Adult alveolar epithelial cells express multiple subtypes of
voltage-gated K+ channels that are located in apical
membrane
So Yeong
Lee1,2,
Peter J.
Maniak1,
David H.
Ingbar3, and
Scott M.
O'Grady1
1 Departments of Physiology and Animal Science and
2 Molecular Veterinary Biosciences Graduate Program,
University of Minnesota, St. Paul 55108; and
3 Department of Medicine, University of Minnesota,
Minneapolis, Minnesota 55455
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ABSTRACT |
Whole cell perforated patch-clamp
experiments were performed with adult rat alveolar epithelial cells.
The holding potential was
60 mV, and depolarizing voltage steps
activated voltage-gated K+ (Kv) channels. The
voltage-activated currents exhibited a mean reversal potential of
32
mV. Complete activation was achieved at
10 mV. The currents exhibited
slow inactivation, with significant variability in the time course
between cells. Tail current analysis revealed cell-to-cell variability
in K+ selectivity, suggesting contributions of multiple Kv
-subunits to the whole cell current. The Kv channels also displayed
steady-state inactivation when the membrane potential was held at
depolarized voltages with a window current between
30 and 5 mV.
Analysis of RNA isolated from these cells by RT-PCR revealed the
presence of eight Kv
-subunits (Kv1.1, Kv1.3, Kv1.4, Kv2.2, Kv4.1,
Kv4.2, Kv4.3, and Kv9.3), three
-subunits (Kv
1.1, Kv
2.1, and
Kv
3.1), and two K+ channel interacting protein (KChIP)
isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv
-subunit antibodies (Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3) showed
labeling of 50-kDa proteins from alveolar epithelial cells grown in
monolayer culture. Immunocytochemical analysis of cells from monolayers
showed that Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were localized to the
apical membrane. We conclude that expression of multiple Kv
-,
-,
and KChIP subunits explains the variability in inactivation gating and
K+ selectivity observed between cells and that Kv channels
in the apical membrane may contribute to basal K+ secretion
across the alveolar epithelium.
voltage-gated potassium channels; potassium ion secretion; oxygen-sensitive potassium channels; alveolar fluid clearance
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INTRODUCTION |
ADULT
ALVEOLAR EPITHELIAL cells play an important role in regulating
the volume and ionic composition of fluid that lines the alveolus.
Previous studies showed that the K+ concentration in adult
human alveolar epithelial lining fluid was more than threefold higher
than in plasma (42). Studies of K+ transport
in resected human lungs from cancer patients have demonstrated net
K+ secretion into the alveolar space (32).
This result was consistent with earlier experiments using perfused
adult rat lungs, in which basal K+ secretion was shown to
be sensitive to ouabain in the lumen perfusate (4).
K+ channel openers such as
2-(3,4-dihydro-2,2-dimethyl-6-nitro-2H-1,4-benzoxazin-4-yl) pyridine N-oxide (YM-934) were previously shown to stimulate
K+ influx into the alveolar space and to increase alveolar
fluid clearance by 60% (33). The results of these studies
suggest that adult alveolar epithelial cells are capable of
K+ secretion and that, in at least one study, increases in
apical membrane K+ conductance could significantly enhance
alveolar fluid clearance. At the present time, little is known about
the transport pathways involved in K+ efflux across the
apical membrane of alveolar epithelial cells.
Voltage-gated K+ (Kv) channels in alveolar epithelial cells
were first identified by DeCoursey et al. (7). Two
distinct K+ currents were identified and shown to be
associated with cells labeled with phosphine, a fluorescent dye that is
concentrated in lamellar bodies. Most of the K+ currents
identified were found to be similar to delayed rectifier K+
channels in excitable cells. However, some cells had a K+
current that was shown to be sensitive to tetraethylammonium (TEA) and
activated at more depolarized membrane potentials. These outward
K+ currents exhibited distinct voltage sensitivities and
were identified as low- and high-threshold types by Peers et al.
(26). In the present study, we also observed
voltage-activated K+ channels in primary cultures of adult
rat alveolar epithelial cells. We observed significant variations in
activation and inactivation gating kinetics, suggesting the presence of
multiple Kv-type K+ channels in these cells. The overall
objective of this study was to identify the major Kv subunits present
in primary cultures of adult rat alveolar epithelial cells and to
determine their localization to either the apical or basolateral
membrane. Interestingly, our results revealed that alveolar epithelial
cells express a remarkable diversity of Kv
-,
-, and
K+ channel interacting protein (KChIP) subunits and that at
least five of the eight
-subunits are present in the apical membrane.
