Dopamine activates amiloride-sensitive sodium channels in alveolar type I cells in lung slice preparations

Active Na+ reabsorption by alveolar epithelial cells generates the driving force used to clear fluids from the air space. Using single-channel methods, we examined epithelial Na+ channel (ENaC) activity of alveolar type I (AT1) cells from live 250- to 300-microm sections of lung tissue, circumventing concerns that protracted cell isolation procedures might compromise the innate transport properties of native lung cells. We used fluorescein-labeled Erythrina crystagalli lectin to positively identify AT1 cells for single-channel patch-clamp analysis. We demonstrated, for the first time, single-channel recordings of highly selective and nonselective amiloride-sensitive ENaC channels (HSC and NSC, respectively) from AT1 cells in situ, with mean conductances of 8.2+/-2.5 and 22+/-3.2 pS, respectively. Additionally, 25 nM amiloride in the patch electrode blocked Na+ channel activity in AT1 cells. Immunohistochemical studies demonstrated the presence of dopamine D1 and D2 receptors on the surface of AT1 cells, and single-channel recordings showed that 10 microM dopamine increased Na+ channel activity [product of the number of channels and single-channel open probability (NPo)] from 0.31+/-0.19 to 0.60+/-0.21 (P<0.001). The D1 receptor antagonist SCH-23390 (10 microM) blocked the stimulatory effect of dopamine on AT1 cells, but the D2 receptor antagonist sulpiride did not.     

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.     

AICAR decreases the activity of two distinct amiloride-sensitive Na+-permeable channels in H441 human lung epithelial cell monolayers

Transepithelial transport of Na(+) across the lung epithelium via amiloride-sensitive Na(+) channels (ENaC) regulates fluid volume in the lung lumen. Activators of AMP-activated protein kinase (AMPK), the adenosine monophosphate mimetic AICAR, and the biguanide metformin decreased amiloride-sensitive apical Na(+) conductance (G(Na+)) in human H441 airway epithelial cell monolayers. Cell-attached patch-clamp recordings identified two distinct constitutively active cation channels in the apical membrane that were likely to contribute to G(Na+): a 5-pS highly Na(+) selective ENaC-like channel (HSC) and an 18-pS nonselective cation channel (NSC). Substituting NaCl with NMDG-Cl in the patch pipette solution shifted the reversal potentials of HSC and NSC, respectively, from +23 mV to -38 mV and 0 mV to -35 mV. Amiloride at 1 microM inhibited HSC activity and 56% of short-circuit current (I(sc)), whereas 10 microM amiloride partially reduced NSC activity and inhibited a further 30% of I(sc). Neither conductance was associated with CNG channels as there was no effect of 10 microM pimoside on I(sc), HSC, or NSC activity, and 8-bromo-cGMP (0.3-0.1 mM) did not induce or increase HSC or NSC activity. Pretreatment of H441 monolayers with 2 mM AICAR inhibited HSC/NSC activity by 90%, and this effect was reversed by the AMPK inhibitor Compound C. All three ENaC proteins were identified in the apical membrane of H441 monolayers, but no change in their abundance was detected after treatment with AICAR. In conclusion, activation of AMPK with AICAR in H441 cell monolayers is associated with inhibition of two distinct amiloride-sensitive Na(+)-permeable channels by a mechanism that likely reduces channel open probability.     

