Unité Mixte de Recherche Centre National de la Recherche Scientifique 6548 Université de Nice-Sophia Antipolis, O6108 Nice Cedex 2, France
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ABSTRACT |
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The role of CFTR in
the control of K+ currents was studied in mouse kidney.
Whole cell clamp was used to identify K+ currents on the
basis of pharmacological sensitivities in primary cultures of proximal
(PCT) and distal convoluted tubule (DCT) and cortical collecting tubule
(CCT) from wild-type (WT) and CFTR knockout (KO) mice. In DCT and CCT
cells, forskolin activated a 293B-sensitive K+ current in
WT, but not in KO, mice. In these cells, a hypotonic shock induced
K+ currents blocked by charybdotoxin in WT, but not in KO,
mice. In PCT cells from WT and KO mice, the hypotonicity-induced
K+ currents were insensitive to these toxins and were
activated at extracellular pH 8.0 and inhibited at pH 6.0, suggesting
that the corresponding channel was TASK2. In conclusion, CFTR is
implicated in the control of KCNQ1 and Ca2+-sensitive
swelling-activated K+ conductances in DCT and CCT, but not
in proximal convoluted tubule, cells. In KO mice, impairment of the
regulatory volume decrease process in DCT and CCT could be due to the
loss of an autocrine mechanism, implicating ATP and adenosine, which
controls swelling-activated Cl and K+ channels.
kidney; cystic fibrosis; regulatory volume decrease; cell volume; calcium level
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INTRODUCTION |
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CYSTIC FIBROSIS
TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) is not only a
Cl channel sensitive to cAMP in the epithelia, but it is
also a modulator of other ion channels or transporters
(1). CFTR interacts with different K+
channels, such as ROMK (11, 19) or KCNQ1 (2),
and probably also with K+ channels implicated in the
control of cell volume (30, 32). However, the nature of
these interactions is often open to discussion. For instance, the
interaction could be direct by protein-protein interaction, as in the
case of ROMK2 (4, 19, 20), or indirect, by control of cAMP
sensitivity, as for KCNQ1 (2). Concerning the
swelling-activated K+ channel, modulation by CFTR could
occur via control of the intracellular Ca2+, because this
K+ channel could be a the Ca2+-dependent
K+ channel (32). It is clear that exact
knowledge of the type of interactions that occur between CFTR and its
partners is important, inasmuch as it facilitates an understanding of
the defects of ion handling in cystic fibrosis (CF) and,
therefore, a possible treatment of the disease. The kidney is a useful
tool for studying CFTR control of K+ conductance, inasmuch
as there is a differential expression of K+ channels along
the nephron, together with a differential expression of CFTR. In the
case of CFTR, despite the presence of cftr transcripts in
proximal (PCT) and distal convoluted tubules (DCT), CFTR expression, along with cAMP-sensitive Cl
conductance, was found in
the distal tubule only (21, 27). However, CFTR-dependent
swelling-activated Cl
channels were recorded in both
segments. It was therefore interesting to study whether different
K+ channels are associated with CFTR-dependent
Cl
channels in PCT and DCT. For this purpose, we carried
out patch-clamp experiments in primary cultures of PCT, DCT, and
cortical collecting tubule (CCT) from wild-type and
cftr
/
mice. The results demonstrate that, in DCT and CCT
cells, CFTR is indispensable for the expression of a cAMP-sensitive
KCNQ1 conductance and for the activation of a
Ca2+-dependent swelling-activated K+
conductance. However, regulation of these currents is quite different, because, in cftr
/
DCT or CCT cells, the cAMP-sensitive
current could not be restored by cAMP application, whereas the
swelling-activated current had been restored by extracellular adenosine
perfusion. This suggests that, in the distal tubule, KCNQ1 function
depends on the integrity of CFTR, whereas the swelling-activated
K+ channel depends on the integrity of an upstream
regulatory mechanism implicating CFTR. As for the swelling-activated
Cl
conductance (25), such a mechanism could
involve an autocrine ATP release (3) followed by
hydrolysis of ATP in adenosine and an adenosine-activated
Ca2+ influx that finally activates
Ca2+-sensitive maxi and small K+ (BK and SK,
respectively) channels. The absence of this cascade in
cftr
/
epithelia explains why DCT and CCT cells are
unable to undergo regulatory volume decrease (RVD) after a hypotonic shock. In contrast, in PCT, CFTR does not control swelling-activated K+ conductance, inasmuch as these channels belong to the
TASK2 family, the expression of which is not related to CFTR. Thus, in
cftr
/
PCT cells, the lack of RVD is mainly due to the
absence of regulation of the swelling-activated Cl
currents.
