1 Department of Physiological
Sciences, We have isolated
ciliated respiratory cells from the nasal epithelium of wild-type and
cystic fibrosis (CF) null mice and used the patch-clamp technique to
investigate their basal conductances. Current-clamp experiments on
unstimulated cells indicated the presence of
K+ and
Cl
nasal epithelial cells; chloride conductance; patch-clamp
technique; cystic fibrosis; transgenic mice; cystic fibrosis
transmembrane conductance regulator
THE PROXIMAL AIRWAYS of the lung are lined with a layer
of fluid that serves to trap airborne particles and bacteria. The composition and thickness of this fluid layer are regulated by the
rates of Na+ absorption and
Cl The airway epithelium contains at least three stimulus-activated
Cl Chan et al. (3) reported that the basal
Cl Animals
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
conductances and, under
certain conditions, a small Na+
conductance. Voltage-clamp experiments revealed three distinct Cl
conductances.
Itv-indep was
time and voltage independent with a linear current-voltage
(I-V)
plot; Iv-act
exhibited activation at potentials greater than ±50 mV, giving an
S-shaped
I-V
plot; and
Ihyp-act was
activated by hyperpolarizing potentials and had an inwardly rectified
I-V
plot. The current density sequence was Ihyp-act = Iv-act
Itv-indep. These
conductances had
Cl
-to-N-methyl-D-glucamine
cation permeability ratios of between 2.8 and 10.3 and were unaffected
by tamoxifen, flufenamate, glibenclamide, DIDS, and
5-nitro-2-(3-phenylpropylamino) benzoic acid but were inhibited by
Zn2+ and
Gd3+.
Itv-indep and
Iv-act were
present in wild-type and CF cells at equal density and frequency.
However, Ihyp-act
was detected in only 3% of CF cells compared with 26% of wild-type
cells, suggesting that this conductance may be modulated by cystic
fibrosis transmembrane conductance regulator (CFTR).
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
secretion across the
airway epithelium and are critical for effective mucocilliary clearance
and bacterial killing (21). In the inherited disease cystic fibrosis
(CF), the airways lack cAMP-stimulated Cl
secretion (1) due to the
absence, or defective functioning, of the cystic fibrosis transmembrane
conductance regulator (CFTR) protein. However, CF airways also exhibit
an increased apical Na+
conductance (1), which is explained by an upregulation of amiloride-sensitive Na+ channels
in response to the absence of CFTR (1, 22). Combined, these alterations
in Cl
and
Na+ conductances are thought to
result in desiccation of the airways, which leads to bacterial
colonization and inflammatory damage and, ultimately, lung failure.
conductance pathways:
1) the CFTR conductance that is
activated by cAMP, 2) a
Ca2+-activated conductance, and
3) a volume-activated conductance (24). The biophysical properties of these stimulus-activated conductances are quite distinct and are similar in both airway cells
(24) and other epithelia that are affected by CF (6, 28). In addition
to these stimulus-activated conductances, there is also evidence for
the existence of a basal (i.e., unstimulated) Cl
conductance in the
airway epithelium (3, 10, 14, 25, 26). This basal
Cl
conductance may act as a
transcellular route for the
Cl
flux that must accompany
Na+ absorption across the airway
epithelium.
