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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -subunits (Kv1.1, Kv1.3, Kv1.4, Kv2.2, Kv4.1, Kv4.2, Kv4.3, and Kv9.3), three beta -subunits (Kvbeta 1.1, Kvbeta 2.1, and Kvbeta 3.1), and two K+ channel interacting protein (KChIP) isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv alpha -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 alpha -, beta -, 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -, beta -, and K+ channel interacting protein (KChIP) subunits and that at least five of the eight alpha -subunits are present in the apical membrane.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega 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 GOmega ) 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); open circle , 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 (open circle , n = 5) 400 ms.

Identification of Kv alpha -, beta -, 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 alpha -, beta -, 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.

                              
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Table 1.   Forward and reverse primers for PCR reactions

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 alpha 1-subunit (1:500 dilution) was identified with a monoclonal antibody obtained from Upstate Biotechnology (Lake Placid, NY), and the rat-specific Kv channel alpha -subunits (1:500 dilution) were identified with affinity-purified polyclonal antibodies from Chemicon. The secondary antibodies used to identify the Na+-K+-ATPase alpha 1-subunit and Kv alpha -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 alpha -subunits and the Na+-K+-ATPase alpha 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -, beta -, 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 alpha -subunits in cultured alveolar epithelial cells. We found that alveolar cell monolayers contain mRNA for eight alpha -subunits representing four distinct families of voltage-gated K+ channels. In addition, three Kv beta -subunits (Kvbeta 1.1, Kvbeta 2.1, and Kvbeta 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 alpha -subunits with RNA isolated from rat alveolar epithelial cells. B: RT-PCR amplification of rat Kv beta - and KChIP subunits with RNA isolated from rat alveolar epithelial cells. C: RT-PCR amplification of rat Kv alpha -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.

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.

Kv alpha -subunit protein expression and localization in cultured alveolar epithelial cells. To determine whether certain Kv alpha -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 alpha -subunits listed above. Preincubation with a 10-fold excess of the peptide antigens for these alpha -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 alpha -subunits of Kv K+ channels.



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Fig. 8.   Identification and localization of Kv1.4, Kv4.2, and Na+-K+-ATPase alpha -subunits in cultured monolayers of alveolar epithelial cells. A: localization of the Kv1.4 and Na+-K+-ATPase alpha -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 alpha 1-subunit was localized to the lateral membrane surface. B: localization of the Kv4.2 and Na+-K+ ATPase alpha -subunits. Kv4.2 immunofluorescence (green) was also associated with the apical membrane.



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Fig. 9.   Kv alpha -subunits were identified in the apical membrane with immunocytochemistry. Preabsorption control experiments involved pretreatment with 10-fold excess of antigenic peptide (Ab + P).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -subunits, two Kv beta -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 alpha -subunit family were able to associate to form heteromultimeric K+ channels. In addition, extensive coassociation of Kv alpha - and Kv beta -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 alpha -subunits, three beta -subunits, and two KChIP proteins in monolayer cultures of these cells. Among the alpha -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 alpha -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 alpha -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 beta -subunits with Kv1.1 can increase the rate of inactivation through an interaction between the inactivation ball domain of the beta -subunit and the Kv1.1 alpha -subunit (30, 37). It has also been shown that coexpression of beta -subunits increases the surface expression of alpha -subunits (38). Similar to beta -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 alpha -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 alpha -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 beta -subunits are also known to confer O2 sensitivity to Kv4.2 in HEK293 cells (27). Therefore, Kv alpha - and beta -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 alpha -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 beta - and KChIP subunits were also identified by RT-PCR analysis. The expression of multiple alpha -, beta -, 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 alpha -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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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