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Antisense oligonucleotides against the alpha -subunit of ENaC decrease lung epithelial cation-channel activity

Lucky Jain1,2, Xi-Juan Chen1, Bela Malik2,3, Otor Al-Khalili2,3, and Douglas C. Eaton2,3

Departments of 1 Pediatrics and 3 Physiology and 2 The Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia 30322


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Amiloride-sensitive Na+ transport by lung epithelia plays a critical role in maintaining alveolar Na+ and water balance. It has been generally assumed that Na+ transport is mediated by the amiloride-sensitive epithelial Na+ channel (ENaC) because molecular biology studies have confirmed the presence of ENaC subunits alpha , beta , and gamma  in lung epithelia. However, the predominant Na+-transporting channel reported from electrophysiological studies by most laboratories is a nonselective, high-conductance channel that is very different from the highly selective, low-conductance ENaC reported in other tissues. In our laboratory, single-channel recordings from apical membrane patches from rat alveolar type II (ATII) cells in primary culture reveal a nonselective cation channel with a conductance of 20.6 ± 1.1 pS and an Na+-to-K+ selectivity of 0.97 ± 0.07. This channel is inhibited by submicromolar concentrations of amiloride. Thus there is some question about the relationship between the gene product observed with single-channel methods and the cloned ENaC subunits. We have employed antisense oligonucleotide methods to block the synthesis of individual ENaC subunit proteins (alpha , beta , and gamma ) and determined the effect of a reduction in the subunit expression on the density of the nonselective cation channel observed in apical membrane patches on ATII cells. Treatment of ATII cells with antisense oligonucleotides inhibited the production of each subunit protein; however, single-channel recordings showed that only the antisense oligonucleotide targeting the alpha -subunit resulted in a significant decrease in the density of nonselective cation channels. Inhibition of the beta - and gamma -subunit proteins alone or together did not cause any changes in the observed channel density. There were no changes in open probability or other channel characteristics. These results support the hypothesis that the alpha -subunit of ENaC alone or in combination with some protein other than the beta - or gamma -subunit protein is the major component of lung alveolar epithelial cation channels.

alveolar type II cells; epithelial sodium channel; single-channel recording


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVE Na+ transport across the pulmonary epithelium drives liquid from the lung lumen to the interstitium, with subsequent absorption of the fluid into the vasculature (1, 22, 23, 27). In the lung, Na+ reabsorption is a two-step process. The first step is passive movement of Na+ from the lumen across the apical membrane into the cell through Na+-permeable ion channels. The second step is active extrusion of Na+ from the cell across the basolateral membrane into the serosal space. Active amiloride-sensitive Na+ transport by lung epithelia has been demonstrated by several investigators (10, 19, 21, 25). Also, all three subunits, alpha , beta , and gamma , of the amiloride-sensitive Na+ channels that have been described in other Na+-transporting epithelia have also been identified in airway epithelial cells (4, 5, 24, 26, 31). Because of this observation, several investigators (1, 13, 20, 22, 23, 31) have suggested that the epithelial Na+ channel (ENaC) subunits known to be responsible for Na+ transport in other epithelial tissues must also be responsible for Na+ transport in the lung. Consistent with this idea is the observation by Hummler et al. (13) that genetically knocking out the alpha -subunit of ENaC leads to defective lung liquid clearance and premature death in perinatal mice. Thus there appears to be direct evidence that, in vivo, the ENaC constitutes the limiting step for Na+ absorption in epithelial cells of, at least, the fetal lung. Furthermore, when observed in other epithelial tissues or if ENaC subunits are expressed in oocytes, single ENaCs are low-conductance (4-5 pS), highly sodium-selective channels. In contrast, most laboratories studying lung ENaCs report nonselective cation channels [Na+-to-K+ permeability ratio (PNa/PK) = 1] with a high conductance (10-45 pS) that are quite different from the highly selective, low-conductance ENaCs. In fetal distal lung epithelial cells from 20-day-gestation rat fetuses, Orser et al. (28) observed, using symmetric solutions and inside-out recordings, single channels with a conductance of 23 ± 1.1 pS and a PNa/PK of 0.9. These channels were blocked by amiloride applied to the apical side of the membrane. Marunaka (18) has described an Na+-permeable, nonselective cation channel with a linear current-voltage relationship and a single-channel conductance of 26.9 ± 0.8 pS in the fetal distal lung epithelium. Feng et al. (8) and Yue et al. (34) have also recently described nonselective cation channels in apical cell-attached and inside-out patches from adult rat alveolar type II (ATII) cells. The channels have a PNa/PK of 1, are voltage independent, and are inhibited by amiloride. In apical membrane patches on adult rat ATII cells in primary culture, Jain et al. (15) have also observed nonselective cation channels similar in conductance and ion selectivity to the ones described by Orser et al. (28). Thus the predominant Na+-permeable channels observed electrophysiologically in both adult and fetal lung cells appear to be nonselective cation channels (although some details of channel characteristics appear to differ between preparations; for a review, see Ref. 20). Although the exact role that these channels play in vivo is unclear, electrophysiological considerations and studies from epithelial cells from other organs suggest a role for this channel in Na+ transport in the lung. For example, Tohda et al. (30) showed that these channels may play a role in the increased reabsorption of fluid by alveolar epithelia in response to beta -agonist stimulation.

