δ-Subunit Confers Novel Biophysical Features to αβγ-Human Epithelial Sodium Channel (ENaC) via a Physical Interaction*

Native amiloride-sensitive Na+ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of δ-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na+ conductances in epithelial cells. For example, the δ-subunit may form multimeric channels with αβγ-ENaC. Reverse transcription-PCR revealed that δ-ENaC is co-expressed with αβγ-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that δ-ENaC is co-expressed with α-, β-, and γ-ENaC in H441 cells at the protein level. Measurement of current-voltage that cation selectivity ratios for the revealed relationships Na+/Li+/K+/Cs+/Ca2+/Mg2+, the apparent dissociation constant (Ki) for amiloride, and unitary conductances for δαβγ-ENaC differed from those of both αβγ- and δβγ-ENaC (n = 6). The contribution of the δ subunit to PLi/PNa ratio and unitary Na+ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC50 for proton activation, mean open and closed times, and the self-inhibition time of δαβγ-ENaC differed from those of αβγ- and δβγ-ENaC. Co-immunoprecipitation of δ-ENaC with α- and γ-subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of δ-ENaC with other subunits could account for heterogeneity of native epithelial channels.

expressed in oocytes. However, the molecular basis of the moderate selective (type II) and nonselective Na ϩ channels (type III) in epithelia, such as the 20-pS channels in A549 (6) and 10-pS channel in H441 cells (2), is unknown.
The ␣-ENaC subunit was originally cloned from the rat colon and has subsequently been identified in a variety of mammalian and amphibian species (1,7). Five ENaC subunits have been cloned to date, namely ␣-, ␤-, ␥-, ␦-, and ⑀-ENaC. Among them, the ␤and ␥-subunits regulate the channel activity of the "conducting" ␣-, ␦-, and ⑀-ENaC subunits when heterologously expressed in oocytes and cell lines (7). Biophysical characterization of ␣␤␥-ENaC channels has revealed a Na ϩ conductance of 4 pS and a Li ϩ conductance of 7 pS, respectively. ␣␤␥-ENaC is highly permeable to Na ϩ but virtually impermeable to K ϩ , with a P Na /P K greater than 20. Amiloride sensitivity and other properties of ␣␤␥-ENaC are reflective of the characterization of highly Na ϩ selective channels in native epithelial cells. In comparison with the ␣-subunit, ␦-ENaC exhibits differences in Na ϩ conductance, amiloride affinity, self-inhibition, and P Na /P Li selectivity ratio (8 -11). However, neither ␣␤␥nor ␦␤␥-ENaC can match all native amiloride-sensitive Na ϩ channels in their biophysical properties. Additionally, many of the cloned, conductive ENaC subunits are not activated by cAMP-elevating agents, which is a hallmark of native epithelial Na ϩ channels.
The biophysical properties of ENaC depend on channel composition. ␣-like conducting subunits can form a channel sharing identical properties to channels composed of ␣-like and ␤␥ subunits (9,10,12,13). In contrast, ␣␤and ␣␥-ENaC channels display variant amiloride sensitivity, conductance, and Na ϩ permeability (14,15). On the other hand, properties of native amiloride-sensitive Na ϩ channels are dependent on culture conditions (16,17). Cells grown on permeable supports have a channel that more closely resembles heterologously expressed ␣␤␥-ENaC channels than the channels expressed by cells grown on coverslips (18). Although it has been reported that a functional ENaC complex is composed of either 2␣/1␤/1␥, or alternatively, 3␣3␤/3␥ (7,19), channel properties may not be determined in a stoichiometric manner.
