8) or to
-subunit-induced activation of
endogenous PLC-X
(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-
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-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.
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ACKNOWLEDGEMENTS |
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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-2. The skillful technical assistance of Erika
Kovács and Irén Veres is highly appreciated.
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FOOTNOTES |
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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.
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REFERENCES |
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---|
1.
Berk, BC.
Angiotensin II signal transduction in vascular smooth muscle: pathways activated by specific tyrosine kinases.
J Am Soc Nephrol
10, Suppl11:
S62-S68,
1999[ISI][Medline].
2.
Bonner TI. New subtypes of muscarinic acetylcholine receptors.
Trends Pharmacol Sci Suppl: 11-15, 1989.
3.
Bonner, TI,
Buckley NJ,
Young AC,
and
Brann MR.
Identification of a family of muscarinic acetylcholine receptor genes.
Science
237:
527-532,
1987[ISI][Medline].
4.
Boyd, DF,
Millar JA,
Watkins CS,
and
Mathie A.
The role of Ca2+ stores in the muscarinic inhibition of the K+ current IK(SO) in neonatal rat cerebellar granule cells.
J Physiol (Lond)
529:
321-331,
2000
5.
Buckler, KJ,
Williams BA,
and
Honore E.
An oxygen-, acid- and anaesthetic-sensitive TASK-like background potassium channel in rat arterial chemoreceptor cells.
J Physiol (Lond)
525:
135-142,
2000
6.
Camps, M,
Carozzi A,
Schnabel P,
Scheer A,
Parker PJ,
and
Gierschik P.
Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits.
Nature
360:
684-686,
1992[ISI][Medline].
7.
Czirják, G,
Fischer T,
Spät A,
Lesage F,
and
Enyedi P.
TASK (TWIK-related acid-sensitive K+ channel) is expressed in glomerulosa cells of rat adrenal cortex and inhibited by angiotensin II.
Mol Endocrinol
14:
863-874,
2000
8.
Davidson, A,
Mengod G,
Matus-Leibovitch N,
and
Oron Y.
Native Xenopus oocytes express two types of muscarinic receptors.
FEBS Lett
284:
252-256,
1991[ISI][Medline].
9.
Duprat, F,
Lesage F,
Fink M,
Reyes R,
Heurteaux C,
and
Lazdunski M.
TASK, a human background K+ channel to sense external pH variations near physiological pH.
EMBO J
16:
5464-5471,
1997
10.
Enyedi, P,
Büki B,
Mucsi I,
and
Spät A.
Polyphosphoinositide metabolism in adrenal glomerulosa cells.
Mol Cell Endocrinol
41:
105-112,
1985[ISI][Medline].
11.
Fernhout, BJ,
Dijcks FA,
Moolenaar WH,
and
Ruigt GS.
Lysophosphatidic acid induces inward currents in Xenopus laevis oocytes; evidence for an extracellular site of action.
Eur J Pharmacol
213:
313-315,
1992[ISI][Medline].
12.
Fink, M,
Duprat F,
Lesage F,
Reyes R,
Romey G,
Heurteaux C,
and
Lazdunski M.
Cloning, functional expression and brain localization of a novelunconventional outward rectifier K+ channel.
EMBO J
15:
6854-6862,
1996[Abstract].
13.
Fink, M,
Lesage F,
Duprat F,
Heurteaux C,
Reyes R,
Fosset M,
and
Lazdunski M.
A neuronal two P domain K+ channel stimulated by arachidonic acid and polyunsaturated fatty acids.
EMBO J
17:
3297-3308,
1998
14.
Huang, XY,
Morielli AD,
and
Peralta EG.
Tyrosine kinase-dependent suppression of a potassium channel by the G protein-coupled m1 muscarinic acetylcholine receptor.
Cell
75:
1145-1156,
1993[ISI][Medline].
15.
Inagami, T,
Kambayashi Y,
Ichiki T,
Tsuzuki S,
Eguchi S,
and
Yamakawa T.
Angiotensin receptors: molecular biology and signalling.
Clin Exp Pharmacol Physiol
26:
544-549,
1999[ISI][Medline].
16.
Kim, Y,
Bang H,
and
Kim D.
TASK-3, a new member of the tandem pore K+ channel family.
J Biol Chem
275:
9340-9347,
2000
17.
Larsson, O,
Barker CJ,
Sj oholm A,
Carlqvist H,
Michell RH,
Bertorello AN-T,
Honkanen RE,
Mayr GW,
Zwiller J,
and
Berggren PO.
Inhibition of phosphatases and increased Ca2+ channel activity by inositolhexakisphosphate.
Science
278:
471-474,
1997
18.
Lee, SB,
and
Rhee SG.
Significance of PIP2 hydrolysis and regulation of phospholipase C isozymes.
Curr Opin Cell Biol
7:
183-189,
1995[ISI][Medline].
19.
Leonoudakis, D,
Gray AT,
Winegar BD,
Kindler CH,
Harada M,
Taylor DMC-R,
Forsayeth JR,
and
Yost CS.