The apparently conflicting information obtained from electrophysiological and molecular biological experiments caused us to investigate the relationship of ENaC to the nonselective cation channels in lung epithelial cells. Antisense oligonucleotides targeting individual subunits were used to manipulate the expression of the respective subunit proteins, and the patch-clamp technique was used to characterize the resulting single channels. Our results suggest that the alpha -subunit is responsible for the nonselective lung epithelial cation channels in rat lung epithelia.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Type II pneumocyte isolation and culture. ATII cells were isolated by enzymatic digestion of lung tissue from adult Sprague-Dawley rats (100-250 g) with the use of published techniques (15). Briefly, the rats were anesthetized with pentobarbital sodium and heparinized (100 U/kg). ATII cells were digested by tracheal installation of elastase (0.4 mg/ml). The lung tissue was minced in DNase (1 mg/ml) and filtered sequentially through 100- and 20-mM nylon mesh. The cells thus obtained were collected, centrifuged, and seeded onto glass coverslips (~2 × 105 cells/cm2) in Dulbecco's modified Eagle's medium-Ham's F-12 medium containing 5% fetal calf serum and antimicrobial agents and supplemented with L-glutamine and sodium bicarbonate. Cells were incubated in 90% air-10% CO2 and used for patch-clamp studies between 24 and 96 h after being plated on glass coverslips. No significant difference in Na+-channel activity or characteristics was observed during this time period. Cell viability (>90%) and ATII cell purity (>95%) has been previously established in our laboratory (15).

Solutions and drugs. All solutions were made with deionized water and then passed through a 0.2-µm filter (Gelman Sciences, Bedford, MA) before use. The bath and pipette solutions used in the cell-attached mode contained (in mM) 140 NaCl, 1 MgCl2, 1 CaCl2, 5 KCl, and 10 HEPES, pH 7.4 with 2 N NaOH. The contents of the bath and pipette solutions were varied as appropriate for specific protocols. All chemicals were obtained from Sigma (St. Louis, MO).

Procedure for single-channel recordings. Patch-clamp experiments were carried out at room temperature. The pipettes were pulled from filamented borosilicate glass capillaries (TW-150, World Precision Instruments) with a two-stage vertical puller (Narishige, Tokyo, Japan). The pipettes were coated with Sylgard (Dow Corning) and fire polished (Narishige). The resistance of these pipettes was 5-8 MOmega when filled with the pipette solution. We used the cell-attached configuration for most of our studies because in this configuration, the cytoplasmic constituents remain intact, thus allowing us to study the role of cytoplasmic second messengers in the regulation of ion-channel activity. Inside-out patches were also used to determine the selectivity of the channel and to determine whether the effects of agents were directly on the channel or mediated by a signaling cascade. After formation of a high-resistance seal (>50 GOmega ) between the pipette and the cell membrane, channel currents were sampled at 5 kHz with a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA) and filtered at 1 kHz with an eight-pole, low-pass Bessel filter. Data were recorded by a computer with pCLAMP 6 software (Axon Instruments). Current-amplitude histograms were made from stable continuously recorded data, and the open and closed current levels were determined from least-squares fitted Gaussian distributions. We used the product (NPo) of the number of channels (N) times the open probability (Po) as a measure of the activity of the channels within a patch. This product could be calculated from the single-channel recording without making any assumptions about the total N in a patch or the Po of a single channel
<IT>NP</IT><SUB>o</SUB> = <LIM><OP>∑</OP><LL><IT>i</IT>=0</LL><UL><IT>N</IT></UL></LIM> <FR><NU><IT>i</IT> ⋅ <IT>t</IT><SUB><IT>i</IT></SUB></NU><DE><IT>T</IT></DE></FR>
where T is the total recording time, i is the number of channels open, and ti is the recording time during which i channels are open. Current-amplitude histograms provide the clearest demonstration of multiple current levels. The total N in a patch was estimated by observing the number of peaks in a current-amplitude histogram over the entire duration of the recording period. The Po of the channels was calculated with FETCHAN in pCLAMP 6 and with locally developed software (17).

