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Department of Physiology and Laboratory of Cellular and Molecular
Physiology (G.C., T.F., A.S., P.E.) Semmelweis University of
Medicine 1444 Budapest, Hungary
Institut de Pharmacologie
Moléculaire et Cellulaire (F.L.) Centre Nationale de la
Recherche Scientifique 06560 Valbonne France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In Xenopus oocytes injected with mRNA prepared from adrenal glomerulosa tissue the expressed K+ current at -100 mV was virtually insensitive to tetraethylammonium (3 mM) and 4-aminopyridine (3 mM). Ba2+ (300 µM) and Cs+ (3 mM) induced voltage-dependent block. Lidocaine (1 mM) and extracellular acidification from pH 7.5 to 6.7 inhibited the current (by 28% and 16%, respectively). This inhibitory profile is similar (although it is not identical) to that of TASK expressed by injecting its cRNA. In oocytes injected with adrenal glomerulosa mRNA, TASK antisense oligonucleotide reduced significantly the expression of K+ current at -100 mV, while the sense oligonucleotide failed to have inhibitory effect. Application of angiotensin II (10 nM) both in isolated glomerulosa cells and in oocytes injected with adrenal glomerulosa mRNA inhibited the K+ current at -100 mV. Similarly, in oocytes coexpressing TASK and AT1a angiotensin II receptor, angiotensin II inhibited the TASK current. These data together indicate that TASK contributes to the generation of high resting potassium permeability of glomerulosa cells, and this background K+ channel may be a target of hormonal regulation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Whereas after ang II stimulation the source of calcium is both the intracellular store and the EC space, the calcium signal evoked by K+ depends exclusively on the influx of Ca2+ (8, 9). Voltage-dependent T- and L-type channels have been detected electrophysiologically (6, 7, 8, 10, 11) and also by molecular biological methods (12). Their contribution to the calcium signal and activation of steroid synthesis is widely accepted both during ang II and K+ stimulation. Considering the significance of voltage-dependent mechanisms, the membrane potential and its alteration during stimulation are of particular interest.
The negative resting membrane potential of glomerulosa cells derives mainly from the high K+ permeability (13). There were several attempts to characterize the K+ channel that contributes to the high resting permeability. Inward rectifiers (14) and more recently a weakly voltage-dependent current (15) were suggested as possible candidates. Inhibition of these potassium currents by ang II was demonstrated (15, 16), but the channels responsible for the currents were not identified or further characterized beyond electophysiological properties.
Recently, a new class of K+ channels with two pore domains has been described (17, 18, 19, 20, 21). Since the permeability of these channels is independent or only slightly dependent on the membrane potential, these background (leak) channels are also good candidates for being involved in the determination of resting membrane potential.
In the present study we demonstrate that TASK, a member of the two-pore domain potassium channel family, is expressed abundantly in glomerulosa cells. Patch clamp results on isolated glomerulosa cells indicated partial pH sensitivity of the potassium conductance. Analysis of the potassium current developing after injection of Xenopus oocytes with rat glomerulosa mRNA suggests that this background channel contributes to the potassium conductance of glomerulosa cells. We show that ang II, via AT1 receptors, inhibits TASK substantially. This mechanism may have a role in the depolarizing effect of ang II in glomerulosa cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(gigaohm) or higher seal resistance was -82 ± 1 mV
(n = 10). To study only those channels that operate around the
resting membrane potential, the cells were clamped at -100 mV. In
normal EC medium (3.6 mM [K+]), the
inward current was 56 ± 20 pA (n = 10). When the EC
[K+] was elevated to 30 mM, the
inward current increased to 307 ± 80 pA (n = 10).
Acidification of the EC medium from pH 7.4 to 6.7 reduced this current
by 17 ± 2% (n = 10). However, changing the EC pH
to 6.7 failed to evolve any further inhibition in cells being
challenged with ang II (10 nM), which caused
significant (61 ± 7%) inward current inhibition at -100 mV (in
30 mM EC [K+], pH 7.4,
n = 6). A biramp voltage protocol (from -100 to +40 mV) was
applied in each bath solution. The current-voltage curves obtained both
at lowered pH (pH 6.7, n = 10) and in the presence of ang II (10
nM, n = 6) crossed over at -38 ± 2%
mV with the control curve (in 30 mM EC
K+, Fig. 1
), which
corresponds to the calculated K+ equilibrium
potential (-39 mV).