An open rectifier potassium channel with two pore domains in tandem cloned from rat cerebellum.
J Neurosci
18:
868-877,
1998
20.
Lesage, F,
and
Lazdunski M.
Molecular and functional properties of two-pore-domain potassium channels.
Am J Physiol Renal Physiol
279:
F793-F801,
2000
21.
Lesage, F,
Terrenoire C,
Romey G,
and
Lazdunski M.
Human TREK2, a 2P domain mechano-sensitive K+ channel with multiple regulations by polyunsaturated fatty acids, lysophospholipids and Gs-, Gi- and Gq-protein-coupled receptors.
J Biol Chem
275:
28398-28405,
2000
22.
Logothetis, DE,
and
Zhang H.
Gating of G protein-sensitive inwardly rectifying K+ channels through phosphatidylinositol 4,5-bisphosphate.
J Physiol (Lond)
520:
630,
1999
23.
Lupu-Meiri, M,
Shapira H,
Matus-Leibovitch N,
and
Oron Y.
Two types of intrinsic muscarinic responses in Xenopus oocytes. I. Differences in latencies and 45Ca efflux kinetics.
Pflügers Arch
417:
391-397,
1990[ISI][Medline].
24.
Ma, HW,
Blitzer RD,
Healy EC,
Premont RT,
Landau EM,
and
Iyengar R.
Receptor-evoked Cl current in Xenopus oocytes is mediated through a beta-type phospholipase C. Cloning of a new form of the enzyme.
J Biol Chem
268:
19915-19918,
1993
25.
Maingret, F,
Lauritzen I,
Patel AJ,
Heurteaux C,
Reyes R,
Lesage F,
Lazdunski M,
and
Honore E.
TREK-1 is a heat-activated background K+ channel.
EMBO J
19:
2483-2491,
2000
26.
Millar, JA,
Barratt L,
Southan AP,
Page KM,
Fyffe RE,
Robertson B,
and
Mathie A.
A functional role for the two-pore domain potassium channel TASK-1 in cerebellar granule neurons.
Proc Natl Acad Sci USA
97:
3614-3618,
2000
27.
Nakanishi, S,
Catt KJ,
and
Balla T.
A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids.
Proc Natl Acad Sci USA
92:
5317-5321,
1995[Abstract].
28.
Park, D,
Jhon DY,
Kriz R,
Knopf J,
and
Rhee SG.
Cloning, sequencing, expression, and Gq-independent activation of phospholipase C-beta 2.
J Biol Chem
267:
16048-16055,
1992
29.
Patel, AJ,
Maingret F,
Magnone V,
Fosset M,
Lazdunski M,
and
Honore E.
TWIK-2, an inactivating 2P domain K+ channel.
J Biol Chem
275:
28722-28730,
2000
30.
Peralta, EG,
Ashkenazi A,
Winslow JW,
Ramachandran J,
and
Capon DJ.
Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes.
Nature
334:
434-437,
1988[ISI][Medline].
31.
Rajan, S,
Wischmeyer E,
Xin LG,
Preisig-Muller R,
Daut J,
Karschin A,
and
Derst C.
TASK-3, a novel tandem pore domain acid-sensitive K+ channel. An extracellular histidine as pH Sensor.
J Biol Chem
275:
16650-16657,
2000
32.
Reyes, R,
Duprat F,
Lesage F,
Fink M,
Salinas M,
Farman N,
and
Lazdunski M.
Cloning and expression of a novel pH-sensitive two pore domain K+ channel from human kidney.
J Biol Chem
273:
30863-30869,
1998
33.
Shyng, SL,
and
Nichols CG.
Membrane phospholipid control of nucleotide sensitivity of KATP channels.
Science
282:
1138-1141,
1998
34.
Sugita, Y,
Nagao T,
and
Urushidani T.
Nonspecific effects of the pharmacological probes commonly used to analyze signal transduction in rabbit parietal cells.
Eur J Pharmacol
365:
77-89,
1999[ISI][Medline].
35.
Talley, EM,
Lei Q,
Sirois JE,
and
Bayliss DA.
TASK-1, a two-pore domain K+ channel, is modulated by multiple neurotransmitters in motoneurons.
Neuron
25:
399-410,
2000[ISI][Medline].
36.
Vajanaphanich, M,
Schultz C,
Rudolf MT,
Wasserman M,
Enyedi P,
Craxton A,
Shears SB,
Tsien RY,
Barrett KE,
and
Traynor-Kaplan A.
Long-term uncoupling of chloride secretion from intracellular calcium levels by Ins(3,4,5,6)P4.
Nature
371:
711-714,
1994[ISI][Medline].
37.
Yoshida, S,
and
Plant S.
Mechanism of release of Ca2+ from intracellular stores in response to ionomycin in oocytes of the frog Xenopus laevis.
J Physiol (Lond)
458:
307-318,
1992[Abstract].