The existence of other subunits may play an important role in determining native channel properties. Indeed, numerous studies demonstrated that culture conditions, cellular protein polarization, subunit composition, and splice variants can partly account for the diversity in channel properties (1,16,17). However, the putative influence of the ␦-subunit co-expression on the ENaC channel properties has not been documented. In the present study, we have tested the hypothesis that ␦-ENaC is co-expressed with ␣␤␥-ENaC subunits in epithelia and that ␦-ENaC influences the biophysical features of ␣␤␥ channels when coexpressed in oocytes. Our results indicated that monovalent and divalent cation selectivities, single channel Na ϩ conductance, amiloride affinity, gating kinetics, and proton activation of ␦␣␤␥-hENaC were intrinsically distinguishable from those of ␣␤␥and ␦␤␥-ENaC. We also demonstrated the presence of ␦-ENaC mRNA in human lung, pancreatic, and colonic epithelial cells using RT-PCR. Also, for the first time, we showed that native ␣and ␥-ENaC co-immunoprecipitate with ␦-ENaC in H441 cells and in transiently transfected COS-7 cells, supporting the idea that these four mammalian ENaC subunits combine to form multimeric channels with novel biophysical properties.

MATERIALS AND METHODS
Oocyte Expression and Whole-cell Recordings-cRNAs for human ␦-, ␣-, ␤-, and ␥-ENaC (hENaC) were prepared as described previously (20). Defolliculated oocytes were cytosolically injected with hENaC cRNAs (25 ng) per oocyte in 50 nl of RNase free water with a ratio of 1␦:1␣:1␤:1␥ subunit (except for cRNA concentration-dependent experiments) and incubated in half-strength L-15 medium. Whole-cell cation currents were measured by the two-electrode voltage clamp technique 2 days after injection. Oocytes were impaled with two electrodes, each having resistances of 0.5-2 megaohms when filled with 3 M KCl. A TEV 200 voltage clamp amplifier was used to clamp the oocytes with concomitant recording of currents. Two reference electrodes were connected to the bath. Sampling protocols were generated by pCLAMP 8.0, and the current at Ϫ60 mV was simultaneously monitored with a chart recorder. The control superfusate was ND-96 (in mM: 96 NaCl, 1 MgCl 2 , 1.8 CaCl 2 , 2 KCl, and 5 HEPES, pH 7.5). Superfusate with pH 6 or below was buffered with MES. pH was adjusted with NaOH and HCl. In experiments designed to measure the ion selectivity, Na ϩ salt was replaced with equal molar amounts of Li ϩ , K ϩ , Cs ϩ , Ca 2ϩ , and Mg 2ϩ for determination of ion selectivity. Switching of bath solutions with neutral or acidic pH was controlled by an SF-77B Perfusion Fast-Step system (Warner Instrument Corp., Hamden, CT). Current-voltage relationships were acquired by stepping the holding potential in 20-mV increments from Ϫ120 to ϩ80 mV after the monitoring currents were stable, with and without the application of amiloride (100 M). Data were sampled at a rate of 1 kHz and filtered at 500 kHz.