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). In oocytes having very negative
Em (-87 mV, or below) in 2 mM
[K+], the shift of reversal potential was
90 ± 1.8 mV (n = 4) when EC [K+] was
increased from 2 mM to 80 mM. This value is
close to the 93 mV shift predicted for
K+-selective channels, indicating that potassium
channels dominate the membrane conductance. (In oocytes having less
negative membrane potential, probably as a consequence of relatively
higher leak current or lower level of expression, the contribution of
other ions to the membrane conductance was more notable.) To correct
for leak and currents carried by ions other than
K+, the difference of inward currents in 80 and 2
mM EC [K+] at -100 mV
(ID80-2) was calculated. Control
ID80-2 (what can be considered as the current of
the endogenous K+ channels of the oocyte) was
111 ± 8 nA in the 10 water-injected or noninjected oocytes having
the largest current differences. ID80-2 was
1882 ± 120 nA in the 10 mRNA injected oocytes of highest
expression.
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), and currents in the presence and
absence of the inhibitor were corrected for the nonspecific current
measured in 2 mM [K+] (as described
above). Neither ImRNA
(ID80-2 in mRNA injected oocytes) nor
ITASK (ID80-2 in TASK cRNA
injected oocytes) was inhibited significantly by the classical
K+-channel blockers tetraethylammonium (3
mM) and 4-aminopyridine (3 mM). Slight
inhibition was observed with Ba2+ (100
µM) at -100 mV, which was absent at more positive
potentials. To study further the characteristics of the
voltage-dependent inhibition, the effect of higher concentration of
Ba2+ (300 µM) was also tested.
Since the Ba2+ block developed slowly, long (2
sec) voltage steps were applied to attain steady state at each membrane
potential. Ba2+ inhibited
ImRNA and ITASK with
similar kinetics (Fig. 3), and the
voltage dependence of steady state inhibition was also similar (Fig. 4, A and B). Applying the model of open
pore channel blockers (22) for ITASK results in a
dissociation constant (KD) of 16.8 ± 2.2
mM and z
of 0.98 ± 0.03 (the product of the charge
of Ba2+ and the fraction of the electrical field
it has to traverse to reach its binding site), which suggests that the
binding site is about halfway in the electrical field.
Cs+ (3 mM) also exerted a
voltage-dependent block of ImRNA and
ITASK (Fig. 4, C and D). Steady state inhibition
by Cs+ was almost instantaneous. In 3
mM [Cs+], the inhibition reached
its maximum below -110 mV but the inhibition was not complete.
ImRNA was reduced by lidocaine (1 mM)
or by changing EC pH from 7.5 to 6.7; however, the degree of inhibition
was weaker than that of ITASK (Table 1).
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To avoid possible nonspecific effects of the oligonucleotides, they
were administered in a second 50-nl injection 2–3 h after the
injection of glomerulosa mRNA. The antisense oligonucleotide reduced
the expression of ImRNA almost to the small
current of the control oocytes injected with water only. The sense
oligonucleotide did not have any effect on its own; furthermore, when
the same amount of sense and antisense oligos were mixed and injected,
the inhibitory effect was partially reverted, confirming the
specificity of the antisense approach (Fig. 5).
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). In oocytes
perifused with high potassium solution ([K+] =
80 mM) alteration of the K+
permeability is expected to influence the current minimally at +20 mV
(as this potential is close to EK). Hence,
current changes at +20 mV were dominated by the
Ca2+-dependent Cl-
current.
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). Thus, during stimulation the Ca2+
signal and the changes of the K+ current could be
detected simultaneously and fairly separately by measuring the current
alternately at -100 and +20 mV in high [K+]
solution. Using this experimental protocol ImRNA
(measured at -100 mV in 80 mM
[K+]) was slightly reduced by ang II mainly in
those oocytes that showed the largest inward current before
stimulation, indicating high level expression (Fig. 6B), while in many
cells the inhibition was barely detectable.
To address the question whether the inhibitory effect of ang II can be
related to the modulation of TASK activity, angiotensin (AT1a)
receptor and TASK cRNAs were coinjected into oocytes. Ang II (10
nM) was applied in the superfusion medium, and its effect
on the currents at -100 and +20 mV was followed for 3–5 min. Ang II
reduced ITASK by 77 ± 2.7% (n = 5,
Fig. 7). Inhibition of TASK was
maintained and only slowly diminished in time after ang II had been
withdrawn while activation of the Ca2+-activated
Cl- current had a transient component and after
reaching a peak value it was quickly reduced to a sustained level.