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MATERIALS AND METHODS |
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Animals
CFTR knockout mice were generated by the gene-targeting methodology described previously (28) at Centre de Développement des Techniques Avancées pour l'Expérimentation Animale (Orléans, France). This strain of mice was originally derived from ES129/Sv cells injected into C57BL/6 embryos. Mice were backcrossed with C57BL/6 mice for three generations and then intercrossed. They were allowed free access to food and water in a facility at 25 ± 1°C with a 12:12-h light-dark cycle. The 4- to 6-wk-old wild-type cftr+/+ mice and cftrPrimary Cell Cultures
PCT, DCT, and CCT were microdissected under sterile conditions. Kidneys were perfused with Hanks' solution (GIBCO) containing 700 kU/l collagenase (Worthington), cut into small pyramids that were incubated for 1 h at room temperature in the perfusion buffer (160 kU/l collagenase, 1% Nuserum, and 1 mM CaCl2), and continuously aerated. The pyramids were then rinsed thoroughly in the same buffer devoid of collagenase. The individual nephrons were dissected by hand in this buffer under binoculars using stainless steel needles mounted on Pasteur pipettes. The criteria used to identify the nephron segments have been described previously (6). Briefly, PCT corresponded to the 1- to 1.5-mm segment of tissue located immediately after the glomerulus. The DCT portion was the segment between the macula densa and the first branching with another tubule [connecting tubule (CNT)]. The CNT segment was discarded. CCT was identified as the straight poorly branched portion that followed the CNT segment. After they were rinsed in the dissecting medium, tubules were transferred to collagen-coated 35-mm petri dishes filled with culture medium composed of equal quantities of DMEM and Ham's F-12 (GIBCO) containing 15 mM NaHCO3, 20 mM HEPES, pH 7.4, 1% serum, 2 mM glutamine, 5 mg/l insulin, 50 nM dexamethasone, 10 µg/l epidermal growth factor, 5 mg/l transferrin, 30 nM sodium selenite, and 10 nM triiodothyronine. Cultures were maintained at 37°C in a 5% CO2-95% air water-saturated atmosphere. The medium was removed 4 days after seeding and then every 2 days.Electrophysiological Studies
Whole cell currents were recorded from 6- to 20-day-old cultured cells grown on collagen-coated supports maintained at 33°C for the duration of the experiments. The ruptured-patch whole cell configuration of the patch-clamp technique was used. Patch pipettes (2- to 3-MData acquisition and analysis.
Voltage-clamp commands, data acquisition, and data analysis were
controlled via a computer equipped with a Digidata 1200 interface (Axon
Instruments). pCLAMP software (versions 5.51 and 6.0, Axon Instruments)
was used to generate whole cell current-voltage (I-V) relationships, with the membrane currents resulting from voltage stimuli filtered at 1 kHz, sampled at 2.5 kHz, and stored directly on
the computer hard disk. Cells were held at 50 mV, and 400-ms pulses
from
100 to +120 mV were applied in 20-mV increments every 2 s.