conductance in cultured
human airway cells was due to a time-dependent current that had either
a linear or outwardly rectified current-voltage (I-V)
in different cells. However, a detailed characterization of the
biophysical and pharmacological properties of the basal Cl
conductance in
respiratory cells has not been performed. Therefore, this was the main
aim of the present work. In particular, we sought to answer the
following questions: 1) is the basal
Cl
conductance a distinct
entity or does it simply represent the background activity of one or
more of the stimulus-activated conductances already known to exist in
the airway epithelium, and 2) is the basal Cl
conductance
affected by the absence of CFTR? To address these questions, we have
used ciliated respiratory cells because they are thought to play an
important role in proximal airway ion transport (1) and have been shown
to express CFTR (2). We decided not to use cultured cells but instead
have developed a procedure for the isolation of ciliated respiratory
cells, which are suitable for patch clamping, from the murine nasal
epithelium (NE). In this study, we demonstrate that, in the absence of
any agonists, one of three distinct spontaneously active
Cl
conductances may be
active in these ciliated cells. In addition, our data provide evidence
suggesting that one of these basal
Cl
conductances is
modulated by CFTR.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Isolation of Ciliated Respiratory Cells From the NE
Ciliated respiratory cells, suitable for patch clamping, were obtained from the NE using a two-stage isolation technique. First, the NE was removed from the mouse using a modification of the method described by Grubb et al. (10). Then, after an overnight incubation in protease, single cells and small groups of cells were teased from the NE. In the mouse, the nasal membrane contains four types of epithelia: respiratory, olfactory, transitional, and squamous (15). Most of the respiratory epithelium is located in the lateral and ventral regions of the nasal cavity (15).Mice were killed by cervical dislocation, and the NE was removed immediately. Throughout the dissection, the preparation was viewed at ×7 magnification using a dissecting microscope. First, the skin over the nose was removed with scissors, and the tissue covering the nasal bones was cut away with a scalpel. The nasal bones were then gripped with forceps at the end closest to the tip of the nose and lifted away, revealing the two pieces of NE on either side of the nasal septum. To remove any blood and debris, the nasal cavity was washed with DMEM containing 1% penicillin-streptomycin (stock contained 103 units penicillin and 10 mg streptomycin/ml; Sigma), a procedure that also lifted the NE away from the nasal cavity, making it easier to remove. Each piece of NE was then dissected from the nasal cavity using forceps and scissors. The NE was first cut at the end closest to the brain and carefully lifted away using forceps. If the NE still appeared to be attached to the septum laterally [where most of the respiratory epithelium is located (15)], it was carefully dissected away with either scissors or a scalpel. Finally, the NE was cut at the end closest to the tip of the nose and removed. The isolated NE was washed in DMEM containing antibiotics and then placed in an Eppendorf tube containing 1 ml of the same solution plus 0.05% wt/vol protease XIV (Sigma) and incubated for 24 h at 4°C. The enzyme activity was stopped by spinning the Eppendorf tube at 14 g for 30 s, removing the supernatant, and resuspending the tissue in DMEM containing antibiotics plus 10% fetal calf serum at 4°C.
To isolate ciliated respiratory cells suitable for patch clamping, the protease-treated NE was viewed under phase-contrast optics (×200), and areas of the tissue that contained cells with beating cilia were identified. Small pieces of NE, containing 50-100 beating cells, were then separated from the main body of the tissue using sharpened stainless steel needles and transferred, using a micropipette, to a microwell containing ~150 µl of DMEM. The microwell was then transferred to a dissecting microscope (×45 magnification), and the small pieces of NE were teased apart using sharpened entomology pins to yield single cells and small groups of cells. The respiratory epithelial cells could easily be distinguished from other cell types by their prominent cilia (Fig. 1). Our criteria for cell viability were as follows: 1) a clear, bright, phase-contrast image and 2) beating cilia. On the basis of these criteria, the majority of isolated respiratory cells were viable.
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To check whether incubating the NE in protease for 24 h at 4°C affected the electrophysiological properties of the isolated cells, we also studied cells isolated from NE that had been exposed to the enzyme for only 1 h. Both groups of cells had similar electrophysiological characteristics, suggesting that the 24-h incubation does not have a detrimental effect. Moreover, the yield of viable cells was much lower when a 1-h protease incubation was employed, so most of our electrophysiological data come from cells isolated using the longer incubation time.
The isolated cells remained viable and suitable for electrophysiological experiments for at least 2 h when bathed in the standard Na+-rich bath solution. However, when a bath solution containing N-methyl-D-glucamine (NMDG) chloride was employed, the cells had to be used within ~30 min. Therefore, under these latter conditions, it was necessary to repeat the last stage of the isolation procedure several times during the day to maintain adequate supplies of fresh cells.