Characterization of a novel splice variant of δ ENaC subunit in human lungs

Salt absorption via apical epithelial sodium channels (ENaC) is a critical rate-limiting process in maintaining airway and lung lining fluid at the physiological level. δ ENaC (termed δ1 in this article) has been detected in human lung epithelial cells in addition to α, β, and γ subunits (Ji HL, Su XF, Kedar S, Li J, Barbry P, Smith PR, Matalon S, Benos DJ. J Biol Chem 281: 8233-8241, 2006; Nie HG, Chen L, Han DY, Li J, Song WF, Wei SP, Fang XH, Gu X, Matalon S, Ji HL, J Physiol 587: 2663-2676, 2009) and may contribute to the differences in the biophysical properties of amiloride-inhibitable cation channels in pulmonary epithelial cells. Here we cloned a splicing variant of the δ1 ENaC, namely, δ2 ENaC in human bronchoalveolar epithelial cells (16HBEo). δ2 ENaC possesses 66 extra amino acids attached to the distal amino terminal tail of the δ1 ENaC. δ2 ENaC was expressed in both alveolar type I and II cells of human lungs as revealed by in situ hybridization and real-time RT-PCR. To characterize the biophysical and pharmacological features of the splicing variant, we injected Xenopus oocytes with human ENaC cRNAs and measured whole cell and single channel currents of δ1βγ, δ2βγ, and αβγ channels. Oocytes injected with δ2βγ cRNAs exhibited whole cell currents significantly greater than those expressing δ1βγ and αβγ channels. Single channel activity, unitary conductance, and open probability of δ2βγ channels were significantly greater compared with δ1βγ and αβγ channels. In addition, δ2βγ and δ1βγ channels displayed significant differences in apparent Na(+) affinity, dissociation constant for amiloride (K(i)(amil)), the EC(50) for capsazepine activation, and gating kinetics by protons. Channels comprising of this novel splice variant may contribute to the diversities of native epithelial Na(+) channels.     

Single-channel recordings from two types of amiloride-sensitive epithelial Na+ channels

We report here the first evidence in intact epithelial cells of unit conductance events from amiloride-sensitive Na+ channels. The events were observed when patch-clamp recordings were made from the apical surface of cultured epithelial kidney cells (A6). Two types of channels were observed: one with a high selectivity to Na+ and one with relatively low selectivity. The characteristics of the low-selectivity channel are as follows: single-channel conductance ranged between 7 and 10 pS (mean = 8.4 +/- 1.3), the current-voltage (I-V) relationship displayed little if any nonlinearity over a range of +/- 80 mV (with respect to the patch pipette) and the channel Na+/K+ selectivity was approximately 3-4:1. Amiloride, a cationic blocker of the channel, reduced channel mean open time and increased channel mean closed times as the voltage of the cell interior was made more negative. Amiloride induced channel flickering at increased negative potentials (intracellular potential with respect to the patch) but did not alter the single-channel conductance or the I-V relationship from that observed in control patches. The characteristics of the high-selectivity channel are: a single-channel conductance of 1-3 pS (mean = 2.8 +/- 1.2), the current-voltage relationship is markedly nonlinear with a Na+/K+ selectivity greater than 20:1. The mean open and closed times for the two types of channels are quite different, the high-selectivity channel being open only about 10% of the time while the low-selectivity channel is open about 30% of the time.     

Characteristics of Kcnn4 channels in the apical membranes of an intestinal epithelial cell line

Intermediate-conductance K(+) (Kcnn4) channels in the apical and basolateral membranes of epithelial cells play important roles in agonist-induced fluid secretion in intestine and colon. Basolateral Kcnn4 channels have been well characterized in situ using patch-clamp methods, but the investigation of Kcnn4 channels in apical membranes in situ has been hampered by a layer of mucus that prevents seal formation. In the present study, we used patch-clamp methods to characterize Kcnn4 channels in the apical membrane of IEC-18 cells, a cell line derived from rat small intestine. A monolayer of IEC-18 cells grown on a permeable support is devoid of mucus, and tight junctions enable selective access to the apical membrane. In inside-out patches, Ca(2+)-dependent K(+) channels observed with iberiotoxin (a Kcnma1/large-conductance, Ca(2+)-activated K(+) channel blocker) and apamin (a Kcnn1-3/small-conductance, Ca(2+)-activated K(+) channel blocker) present in the pipette solution exhibited a single-channel conductance of 31 pS with inward rectification. The currents were reversibly blocked by TRAM-34 (a Kcnn4 blocker) with an IC(50) of 8.7 ± 2.0 μM. The channels were not observed when charybdotoxin, a peptide inhibitor of Kcnn4 channels, was added to the pipette solution. TRAM-34 was less potent in inhibiting Kcnn4 channels in patches from apical membranes than in patches from basolateral membranes, which was consistent with a preferential expression of Kcnn4c and Kcnn4b isoforms in apical and basolateral membranes, respectively. The expression of both isoforms in IEC-18 cells was confirmed by RT-PCR and Western blot analyses. This is the first characterization of Kcnn4 channels in the apical membrane of intestinal epithelial cells.     