Expression in Cultured Cells
The cDNA encoding CFTR was introduced into a polycistronic expression vector derived from the pIRESneo plasmid (cytomegalovirus promoter; Clontech), in which the neomycin resistance gene had been replaced by cDNA encoding the chain of the human CD8 cell surface antigen. Distal cells were transfected using the DAC-30 method according to the manufacturer's instructions (Eurogentec, Herstal, Belgium). The 6-day-old cultured cells grown on a 35-mm-diameter petri dish were serum starved for 24 h before transfection. Transfected cells with 2 µg of CD8-CFTR coexpress CFTR and CD8 at their plasma membrane and can be visualized using anti-CD8 antibody-coated beads (Dynabeads M-450, Dynal, Oslo, Norway) (13a). Cells were electrophysiologically tested 48 h after transfection.Chemicals
5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; Calbiochem) was prepared at 100 mM in DMSO and used at 0.1 mM in final solutions. Forskolin and ionomycin were prepared at 10 and 2 mM, respectively, in ethanol and used at 10 and 2 µM, respectively, in bath medium. Tetraethylammonium (TEA), charybdotoxin (CTX), quinidine, 6-N,N-diethyl- ![]() |
RESULTS |
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K+ Currents Activated by Forskolin
Experiments were performed in a hyperosmotic extracellular solution (350 mosmol/kgH2O) to characterize K+ currents activated by 10 µM forskolin in PCT, DCT, and CCT cells. Under these conditions, volume-activated K+ currents could not be detected. Moreover, to eliminate the Cl
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The forskolin-sensitive K+ currents measured at +100 mV are
compared in PCT, DCT, and CCT cells from cftr+/+ and
cftr/
mice in Fig. 1B. Only DCT and CCT from
wild-type mice exhibited forskolin-activated K+
conductance. In these cells, application of 10 µM 293B, 1 mM TEA, and
0.5 mM quinidine blocked this current by 80 ± 2 (n = 10), 55 ± 5 (n = 18), and
85 ± 4% (n = 9), respectively.
K+ Currents Induced by Hypotonic Shock
To prevent development of Cl
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For the cftr/
mice, the effect of hypotonic shock was
different between PCT and DCT or CCT cells. Hypotonic shock always induced swelling-activated K+ currents in PCT cells
(n = 9), whereas it did not significantly modify the
K+ conductance in DCT (n = 19) and CCT
(n = 9) cells (Fig. 2). Current intensity measured at
+100 mV for all types of cells from cftr+/+ and
cftr
/
mice is shown in Fig. 2B. The inability
of hypotonic shock to trigger swelling-activated K+
currents in DCT and CCT cells was observed in 100% of the 28 trials.
Overall, the results indicate that CFTR protein could be implicated in
the control of swelling-activated K+ conductances in
terminal, but not in proximal, segments of the nephron.
The results reported on DCT and CCT cells show that swelling-activated K+ conductances were roughly similar in these two cell types. For this reason, no further distinction was made between DCT and CCT in the following experimental series.
Pharmacology of Swelling-Activated K+ Channels
To further characterize the swelling-activated K+ currents, we tested the effect of various K+ channel blockers added separately to the bathing hypotonic solution. The swelling-activated outward K+ current measured at +100 mV in PCT cells from cftr+/+ mice is shown in Fig. 3. Perfusion of 1 mM TEA, 10 nM CTX, and 10 µM 293B did not significantly modify the swelling-activated K+ currents. In contrast, 0.5 mM quinidine and 10 µM clofilium decreased K+ current amplitude by 64 ± 6 (n = 6) and 55 ± 5% (n = 5), respectively (Fig. 3, A and B). Similar results were obtained in PCT from cftr
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The effects of the same channel blockers in DCT and CCT cells from
cftr+/+ mice are illustrated in Fig.
4. The noticeable difference from the PCT
cells was the strong reduction of swelling-activated K+
conductance in the presence of TEA and CTX (Fig. 4A):
inhibition = 82 ± 7 (n = 6) and 71 ± 3% (n = 18) for TEA and CTX, respectively. As
expected, quinidine was also very efficient in blocking the K+ currents (70 ± 5%, n = 5). In
contrast, 10 nM apamin was less efficient in blocking these currents
(26 ± 3%, n = 18; Fig. 4, A and
C). Finally, as observed in PCT cells, 293B did not
significantly decrease swelling-activated K+ currents in
DCT and CCT cells.
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The relative insensitivity to a number of known K+ channel inhibitors of the swelling-activated K+ current of PCT cells led us to carry out a further experiment to obtain more information concerning the nature of this channel.