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), which difference was
statistically significant (P < 0.05).
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). In one
experiment another TASK-specific probe (PstI-SacI
fragment of human TASK), homologous to 1016–1228 nucleotides of
rat TASK coding region was used and gave similar results (data not
shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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While these channels may have a role in restoring the resting condition from a stimulated state, they presumably are not involved in generation of the negative Em, since they require depolarization and/or high intracellular [Ca2+] for opening. Only the inwardly rectifying K+ channel would be appropriate for maintaining the very negative Em; however, it could not be detected at the macroscopic current level (15, 29). A challenging question is what is behind the discrepancy between single-channel and macroscopic current results. Lotshaw (15) described a weakly voltage-dependent (background) potassium channel in nystatin- perforated cells and suggested that it has a major role in the control of the resting membrane potential. Rapid run down may have hampered the detection of this current in single-channel measurements; nevertheless, the characteristics of a single-channel conductance described and interpreted as delayed rectifier (29) are consistent with this background channel. Our results also provide evidence that during hyperpolarization (at -100 mV) potassium channels, which do not show inward rectification, are mainly responsible for the membrane conductance. This conductance is pH sensitive, in accordance with the previously observed stimulation of aldosterone production by acidic pH (30, 31). In an attempt to gather more information about the channels responsible for this conductance, more detailed characterization was performed in a heterologous expression system.
A wide range of K+ channels, including several inwardly rectifying ones, were successfully expressed and studied in oocytes. Although the rate and the time dependence of expression of distinct channels may be different, this system provides significant advantages and may be optimal for characterizing the dominant K+ conductance of a cell type by injecting its mRNA into oocytes. It should be recalled, however, that endogenous Xenopus channels may influence the membrane conductance. Oocytes have usually only small endogenous currents at membrane potentials below -40 mV; however, Cl- channels, activating slowly in response to hyperpolarization and sometimes conducting substantial currents, were described to be present occasionally in particular oocyte batches (32). In our experiments, some oocyte batches also showed this type of current; however, it could be corrected for by calculating the difference of the inward currents in 80 and 2 mM EC [K+] (ID80-2). The effect of inhibitors was measured in oocytes, where the hyperpolarization-activated current was negligible.
The K+ current measured at -100 mV in oocytes injected with mRNA prepared from adrenal glomerulosa or with TASK cRNA (ImRNA and ITASK, respectively) was not affected by the conventional K+ channel blockers. It was minimally inhibited by 100 µM Ba2+, which argues against the significant contribution of most of the inwardly rectifying K+ channels to the current. Voltage dependence of inhibition by higher concentration of Ba2+ (300 µM) and Cs+ (3 mM) was similar in the case of ImRNA and ITASK. A further similarity between ITASK and ImRNA is their slowly developing inhibition by 300 µM Ba2+, the time dependence of which is not general even among the members of the tandem pore domain K+ channel family (33). While the parameters of the Ba2+ binding site can be calculated according to the original open-channel block model (22), the steep voltage dependence of the Cs+ inhibition, which cannot be explained by this model, indicates a more compound mechanism of blockage (34). Accordingly, TASK probably has a multiion conducting pore (35) similar to many other K+ channels (34). Local anesthetics and decreased EC pH inhibit human and rat TASK (18, 23). Calculation with the inhibition of ImRNA and ITASK by lidocaine (1 mM) or by EC acidification indicates that ITASK is responsible for at least 25% of ImRNA. If we consider this minimum contribution of TASK to ImRNA suggested by the EC acidification, then the additional component of ImRNA shows significant pharmacological similarities to the two-pore domain background potassium channels.
The TASK antisense oligonucleotide prevented the expression of ITASK (data not shown) and also reduced the expression of ImRNA by 85%. Control experiments confirmed the specificity of the antisense effect. The sense oligo failed to inhibit the expression of ImRNA; moreover, when coinjected with the antisense one, it reduced the inhibitory effect of the latter. Considering that the sense and the antisense oligonucleotides were complementary, this means that only the single-stranded form of the antisense was effective, which indicates its specificity. Partial reversal of the inhibition is probably due to incomplete formation of oligonucleotide dimers (although theoretically a limited nonspecific effect cannot be ruled out). Almost complete inhibition of ImRNA by TASK antisense oligonucleotide raises the possibility that TASK expressed by injection of glomerulosa mRNA might have partially different pharmacological properties from pure ITASK. It would be conceivable if another pore-forming or auxiliary subunit cooperating with TASK was presumed.