K(+) channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells

Active Na(+) absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and K(ATP) K(+) channel activities exerts sustained control in Na(+) transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the α-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K(+) channels, and 2) to determine the physiological impact of K(+) channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and K(ATP) channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24h) increased α-ENaC expression, similarly to K(ATP) activation by pinacidil. Conversely, pharmacological KvLQT1 and K(ATP) inhibition or silencing with siRNAs down-regulated α-ENaC expression. Furthermore, K(+) channel blockers significantly decreased α-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and K(ATP) activation dose-dependently enhanced α-ENaC promoter activity. Finally, we noted a physiological impact of changes in K(+) channel functions on ERK activity, α-, β-, γ-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K(+) channels regulate α-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.     

Interaction of syntaxins with the amiloride-sensitive epithelial sodium channel.

Amiloride-sensitive sodium channels mediate sodium entry across the apical membrane of epithelial cells in variety of tissues. The rate of Na(+) entry is controlled by the regulation of the epithelial sodium channel (ENaC) complex. Insertion/retrieval of the ENaC complex into the apical membrane as well as direct kinetic effects at the single channel level are recognized mechanisms of regulation. Recent data suggest that the syntaxin family of targeting proteins interact with and functionally regulate a number of ion channels and pumps. To evaluate the role of these proteins in regulating ENaC activity, we co-expressed rat ENaC cRNA (alpha, beta, gamma subunits) with syntaxin 1A or 3 cRNAs in Xenopus oocytes. Basal ENaC currents were inhibited by syntaxin 1A and stimulated by syntaxin 3. Both syntaxin 1A and syntaxin 3 could be co-immunoprecipitated with ENaC subunit proteins, suggesting physical interaction. Interestingly, immunofluorescence data suggest that with either syntaxin isoform the ENaC-associated epifluorescence on the oocyte surface is enhanced. These data indicate that (i) both syntaxin isoforms increase the net externalization of the ENaC channel complex, (ii) that the functional regulation is isoform specific, and (iii) suggest that ENaC may be regulated through mechanisms involving protein-protein interactions.     

Serine protease activation of near-silent epithelial Na+ channels

The regulation of epithelial Na+ channel (ENaC) function is critical for normal salt and water balance. This regulation is achieved through cell surface insertion/retrieval of channels, by changes in channel open probability (Po), or through a combination of these processes. Epithelium-derived serine proteases, including channel activating protease (CAP) and prostasin, regulate epithelial Na+ transport, but the molecular mechanism is unknown. We tested the hypothesis that extracellular serine proteases activate a near-silent ENaC population resident in the plasma membrane. Single-channel events were recorded in outside-out patches from fibroblasts (NIH/3T3) stably expressing rat alpha-, beta-, and gamma-subunits (rENaC), before and during exposure to trypsin, a serine protease homologous to CAP and prostasin. Under baseline conditions, near-silent patches were defined as having rENaC activity (NPo) < 0.03, where N is the number of channels. Within 1-5 min of 3 microg/ml bath trypsin superfusion, NPo increased approximately 66-fold (n = 7). In patches observed to contain a single functional channel, trypsin increased Po from 0.02 +/- 0.01 to 0.57 +/- 0.03 (n = 3, mean +/- SE), resulting from the combination of an increased channel open time and decreased channel closed time. Catalytic activity was required for activation of near-silent ENaC. Channel conductance and the Na+/Li+ current ratio with trypsin were similar to control values. Modulation of ENaC Po by endogenous epithelial serine proteases is a potentially important regulator of epithelial Na+ transport, distinct from the regulation achieved by hormone-induced plasma membrane insertion of channels.     