Regulation of the K+ Conductance Induced by Hypotonic Shock in cftr+/+ PCT Cells
Role of extracellular pH.
To study the modulation of the swelling-activated K+
currents by extracellular pH (pHe), PCT cells were swollen
in hypotonic solutions (270 mosmol/kgH2O) adjusted to
pHe 6.0-8.0. Trace recordings at three different pH
values are shown in Fig. 5A.
Compared with the control K+ currents measured at
pHe 7.4, K+ currents at pHe 6.0 were reduced by 53 ± 3% (n = 9), whereas currents at pHe 8.0 were increased by 44 ± 10%
(n = 9). The corresponding I-V curves are
reported in Fig. 5B. Change in pHe did not
significantly modify Erev: 73 ± 8,
73 ± 2, and
74 ± 4 mV at pHe 6.0, 7.4, and
8.0, respectively (n = 9). Inhibition of the
K+ current at acidic pH became significant for membrane
potential of +20 mV [K+ current = 254 ± 53 (n = 9) and 436 ± 60 pA (n = 10)
at pHe 6.0 and 7.4, respectively (unpaired
t-test = 2.27, P < 0.039)], and stimulation of currents at alkaline pH became significant for membrane
potential of
20 mV [K+ current = 358 ± 75 (n = 6) and 174 ± 37 pA (n = 10)
at pHe 8.0 and 7.4, respectively (unpaired
t-test = 2.19, P < 0.047)].
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Role of extracellular Ca2+.
To eliminate the possibility that cytosolic Ca2+ is
involved in the development of hypotonicity-induced K+
currents, experiments were generally performed using pipette solutions
containing 5 mM EGTA without additional Ca2+. The effects
of extracellular Ca2+ on the development of
hypotonicity-induced K+ currents were also tested in
cftr+/+ PCT cells. When the hypotonic shock was carried out
in the absence of bath Ca2+, development of the
K+ current was not significantly modified (Fig.
6, A and B). The channel implicated in this response was clearly blocked by
hypertonicity and 0.5 mM quinidine (Fig. 6, C-E). These
results, which were also obtained using cftr/
PCT cells
(data not shown), indicate that Ca2+ is not involved in
control of the swelling-activated K+ conductance measured
in PCT cells in primary culture.
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Regulation of the K+ Conductance
Induced by Hypotonic Shock in
cftr+/+ and
cftr/
DCT Cells
Role of external Ca2+.
As reported above, the experiments were carried out in the presence of
5 mM EGTA in the pipette solutions. In DCT cells from cftr+/+ mice, the absence of bath Ca2+
completely prevented hypotonicity from inducing K+ currents
(Fig. 7, A and B).
Conversely, perfusion of a solution containing 1 mM free
Ca2+ restored the response to hypotonicity (Fig.
7C). As expected, this swelling-activated K+
conductance was blocked by hypertonicity and by CTX + apamin (10 nM; Fig. 7, D and E). As previously described for
swelling-activated Cl currents in DCT cells
(1), it appears that an influx of Ca2+ is
required to permit the development of swelling-activated K+
currents in cftr+/+ DCT cells. It was therefore interesting
to study the role of extracellular Ca2+ in
cftr
/
DCT cells. Whole cell currents were recorded in
the presence of 20 mM EGTA in the pipette solution and 1 mM free
Ca2+ in the bath (Fig. 8). In
the absence of ionomycin in the bath solution, the hypotonic shock
remained inefficient for triggering K+ currents in
cftr
/
cells (Fig. 8A). However, when the
hypotonic shock was performed in the presence of 2 µM ionomycin,
K+ currents were activated within 5 min (Fig.
8B). These currents were clearly due to K+
movements, because they were blocked by CTX + apamin and TEA (Fig.
8, C and D). Moreover, analysis of the
I-V curves (Fig. 8E) indicated that the
instantaneous outwardly rectifying currents reversed at
79.5 ± 1.0 mV (n = 10). Finally, when the cells were reexposed
to the hyperosmotic solution, the currents returned toward control
level within 2-3 min. Overall, the ionomycin-induced K+ currents developed during hypotonicity in DCT cells from
cftr
/
mice were quite similar to the swelling-activated
K+ currents measured in cftr+/+ mice.