Reverse Transcription-PCR-Confluent H441, A549, CFPAC, and Caco-2 cells were scraped into ice-cold PBS. Following pelleting in a microcentrifuge, the PBS was discarded, and the cells were snap-frozen in a dry ice-methanol mixture and stored at Ϫ80°C. Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA was dissolved in RNase-free water and stored at Ϫ80°C. RNA concentration was assessed by spectrophotometry at 260 nm. RNA quality was confirmed by running 5 g of total RNA from each preparation on a 1.2% denatured agarose gel. The specific oligonucleotide primers for the RT-PCR analysis of the ␣-, ␤-, ␦-, and ␥-ENaC subunits were as follows: ␣-ENaC, 5Ј-AAC AAA TCG GAC TGC TTC TAC-3Ј (sense), 5Ј-AGC CAC CAT CAT CCA TAA A-3Ј(antisense); ␤-ENaC, 5Ј-GGG ACC AAA GCA CCA AT-3Ј (sense), 5Ј-CAG ACG CAG GGA GTC ATAG-3Ј (antisense); ␦-ENaC, 5Ј-CGA AGC ATG GAC GGG AGA ATG-3Ј (sense), 5Ј-GGT GCC AGT GAC GCT CAA AGA-3Ј (antisense); ␥-ENaC, 5Ј-GCA CCG TTC GCC ACC TTC TA-3Ј (sense), 5Ј-AGG TCA CCA GCA GCT CCT CA-3Ј (antisense). Sequence accession numbers in GenBank TM are 38197484, 4506816, 34101281, and 42476332 for the ␣-, ␤-, ␦-, and ␥-subunits, respectively. The corresponding product sizes are 406 bp (␣), 440 bp (␤), 325 bp (␦), and 456 bp (␥). Custom primers specific for ␦-, ␣-, ␤-, and ␥-ENaC were synthesized by Sigma Genosys and used at a final concentration of 0.4 M. RT-PCR was performed using the OneStep RT-PCR kit (Qiagen) in a Mastercycler gradient thermocycler (Eppendorf Scientific). One microgram of total RNA was added as a template for each reaction. Reverse transcription was carried out using a single cycle of 50°C for 30 min. This was followed by a single cycle of 95°C for 15 min to inactivate the reverse transcriptase while activating the HotStart TaqDNA polymerase, and then 30 cycles of 94°C for 0.5 min, 56°C for 1 min, 72°C for 1.5 min were performed followed by a single 10-min cycle at 72°C for extension. RT-PCR products were electrophoresed on a 2% agarose gel using 100-bp PCR markers (Promega) as a standard to determine the molecular size. Products of the predicted molecular size were excised and purified from the gel using the QIAquick gel extraction kit (Qiagen) and subcloned into the pCR-2.1 vector (Invitrogen). Positive clones were selected by blue/white screening and followed by digestion with EcoRI (Promega) to verify incorporation of insert of the correct size. Authenticity of PCR products in the pCR2.1 vector were verified by automated DNA sequencing (DNA Sequencing Facility, Iowa State University).
Immunofluorescence Microscopy-Confluent H441 cell monolayers were grown on 12-mm diameter polycarbonate filters (Corning Costar) for approximately 2 weeks. Following fixation in 3% buffered formaldehyde for 20 min, cells were permeabilized in 0.01% Triton X-100 in PBS for 5 min, and nonspecific protein binding sites were blocked using 5% bovine serum albumin in PBS for 30 min. Anti-␦ENaC (Open Biosystems, Huntsville AL) as well as ␣-, ␤-, and ␥-ENaC polyclonal antibodies (prepared by Pascal Barbry) at 5-20 g/ml for 2 h at room temperature) was used to detect ENaC protein. An 18-amino acid peptide in extracellular loop (RESAFKLSTGTSRWPSAK) was antigen for ␦-ENaC antibody preparation. Cells incubated with PBS in the absence of ENaC antibodies serve as control. Nuclei were stained with Vectashield-4Ј,6diamidino-2-phenylindole (Vector Labs). Images were captured on an Olympus IX170 inverted epifluorescence microscope using SenSyscooled charge-coupled high resolution camera (Photometrics, Tucson, AZ). Images were assembled using Adobe PhotoShop.