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MATERIALS AND METHODS |
Materials.
Male pathogen-free Sprague-Dawley rats weighing 150-174 g were
purchased from Harlan (Indianapolis, IN). Elastase was purchased from
Worthington Biochemical (Freehold, NJ). Rat immunoglobulin G (IgG),
deoxyribonuclease I (DNAase I), nonessential amino acids, bovine serum
albumin (BSA), L-glutamine, HEPES, and trypsin inhibitor were obtained
from Sigma Chemical (St. Louis, MO). Dulbecco's modified Eagle's
medium (DMEM)-Ham's F-12 nutrient mixture (DMEM-F-12) in a 1:1 ratio,
penicillin-streptomycin, and phosphate-buffered saline (PBS) were
purchased from GIBCO-BRL (Grand Island, NY). Nitex mesh (120 and 40 µm) was purchased from Tetko (Elmsford, NY). Tissue culture-treated
Transwell polycarbonate filters were purchased from Corning Costar
(Cambridge, MA).
Cell preparation and culture.
Alveolar epithelial cells were isolated from adult rat lungs with a
protocol described previously (5, 15) and approved by the
University of Minnesota Institutional Animal Care and Use Committee. Briefly, the lungs were removed, flushed with
saline, and then filled with elastase-containing solution (2.7 IU/ml) and incubated at 37°C for 30 min in a shaker bath. Finely minced tissues were filtered through 120- and 40-µm Nitex mesh. Cells were
further purified by panning on IgG-coated petri dishes to remove
remnant macrophages and suspended directly in serum-free DMEM-F-12
medium supplemented with 1.25 mg/ml BSA, 0.1% nonessential amino
acids, 2.0 mM glutamine, 100 U/ml sodium penicillin G, and 100 µg/ml
streptomycin. Cells were seeded onto Transwell membrane filters (4.52 cm2, 0.4-µm pore size) at a density of 1.5 × 106 cells/cm2 to prepare confluent monolayers.
This method resulted in isolation of alveolar epithelial cells with
characteristics consistent with type II cells (presence of microvilli
and lamellar bodies) (14).
Patch-clamp recording.
Patch-clamp experiments were performed on cells maintained in monolayer
culture between 5 and 7 days. The amphotericin-perforated whole cell
patch configuration was used in all experiments as a means to retain
regulatory components within the cells that may be lost when the
standard whole cell recording technique is used. Pipette electrodes
were pulled to a resistance of 2-4 M
from 7052 glass (Garner
Glass, Claremont, CA). The pipette tip was filled with
KMeSO4 saline solution consisting of (in mM) 130 KMeSO4, 5 KCl, 1 CaCl2, and 10 HEPES, pH 7.2. The pipette was then back-filled with the same solution containing 10 µM amphotericin B. High-resistance seals (>5 G
) were formed
between the pipette and cell membrane as amphotericin B was allowed to
partition into the membrane to obtain the whole cell configuration
before currents were recorded. In most cases, the bath solution
contained serum-free DMEM-F-12 medium (in mM: 120 NaCl, 4 KCl, 1 CaCl2, 0.3 MgCl2, 0.4 MgSO4, pH
7.4) or symmetrical K+ saline solution (in mM: 135 KCl, 1 CaCl2, 10 HEPES, pH 7.2). An Axopatch 1D amplifier and a
Digidata 1322A interface were used (both from Axon Instruments, Union
City, CA). pCLAMP 8 software was used to generate voltage step commands
and to record the resulting currents. Voltage step protocols used in
the experiments are described in the legends to Figs. 1-4.

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Fig. 1.
Identification of voltage-gated K+ (Kv)
currents with the whole cell perforated patch-clamp technique.
A: representative current traces for voltage-activated Kv
currents in isolated rat alveolar epithelial cells bathed in
Dulbecco's modified Eagle's medium (DMEM)-F-12 medium.
Inset: whole cell currents recorded in symmetrical
K+ saline solution. Cells were held at 60 mV for at least
3 min and then stepped through a series of depolarizing voltage steps
from 60 to +10 mV in 5-mV increments. hp, Holding potential.