Electrophysiology
The cell preparations were transferred to a tissue bath (volume of 1.5 ml) mounted on a Nikon Diaphot inverted microscope and viewed using phase-contrast optics. Because isolated respiratory cells retain their morphology (Fig. 1), have beating cilia, and are often motile, they could easily be distinguished from other cell types. Pipettes were pulled from borosilicate glass (Clarke Electromedical) and had resistances, after fire polishing, of between 2 and 4 MVm (current-clamp experiments) and whole cell currents (voltage-clamp experiments) were recorded with an EPC-7 patch-clamp amplifier (List Electronic, Darmstadt, Germany). To obtain I-V relationships, the Vm was held at 0 mV and then voltage clamped over the range ±100 mV in steps of 20 mV. Each voltage step lasted 500 ms, and there was an 800-ms interval at the holding potential between steps. Data were filtered at 1 kHz and sampled at 2 kHz with a Cambridge electronic design 1401 interface (CED, Cambridge, UK) and stored on the computer hard disk. I-V plots for the time-independent current (Itv-indep) were constructed using the average current measured over a 200-ms period starting 150 ms into the voltage pulse. I-V plots for the two other currents we identified (Iv-act and Ihyp-act) were constructed using the average current measured over a 2-ms period starting 495 ms into the voltage pulse. The currents were not leak corrected. Series resistance (Rs) was typically two to three times the pipette resistance, and Rs compensation (40-50%) was routinely used. Vm have been corrected for current flow across the uncompensated fraction of Rs using the relationship Vm = Vp - IRs, where Vp is the pipette potential and I is current flow. Reversal potentials (Erev) were obtained from I-V plots by interpolation after fitting a third- or fourth-order polynomial using least squares regression analysis. The input capacitance (Ci) of the cells was routinely measured using the analog circuitry of the EPC-7 amplifier and compensated before the recording was started. Ci values were used to calculate current density (which is expressed as pA/pF). Junction potentials were measured, and the appropriate corrections were applied to Vm (27).
Solutions and Chemicals
For measurement of Vm under current-clamp conditions, the pipette solution contained (in mM) 120 KCl, 2.0 EGTA, 2.0 MgCl2, 1.0 ATP, and 10.0 HEPES, pH 7.2 (calculated free Ca2+ concentration of <1 nM). The standard bath solution contained (in mM) 145 NaCl, 4.5 KCl, 2.0 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4. The high K+ and sodium aspartate bath solutions contained 145 mM of either KCl or sodium aspartate instead of NaCl.To isolate anion-selective currents in the voltage-clamp experiments,
cation conductances were blocked with NMDG. To prevent the development
of swelling-induced Cl
currents, the pipette solution was made 45 mosM hypotonic
to the bath solution. The pipette solution contained (in mM) 120 NMDGCl, 2.0 MgCl2, 2.0 EGTA, 1.0 ATP, and 10.0 HEPES, pH 7.2 (calculated free
Ca2+ concentration of <1 nM).
The standard bath solution contained (in mM) 149.5 NMDGCl, 2.0 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES, pH
7.4. Cl
selectivity of the
currents was assessed by
Erev shifts
following replacement of extracellular
Cl
. In the
Cl
-replacement bath
solution, 100 mM NMDGCl was replaced by an isosmotic amount of
mannitol. The osmolarities of all solutions were checked using a
freezing-point depression osmometer (Roebling, model 10B), and all
pipette solutions were filtered (10 µm pore size) before use.
Tamoxifen (10 µM), flufenamic acid (50 µM), glibenclamide (100 µM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 µM) were made up daily as stock solutions in dimethyl sulfoxide and then diluted 1,000-fold to give the final concentrations employed. DIDS (0.5 mM), tetraethylammonium (TEA; 10 mM), ZnCl2 (0.5 mM), and GdCl3 (0.5 mM) were dissolved directly in the standard bath solution.
Statistics
Significance of difference between means was determined using either analysis of variance followed by Dunn's multiple comparison test (ANOVA + DMCT), the Mann-Whitney U test (M-WUT, nonparametric equivalent of unpaired t-test), or the Wilcoxon's signed rank test (WSRT, nonparametric equivalent of paired t-test) as appropriate. Significance of difference between the number of cells responding to a particular maneuver was assessed using the ![]() |
RESULTS |
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Electrical Coupling Between Respiratory Cells
About 50% of the whole cell recordings made in this study were obtained from single ciliated respiratory cells. The Ci values for single WT and CF cells were 17.3 ± 1.3 pF (n = 20 cells from 15 mice) and 18.4 ± 1.5 pF (n = 21 cells from 6 mice), respectively, values that are not statistically different (P = 0.70; M-WUT) (Table 1). The remainder of the experiments were performed on respiratory cells joined together in groups, with up to a maximum of seven cells per group. Table 1 shows that, for both genotypes, cells in groups were not electrically coupled to their neighbors, since there was no correlation between the Ci and the number of cells in a group (P = 0.35; ANOVA). For this reason, electrophysiological data obtained from single isolated cells and from cells in groups have been combined.