Co-immunoprecipitation-COS-7 or H441 cells were grown in low bicarbonate Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin-streptomycin. Cells were maintained at 37°C and 5% CO 2 . COS-7 cells were plated at a density of 2.4 ϫ 10 6 cells in 100-mm plates 24 h prior to transfection. Cells were transfected with ϳ24 g of each cDNA construct or vector alone using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. COS-7 and H441 cells were lysed in a lysis buffer (in mM: 120 NaCl, 100 KCl, 20 EDTA, 1 dithiothreitol, 20 Tris-HCl, 1% Triton X-100, pH 7.5) supplemented with one protease inhibitor mixture tablet (Roche Applied Science)/10 ml of lysis buffer at 4°C for 1 h. The lysate was centrifuged at 14,000 rpm for 10 min, and the pellet was discarded. The supernatant was incubated with 10 g of anti-␣ENaC (Affinity Bioreagents) or anti-HA antibody (Sigma) to immunoprecipitate the specific proteins for 2 h at 4°C. The supernatant of H441 cell lysates was incubated with 10 g of anti-␣ENaC (Affinity Bioreagents) or anti-␦ENaC antibody (Open Biosystems) to immunoprecipitate the specific proteins for 2 h at 4°C. Subsequently, 50 l of protein A/G-plus Agarose beads was added and incubated for 1 h at 4°C. The beads were washed for five times with the lysis buffer, resuspended in 2ϫ SDS sample buffer, and boiled at 95°C for 5 min. The samples were analyzed by Western blotting (8% SDS-PAGE gel) using anti-HA monoclonal (Sigma), anti-␥ENaC (Affinity Bioreagents), and anti-␣ENaC polyclonal antibodies (Affinity Bioreagents).
Single Channel Patch Clamp Assay-Unitary Na ϩ and Li ϩ conductance in oocytes was digitized from on-cell and inside-out patches isolated from vitelline membrane-stripped eggs as described previously (21). Linear regression analysis was applied to the single channel current-voltage curves to compute the slope conductance.
Data Analysis-Data were primarily analyzed with the Axon Clampfit 9.2 and further statistically computed and plotted with Origin 7.5 (OriginLab). Proton-activated Na ϩ current at each pH was computed by subtracting the basal current from the peak current in the presence of protons. The EC 50 of external pH was calculated by fitting the acidactivated current with the Hill equation where I Na is the proton-activated currents, I max is the maximal acidactivated current, pH o stands for the extraoocyte pH, EC 50 represents the value of pH o that results in half of the maximal current of the ENaC channel, and n is the Hill coefficient. Mean open time (MOT) and mean closed time (MCT) were calculated using the equations and where T is the total recording time, e is the event number, and NP o have their usual meanings (22,23). Because the heterologously expressed ENaC in oocytes exhibit multiple channels in each patch, the real baseline at which all of channels are silent is difficult to identify; therefore, the values of MOT and MCT were calculated based on the assumption that the lowest level during the recording period represents the closed level.
All results are presented as mean Ϯ S.E. of amiloride-sensitive currents. Amiloride-sensitive currents are defined as the difference between the total current and the amiloride-resistant current. One-way analysis of variance computation combined with the Bonferroni test was used to analyze data with unequal variance between each group. A probability level of 0.05 was considered significant.

Expression of ENaC mRNA in Human
Epithelia-To examine whether ␦-ENaC is co-expressed with the ␣␤␥-subunits in human epithelial cells at the mRNA level, cells that have previously been shown to have an functional amiloride-sensitive Na ϩ conductance were chosen for RT-PCR. As shown in Fig. 1, a 419-bp product was detected in human lung epithelial cells (H441), human pancreatic epithelial cells (CFPAC), human colonic epithelial cells (Caco-2), and human lung epithelial cells (A549). Sequencing of this product from each cell line indicated that the band corresponded to ␦-ENaC originally cloned from human testis (GI 21752051), encoding a 714-amino acid protein. An additional 325-bp product was also present in A549, CFPAC, and Caco-2 but not H441 cells. Sequencing of this product revealed that it corresponds to a ␦-ENaC isoform (GI34101282) initially cloned from human renal epithelia (9), which encodes a protein of 638-amino acid residues. Expression of ␣and ␥-ENaC was also detected in these four human epithelial cells, whereas the ␤-subunit was only weakly expressed in Caco-2 cells and was absent in CFPAC cells (Fig. 1).