B: current-voltage relationships for the Kv current in
DMEM-F-12 medium (n = 6) and in symmetrical
K+ saline solution (n = 5) obtained 200 ms
after the voltage step from the holding potential ( 60 mV). The data
for cells bathed in DMEM-F-12 medium were fit with the Boltzmann
equation. The data for the symmetrical K+ condition were
fit with a cubic spline function. C: conductance
(G)-voltage relationship determined 200 ms after the voltage
step (n = 6). Gmax, maximum
G.
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Fig. 2.
Ion selectivity variations detected by tail current
analysis. A: tail currents obtained from alveolar epithelial
cells bathed in DMEM-F-12 medium. Cells were held at 60 mV and then
stepped to +20 mV for either 25 (top) or 15 (bottom) ms to maximally activate the channels. Once
activated, cells were stepped through a series of hyperpolarizing
voltages from 100 to 20 mV in 10-mV increments for 50 ms.
B: open channel current-voltage relationships for 2 clearly
distinguishable current responses with significantly different reversal
potentials (RP). , RP = 60 ± 3 mV
(n = 5); , RP = 48 ± 4 mV
(n = 6). Arrows indicate the times at which the
currents were measured.
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Fig. 3.
Activation ( , n = 5) and
inactivation ( , n = 5) curves showing
the range of voltages at which Kv channels have significant open
probability. Cells were bathed in DMEM-F-12 medium. The steady-state
inactivation curve was produced by holding the cell at 60 mV for 3 min and then stepping the membrane voltage to +40 mV for 1 s to
maximally activate the channels. The peak current was then recorded.
This protocol was repeated at holding potentials of 40, 20, 0, and
+20 mV to generate the steady-state inactivation curve. Current/maximum
current (I/Imax) values were
normalized to a holding potential of 60 mV for the steady-state
inactivation curve and +20 mV for the activation curve.
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Fig. 4.
Differences in inactivation gating kinetics observed
between cells. A: representative current traces from 2 cells
illustrating differences in inactivation gating kinetics in response to
a voltage step from 60 to 0 mV. B: measurements of
inactivation time constants fit to a single-exponential function at
voltages between 10 and +10 mV. Cells were divided into 2 groups
based on inactivation time constants that were either greater than
( , n = 5) or less than
( , n = 5) 400 ms.
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Identification of Kv
-,
-, and KChIP subunits from rat
alveolar cells by reverse transcription-polymerase chain reaction.
Reverse transcription-polymerase chain reaction (RT-PCR) was used to
identify Kv
-,
-, and KChIP subunits in rat alveolar epithelial
cells maintained in culture between 5 and 7 days. Total RNA was
extracted from rat alveolar cells, rat brain, and rat atrium with
TRIzol reagent (Life Technologies, Rockville, MD). RNA was treated with
DNAase I (2 IU) for a period of 10 min to reduce the possibility of
genomic sequence contamination. Total RNA (2 µg) was reverse
transcribed with random hexamer primers and the Superscript II reverse
transcription kit (Life Technologies). Primers used in this study are
shown in Table 1. The initial denaturation condition was 94°C for 4 min, followed by 94°C for 45 s, annealing temperature (Table 1) for 45 s, and 72°C
for 1 min for 30 cycles. All of the PCR products were gel purified with
the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA), and purified
products were sequenced with gene-specific primers to confirm the
amplified sequences. DNA sequencing was performed at the Advanced
Genetics Analysis Center at the University of Minnesota.
Western blot analysis.
Cell monolayers were disrupted with TRIzol reagent, homogenized, and
resuspended in cold 1% sodium dodecyl sulfate (SDS), EDTA (1 mM) plus
protease inhibitors [in µg/ml: 50 phenylmethylsulfonyl fluoride
(PMSF), 1 aprotinin, 1 pepstatin, 1 leupeptin; Sigma Chemical]. A
protein assay was performed with a bicinchoninic acid (BCA) protein
assay kit (Pierce, Rockford, IL). Proteins were separated by
polyacrylamide gel electrophoresis (8%). Electroblotting was done with
Immobilon-P (Millipore, Bedford, MA). After washing, blots were reacted
overnight in primary Kv antibody (Chemicon, Temecula, CA) in freshly
prepared 1× Tris-buffered saline (TBS)-Tween 20 containing 3% nonfat
dry milk. The next day, blots were washed and reacted with goat
anti-rabbit alkaline phosphatase-labeled (GAR-AP) antibody. After
washing, alkaline phosphatase color reagent was added to 100 ml of 1×
alkaline phosphatase color development buffer at room temperature.
