TASK (TWIKRelated Acid-Sensitive K+ Channel) Is Expressed in Glomerulosa Cells of Rat Adrenal Cortex and Inhibited by Angiotensin II
Gábor Czirják,
Tamás Fischer,
András Spät,
Florian Lesage and
Péter Enyedi
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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The present study was conducted to explore the
possible contribution of a recently described leak
K+ channel, TASK (TWIK-related acid-sensitive
K+ channel), to the high resting
K+ conductance of adrenal glomerulosa cells.
Northern blot analysis showed the strongest TASK message in adrenal
glomerulosa (capsular) tissue among the examined tissues including
heart and brain. Single-cell PCR demonstrated TASK expression in
glomerulosa cells. In patch-clamp experiments performed on isolated
glomerulosa cells the inward current at -100 mV in 30
mM [K+] (reflecting
mainly potassium conductance) was pH sensitive (17 ± 2%
reduction when the pH changed from 7.4 to 6.7).
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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The most important physiological activators of adrenal glomerulosa
cells are angiotensin II (ang II), elevated extracellular (EC)
[K+], and ACTH. While ACTH acts primarily via
cAMP, the other two stimuli elevate cytoplasmic
[Ca2+]. Binding of ang II to AT1 receptors
rapidly activates phosholipase C, which is followed by inositol 1,4,5
trisphosphate (InsP3) generation and release of
Ca2+ from intracellular stores. This early phase
of the signal is followed by a sustained stimulation that is dependent
on Ca2+ influx. While observations by different
groups indicate the role of capacitative Ca2+
entry in the sustained phase (1, 2), in addition to this store-operated
mechanism, a further dihydropiridine-sensitive influx component has
also been well documented (cf Ref. 3). In addition,
InsP3 generation ang II depolarizes glomerulosa
cells (4, 5), which explains the activation of voltage-dependent
calcium channels and partial dihydropyridine sensitivity of the calcium
and aldosterone response (3, 6, 7).
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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pH and Angiotensin II Sensitivity of the Potassium Conductance of
Glomerulosa Cells
The potassium conductance of adrenal glomerulosa cells was studied
by the patch clamp technique. The membrane potential of cells showing
10 G
(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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Figure 1. Inhibition of the Currents of a Rat Adrenal
Glomerulosa Cell by Acidification and/or Angiotensin II
A, 3.6 mM EC [K+], pH 7.4;
B, 30 mM EC [K+], pH
7.4; C, 30 mM EC [K+], pH 6.7;
D, 30 mM EC [K+], pH
7.4, 10 nM angiotensin II; E, 30 mM
EC [K+], pH 6.7, 10 nM angiotensin
II. (Biramp depolarizations from -100 mV to +40 mV (see
inset), the holding potential was -80 mV).
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Expression of High Resting K+ Permeability
in Xenopus Oocytes
The resting membrane potential (Em) of
oocytes injected with mRNA of rat glomerulosa tissue was more negative
than that of water-injected or noninjected oocytes (-80.9 ± 1.6
mV and -44.3 ± 1.7 mV for the 10 most polarized oocytes of the
respective group). To characterize the expressed ion conductances
responsible for driving Em close to the
K+ equilibrium potential
(EK), current-voltage (I-V) relationship was
measured in solutions containing 2 and 80 mM
[K+]. Changing of EC
[K+] from 2 to 80 mM induced the
appearance of a noninactivating current in oocytes injected with mRNA
(Fig. 2
). 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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Figure 2. Currents of Xenopus Oocytes Injected
with mRNA Purified from Adrenal Capsular Tissue
A, Currents elicited by 300-msec depolarizing voltage steps from
-120 to +20 mV in 10-mV increments in 80 mM EC
[K+]. B, Steady-state current voltage
relationship of ImRNA (measured at the end of the
300-msec voltage steps) in 2 mM ( ), 80
mM ( ) EC [K+] and
again in 2 mM EC [K+] (X)
after washout of the high potassium solution.
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Comparison of Pharmacology of ImRNA and
ITASK
The effect of inhibitors on the inward K+
current in oocytes injected with glomerulosa mRNA or TASK cRNA was
measured in 80 mM [K+] at -100 mV.
Inhibition was expressed in per cent of the control current (80
mM [K+] at -100 mV (Table 1
), 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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Figure 3. Time Dependence of the Ba2+ Block
ImRNA (A and C) and ITASK (B and D) in
high (80 mM) [K+] solution in the
absence (A and B) and in the presence (C and D) of 300 µm
Ba2+. Oocytes were held at 0 mV and currents were recorded
during 2 s voltage steps from -120 to +20 mV in 10-mV increments.