Expression of ␦-ENaC Protein in Lung Epithelial Cells-To detect ␦-ENaC expression at the protein level, H441 monolayers grown on filters were immunostained with a specific anti-␦-ENaC antibody followed by a fluorescein isothiocyanate-conjugated secondary antibody. ␦-ENaC expression was observed (Fig. 2). ␣-, ␤-, and ␥-ENaC expres-sion were also detected in H441 monolayers, in agreement with other studies (2,24). Cells incubated with nonimmune IgG and the secondary antibody did not exhibit immunofluorescence. Differences in fluorescence intensity may reflect variable ENaC subunit expression at the protein level and different affinity among antibodies against ENaC proteins. Similar results were seen in A549 cells. These results suggest that ␦-ENaC is co-expressed with ␣-, ␤-, and ␥-ENaC protein in native human lung epithelial cells.
Co-immunoprecipitation of ␣and ␥-ENaC with ␦-ENaC in H441 and COS-7 Cells-Co-expression of ␦-ENaC with ␣-, ␤-, and ␥-ENaC indicated that these four ENaC subunits may associate with each other to form a multiple subunit channel protein complex. To test this hypothesis, we first detected native ␦-ENaC protein expression in H441 cells. As shown in Fig. 3A and in the right lane in Fig. 3C, ␦-, ␣-, and ␥-ENaC proteins were detected as 85-90 kb for ␦and ␣-ENaC and 150 kb for ␥-ENaC. ␥-ENaC was found to co-immunoprecipitate with ␦and ␣-ENaC (Fig. 3B). Specificity was demonstrated by Western analysis with anti-␥ENaC following pretreatment of the anti-␥ENaC antibody with excess neutralizing peptide (Fig. 3B). In addition, ␦-ENaC also precipitated with ␣-subunit in H441 cells (Fig. 3C) and in COS-7 cells following transient transfection of HA-tagged ␦-ENaC and ␣-ENaC.
Cation Permeability-In this set of experiments, we tested the hypothesis that co-expression of ␦-ENaC alters the ion selectivity of ␣␤␥-ENaC. As shown in Fig. 3A, the inward amiloride-sensitive Na ϩ current was greater than that carried by Li ϩ ions. Much less inward and relative greater outward currents were detected when K ϩ , Cs ϩ , Ca 2ϩ ,    were detected by immunoprecipitation (IP) followed by immunoblotting (IB) with the appropriate antibodies. Five l of serum for ␦-ENaC antibody or 1 g/ml for ␣ and ␥-ENaC antibodies was used for both immunoprecipitation and immunoblotting. B, co-immunoprecipitation of ␥with ␦-ENaC, and ␥with ␣-ENaC. ␥-ENaC was detected by immunoblotting with ␥-ENaC antibody on proteins co-immunoprecipitated with both ␦and ␣-ENaC antibodies. The specificity of ␥-ENaC signal was established by the lack of signal when immunoblotting was performed in the presence of immunogenic peptide (neutralizing peptide,1 g/ml). C, co-immunoprecipitation of ␦with ␣-ENaC. Cell lysates were immunoprecipitated using a specific antibody against ␦-ENaC, and immunoblots of the precipitated proteins were probed with ␣-ENaC antibody. As a control, an aliquot of the H441 cell lysates was also probed with ␣-ENaC antibody without precipitation of ␦-ENaC antibody. These experiments were repeated three times with identical results.
and Mg 2ϩ were used as charge carriers. Meanwhile, the reversal potential shifted leftward from depolarization voltages (10 -20 mV) by ϳ50 -150 mV. Additionally, the resting membrane potentials, which were generally above 0 mV under current clamp configuration in oocytes perfused with Na ϩ (or Li ϩ ), showed hyperpolarization to a variable extent (Fig. 3A).