A new subunit of the epithelial Na+ channel identifies regions involved in Na+ self-inhibition

The epithelial Na+ channel (ENaC) is the apical entry pathway for Na+ in many Na+-reabsorbing epithelia. ENaC is a heterotetrameric protein composed of homologous alpha, beta, and gamma subunits. Mutations in ENaC cause severe hypertension or salt wasting in humans; and consequently, ENaC activity is tightly controlled. According to the concept of Na+ self-inhibition, the extracellular Na+ ion itself can reduce ENaC activity. The molecular basis for Na+ self-inhibition is unknown. Here, we describe cloning of a new ENaC subunit from Xenopus laevis (epsilonxENaC). epsilonxENaC can replace alphaxENaC and formed functional, highly selective, amiloride-sensitive Na+ channels when coexpressed with betaxENaC and gammaxENaC. Channels containing epsilonxENaC showed strong inhibition by extracellular Na+. This Na+ self-inhibition was significantly slower than for alphaxENaC-containing channels. Using site-directed mutagenesis, we show that the proximal part of the large extracellular domain controls the speed of self-inhibition. This suggests that this region is involved in conformational changes during Na+ self-inhibition.     

Distinct pools of epithelial sodium channels are expressed at the plasma membrane

The epithelial Na+ channel (ENaC) is assembled in the endoplasmic reticulum from three structurally related subunits (alpha, beta, and gamma). Channel maturation within the biosynthetic pathway involves cleavage of the alpha and gamma subunits by furin and processing of N-linked glycans on alpha, beta, and gamma to complex type. Both mature and immature subunits have been observed at the surface of stably transfected Madin-Darby canine kidney cells. We have examined whether channel maturation is an all-or-none event or whether heterogeneous processing of channel subunits occurs within an individual channel complex. Using an immobilized lectin to isolate proteins with complex type N-glycans, we found that individual channel complexes with mature subunits lack immature subunits. Furthermore, terminal processing of N-glycans on ENaC subunits was not dependent on cleavage of ENaC subunits, and proteolysis of channel subunits was not dependent on prior processing of N-glycans. Our results suggest that processing of subunits within an individual channel complex is an all-or-none event such that channels present on the cell surface contain either all mature or all immature subunits. The presence of immature channel complexes at the plasma membrane provides epithelial cells with a reserve of poorly functional channels that can be activated by proteases in post-Golgi compartments.     

Influenza virus inhibits ENaC and lung fluid clearance

Fluid-free alveolar space is critical for normal gas exchange. Influenza virus alters fluid transport across respiratory epithelia producing rhinorrhea, middle ear effusions, and alveolar flooding. However, the mechanism of fluid retention remains unclear. We investigated influenza virus strain A/PR/8/34, which can attach and enter mammalian cells but is incapable of viral replication and productive infection in mammalian epithelia, on epithelial sodium channels (ENaC) in rat alveolar type II (ATII) cells. In parallel, we determined the effects of virus on amiloride-sensitive (i.e., ENaC-mediated) fluid clearance in rat lungs in vivo. Although influenza virus did not change the inulin permeability of ATII monolayers, it rapidly reduced the net volume transport across monolayers. Virus reduced the open probability of single ENaC channels in apical cell-attached patches. U-73122, a phospholipase C (PLC) inhibitor, and PP2, a Src inhibitor, blocked the effect of virus on ENaC. GF-109203X, a protein kinase C (PKC) inhibitor, also blocked the effect, suggesting a PKC-mediated mechanism. In parallel, intratracheal administration of influenza virus produced a rapid inhibition of amiloride-sensitive (i.e., ENaC-dependent) lung fluid transport. Together, these results show that influenza virus rapidly inhibits ENaC in ATII cells via a PLC- and Src-mediated activation of PKC but does not increase epithelial permeability in this same rapid time course. We speculate that this rapid inhibition of ENaC and formation of edema when the virus first attaches to the alveolar epithelium might facilitate subsequent influenza infection and may exacerbate influenza-mediated alveolar flooding that can lead to acute respiratory failure and death.     

cAMP increases density of ENaC subunits in the apical membrane of MDCK cells in direct proportion to amiloride-sensitive Na(+) transport