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Current-Clamp Experiments
The mean resting Vm of the isolated respiratory epithelial cells was 32.2 ± 3.8 mV (n = 22 cells from 3 mice). Figure 2A shows a plot of whole cell conductance (G) against Vm for 22 cells. These data suggest that there might be two populations of respiratory cells in the NE: a group that has a high Vm and a low G (type 1 cells, n = 13/22 cells from 3 mice, 59.1%) and a group that has a low Vm and a high G (type 2 cells, n = 9/22 cells from 3 mice, 40.9%). Type 1 cells had an average Vm and inward G of
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Initially, we thought that the low
Vm and high
G values of the type
2 cells might simply be explained by a leaky plasma
membrane caused by damage during the isolation procedure. To check for this, we tested the effect of changes in extracellular
K+ and
Cl concentrations, and of
various ion channel blockers, on the
Vm of the
isolated cells. Figure
3A shows
data for type 1 cells. Note that both
increasing extracellular K+
concentration
([K+]o)
from 4.5 to 149.5 mM and exposing the cells to 10 mM TEACl (a
K+ channel blocker) caused a
marked depolarization of
Vm. Replacing most of the extracellular
Cl
with aspartate also
caused a small but statistically significant depolarization, whereas
the metal ions Gd3+ and
Zn2+ had no effect. Amiloride (100 µM) alone also had no effect on Vm in
type 1 cells (Fig.
3A); however, if amiloride was
tested after exposure to TEACl and
Zn2+, then we observed small
hyperpolarizations in three out of four cells (individual values were
8, 0,
1.5, and
1.5 mV). Together, these data
suggest that a K+ conductance
dominates the membrane of type 1 cells
but that the cells also have a small
Na+ conductance. The results of
similar experiments performed on type
2 cells are shown in Fig.
3B. High
[K+]o,
TEACl, and replacement of extracellular
Cl
with aspartate all
caused a significant depolarization of
Vm. However, in
contrast to type 1 cells, the
Vm of
type 2 cells was markedly
hyperpolarized by Gd3+ and
Zn2+. Again, amiloride alone had
no effect on Vm
(Fig. 3B) but did cause a small
hyperpolarization in three out of four type
2 cells that had been previously exposed to TEACl and
Zn2+ (individual values were
6,
2, 0, and
0.5 mV). These data suggest that the
membrane of type 2 cells possesses
both K+ and
Cl
conductances plus a
small Na+ conductance. The
presence of a substantial
Cl
conductance most likely
explains why type 2 cells have a
relatively low resting
Vm.
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Voltage-Clamp Experiments
Spontaneously active whole cell anion currents.
The current-clamp experiments suggested that a proportion of the
isolated respiratory cells (the type 2 cells that make up 40.9% of the total) possesses a resting
Cl conductance. We sought
to establish the biophysical characteristics of this conductance by
measuring whole cell anion currents in voltage-clamp experiments.
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Densities of spontaneously active currents in WT and CF cells.
Figure 5 compares the densities of the
various spontaneously active currents, measured on average 44 ± 3 s after a whole cell recording was established, in WT and
CF nasal cells. The current density sequence for both WT and CF cells
was the same,
Ihyp-act = Iv-act Itv-indep > Ins (Fig. 5).
Densities for
Ihyp-act and Iv-act were in
the range 60-100 pA/pF, whereas those for
Itv-indep and
Ins were much
smaller at ~6 and 1 pA/pF, respectively. There were no statistical
differences in the magnitude of the currents between WT and CF cells,
at least for
Iv-act,
Itv-indep, and
Ins. The mean
density of the
Ihyp-act current
in the two genotypes was not markedly different (Fig. 5); however, this
current was observed so infrequently in CF cells (see below) that a
statistical comparison with WT was not possible.
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Frequency of spontaneously active currents in WT and CF cells.
Figure 6 shows the frequency at which each
of the four different spontaneously active currents were detected in WT
and CF cells. These data were derived from
I-V
plots that were performed on average 44 ± 3 s after the onset of
whole cell recording. Of the four identified currents,
Itv-indep and
Iv-act were
observed at the same frequency in WT and CF genotypes
(P > 0.05;
2). However,
Ihyp-act was
present at a significantly lower frequency in the CF cells (WT, 13/50
cells from 15 mice vs. CF, 1/29 cells from 6 mice;
P = 0.01;
2) (Fig. 6). This is unlikely
to result from a slow activation of
Ihyp-act in CF
cells following the onset of whole cell recording, because after 5 min
of recording the frequency of detecting
Ihyp-act remained
significantly lower in the CF genotype (WT, 21/47 cells from 15 mice
vs. CF, 3/23 cells from 3 mice; P = 0.01;
2). The reduced
occurrence of
Ihyp-act in CF
cells was associated with an increased frequency of the
Ins (WT, 11/50
cells from 15 mice vs. CF, 13/29 cells from 3 mice;
P = 0.04;
2).