RT-PCR and Western blotting. Semiquantitative RT-PCR of total RNA obtained from rat ATII cells was performed with subunit-specific primers based on published sequences. PCR reactions were run in the linear range of amplification, with glyceraldehyde-3-phosphate dehydrogenase amplified as standard for comparison. These primers amplified a single fragment of the expected size for each RNA [579 bp for rat (r) alpha -ENaC, 548 bp for beta -rENaC, and 558 bp for gamma -rENaC]. The PCR products were not seen in control reactions without RNA. The sequences of the amplified fragments are identical to the published sequences (31). The sequences of the primers are given in Table 1.

                              
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Table 1.   Characteristics of RT-PCR primers

To determine whether protein for each of the subunits was present, we used Western blot experiments to confirm the presence of protein for all three subunits in ATII cells in primary culture. Polyclonal antibodies were produced by identifying unique antigenic 20-amino acid sequences in each subunit. The peptides were produced in Emory's microchemical facility and sent to Lofstrand Laboratories where the peptides were coupled to KLH and used to immunize at least two rabbits with each peptide. This provided at least one anti-peptide antibody specific for each subunit.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Characteristics of single nonselective cation channels in the apical membrane of ATII cells. Jain et al. (15) and others (18, 28) have previously reported that the predominant Na+-permeable channel observed in cell-attached patches on the apical membrane of ATII cells with 140 mM NaCl in the bath and pipette is a high-conductance (20.6 ± 1.1 pS) channel (Fig. 1A). The single-channel current has a linear current-voltage relationship with no detectable rectification (Fig. 1B). The pipette potential at which current polarity reversed was estimated to be -37 mV. Because the resting membrane potential of alveolar epithelium has previously (28) been shown to be approximately -30 to -40 mV, the reversal potential appears to be close to 0 mV, which would be expected for a nonselective cation channel.


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Fig. 1.   Characteristics of nonselective cation channel. A: single-channel recordings of a nonselective cation channel from a cell-attached patch in alveolar type II cell. C, closed state. Nos. on right, mV. B: current-voltage relationship showing that unit conductance of channel is 20.6 ± 1.1 pS and that current-voltage relationship is linear, with no detectable rectification. Vp, membrane potential. C: amplitude histogram of patch in A showing that open probability is ~0.2.

Ion selectivity was determined with excised inside-out patches and solutions of varying ionic compositions. The channel had a similar permeability to Na+ and K+ (PNa/PK = 0.97 ± 0.07; n = 7 patches). The channel Po was decreased by amiloride (0.1-1 mM) applied to the extracellular side (i.e., in the micropipette). The Po of untreated channels was 0.31 ± 0.01 vs. 0.03 ± 0.01 after 100 nM amiloride (P < 0.01; n = 7 patches). More than one current level was observed in 85.7% of the active patches.

Functional importance of Na+-permeable nonselective cation channels and their relationship to ENaC. We examined the expression of mRNA for the three subunits of the ENaC by RT-PCR experiments. Semiquantitative RT-PCR of total RNA obtained from rat type II cells was performed with subunit-specific primers. PCRs were run in the linear range of amplification, with glyceraldehyde-3-phosphate dehydrogenase amplified as a standard for comparison. These primers amplified a single fragment of the expected size for each RNA (579 bp for alpha -rENaC, 548 bp for beta -rENaC, and 558 bp for gamma -rENaC; Fig. 2A). The PCR products were not seen in the control reactions without RNA. The sequences of the amplified fragments are identical to the published sequences (31). Thus ATII cells, at least, have an RNA message for all of the ENaC subunits. To determine whether protein for each of the subunits was present, we used Western blot experiments to confirm the presence of protein for all three subunits in ATII cells in primary culture (Fig. 2B). These results show that even under conditions when the predominant Na+-permeable channel is the nonselective cation channel, there is an easily detectable message for all ENaC subunits and protein for the alpha - and beta -subunits (we have yet to perform Western blot experiments for the gamma -subunit). These results, of course, in no way prove that the nonselective cation channel is related to ENaC.