Immunocytochemistry.
Cultured alveolar epithelial cells were grown on 12-mm Costar filters
for 7 days. The filter membranes were cut away and placed in 1 ml of
methanol for 15 min. The methanol was removed, and primary antibody
[1:500 dilution in PBS with 0.1% Triton-X and 1% normal donkey serum
(NDS); Chemicon, Temecula, CA] was added for 1 h at room
temperature. The antibody was then removed, and the filters were rinsed
twice with PBS. For the double-label experiments shown in Fig. 8, the
rat-specific Na+-K+-ATPase
1-subunit (1:500 dilution) was identified with a
monoclonal antibody obtained from Upstate Biotechnology (Lake Placid,
NY), and the rat-specific Kv channel
-subunits (1:500 dilution) were identified with affinity-purified polyclonal antibodies from Chemicon. The secondary antibodies used to identify the
Na+-K+-ATPase
1-subunit and Kv
-subunits were DaM-Cy3 (1:500 dilution in PBS-0.1% Triton-X-1%
NDS) and DaRab-Cy2 (1:100 dilution in PBS-0.1% Triton-X-1% NDS),
respectively (Jackson Laboratories, West Grove, PA). The secondary
antibodies used to label the Kv
-subunits and the
Na+-K+-ATPase
1-subunits were
added for 1 h at room temperature in the dark. The antibody was
removed, and the filters were rinsed with PBS. The filters were then
placed on microscope slides, mounted with Vectashield (Vector
Laboratories, Burlingame, CA) and a 24 × 50-mm coverslip, and
sealed with nail polish. Absorption control experiments were performed
by preincubation with a 10-fold excess of antigenic peptide (also
available from Chemicon) in PBS at 4°C for 30 min before addition to
filters. In Fig. 8, the images were acquired with a Zeiss Axiovert 200M
inverted fluorescent microscope equipped with a spinning disk confocal
system (Atto Instruments, Rockville, MD). Images were collected at
×320 with a Hamamatsu Orca digital camera. The confocal images shown
in Fig. 9 were acquired with a MRC 1024 confocal microscope and
Confocal Assistant software (BioRad, Hercules, CA) with a ×60 oil
objective. Optical sections were acquired in 0.5-µm steps.
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RESULTS |
Functional characteristics of Kv channels expressed in cultured
alveolar epithelial cells.
The data presented in Fig. 1A
show representative current traces evoked by step depolarization of the
membrane potential from
60 mV to 10 mV in 5-mV increments. Inward
currents over a range from
30 to 0 mV were detected when the
extracellular solution was changed from DMEM-F-12 medium to symmetrical
K+ saline solution. Figure 1B shows a plot of
the current-voltage relationship for both DMEM-F-12 medium and
symmetrical K+ saline solution. The activation threshold
was
30 mV, and the reversal potential shifted to ~0 mV when
symmetrical K+ solution was used to bathe the cells. This
result was similar to the properties of Kv current-voltage plots
previously reported by DeCoursey et al. (7).
Figure 1C shows the conductance-voltage relationship
normalized to the maximum conductance (Gmax) for each cell (n = 6). Conductance was calculated as the
ratio of current at each voltage step divided by the difference between the command potential and the K+ equilibrium potential
(estimated from intracellular and extracellular K+
concentrations to be
82 mV).
Figure 2 shows the open channel
current-voltage relationships obtained from tail current analyses of
cells bathed in DMEM-F-12 medium. Two distinct current responses were
identified that exhibited different time courses for activation and
deactivation gating (Fig. 2A). In addition, reversal
potentials for the open channel current-voltage relationships were also
significantly different, suggesting differences in K+
selectivity. It is also worth noting that cells exhibiting currents with more negative reversal potentials also had faster inactivation rates. In at least three cells, we observed a combination of the two
currents shown in Fig. 2A with intermediate reversal
potentials. These findings suggest the possibility that alveolar
epithelial cells express different combinations of Kv channel subunits
that could account for the variations in activation gating and
selectivity behavior observed in these cells.