Depolarization of glomerulosa cells by ang II has been demonstrated both by fluorimetric (5, 36) and electrophysiological methods (4, 37). The depolarization may be attributed principally to sustained inhibition of K+ permeability (38, 39), while inhibition of the Na+,K+-ATPase (40), as well as opening of a nonspecific cation channel (41), may contribute to the effect. As to K+ permeability, patch-clamp studies revealed ang II-induced inhibition of inward rectifier (14), delayed rectifier K+ (14, 15), and weakly voltage-dependent currents (15). While inhibition of delayed rectifying K+ channels may prolong but may not initiate depolarization, the significance of inward rectifiers is questionable (see above). Therefore, inhibition of the weakly voltage-dependent K+ current, which was confirmed in the present experiments, may account predominantly for the depolarizing action of ang II in rat glomerulosa cells.
In oocytes injected with mRNA prepared from glomerulosa tissue, ang II also reduced the inward current at -100 mV in 80 mM [K+], which indicates the inhibition of the expressed ImRNA. The possible reason why the degree of the detected inhibition was smaller than that observed in glomerulosa cells could be the concomitant activation of the calcium-activated Cl- and/or K+ channels (the latter possibly introduced by adrenal glomerulosa mRNA). This may have partially masked the inhibitory effect mainly in oocytes where the expression of ImRNA was moderate. Toxicity of glomerulosa mRNA limited the achievable ImRNA expression, and coinjection of ang II receptor cRNA with mRNA failed to increase the apparent inhibition at -100 mV by ang II (result not shown). We examined whether such regulation could be exerted via inhibition of TASK. When TASK and AT1a angiotensin II receptor were coexpressed, a dramatic inhibition of ITASK was observed as a result of ang II stimulation.
In conclusion, we demonstrated that TASK, a background potassium channel, is abundantly expressed in adrenal glomerulosa cells. TASK is a significant component of the potassium conductance expressed in oocytes after injection of glomerulosa mRNA; thus, it may contribute to the maintenance of the highly negative membrane potential in adrenal glomerulosa cells. Activation of angiotensin II (AT1a) receptor inhibits TASK; therefore, this channel is a target for the depolarizing action of ang II and it may be a component of the complex signal transduction routes used by the peptide in vivo. To our knowledge this is the first demonstration that a K+ channel of the tandem pore domain family is inhibited by a Ca2+-mobilizing hormone. The signaling pathway of this inhibition and its contribution to the physiological function of intact glomerulosa cells remain to be established.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-32P]dCTP,
[-35S]dCTP, and
[-32P]dATP were from Izinta (Budapest,
Hungary).
Animals and Tissue Preparation
Mature female Xenopus laevis frogs were obtained from
Amrep Reptielen (Breda, The Netherlands). Frogs were anesthetized by
immersing them into benzocaine solution (0.03%). Ovarian lobes were
removed, the tissue was dissected and treated with collagenase (1.45
mg/ml, 148 U/mg, type I, Worthington Biochemical Corp.
(Freehold, NJ) followed by continuous mechanical agitation in
Ca2+-free OR2 solution containing (in
mM): NaCl, 82.5; KCl, 2;
MgCl2, 1; HEPES, 5; pH 7.5) for 1.5–2 h. Stage V
and VI oocytes were defolliculated manually and kept at 19 C in
modified Barth’s saline containing (in mM):
NaCl, 88; KCl, 1; NaHCO3, 2.4;
MgSO4, 0.82;
Ca(NO3)2, 0.33;
CaCl2, 0.41; HEPES, 20, buffered to pH 7.5 with
NaOH and supplemented with penicillin (100 U/ml), streptomycin (100
µg/ml), sodium pyruvate (4.5 mM), and
theophyllin (0.5 mM).