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Fig. 2.   RT-PCR and Western blot demonstrate presence of epithelial Na+ channel (ENaC) subunits. A: RT-PCR is capable of amplifying appropriate size cDNA for each subunit. B: Western blot demonstrates presence of alpha - and beta -subunits. gamma -Subunit has also been demonstrated in alveolar type II cells by others (21a).

Antisense oligonucleotides against alpha -, beta -, and gamma -subunits of ENaC decrease the corresponding protein. To address directly the relationship between the nonselective cation channel and ENaC, we used antisense oligonucleotides directed against the sequences around the translation start site of each subunit. Cultured cells were exposed to oligonucleotides overnight. Using quantitative densitometry of Western blots, we found that in cells treated overnight with antisense oligonucleotides, the corresponding protein was more than twofold lower. Figure 3 shows results for the alpha - and beta -subunits. We also used RT-PCR to examine the mRNA levels after oligonucleotide treatment. Contrary to our expectations, we found that mRNA levels for the subunits were unchanged or even slightly increased after oligonucleotide treatment. We believe that this represents transcriptional control of ENaC mRNA in response to reduced Na+ transport as described for several other gene products (11, 12, 16).


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Fig. 3.   Antisense oligonucleotide induced inhibition of ENaC subunit expression. Top: Western blot analysis (n = 1) for alpha -subunit (A) and beta -subunit (B) of ENaC in rat alveolar type II cells treated with sense and antisense oligonucleotides. Bottom: densitometric estimation of protein concentration (conc) for corresponding subunit. Expression of all subunits was significantly reduced by treatment with antisense oligonucleotides.

Antisense oligonucleotide against alpha -subunit of ENaC decreases lung epithelial cation-channel activity. Because antisense oligonucleotides reduce the expression of ENaC subunit proteins, we also determined the effects of antisense oligonucleotides on the expression of cation channels by using the patch-clamp technique to measure single-channel activity in ATII cells treated with oligonucleotides. Cultured ATII cells were exposed to oligonucleotides for 24 h. Addition of the antisense oligonucleotide to the alpha -subunit to the culture medium produced a marked reduction in the number of apical membrane cell-attached patches with cation channels (Fig. 4) compared with those exposed to the sense oligonucleotide as a control. In 62 patches on cells that had been treated with sense oligonucleotides, 69.4% of the patches had one or more nonselective cation channels; in 63 patches treated with antisense oligonucleotides, only 34.9% of the patches contained a nonselective cation channel (with the z-test for statistical ratios, the probability of observing such different frequencies is <0.0001). On the other hand, similar experiments with antisense oligonucleotides targeting the beta - or gamma -subunits did not alter the channel number. There were no significant changes in Po or other channel characteristics [Po of channels from alpha -sense-treated cells = 0.183 ± 0.03 (n = 42 patches) vs. 0.257 ± 0.05 (n = 21 patches) in alpha -antisense-treated cells; Po of channels from beta -sense-treated cells = 0.239 ± 0.037 (n = 30 patches) vs. 0.227 ± 0.045 (n = 29 patches) in beta -antisense-treated cells; Po of channels from gamma -sense-treated cells = 0.148 ± 0.02 (n = 40 patches) vs. 0.141 ± 0.03 (n = 34 patches) in gamma -antisense-treated cells; none of the treatments produced a significant difference]. We thought that the beta - or gamma -subunit might substitute for each other in formation of the cation channel so we simultaneously inhibited both the beta - and gamma -subunits with antisense oligonucleotides. This treatment also did not affect the level of expression and functional characteristics of the channels.


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Fig. 4.   Treatment with antisense oligonucleotide directed against alpha -subunit of ENaC produced a significant reduction in number of apical membrane cell-attached patches with cation channels. Addition of either beta - or gamma -antisense oligonucleotide separately or together or sense oligonucleotide for any of the subunits (as a control) did not change cation-channel activity.