Figure 3 shows normalized activation and
steady-state inactivation curves that identify the range of voltages in
which significant open probability exists for the Kv channels expressed
in alveolar epithelial cells. A window current exists between
35 and
5 mV with a peak at
15 mV. Significant variability in inactivation gating was observed in 25 cells, with 20% of the cells exhibiting more
rapid inactivation gating in response to maximum activation at voltages
above
10 mV (Fig. 4A). Mean
time constants for single-exponential fits of the inactivation time
courses were plotted as a function of voltage (between
10 and +10 mV)
and are shown in Fig. 4B.
Identification of Kv
-,
-, and KChIP subunits in cultured
alveolar epithelial cells.
The results presented in Fig. 5 show
RT-PCR products obtained with rat-specific PCR primers (Table 1)
designed to detect mRNA for various Kv
-subunits in cultured
alveolar epithelial cells. We found that alveolar cell monolayers
contain mRNA for eight
-subunits representing four distinct families
of voltage-gated K+ channels. In addition, three Kv
-subunits (Kv
1.1, Kv
2.1, and Kv
3.1) as well as two KChIP
subunits (KChIP2 and KChIP3) were also identified (Fig. 5B).
As a positive control, we tested primers that failed to detect mRNA
transcripts in alveolar epithelial cell samples against mRNA isolated
from rat brain. In Fig. 5C we show that the primers could
detect Kv PCR products from brain, supporting the conclusion that these
Kv channel subunits are not expressed in cultured alveolar epithelial
cells. Note that each PCR product obtained from brain and cultured
alveolar cells was purified and sequenced to verify its molecular
identity.

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Fig. 5.
Kv
subunit expression was detected with RT-PCR. A: RT-PCR
amplification of rat Kv -subunits with RNA isolated from rat
alveolar epithelial cells. B: RT-PCR amplification of rat Kv
- and KChIP subunits with RNA isolated from rat alveolar epithelial
cells. C: RT-PCR amplification of rat Kv -subunits with
RNA isolated from rat brain. D-F: negative controls in
which RT reactions were performed without reverse transcriptase.
Primers used for these experiments and predicted sizes of PCR products
are shown in Table 1.
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In addition to probing for Kv channel subunits, we attempted to detect
components of ATP-sensitive K+ (KATP) channels
(Kir6.1, Kir6.2, SUR1, and SUR2) in rat alveolar epithelial cells by
RT-PCR (Fig. 6A). Although
some nonspecific bands were observed, none of the KATP
channel subunits could be detected even though the primers for Kir6.1,
Kir6.2, SUR1, and SUR2 subunits could detect mRNA for these proteins in
rat atrium, which was used as a positive control (Fig.
6B).

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Fig. 6.
Detection of ATP-sensitive K+
(KATP) channels with RT-PCR. A: RT-PCR
amplification of rat KATP channels with RNA isolated from
rat alveolar epithelial cells. B: RT-PCR amplification of
rat KATP channels with RNA isolated from rat atrium. Lane
marked SUR2A/2B shows both SUR2A (387 bp) and SUR2B (211 bp). The band
detected near 500 bp was nonspecific. Primers used in this
experiment and predicted sizes of PCR products are shown in Table
1.
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Kv
-subunit protein expression and localization in cultured
alveolar epithelial cells.
To determine whether certain Kv
-subunits are expressed as protein,
Western blot experiments were conducted with commercially available
antibodies directed against Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3. A
representative blot (Fig. 7) identifies
proteins with estimated molecular masses of 50 kDa that were
specifically labeled with antibodies to the Kv
-subunits listed
above. Preincubation with a 10-fold excess of the peptide
antigens for these
-subunits blocked labeling of the channel
subunits. Laser confocal microscopy of immunocytochemistry experiments
using these antibodies on confluent monolayers of alveolar epithelial
cells showed clear punctate labeling patterns associated with the
apical membrane (Figs. 8 and 9). Note
that no labeling was found along the basolateral membrane surfaces
(Fig. 9). When the antibodies were
preincubated with a 10-fold excess of antigenic peptide, specific
labeling of the apical membrane was not detected. Confocal vertical
sections taken from antibody-labeled monolayers showed that the
fluorescently labeled antibody is present at the apical surface and
does not appear to exhibit association with the basolateral membrane.

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Fig. 7.
Western blot showing specific antibody labeling to
membrane proteins with molecular masses that correspond to -subunits
of Kv K+ channels.