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Role of extracellular adenosine.
The results described above strongly suggest that swelling-activated
K+ and Cl currents in DCT cells are regulated
by an identical mechanism involving a Ca2+ influx. Inasmuch
as we previously demonstrated that this Ca2+ influx could
be due to stimulation of A1 receptors by extracellular adenosine, experiments were performed to determine the role of adenosine in the K+ permeability of DCT cells from
cftr+/+ and cftr
/
mice. Adenosine (10 µM)
activated an outwardly rectifying K+ conductance with
Erev of
82.1 ± 4.1 (n = 17 cells) and
78.1 ± 2.0 mV (n = 18) for
cftr+/+ and cftr
/
DCT cells, respectively (Fig. 9, A and B). In the
presence of adenosine, the maximal slope conductances reached 18.0 ± 1.4 (n = 17) and 18.7 ± 1.0 nS
(n = 18) in cftr+/+ and cftr
/
DCT cells, respectively. These adenosine-sensitive K+
currents were decreased in the presence of CTX + apamin (10 nM) by
84 ± 3% (n = 9) in both types of DCT monolayers.
To further study the influence of external Ca2+ on
adenosine-sensitive K+ currents, experiments were performed
in the absence of bath Ca2+. The results are illustrated in
Fig. 10. As expected, removal of
external Ca2+ completely prevented adenosine from inducing
K+ currents (Fig. 10, A and B).
Conversely, perfusion of a solution containing 1 mM free
Ca2+ restored the response to adenosine (Fig.
10C).
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K+ Currents Activated by an Osmotic
Shock in Cultured DCT Cells From
cftr/
Mice Transfected With
CFTR cDNA
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DISCUSSION |
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In the majority of mammalian cells, an increase in cell volume
activates Cl currents. In renal tissue, we previously
showed the presence of swelling-activated Cl
currents
responsible for the RVD after a hypotonic shock (26, 27).
Such currents were also found in primary cultures of PCT, DCT, and CCT
from wild-type mice (1). Moreover, the cftr
knockout strongly decreased these currents in PCT, DCT, and cortical
collecting duct cells (CCD). In this study, we were interested in the
consequences of the cftr knockout on the K+
currents activated by cAMP and on swelling-activated K+
currents activated by a hypotonic shock.
In the present work, we showed the presence of cAMP-activated
K+ currents in DCT and CCD cells from wild-type mice only.
K+ channel activity was observed in >90% of the trials
only when the monolayers were treated with forskolin for 10 min before
the seal formation. This could indicate that cAMP induced incorporation of new channels in the membrane of DCT or CCT cells. These
K+ currents were completely inhibited by the chromanol
analog 293B. Because this antiarrhythmic is known to block mainly the
KCNQ1 channels (15, 34), it is probable that the
cAMP-sensitive K+ currents in mouse distal cells belong to
the KCNQ1 family of channels. Activation of KCNQ1 channels by the cAMP
pathway has been reported in epithelial cells from many tissues,
including inner ear, colon, small intestine, and airways (1a, 8, 33).
In the kidney, although the presence of KCNQ1 transcripts has been demonstrated in the CCD of the outer medulla and the DCT and CCT of the
cortex (8), the role of the protein remains unclear. In
our work, the putative presence of KCNQ1 in DCT and CCT cells raised
the problem of its coexpression with a KCNE protein. In epithelia,
KCNQ1 associates with the small regulatory -subunit KCNE1 or KCNE3
to form a K+ channel that could be implicated in the
cAMP-stimulated Cl
secretion (1a). Using RT-PCR and
Southern blotting experiments, we previously identified transcripts
encoding the KCNQ1 and KCNE1 sequence in cultured PCT cells from
wild-type mice (unpublished observations). In contrast, DCT cells also
expressed KCNQ1, but not KCNE1, excluding a role for this subunit in
the cAMP-sensitive K+ channel in this part of the nephron.
Whether KCNQ1 expression in DCT and CCT cells required an additional
subunit such as KCNE3 remains to be elucidated.