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Pharmacology of spontaneously active anion currents.
We tested the following putative
Cl channel blockers on the
three types of anion currents in WT and CF cells
(n = 1-4 experiments with each
blocker on each current): tamoxifen (10 µM), flufenamic acid (50 µM), glibenclamide (100 µM), DIDS (500 µM), and NPPB (10 µM).
At the concentrations tested, none of these compounds had an inhibitory
effect. However, Zn2+ and
Gd3+ did block the currents.
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![]() |
DISCUSSION |
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To date, all the patch-clamp studies that have been performed on the respiratory epithelium have utilized cultured cells (Refs. 11, 14 and see Ref. 3 for additional references). This means that 1) the cells may not be expressing a full channel complement, 2) channels cannot be localized to specific cell types, and 3) the membrane localization of the channels is uncertain if the cultured cells are not polarized and transporting. In this paper, we have described a new method for isolating viable ciliated respiratory cells from murine NE that are suitable for patch clamping. These cells are clearly polarized and retain the distinct morphological characteristic of respiratory cells in the native tissue: abundant cilia on their apical membrane. We took the fact that the cilia continued to beat and that beat frequency was increased by mechanical stimulation (19) as an indication that the cells were viable. Our isolated cells did not, however, appear to possess functioning gap junctions, a property that has previously been described for ciliated respiratory cells (19). To our knowledge, this is the first patch-clamp study to be performed on freshly isolated nasal respiratory cells.
Vm of WT Nasal Cells
The mean Vm of the isolated respiratory cells, measured in current-clamp experiments, wasExperiments in which we tested the effects of extracellular ion
substitutions and of various channel blockers on
Vm suggested that
the membrane of type 1 cells was
dominated by a K+ conductance,
whereas type 2 cells possessed both
K+ and
Cl conductances. The
relatively high Cl
conductance in the type 2 cells would
explain why these cells have a low
Vm. Exposure of
the in vitro murine NE to 100 µM amiloride caused a 37% inhibition
of equivalent short-circuit current (10), and 54% of freshly isolated
human nasal respiratory cells had a detectable
Na+ conductance, as judged by a
hyperpolarization in response to amiloride and the removal of
extracellular Na+ (14). We could
not detect an effect of amiloride alone on
Vm in the
isolated murine respiratory cells. However, small hyperpolarizations were observed if cells were exposed to amiloride after block of the
K+ and
Cl
conductances, suggesting
that a small Na+ conductance is
present.
Basal Cl Conductances in WT and CF
Nasal Cells
On the basis of their biophysical characteristics, three distinct basal
Cl conductances were
identified in the nasal respiratory cells. All three conductances were
detected at about the same frequency in WT cells; however, there were
differences in their densities. Densities for
Ihyp-act and
Iv-act were in
the range of 60-100 pA/pF, whereas that for
Itv-indep was
much smaller at ~6 pA/pF. This difference in current density may
explain why the proportion of cells exhibiting a basal
Cl
conductance in the whole
cell experiments (75%) is substantially higher than the proportion of
type 2 cells (40.9%) that we detected in the current-clamp experiments. If the low-density
Itv-indep current
is omitted, then the proportion of cells with a
Cl
conductance falls to
~50%, which is much closer to the proportion of
type 2 cells.
Whereas a panel of compounds (NPPB, DIDS, glibenclamide, tamoxifen,
flufenamic acid) that have been shown to act as
Cl channel blockers in
other cell types had no effect on the conductances in nasal respiratory
cells, these conductances were blocked by ZnCl2 and
GdCl3. At the dose we employed
(500 µM), GdCl3 blocked all
three conductances with equal efficacy. We were surprised by this
blocking effect of Gd3+, since
this trivalent cation is usually considered to be an inhibitor of
stretch-activated cation channels (29). However,
Gd3+ has recently been reported to
block a volume-activated Cl
conductance in proximal tubule cells (18).
ZnCl2 was a more effective blocker
of Iv-act
compared with
Itv-indep and
Ihyp-act. Previously, ZnCl2 has been
reported to inhibit a hyperpolarization-activated Cl
conductance in rat
superior cervical ganglion cells (5). Thus the pharmacological
properties of the channels we have identified in the nasal respiratory
epithelial cells are unusual, in that "traditional"
Cl
channel blockers have no
effect, whereas the channels are blocked by
Zn2+ and
Gd3+.