(In C and D only every second trace is shown.) The inhibition is
calculated by subtracting the currents in the presence of
Ba2+ (C and D) from the currents in the absence of
Ba2+ (A and B), respectively, and the difference is plotted
[ImRNA (panel E) and ITASK (panel F)].
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Inhibition of ImRNA Expression by TASK
Antisense Oligonucleotide
The contribution of TASK to the potassium conductance induced by
injection of glomerulosa mRNA into Xenopus oocytes was
addressed also by an antisense method. The antisense (TASK5'a, 22-mer)
oligonucleotide was designed to anneal with the start ATG codon of the
TASK mRNA extending 4 bases upstream and 18 bases downstream. The
25-nucleotide long sense oligonucleotide (TASK5's) was used as the
negative control being complementary with the antisense nucleotide in a
18-bp segment.
To avoid possible nonspecific effects of the oligonucleotides, they
were administered in a second 50-nl injection 23 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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Figure 5. Effects of the Sense and Antisense Oligonucleotides
on the Expression of ImRNA
Currents were recorded at the end of 300-msec voltage pulses to -100
mV from a holding potential of -30 mV in every 3 sec.
ID80-2 was estimated as the difference of the maximum of
current in 80 mM [K+] and the average of
the currents in 2 mM [K+] before and
after the high K+ solution (see inset).
Numbers in the bars represent number of oocytes deriving
from two frogs. (*, P < 10- 5; **,
P < 0.0015).
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Ang II inhibits ImRNA and
ITASK
Oocytes injected with glomerulosa mRNA or ang II receptor cRNA
responded to ang II (10 nM) challenge with a calcium signal
that was detected by following the activation of the endogenous
Ca2+-activated Cl- channel
of the oocyte. In standard EC solution ([K+] =
2 mM) at negative membrane potentials (-80 or -100 mV),
the activation of the channel appears only as a small inward current,
while it is represented as a much larger increase of the outward
current at +20 mV (Fig. 6A
). 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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Figure 6. Effect of Angiotensin II (10 nM)
on the Current Measured at -100 (or -80) and +20 mV in 2 and 80
mM EC [K+] in Oocytes Injected with
Adrenal Capsular mRNA
A, EC [K+] was 2 mM. The holding
potential was -80 mV, and 300-msec depolarizing voltage steps (to +20
mV) were applied every 3 sec. Currents at -80 mV measured before the
step and currents measured at the end of the step were plotted. B, EC
[K+] was 80 mM. Every 3 sec the voltage
protocol (-100 mV for 300 msec, 0 mV for 250 msec, +20 mV for 300
msec, depicted in Fig. 7 ) was applied from a holding potential of 0 mV.
Currents at the end of the steps to -100 mV and +20 mV were plotted.
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On the other hand, in oocytes having robust ImRNA
or ITASK expression, the major component of the
inward current at -100 mV in high [K+]
solution was the potassium influx, since the
Ca2+-activated Cl- current
was relatively small at -100 mV (even during ang II stimulation; see
Fig. 6A
). 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 35 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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Figure 7. Inhibition of TASK by Angiotensin II (10
nM) in an Oocyte Coinjected with TASK and Angiotensin
II Receptor cRNAs
A, Every 3 sec the voltage protocol (see inset)
was applied from a holding potential of 0 mV. Currents before ang II
stimulus (0 sec), at the peak of the Ca2+-activated current
(75 sec) and after the developing of the maximum inhibition of
ITASK (105 sec) are shown. B, Currents at the end of the
step to -100 mV and at the end of the step to +20 mV were plotted as
the function of time.
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Molecular Biological Verification of TASK Expression in Glomerulosa
Cells
The PCR product generated by TASK1s and TASK1a (two degenerate
primers for both TASK and TWIK, flanking a nonconserved region of the
two mRNAs) was cloned, sequenced, and identified as the appropriate
fragment of rat TASK. In addition to this fragment the total coding
region of TASK in two separate overlapping (5' and 3') parts was
amplified (TASK5's-TASK1a, 1608 n and TASK1s-TASK3'a 249-1233 n) and
sequenced. The sequence of these products corresponded to that of rat
TASK. TASK-specific PCR product was amplified (in two independent
experiments) from single glomerulosa and fasciculata- reticularis
cells, which derived from capsular and decapsular cell cultures,
respectively. Under identical conditions in nested PCR reaction, the
ratio of TASK-positive cells was higher in glomerulosa (28 positive of
29 cells) than in fasciculata-reticularis cells (21 positive of 29
cells, Fig. 8
), which difference was
statistically significant (P < 0.05).