To further investigate the voltage dependence of amiloride inhibition for ␦␣␤␥-hENaC, the values for K i amil were plotted against the applied membrane potentials (Fig. 5B). The more depolarizing the membrane potential, the greater the value of K i amil . As we have previously described for ␣␤␥and ␦␤␥-ENaC, positively charged amiloride interacts with ENaC in a voltage-dependent manner (21). The K i of amiloride at 0 mV estimated by fitting the voltage-dependent plot with the Woodhill equation was 3.79 Ϯ 0.2 M for ␦␣␤␥-ENaC, whereas it was 13.1 and 0.33 M for ␦␤␥and ␣␤␥-ENaC, respectively (21). The voltage-sensitive fractional distance for amiloride was 0.41. In comparison, it was 0.34 for ␣␤␥-ENaC and 0.48 for ␦␤␥-ENaC, respectively (21).
Unitary Conductance-Because the ion permeability ratio (P Na /P Li ) and apparent equivalent dissociation constant of amiloride for the ␦␣␤␥-ENaC channel are the arithmetic mean of those of ␣␤␥and ␦␤␥-ENaC, the question arose whether oocytes injected with ␦␣␤␥-ENaC subunits express two separate populations of ENaC, that is, ␣␤␥and ␦␤␥-ENaC channels or one population of ␦␣␤␥-channels with novel biophysical characteristics. To address this question, on-cell patches were used to measure single channel conductances (Fig. 6C). Only one unitary Na ϩ or Li ϩ current level was observed (Fig. 5D), and the corresponding slope conductances, respectively, were 8 Ϯ 0.2 pS for Na ϩ and 7.5 Ϯ 0.1 pS for Li ϩ (n ϭ 6). No amiloride-sensitive subconductance was observed.
Self-inhibition-Self-inhibition by extracellular Na ϩ ions is a biophysical hallmark of the ENaC channels (1). To characterize this inherent biophysical property, the self-inhibition time of Na ϩ for ␦␣␤␥-ENaC was examined (Fig. 8). To calculate self-inhibition time, the currents at Ϫ60 mV were digitized while switching bath solutions from low Na ϩ ND-96 medium (1 mM Na ϩ ) to ND-96 (96 mM Na ϩ ) medium. The current fraction from the time point for the first time to switch bath solution (from 1 to 100 mM) to the peak of current and from the peak of current to the time point for the second time to switch bath solution (from 100 to 1 mM) was fitted with the first-order exponential function for estimating a and si , respectively (Fig. 8). Fig. 9 shows that the inward current reached its peak in less than 1 s followed by a run-down in the presence of constant external Na ϩ level. The self-inhibition time ( si ) for ␦␣␤␥-ENaC was 3427 Ϯ 217 ms (n ϭ 13), which was significantly greater than that of ␣␤␥-ENaC (2584 Ϯ 72 ms, n ϭ 47, p Ͻ 0.05) but less than that of ␦␤␥-ENaC (8626 Ϯ 1541 ms, n ϭ 23, p Ͻ 0.01). In contrast, the activation ( a ) was not changed significantly. The ␣␤␥-ENaC current displayed a steep run-down (45% of the peak current in 12.5 s), whereas the ␦␤␥-ENaC current level decreased to a lesser extent (12% of peak current in 12.5 s). These results are consistent with measurements of rat and mouse ␣␤␥-ENaC expressed in oocytes (26,27). Therefore, the corresponding ratio of the peak and sustained currents was much less for ␣␤␥-ENAC when compared with those of ␦␤␥ and ␦␣␤␥-ENaC (p Ͻ 0.01).
Dependence of P NA /P Li Ratio and Unitary Na ϩ Conductance on Injected cRNA Concentrations-To determine the expression level of ␦-ENaC needed to confer the biophysical features of ␣␤␥-ENaC, we measured the P Li /P Na ratios and unitary Na ϩ conductance in oocytes co-injected with various ratios of ␦and ␣␤␥-ENaC cRNAs (Fig. 9). The P Li /P Na ratio (left panel) in oocytes co-injected with an equivalent concentration (1␦:1␣) of ␦and ␣-ENaC cRNAs was significantly lower when compared with that of ␣␤␥-ENaC (0.7 versus 2.0, p Ͻ 0.05). Even with the cRNA ratio of 1␦:10␣, the relative Li ϩ permeability reduced markedly. Meanwhile, the unitary Na ϩ conductance increased in onethird of patches in oocytes expressing 1␦:10␣-ENaC (right panel). A further increase (10␦:1␣) in the ␦-subunit cRNA co-injected elevated the conductance closing to the level of ␦␤␥-ENaC.