Wistar rats (250–350 g, Charles River Kft., Budapest, Hungary) were stunned before decapitation, and the adrenal glands were removed. Capsular tissue (containing the zona glomerulosa with the fibrous capsule) and decapsulated tissue (containing the inner cortical zones and the adrenal medulla) were separated macroscopically according to standard methods (42). Glomerulosa and fasciculata-reticularis cells, respectively, were prepared using a collagenase digestion technique as previously described (43). The contamination of this type of glomerulosa cell preparation was tested previously by electron microscopic analysis and was found to be less than 5%. Isolated cells were plated onto poly-L-lysine-coated (1 µg/cm2) Petri dishes and were kept in a CO2 (5%) incubator at 37 C in a mixture (38:62, vol/vol) of modified Krebs-Ringer-bicarbonate-glucose solution and Medium 199 (K+, 3.6 mM; Ca2+, 1.2 mM; Mg2+, 0.5 mM). The medium was completed with 100 U/ml penicillin and 100 µg/ml streptomycin. Glomerulosa and fasciculata-reticularis cells for molecular biological studies were selected according to standard criteria [size and lipid droplets (44)] from the respective capsular and decapsular preparations after one-day culturing.
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 Xenopus laevis Oocytes
Oocytes were injected 1 day after defolliculation. Fifty
nanoliters of the appropriate RNA solution were delivered with
Nanoliter Injector (World Precision Instruments, Saratosa, FL). In
experiments designed to test the effect of sense and antisense TASK
oligonucleotides on the expression of glomerulosa tissue mRNA-induced
inward current, the oligonucleotides were administered in a second
injection (TASK5'a: 5'-CACATTCTGCCGCTTCATCGTC-3' (70 µM,
50 nl) and TASK5's: 5'-GGCATATGAAGCGGCAGAATGTGCG-3' (90
µM, 50 nl)) 2–3 h after the injection of mRNA. Currents
were measured 3 or 4 days after the injection(s).
Electrophysiology
Patch-Clamp Recordings
For ion current measurements on adrenal glomerulosa cells, the
whole-cell patch-clamp technique (45) was applied. The standard EC
solution had the following composition (mM): NaCl, 137;
KCl, 3.6; MgCl2, 0.5;
CaCl2, 2; glucose, 11; HEPES, 10;
piperazine-N,N'-bis[2-ethanesulfonic acid] (PIPES),
3.3 [pH 7.4 or pH 6.7 (NaOH)]. Pipettes were pulled from borosilicate
glass Clark GC120TF-10 (Clark Electromedical, Pangburne, Reading, UK)
by a P-87 puller (Sutter Instrument Co., Novato, CA) and fire
polished. Pipette resistance ranged between 4 and 6 M when filled
with the intracellular solution, containing (mM): KCl, 135;
MgCl2, 2; CaCl2, 0.05;
EGTA, 1; Na-ATP, 2; HEPES, 10; pH 7.3 (KOH). The pipette was connected
to the headstage of a patch-clamp amplifier [Axopatch-1D (Axon
Instruments, Inc., Foster City, CA) or RK-400 (Biological,
Claix, France)] which was mounted on a PCS-750/1000 manipulator
(Burleigh Instruments, Inc., Fishers, NY). Seal resistance was about 10
G. The capacitance of the selected glomerulosa cells amounted to
5–10 pF. Series resistance was about 10 M. Data were filtered at 1
kHz (-3 dB; 4-pole, low-pass Bessel filter) and digitally sampled at 4
kHz by a Digidata 1200 interface board (Axon Instruments, Inc.),
stored, and later analyzed by PC/AT computer. Experiments, data
storage, and analysis were performed with pClamp software, version 6.0
(Axon Instruments, Inc.). Solutions were applied by a gravity-driven
perfusion system.
Two-Electrode Voltage Clamp
Membrane currents of oocytes were recorded by two-electrode voltage
clamp (OC-725-C, Warner Instrument Corp., 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 1–2.5 kHz with a Digidata Interface (Axon Instruments,
Inc.), and stored on a PC/AT computer. Recording and data analysis were
performed using pCLAMP software 6.0.4 (Axon Instruments, Inc.).