Antidiuretic hormone and/or cAMP increase Na(+) transport in the rat renal collecting duct and similar epithelia, including Madin-Darby canine kidney (MDCK) cell monolayers grown in culture. This study was undertaken to determine if that increment in Na(+) transport could be explained quantitatively by an increased density of ENaC Na(+) channels in the apical membrane. MDCK cells with no endogenous ENaC expression were retrovirally transfected with rat alpha-, beta-, and gammaENaC subunits, each of which were labeled with the FLAG epitope in their extracellular loop as described previously (Firsov, D., L. Schild, I. Gautschi, A.-M. Mérillat, E. Schneeberger, and B.C. Rossier. 1996. PROC: Natl. Acad. Sci. USA. 93:15370-15375). The density of ENaC subunits was quantified by specific binding of (125)I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a saturable function of M2 concentration with half-maximal binding at 4-8 nM. Transepithelial Na(+) transport was measured as the amiloride-sensitive short-circuit current (AS-I(sc)) across MDCK cells grown on permeable supports. Specific M2 binding was positively correlated with AS-I(sc) measured in the same experiments. Stimulation with cAMP (20 microM 8-p-chlorothio-cAMP plus 200 microM IBMX) significantly increased AS-I(sc) from 11.2 +/- 1.3 to 18.1 +/- 1.3 microA/cm(2). M2 binding (at 1.7 nM M2) increased in direct proportion to AS-I(sc) from 0.62 +/- 0.13 to 1.16 +/- 0.18 fmol/cm(2). Based on the concentration dependence of M2 binding, the quantity of Na(+) channels per unit of AS-I(sc) was calculated to be the same in the presence and absence of cAMP, 0.23 +/- 0.04 and 0.21 +/-0.05 fmol/microA, respectively. These values would be consistent with a single channel conductance of approximately 5 pS (typically reported for ENaC channels) only if the open probability is <0.02, i.e., less than one-tenth of the typical value. We interpret the proportional increases in binding and AS-I(sc) to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the I(sc) increase produced by cAMP.     

The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel

The epithelial sodium channel (ENaC) is ubiquitinated by the E3 ligase Nedd4-2 at the apical membranes of polarized cortical collecting duct (CCD) epithelial cells. This leads to ENaC endocytosis and possible degradation. Because ENaC is known to recycle at the apical membranes of CCD cells, deubiquitinating enzymes (DUBs) are likely involved in regulating ENaC surface density by facilitating ENaC recycling as opposed to degradation. Using a chemical probe approach to tag active DUBs, we identified ubiquitin C-terminal hydrolase (UCH) isoform L3 as the predominant DUB in endosomal compartments of CCD cells. Blocking UCH-L3 activity or reducing its expression by selective knockdown increased ENaC ubiquitination and resulted in its removal from the apical membranes of CCD cells. Functionally this caused a rapid reduction in transepithelial Na(+) currents across the CCD epithelia. Surface biotinylation demonstrated the loss of ENaC from the apical surface when UCH-L3 was inhibited. Whole cell or apical surface immunoprecipitation demonstrated increased ENaC ubiquitination with UCH-L3 inhibition. This constitutes a novel function for UCH in epithelia and in the regulation of ion channels and demonstrates the dynamic regulation of apically located ENaC by recycling, which is facilitated by this DUB.     

Potassium channel activity recorded from the apical membrane of freshly isolated epithelial cells in rat caudal epididymis.

K+ channels were recorded in excised, inside-out patches from the apical membrane of the freshly isolated tubule of the caudal portion of the rat epididymis. With asymmetric K+ concentrations in bath and pipette (140 mM K+in/6 mM K+out), the channels had a slope conductance of 54.2 pS at 0 mV. The relative permeability of K+ over Na+ was about 171 to 1. The channels were activated by intracellular Ca2+ and by membrane depolarization. These channels belong to a class defined as "intermediate-conductance Ca2+-activated K+ channel. " External tetraethylammonium ions (TEA+) caused a flickery block of the channel with reduction in single-channel current amplitude measured at a range of holding membrane potentials (-40 to 60 mV). Activity of the K+ channels was inhibited by intracellular ATP (KD =1.188 mM). The channel activity was detected only occasionally in patches from the apical membrane (about 1 in 17 patches containing active channels). The presence of the intermediate-conductance Ca2+-activated K+ channels indicates that they could provide a route for K+ secretion in a Ca2+-dependent process responsible for a high luminal K+ concentration found in the epididymal duct of the rat.     