In previous studies (26), we clearly established that CFTR
functions as a cAMP-activated Cl channel in the apical
membrane of DCT cells. Although from these results the localization of
the cAMP-sensitive K+ secretion cannot be specified, this
channel might participate in the driving force of apical
Cl
secretion (15, 17). Our results show that
forskolin did not activate K+ current in DCT and CCT cells
from cftr
/
mice. Although the role of CFTR in the
control of K+ channels is well established in various
tissues (2, 16, 20), the mechanism of the interaction
between CFTR and K+ channel protein is far from being
completely understood. This mechanism probably depends on the nature of
the K+ channel, because, in addition to KCNQ1, other types
of channels, such as the ROMK family, could interact directly with CFTR
(2, 11, 20). The data reported in the literature for the
relation between KCNQ1 and CFTR are conflicting. Some studies lead to
the conclusion that KCNQ1 currents were activated by the cAMP pathway, independent of the presence of CFTR (2, 18, 34); other studies demonstrate that CFTR directly activates KCNQ1 conductances (7, 17) and that this conductance was not detected in the epithelial cell line CFPAC expressing
F508-CFTR (16).
This last finding corroborates our own observations and suggests that, in the distal tubule, KCNQ1 K+ currents are dependent on
CFTR expression.
As expected, a hypotonic shock activated K+ currents in
PCT, DCT, and CCD cells from wild-type mice. In the monolayers from cftr/
mice, development of these K+ currents
was completely impaired in DCT and CCT cells but was not modified in
PCT cells. This suggests two different types of swelling-activated
K+ currents in PCT and DCT cells. The pharmacological study
performed on these channels confirmed this hypothesis. In PCT cells,
the swelling-activated K+ conductance shared some
properties with the K+ currents activated by cell swelling
that have been reported in gallbladder epithelium (31) and
in Ehrlich ascites tumor cells (12, 22). It was
insensitive to TEA but was strongly blocked by quinidine and clofilium.
Moreover, this K+ conductance was not affected by
intracellular Ca2+ concentration but was sensitive to
external pH variations, with activation at alkaline pH and inhibition
at acidic pH. Finally, this pharmacological profile is consistent with
TASK2 channels. Further experiments are required to definitively
characterize the nature of the swelling-activated K+
channels in PCT. However, it is now well established that TASK2 is
strongly expressed in the kidney (23) and, more precisely, in mouse proximal tubule, in situ or in primary culture
(29).
In DCT and CCT cells, the swelling-activated K+ conductances exhibited a different pharmacology: they were inhibited by TEA and CTX and were dependent on extracellular Ca2+. The inhibition by CTX indicates that Ca2+ activated BK and intermediate K+ (IK) channels. Furthermore, a minor role of SK channels cannot be excluded, because this toxin inhibited the swelling-activated K+ currents by 27% and has been shown to be additive to the CTX effect.
These K+ conductances were activated by hypotonicity in the presence of a high concentration of EGTA in the pipette solution. The simplest interpretation of this finding could be that an increase in intracellular Ca2+ is not necessarily required to activate K+ currents and that Ca2+ influx is sufficient to increase the K+ conductance during hypotonicity (25, 27). This conclusion is supported by the recent data of Grunnet et al. (9), who reported that stimulation of BK and SK channels during swelling was not mediated by a change in intracellular Ca2+.