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Fig. 8.
Identification and localization of Kv1.4, Kv4.2, and
Na+-K+-ATPase -subunits in cultured
monolayers of alveolar epithelial cells. A: localization of
the Kv1.4 and Na+-K+-ATPase -subunits.
Vertical sections (0.5 µm) showed that Kv1.4 immunofluorescence
(green) was associated with the apical, but not basolateral, surface of
the cell. Immunofluorescence (red) associated with the
Na+-K+-ATPase 1-subunit was
localized to the lateral membrane surface. B: localization
of the Kv4.2 and Na+-K+ ATPase -subunits.
Kv4.2 immunofluorescence (green) was also associated with the apical
membrane.
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Fig. 9.
Kv
-subunits were identified in the apical membrane with
immunocytochemistry. Preabsorption control experiments involved
pretreatment with 10-fold excess of antigenic peptide (Ab + P).
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DISCUSSION |
Voltage-sensitive K+ channels have been described
previously in several epithelia including rat prostate epithelial cells
(24), intestinal epithelial cells (29), rat
spiral prominence epithelial cells of cochlea (17), rabbit
renal papillary epithelial cells (43), and flounder
intestinal epithelial cells (22). Although Kv-type
K+ channels are known to be associated with excitable
cells, these earlier studies demonstrated that they play important
roles in controlling membrane potential and K+ transport in
epithelia. At this time the molecular identification of multiple Kv
channel subunits has not been described. In a recent study of porcine
granulosa cells, six Kv
-subunits, two Kv
-subunits, and KCNQ1
and KCNE1 channels were identified by RT-PCR and Western blot analysis
(19). The study showed that members of the same Kv
-subunit family were able to associate to form heteromultimeric K+ channels. In addition, extensive coassociation of Kv
- and Kv
-subunits was demonstrated. Expression of multiple Kv
channel proteins in these cells explained the diverse
electrophysiological and pharmacological properties of whole cell
membrane currents recorded from these cells (19).
In previous studies of alveolar type II cells, two distinct types of
K+ channels were identified (7). These
channels were described as either n (normal)- or l (large)-type
channels depending on their voltage dependence and TEA sensitivity. The
n-type channel was activated at more negative potentials (
30 mV)
compared with the l-type channel, which was activated at
10 mV. Most
alveolar type II cells possessed the n-type K+ channel;
however, a small number of cells possessed K+ channels with
l-type characteristics. Peers et al. (26) also reported
two different types of K+ channels, low-threshold currents
and high-threshold currents from type II pneumocytes, that had
different voltage dependence and blocker sensitivities. Low-threshold
currents were activated at more depolarized voltages than
40 mV, and
these currents were blocked by 2 mM 4-aminopyridine (4-AP). In
contrast, high-threshold currents were activated at
20 mV and were
not blocked by 4-AP. In the present study, Kv currents were also
identified, but unlike previous studies, we observed that some cells
possessed more rapidly inactivating currents, suggesting the presence
of channels with N-type inactivation. We established the molecular
identity of these K+ channels by RT-PCR and discovered
eight distinct
-subunits, three
-subunits, and two KChIP proteins
in monolayer cultures of these cells. Among the
-subunits, Kv1.1,
Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were identified at the protein level by
Western blot analysis and immunocytochemistry with commercially
available antibodies. The immunocytochemistry results indicate that at
least five different Kv
-subunits are present in the apical membrane
of adult rat alveolar epithelial cells.
Previous studies of Kv channel structure and function have shown that
Kv1.4 and the Kv4 channel
-subunits exhibit N-type inactivation, and
these are often referred to as A-type currents in both native cells and
heterologous expression systems (35, 36, 41). In contrast,
the K+ currents from Kv1.1, Kv1.3, and Kv2.2 exhibit slow
inactivation properties when expressed by themselves in expression
systems (10, 13, 16, 18). Coexpression of Kv
-subunits
with Kv1.1 can increase the rate of inactivation through an interaction
between the inactivation ball domain of the
-subunit and the Kv1.1
-subunit (30, 37). It has also been shown that
coexpression of
-subunits increases the surface expression of
-subunits (38). Similar to
-subunits, KChIPs also
increase the cell surface expression of A-type K+ currents,
especially members of the Kv4 subfamily (25-27).