Mechanisms of increased Na(+) transport in ATII cells by cAMP: we agree to disagree and do more experiments

Existing evidence supports the presence of active transport of Na(+) across the mammalian alveolar epithelium and its upregulation by agents that increase cytoplasmic cAMP levels. However, there is controversy regarding the mechanisms responsible for this upregulation. Herein we present the results of various patch-clamp studies indicating the presence of 25- to 27-pS, amiloride-sensitive, moderately selective Na(+) channels (Na(+)-to-K(+) permeability ratio = 7:1) located on the apical membranes of rat alveolar type II (ATII) cells maintained in primary culture. The addition of terbutaline to the bath solution increased the open probability of single channels present in cell-attached patches of ATII cells without affecting their conductance. A similar increase in open probability was seen after the addition of protein kinase A, ATP, and Mg(2+) to the cytoplasmic side of inside-out patches. Measurement of short-circuit currents across confluent monolayers of rat or rabbit ATII cells indicates that terbutaline and 8-(4-chlorophenylthio)-cAMP increase vectorial Na(+) transport and activate Cl(-) channels. Currently, there is a controversy as to whether the cAMP-induced increase in Na(+) transport is due solely to hyperpolarization of the cytoplasmic side of the ATII cell membrane due to Cl(-) influx or whether it results from simultaneous stimulation of both Cl(-) and Na(+) conductive pathways. Additional studies are needed to resolve this issue.     

The epithelial sodium channel δ-subunit: new notes for an old song

Amiloride-sensitive epithelial Na(+) channels (ENaCs) can be formed by different combinations of four homologous subunits, named α, β, γ, and δ. In addition to providing an apical entry pathway for transepithelial Na(+) reabsorption in tight epithelia such as the kidney distal tubule and collecting duct, ENaCs are also expressed in nonepithelial cells, where they may play different functional roles. The δ-subunit of ENaC was originally identified in humans and is able to form amiloride-sensitive Na(+) channels alone or in combination with β and γ, generally resembling the canonical kidney ENaC formed by α, β, and γ. However, δ differs from α in its tissue distribution and channel properties. Despite the low sequence conservation between α and δ (37% identity), their similar functional characteristics provide an excellent model for exploring structural correlates of specific ENaC biophysical and pharmacological properties. Moreover, the study of cellular mechanisms modulating the activity of different ENaC subunit combinations provides an opportunity to gain insight into the regulation of the channel. In this review, we examine the evolution of ENaC genes, channel subunit composition, the distinct functional and pharmacological features that δ confers to ENaC, and how this can be exploited to better understand this ion channel. Finally, we briefly consider possible functional roles of the ENaC δ-subunit.     

Biophysical properties and molecular characterization of amiloride-sensitive sodium channels in A549 cells

Amiloride-sensitive Na(+) channels, present in fetal and adult alveolar epithelial type II (ATII) cells, play a critical role in the reabsorption of fetal fluid shortly after birth and in limiting the extent of alveolar edema across the adult lung. Because of the difficulty in isolating and culturing ATII cells, there is considerable interest in characterizing the properties of ion channels and their response to injury of ATII cell-like cell lines such as A549 that derive from a human alveolar cell carcinoma. A549 cells were shown to contain alpha-, beta-, and gamma-epithelial Na(+) channel mRNAs. In the whole cell mode of the patch-clamp technique (bath, 145 mM Na(+); pipette, 145 mM K(+)), A549 cells exhibited inward Na(+) currents reversibly inhibited by amiloride, with an inhibition constant of 0.83 microM. Ion substitution studies showed that these channels were moderately selective for Na(+) (Na(+)-to-K(+) permeability ratio = 6:1). Inward Na(+) currents were activated by forskolin (10 microM) and inhibited by nitric oxide (300 nM) and cGMP. Recordings in cell-attached mode revealed the presence of an amiloride-sensitive Na(+) channel with a unitary conductance of 8.6 +/- 0.04 (SE) pS. Channel activity was increased by forskolin and decreased by nitric oxide and the cGMP analog 8-bromo-cGMP. These data demonstrate that A549 cells contain amiloride-sensitive Na(+) channels with biophysical properties similar to those of ATII cells.