DISCUSSION
The aim of the present study was to test the hypothesis that the ␦-ENaC subunit may combine with ␣␤␥-subunits to form a channel complex exhibiting unique biophysical properties. ␦␣␤␥-hENaC heterologously expressed in oocytes was characterized by ion selectivity, amiloride sensitivity, unitary Na ϩ conductance, gating kinetics, proton activation, and self-inhibition that differed from those of both ␣␤␥and ␦␤␥-hENaC. We have provided evidence for the presence of ␦-, ␣-, ␤-, and ␥-ENaC mRNA and protein in human pulmonary epithelial cells (H441 and A549). Our immunocytochemical and co-immunoprecipitation data from H441 cells suggest that the ENaC channel complex in native epithelial cells may be maximally composed of all four mammalian ENaC subunits.
Transcripts for ␣-, ␤-, and/or ␥-ENaC subunits have been detected in untreated and dexamethasone-treated H441 (2, 28 -30) and A549 cells (2,6,24,31) by RT-PCR, Northern blot, and ribonuclease protection assays. Expression of ␣-, ␤-, and ␥-ENaC in human lung epithelial cells reported in the present study agreed with these observations. Furthermore, an amiloride-sensitive channel has been identified in H441 and A549 cells using patch clamp and Ussing chamber techniques by several groups (2,3,6,28,(31)(32)(33). This is the first report of ENaC mRNA expression in Caco-2 cells, although a benzamil-sensitive short circuit current has been reported (34,35). A novel finding of this study was that the ␦-subunit is co-expressed with ␣-, ␤-, and ␥-ENaC in the native human epithelial cells we examined. Consistent with the previous reports of human ␣and ␦-ENaC transcripts in pancreatic cells (9, 10), ␣and ␦-ENaC transcripts were detected in CFPAC cells. However, a functional amiloride-sensitive conductance has not been successfully detected in rat, bovine, or human pancreatic duct cells (36 -38). Recently, Mall et al. (39) reported that ␤rather than ␣-(␦-) ENaC is a rate-limiting element for salt absorption via airway epithelia. Absence of the essential ␤-subunit and weakly expression of the splicing variant ␦-ENaC in CFPAC and Coca-2 cells may cause undetectable electrical activity (Fig. 1) (36). The possible physiologic function of ENaC, therefore, in pancreatic epithelial cells remains obscure.  Oocytes were held at 0 mV and stepped to Ϫ60 mV while acidic bath solutions were applied. B, dose response curve for protonactivation. The value of EC 50 for proton activation is pH 6.5 Ϯ 0.1, n ϭ 8, which is greater than that of ␦␤␥-hENaC (pH 6.1, p Ͻ 0.05) (25).
To address the question whether Ca 2ϩ and Mg 2ϩ can be transported by ENaC, we substituted Na ϩ with equamolar Ca 2ϩ or Mg 2ϩ in the bath solution. Surprisingly, amiloride-sensitive Ca 2ϩ and Mg 2ϩ currents at hyperpolarization potentials were even greater than that carried by K ϩ . Our observations suggest that ENaC is permeable to divalent cations. The physiological impact of Ca 2ϩ and Mg 2ϩ permeability is unknown because the normal extracellular concentration for Ca 2ϩ and Mg 2ϩ is a few micromoles. Assuming that Ca 2ϩ and Mg 2ϩ competitively bind to the channel pore, the driving force would be too weak for Ca 2ϩ and Mg 2ϩ under physiologic conditions in which sodium is the predominant salt.
showed a mixture of those of ␣␤␥and ␦␤␥-ENaC channels. Based on the assumption that there are two separate ENaC channels with distinguishable intrinsic features, two unitary Na ϩ conductances should be detected in single channel experiments. However, only one Na ϩ conductance of 8 pS, which is the arithmetic mean of ␣␤␥and ␦␤␥-ENaC, was recorded. Moreover, analysis of the gating kinetics should demonstrate that the MOT value should be an average of ␣␤␥or ␦␤␥-ENaC or should not differ from either one. However, the MOT value of ␦␣␤␥-ENaC is markedly less than both.