Although they belong to a distinct channel family, the
K+ channels activated by hypotonicity in PCT and DCT cells
of wild-type mice participate in RVD, together with swelling-activated
Cl conductances (27). In both monolayers,
the swelling-activated K+ currents were insensitive to the
chromanol derivative 293B, indicating that KCNQ1 is not implicated in
this conductance. This conclusion is at variance with that reached by
Lock and Valverde (14), who proposed that the KCNQ1-KCNE1
complex was implicated in the maintenance of K+ secretion
linked to RVD in murine airway cells. As we previously demonstrated
(1), null mutation of CFTR strongly impaired the RVD in
PCT and DCT cells. Obviously, in PCT cells this inhibition was due to a
defect in swelling-activated Cl
conductances, because the
accompanying activated K+ channels remained functional in
cftr
/
monolayers. In contrast, in DCT cells,
swelling-activated Cl
and K+ conductances
were affected by the knockout of CFTR. At this step in our work, the
question arose as to what extent the Cl
and
K+ channels in DCT cells were modulated by a common
mechanism that was altered in the absence of CFTR. As demonstrated for
swelling-activated Cl
conductance, ionomycin in the
presence of a high concentration of EGTA inside the cells restored the
swelling-activated K+ currents in the DCT and CCT cells
from cftr
/
mice, confirming the role of Ca2+
entry in these cells. Moreover, adenosine mimicked the effect of
ionomycin in the presence of external Ca2+ only. Therefore,
in the case of the Cl
channels, this Ca2+
influx might be due to stimulation of A1 receptors by
adenosine generated by the degradation of ATP by membrane ectoenzymes.
According to Braunstein et al. (3), lack of CFTR leads to
impairment of ATP release during hypotonic shock (5, 10,
24). The final result is loss of the RVD process, because the
first element (ATP release) of the cascade leading to activation of
Cl
and K+ channels is missing.
The results of these experiments are summarized in Fig.
13. In PCT and DCT cells, RVD after
hypotonic challenge is dependent on CFTR. However, in PCT cells, only
the swelling-activated Cl conductance is affected by this
regulation. In DCT cells, swelling-activated Cl
and K+ currents are regulated by CFTR during hypotonic
shock. Although swelling-activated Cl
currents share the
same properties in PCT and DCT cells, this is not the case for
swelling-activated K+ currents: in PCT cells the
K+ current could be due to TASK2 channels, whereas in DCT
cells the K+ current could flow through
Ca2+-dependent BK and SK channels. With regard to the
cAMP-sensitive conductances, the data strengthen the hypothesis that
CFTR mediates forskolin-activated Cl
and K+
currents in DCT cells. The K+ conductance could be formed
by KCNQ1-like channels. In contrast, forskolin-activated
Cl
and K+ currents are not detectable in PCT
cells. Therefore, it appears that, in renal epithelium, CFTR not only
functions as a cAMP-activated Cl
channel but also
participates in regulation of cell volume. These two functions are
probably independent, because PCT exhibits only the latter, whereas DCT
exhibits both functions. This suggests that the form of CFTR expressed
in PCT is different from that in DCT. We have no evidence of such
differences. However, it may be recalled that an alternative splice
form of CFTR (TNR-CFTR) was found in inner medullary collecting duct,
indicating a possible tissue-specific processing of CFTR
(13).
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The question arises of whether the inhibition of swelling-activated
K+ and Cl currents in cftr
/
has physiological consequences. The cftr
/
mice exhibit
drastic growth and intestinal dysfunctions and generally die 6 wk after
birth. To optimize their lifetime, the animals are fed a special liquid
diet. Despite these precautions, the animals remain very weak, and
their renal function has not been investigated. Therefore, kidney
function could be impaired, but the very poor physiological state of
the animals could mask this specific alteration. There is no apparent
difference in growth between cell cultures from cftr
/
and control cells from cftr+/+ mice, at least when they are
kept in constant control conditions, such as an incubator. However,
preliminary experiments (data not shown) indicate that a hypotonic
shock stopped division of the cftr
/
, but not the
cftr+/+, cells.
The situation is quite different in humans, because the dysfunction of
CFTR was mainly due to F508 mutation. However, according to
Braunstein et al. (3), it is possible that the
F508
mutation resulted in an alteration of RVD. Thus we can suppose that the human kidney from CF patients will also exhibit altered RVD. The consequences of such a modification remain to be analyzed in terms of a
renal role of CFTR.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Poujeol, UMR CNRS 6548, Bâtiment Sciences Naturelles Université de Nice-Sophia Antipolis, Parc Valrose, O6108 Nice Cedex 2, France (E-mail:poujeol{at}unice.fr).
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.
First published December 10, 2002;10.1152/ajprenal.00238.2002
Received 26 June 2002; accepted in final form 3 December 2002.
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