Experiments were carried out at room temperature, and solutions were
applied by a gravity-driven perfusion system. Low
[K+] solution contained (in mM):
NaCl, 95.4; KCl, 2; CaCl2,1.8; HEPES, 5. High
[K+] solution contained 80 mM
K+ (78 mM Na+
of the low [K+] solution was replaced with
K+). Unless otherwise stated, the pH of every
solution was adjusted to 7.5 with NaOH.
mRNA Purification and cRNA Synthesis
Total RNA was extracted from different rat tissues as previously
described, using the phenol-chloroform-guanidium isothiocyanate method
(12). mRNA was purified on oligo dT-cellulose (Pharmacia Biotech or New England Biolabs, Inc.), aliquoted,
and stored at -70 C. TASK and ang II receptor cRNA were synthesized
in vitro according to the manufacturer’s instructions
(Ambion, Inc. mMESSAGE mMACHINE T7 In vitro
Transcription Kit) using the XhoI-linearized pEXO-TASK
construction (18), which contained the total coding region of human
TASK and a NotI-linearized plasmid construction comprising
the coding sequence and 5'- untranslated region of rat AT1a
receptor (a gift from Dr. L. Hunyady).
RT-PCR and Single-Cell PCR
TASK cDNA fragments were amplified by Taq DNA
polymerase using TASK1s (5'-SYTCTWCTTCGCCAKCACCG-3') or TASK5's sense
and TASK1a (5'-CCSARGCCRATGGTGSTSAG-3') or TASK3'a
(CACKGAGCTCCTGCGCTTCATG) antisense oligonucleotides after reverse
transcription (MMLV-RT, random hexamers from Promega Corp.) of 1 µg of total RNA prepared from rat glomerulosa
tissue. TASK1s and TASK1a were designed to amplify not only TASK but
also the cDNA of TWIK (17). The first denaturing step (94 C, 120 sec)
was followed by 35 cycles of denaturation (30 sec at 94 C), annealation
(60 sec at 50 C), and extension (90 sec at 72 C). The TASK1s-TASK1a
product was cloned into pBluescript KS- (Stratagene, La
Jolla, CA) vector with blunt ends and sequenced according to standard
methods (Sequenase II kit, United States Biochemical Corp., Cleveland, OH). 5'- and 3'-parts of the mRNA were
amplified by TASK5's-TASK1a and TASK1s-Task3'a primers, respectively.
For single-cell PCR, individual glomerulosa or fasciculata-reticularis
cells were selected microscopically from the appropriate capsular or
decapsular cell preparation, respectively, and were placed into RT
reactions after freezing and thawing. Nested PCR was performed with
primer pairs TASK1s and 5'-TCCTTCTGCAGCGCCACGTAG-3' in the first, and
5'-ACGGACGGAGGCAAGGTGTTC-3' and TASK1a in the second reaction. Tissue
culture medium after RT or cells without RT were used as controls. PCR
conditions were the same as above both in the first and the second
reaction.
Northern Blot Analysis
Ten micrograms of total RNA from different tissues were loaded
and run on 1% agarose formaldehyde gel after denaturation.
Electrophoretic separation of the RNA was followed by its transfer to
Hybond nylon membrane (Amersham Pharmacia Biotech). TASK
probe was generated by random primer labeling the 360-bp TASK1s-TASK1a
PCR product or the 212-bp PstI-SacI fragment of
the human TASK clone (18) with [32P]dCTP.
Hybridization was carried out at 42 C for 24 h. After
hybridization the blot was washed successively in buffers of 1x SSPE +
0.1% SDS twice for 30 min at room temperature and 0.1x SSPE + 0.1%
SDS twice for 30 min at 65 C (46). After detection of the radioactivity
by Phosphor-imager (model GS-525, Bio-Rad Laboratories, Inc. Hercules, CA), the membrane was stripped and reprobed for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference signal as
previously described (12).
Statistics
Data are expressed as means ± SEM. Statistical
significance was estimated by the nonparametric Mann-Whitney
U test, or the nonparametric Fisher exact test [STATISTICA
program package (StatSoft, Tulsa, OK)].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by the Hungarian National Research Fund (OTKA T019983), by the Hungarian National Academy of Sciences (AKP 97–16 3,2/49), and by the Hungarian Medical Research Council (ETT- 528/96).
Received for publication July 16, 1999. Revision received January 20, 2000. Accepted for publication February 23, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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G. Czirjak, Z. E. Toth, and P. Enyedi The Two-pore Domain K+ Channel, TRESK, Is Activated by the Cytoplasmic Calcium Signal through Calcineurin J. Biol. Chem., April 30, 2004; 279(18): 18550 - 18558. [Abstract] [Full Text] [PDF]
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), 80
mM (
) EC [K+] and
again in 2 mM EC [K+] (X)
after washout of the high potassium solution.
quotient).
Dividing the quotients of different tissues by the quotient of capsular
tissue in the same blot resulted in the following values: capsular
tissue (