The simplest interpretation of our data is that 75% of WT nasal
respiratory cells contain one of three types of
Cl channels that are open
under unstimulated conditions. Although we cannot entirely exclude the
possibility that
Itv-indep (which had the lowest density) was also present in cells that exhibited Iv-act and
Ihyp-act, our
data from the CF cells argue against this. In the CF cells, the
occurrence of
Ihyp-act was
markedly reduced and yet there was no increase in the frequency of
either Itv-indep
or Iv-act (only
in Ins; see Fig.
6). Moreover, we have recently found that
Ihyp-act can be
completely and selectively inhibited by
low-Cl
bath solutions and
that no other Cl
currents
are revealed under these conditions. Why individual ciliated cells
should express only one type of basal
Cl
conductance is unclear,
but it could reflect either developmental or functional differences
within the respiratory epithelium. The three
Cl
conductances were
observed at about the same frequency in WT cells. Therefore, it is
probable that
Iv-act and
Ihyp-act make up
the unstimulated cellular
Cl
permeability of the
airway epithelium, since they both have much higher densities than
Itv-indep, and
their voltage-dependencies suggest that they would be open at
physiological Vm
values.
Comparison With Other Cl
Conductances
Itv-indep.
A conductance with similar time-independent kinetics to
Itv-indep has
previously been described in the basolateral membrane of gastric
parietal cells where it is thought to play a "housekeeping" role,
such as the regulation of
Vm (13). However,
unlike Itv-indep, the parietal cell Cl
conductance was rapidly blocked by NPPB with an inhibition
constant of 12 µM (13).
Iv-act.
To our knowledge, a conductance with similar properties to
Iv-act, i.e.,
time-dependent activation at potentials greater than ±50 mV, has
not been described previously.
Iv-act was poorly
selective for Cl, having a
Cl
permeability that was
only 2.8-fold higher than that to
NMDG+. Because
NMDG+ is a large organic cation,
the Cl
selectivity against
Na+ is likely to be even lower.
Thus, in a physiological situation, this channel may not select for
anions over cations. Previously, a nonselective cation conductance with
a relatively low selectivity for cations over anions
(Na+-to-Cl
permeability ratio = 1.68), and which exhibits slight activation at
depolarizing potentials, has been described in Caco-2 cells (16). This
conductance is stimulated by protein kinase C and by cell shrinkage,
effects that are thought to require participation of a regulatory
protein that contains an ATP-binding Walker motif (16).
Ihyp-act.
In terms of its biophysical and pharmacological characteristics,
Ihyp-act bears a
close resemblance to the ClC-2
Cl channel that also
activates at hyperpolarizing potentials and is insensitive to DIDS
(23). ClC-2 is expressed in muscle, nerve, and epithelial tissues of
the rat (23). A ClC-2-like conductance has also been identified in the
granular duct cells of mouse mandibular glands (7). The salivary gland
conductance is activated by hyperpolarizing potentials, inactivates at
depolarizing potentials, and is unaffected by a variety of
Cl
channel blockers,
including NPPB, DIDS, and glibenclamide (7). A human ClC-2 analog
(called ClC-2G or ClC-2
) has recently been cloned from T84 cells (4)
and has also been identified in both fetal and adult human lung (20).
One particularly interesting aspect of our study was that
Ihyp-act was
present at a markedly reduced frequency in the CF nulls, indicating
that this conductance may be modulated by CFTR in respiratory cells.
CFTR is known to have an inhibitory effect on epithelial
Na+ channels (22), and an
outwardly rectifying Cl
channel in airway epithelium also requires CFTR for proper activation by agonists (8, 9).
Physiological Role of the Basal Cl
Conductances in Nasal Cells
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ACKNOWLEDGEMENTS |
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We thank David Stephenson for skilled technical assistance.
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FOOTNOTES |
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This work was funded by the Biotechnology and Biological Sciences Research Council.
Present addresses: W. H. Colledge and R. Ratcliff, Physiological Laboratory, Univ. of Cambridge, Downing Street, Cambridge CB2 3EG, UK; R. Tarran, Dept. of Medicine, Univ. of North Carolina at Chapel Hill, 6017 Thurston-Bowles Bldg., Chapel Hill, NC 27599-7248.
Address for reprint requests: B. E. Argent, Dept. of Physiological Sciences, Univ. Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Received 25 April 1997; accepted in final form 10 December 1997.
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