KChIPs also modulate the voltage dependence, inactivation kinetics, and recovery from inactivation of Kv4
-subunits in various expression systems (1, 3, 6, 20). KChIP2 and KChIP3 were previously detected in brain, heart, kidney, and lung tissues (1, 23, 31). In this study KChIP2 and KChIP3 were localized to alveolar epithelial cells, and they may modulate the properties of the Kv4
channels that are also expressed in these cells.
Interestingly, we identified Kv9.3 in cultured alveolar cells, which
was previously shown to be an electrically silent K+
channel
-subunit (39, 40). Kv2.1/Kv9.3 heteromeric
channels have been shown to be significantly inhibited by hypoxia in
COS cells (25) and in mouse L cells (12). In
addition, chronic hypoxia downregulates mRNA expression of Kv1.1,
Kv4.3, and Kv9.3 in pulmonary artery smooth muscle cells
(28). Kv
-subunits are also known to confer
O2 sensitivity to Kv4.2 in HEK293 cells (27).
Therefore, Kv
- and
-subunits expressed in rat alveolar epithelial cells may play a role in oxygen sensing; however, it is
still unclear whether Kv channels are intrinsically O2
sensitive or are under the control of an independent
O2-sensing mechanism (2, 11, 21, 44).
In an earlier study by Sakuma et al. (33), the presence of
KATP channels in human alveolar cells was suggested from
experiments with YM-934, which increased both K+ influx
into the alveolar space and alveolar fluid clearance in human lung.
Addition of glibenclamide, a KATP channel blocker, inhibited the YM-934-stimulated increase in alveolar fluid clearance, providing additional evidence to suggest a role for KATP
channels. Therefore, we attempted to detect the components of
KATP channels (Kir6.1, Kir6.2, SUR1, and SUR2) in rat
alveolar epithelial cells by RT-PCR. None of these subunits could be
detected even though the primers for Kir6.1, Kir6.2, SUR1, and SUR2
subunits could detect mRNA for these proteins in rat heart, which was
used as a positive control (Fig. 6). Thus we could not establish a
molecular basis for the presence of KATP channels in
cultured rat alveolar epithelial cells.
Our results from immunocytochemistry showed that at least five
different Kv channel
-subunits, Kv1.1, Kv1.3, Kv1.4, Kv4.2, and
Kv4.3, were located in the vicinity of the apical membrane. It is
possible that some of the channels are located in subapical vesicles;
however, the fluorescence signal is diffuse and does not allow us to
distinguish differences between surface and subapical vesicle labeling.
In vivo experiments have shown that K+ concentration in the
alveolar epithelial lining fluid is higher than in plasma, suggesting
that alveolar epithelial cells are capable of K+ secretion
(8, 9, 21, 34, 42). Therefore, Kv channels present in the
apical membrane of the alveolar epithelial cells may be involved in
K+ secretion into the alveolar space. The window current
between
30 and 0 mV indicates that Kv channels possess significant
open probability at depolarized voltages and could contribute to basal K+ secretion when the resting voltage is within this range.
In conclusion, the results of this study demonstrate that cultured
adult rat alveolar epithelial cells express a variety of Kv-type
channels encompassing four distinct families. In addition, auxiliary
- and KChIP subunits were also identified by RT-PCR analysis. The
expression of multiple
-,
-, and KChIP subunits provides for
significant variations in channel function that presumably explain the
differences in gating, voltage dependence, and blocker pharmacology
observed in this study and previously reported by DeCoursey et al.
(7) and Peers et al. (26). Moreover, the localization of at least five
-subunits to the apical membrane suggests that these channels participate in transepithelial
K+ secretion and presumably have a significant influence on
apical membrane voltage.
 |
ACKNOWLEDGEMENTS |
We thank Pat Jung for help with immunocytochemistry experiments.
 |
FOOTNOTES |
This work was supported by a Specialized Center of Research Grant from
the National Heart, Lung, and Blood Institute (HL-50152).
Address for reprint requests and other correspondence:
S. M. O'Grady, Depts. of Physiology and Animal Science,
Univ. of Minnesota, 495 Animal Science/Veterinary Medicine Bldg., 1988 Fitch Ave., St. Paul, MN 55108 (E-mail:
ograd001{at}umn.edu).
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 February 26, 2003;10.1152/ajpcell.00429.2002
Received 18 September 2002; accepted in final form 18 February
2003.
 |
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