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Figure 8. Single-Cell PCR for Detection of TASK mRNA
Nested RT-PCR was performed from single adrenal glomerulosa cells (A),
tissue culture medium of glomerulosa cells (B), single adrenal
glomerulosa cells without reverse transcription (C), single
adrenal fasciculata/reticularis cells (D), tissue culture medium of
fasciculata/reticularis cells (E) and single adrenal
fasciculata/reticularis cells without reverse transcription (F). The
expected size of the PCR product is 302 bp. (Representative one of two
experiments).
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Northern blots were performed for comparing the TASK mRNA content of
rat glomerulosa tissue to that of other rat tissues using the labeled
TASK1s-TASK1a fragment (249608 nucleotides) as a probe. Since
the heart was found to be the most abundant source of rat TASK (23),
and TASK in the mouse was shown to be expressed in the atria rather
than in the ventricles of the heart (18), we prepared RNA from rat
atria. TASK expression in rat adrenal capsule was the highest among the
examined tissues (Fig. 9
). In one
experiment another TASK-specific probe (PstI-SacI
fragment of human TASK), homologous to 10161228 nucleotides of
rat TASK coding region was used and gave similar results (data not
shown).

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Figure 9. Expression of TASK mRNA in Different Rat Tissues
A, Representative Northern blot (10 µg RNA) with the rat
TASK1s-TASK1a probe. B, GAPDH was used as the reference
signal. After subtracting background counts of the TASK bands were
divided by the count of the corresponding GAPDH band ( quotient).
Dividing the quotients of different tissues by the quotient of capsular
tissue in the same blot resulted in the following values: capsular
tissue (1 ); decapsular tissue (0.52 ± 0.07); cerebellum
(0.08 ± 0.03); atria of heart (0.07 ± 0.01); liver and
brain below 0.05 (n = 5 Northern blots).
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DISCUSSION
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Sensitivity to physiological changes of EC
[K+] (24, 25), maintenance of highly negative
membrane potential (13, 15, 26), and the importance of
voltage-sensitive mechanisms in the action of physiological stimuli on
glomerulosa cells (27) urged several researchers to study the potassium
channels of this tissue. The first detailed analysis of electrical
properties of rat adrenal glomerulosa cells was performed by Quinn and
co-workers (13) using whole-cell patch-clamp methods (13). While around
and below the resting membrane potential the voltage-current
relationship followed the constant field equation, it showed an outward
rectification under depolarized conditions. The increased conductance
in the depolarized state was assigned to opening of voltage- and/or
Ca2+-activated K+ channels.
In addition to the noninactivating delayed rectifier
K+ currents (which were present in all species) a
transient (A type) current was also detected in human (16, 28) and
bovine (16) glomerulosa cells. Studies at the single-channel level
revealed further members of the channel repertoire. Inward rectifier
K+ channels were observed frequently in membrane
patches of rat (and bovine) glomerulosa cells (29). Delayed rectifiers
(29) and Ca2+-activated
maxi-K+ channels (15) were also well
distinguished.
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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Chemicals
Enzymes and kits for molecular biological studies were purchased
from Ambion, Inc. (Austin, TX), Amersham Pharmacia Biotech (Little Chalfont, UK), Fermentas (Vilnius, Lithuania),
New England Biolabs, Inc. (Beverly, MA), Pharmacia Biotech (Uppsala, Sweden), and Promega Corp.
(Madison, WI). Fine chemicals of analytical grade were obtained from
Fluka Chemical Co. (Buchs, Switzerland), Promega Corp., and Sigma (St. Louis, MO).
[
-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 1.52 h. Stage V
and VI oocytes were defolliculated manually and kept at 19 C in
modified Barths 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 (250350 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)) 23 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
510 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 0.33 M
when filled
with 3 M KCl. Currents were filtered at 1 kHz, digitally
sampled at 12.5 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 manufacturers 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)].
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Dr. László Hunyady for the
angiotensin II (AT1a) receptor plasmid construct. The skillful
technical assistance of Miss Erika Kovács and Mrs. Irén
Veres is greatly appreciated.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Péter Enyedi, M.D., Ph.D., Department of Physiology, Semmelweis University of Medicine, P.O. Box 259, H-1444 Budapest, Hungary.
This work was supported by the Hungarian National Research Fund (OTKA
T019983), by the Hungarian National Academy of Sciences (AKP 9716
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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