These results suggest that the lung epithelial cation channel belongs to the ENaC family of Na+ channels and is formed from the alpha -subunit protein alone or the alpha -subunit with some hitherto unidentified subunits other than either the beta - or gamma -subunit.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Whole tissue experiments on a variety of epithelial tissues including the lung imply a simple picture of Na+ channels. This picture is consistent with tissue reabsorption of Na+ and, at a single-channel level, implies that the channels should be highly selective for Na+ over K+, should open and close infrequently, and should be blocked by low concentrations of the drug amiloride (2). Indeed, channels with such properties can be observed in several epithelial preparations (9), but examination of single channels in several Na+-transporting epithelial cells suggests a more complicated picture of amiloride-blockable channels. Single-channel studies (3, 20) have identified at least four different amiloride-blockable channels with either high selectivity, low selectivity, or no selectivity for Na+ over K+ in the apical membranes of a variety of cultured and native epithelial cells. These channels differ in their ion selectivity, unitary conductance, and other characteristics, but they all have been proposed to play some role in Na+ absorption. Benos et al. (3) recently compiled and plotted selectivity versus conductance of epithelial Na+ channels from the published literature and showed the wide variability in these characteristics. Few of these reported channels match the characteristics of the prototypical native amiloride-sensitive Na+ channel that has a low conductance (4-6 pS) and high selectivity for Na+ (PNa/PK > 10). In fact, only one study (6) has reported a channel fitting this description from the lung. In this study, the investigators recorded a 4-pS amiloride-sensitive channel from outside-out patches excised from apical membrane. The channel was highly selective for Na+ (PNa/PK > 10). However, no other laboratory has subsequently reported observing this channel in lung epithelia on a consistent basis. For epithelial cells in culture, culture conditions are apparently the primary determinant of the frequency with which these different channel types are found in the apical membrane (7, 33), but ultimately, the observed variations in single-channel properties must be due to variations in subunit stoichiometry, variations in subunit types combining to form channels, or posttranslational modifications of the subunit proteins.

Molecular basis for Na+ transport in lung epithelial cells. The differences in the properties of Na+-permeable channels make a determination of the molecular basis for Na+ transport an important issue. Several investigators and experiments with transgenic animals (1, 13, 20, 21, 22, 23) have suggested that alpha -, beta -, and gamma -ENaC subunits known to be responsible for Na+ transport in other epithelial tissues are also responsible for Na+ transport in the lung. This would be consistent with the observation that rat adult and fetal lungs have been shown to express all three subunits of the ENaC (13). However, electrophysiological measurements suggest that if ATII cells do express ENaCs, then the functional characteristics are quite different from the highly selective, low-conductance (4-5 pS) ENaCs observed in other tissues.

In this study, we have used antisense oligonucleotides targeting each of the three subunits to manipulate the expression of the respective protein and the patch-clamp technique to study the changes resulting from these manipulations in single-channel characteristics. Our results support the hypothesis that nonselective lung epithelial cation channels are primarily composed of the alpha -subunit. This is the first evidence to show that nonselective cation channels belong to the ENaC family of channels. The different cation channels may represent different forms of ENaC, with at least one protein (alpha -subunit) in common. This conclusion is consistent with the work of Hummler et al. (13), who showed that inactivating the mouse alpha -ENaC gene leads to early death of newborns due to defective neonatal lung liquid clearance. alpha -ENaC knockout completely abolished amiloride-sensitive Na+ transport in the airway epithelia, resulting in respiratory distress in neonates and death within 40 h of birth. Furthermore, Canessa et al. (5) and Ismailov et al. (14) have shown that the alpha -subunit alone can form fully functional amiloride-sensitive Na+ channels, whereas the beta - and gamma -subunits individually or in combination cannot. In fact, Kizer et al. (16a) have recently shown that expression of the alpha -subunit of the ENaCs from osteoblasts into a null cell line (LM TK-) resulted in a nonselective cation channel (PNa/PK = 1.1 ± 0.1) with a conductance of 24.2 ± 1.0 pS. These results are consistent with our findings that inhibition of the alpha -subunit reduces cation-channel number, whereas inhibition of the beta - and gamma -subunits does not.

In summary, our results support the hypothesis that expression of the alpha -subunit of ENaC alone or in combination with some protein other than the beta - or gamma -subunit protein is the major component of lung epithelial cation channels.


    ACKNOWLEDGEMENTS

This work was supported by American Lung Association Grant RG-133-N (to L. Jain), National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-37963 and P05-DK-50268 (to D. C. Eaton), and the Center for Cell and Molecular Signaling (Emory University School of Medicine, Atlanta, GA).


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. Jain, Dept. of Pediatrics, Emory Univ. School of Medicine, 2040 Ridgewood Dr., NE, Atlanta, GA 30322 (E-mail: ljain{at}emory.edu).

Received 13 January 1999; accepted in final form 11 March 1999.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 276(6):L1046-L1051
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