It is unlikely that either ␣␤␥or ␦␤␥-ENaC is predominately expressed in oocytes due to the competitive exclusion between the two conducting subunits, ␣and ␦-ENaC. In addition to ion selectivity and amiloride sensitivity, the EC 50 of the external pH for proton activation differs from that of ␦␤␥-ENaC (25). ␣␤␥-ENaC expressed in oocytes is not activated by extraoocyte acidosis (25). Furthermore, the self-inhibition experiments are not supportive of either ␣␤␥or ␦␤␥-ENaC channels being expressed. The self-inhibition time and ratio of the sustained and peak currents are significantly greater than those of ␣␤␥but less than those of ␦␤␥-ENaC.
Heterologously expressed ␦␣␤␥-ENaC has a unitary Na ϩ conductance differing from those expressed in A549 cells (8 pS versus 4 and 20 pS) (6). The single channel Li ϩ conductance of ␦␣␤␥-ENaC diverges from that in H441 cells (7 versus 11 pS) (2). It is not surprising to see differences in the biophysical characteristics between heterologously expressed ␦␣␤␥-ENaC and the native amiloride-sensitive Na ϩ conductances in H441 and A549 cells. H441 cells only express a splicing variant of ␦-ENaC (Fig. 1), the biophysical properties of which have not been characterized, and may contribute to these differences. Although we did not examine changes in unitary Li ϩ conductance in oocytes expressing various ratios of variant ␦and ␣-ENaC, channels composed of the splicing variant may have a similar Li ϩ conductance to that in H441 cells. On the other hand, culture conditions influence the biophysical properties and expression level of individual ENaC subunits (16,18,40). As our results show, the expression ratio between ENaC subunits governs the single channel features (Fig. 9). In addition, unknown ENaC subunits that have not been cloned or the splicing variants of ␦-, ␣-, ␤-, ␥-ENaC subunits as well as other amiloride-sensitive cation channels expressed in epithelial cells, for example, cyclic nucleotide-gated Na ϩ channels, may contribute to the discrepancy between cloned ENaC and native epithelial Na ϩ channels. Even for heterologously expressed ␣␤␥-ENaC, the absence of cyclic AMP sensitivity and the differences in their whole-cell and single channel properties, when compared with the variety of amiloride-sensitive Na ϩ channels expressed in number of native epithelia, are still unsolved questions. Therefore, ␦-ENaC co-expression may contribute at least in part to the diverse properties of native epithe-lial Na ϩ channels. Genetic manipulation of ␦-ENaC expression, such as small interfering RNA, is required to further verify the contribution of ␦-ENaC to native Na ϩ transport across epithelia.
Our immunocytochemical results combined with the RT-PCR data demonstrate that ␦-ENaC is co-expressed with ␣-, ␤-, and ␥-ENaC in human pulmonary epithelial cells (Figs. 1-3). The question raised from these results is what level of ␦-ENaC expression is required to alter the biophysical properties of ␣␤␥-ENaC? Our results from heterologous co-expression of ENaC subunits in oocytes supported the notion that only a small amount of ␦may significantly impact on ␣␤␥-ENaC properties. Existence of two subpopulations in oocytes co-injected 1␦:10␣ ENaC cRNAs indicated that they may have variable subunit composition. At present, the stoichiometry of ␦␣␤␥-ENaC is unknown.