Ion channels of the lung and their role in disease pathogenesis

Abstract

Maintenance of normal epithelial ion and water transport in the lungs includes providing a thin layer of surface liquid that coats the conducting airways. This airway surface liquid is critical for normal lung function in a number of ways but, perhaps most importantly, is required for normal mucociliary clearance and bacterial removal. Preservation of the appropriate level of hydration, pH, and viscosity for the airway surface liquid requires the proper regulation and function of a battery of different types of ion channels and transporters. Here we discuss how alterations in ion channel/transporter function often lead to lung pathologies.

a thin fluid layer that forms a continuous barrier between the organism and external environment covers the lining of the lung. Although airway epithelia consist of various cell types with different morphologies and functions, one critical function is to control the volume, pH, and viscosity of this fluid layer to maintain normal lung function. This process requires that the airway epithelial cells must somehow govern the coordinated transport of solutes, ions, and water at their basal and apical surfaces. This organized transport is achieved by equipping epithelial cells with a variety of membrane solute, ion, and water transporters, differentially distributed along the apico-basal axis, among which sodium, chloride, and potassium channels/transporters play a critical role in maintaining lung homeostasis (Fig. 1). The functional significance of these channels is highlighted by realization that impairment of epithelial ion transport processes that alter the fluid composition/content are associated with a number of human diseases/pathologies, including cystic fibrosis (CF) (129), chronic bronchitis (22), chronic obstructive pulmonary disease (COPD) (249), and pulmonary edema (160). In this review, we focus on recent developments in understanding how ion channels maintain normal lung homeostasis.

Fig. 1.

Ion channels and transporters that participate in regulation of lung periciliary liquid (PCL) homeostasis and mucociliary clearance by ciliated airway epithelia. Apical membrane sodium reabsorption, accompanied by chloride secretion along with passive H₂O transport, provides the main mechanism responsible for PCL composition. The sodium reabsorption is mediated by coordinated function of epithelial sodium channel (ENaC, apical) and Na⁺-K⁺-ATPase (basal). However, apical cyclic nucleotide-gated channels (CNG) may contribute to sodium uptake as well. Furthermore, basal membrane located. CaCC, Ca²⁺-activated chloride channels; CFTR, cystic fibrosis transmembrane conductance regulator; TMEM, transmembrane domain; BK_Ca, large-conductance Ca²⁺-activated, voltage-dependent potassium channel; SK4, small-conductance Ca²⁺-activated potassium channels; NHE, Na⁺/H⁺ exchanger; NKCC, Na⁺-K⁺-Cl⁻ cotransporter; AE, anion exchanger; BORC, basolateral outward rectifying channel; BIRC, basolateral inward rectifying channel; BCFTR, basolateral CFTR-like channel; Kv7.1, α-subunit of a voltage-dependent potassium channel; K_Ca3.1, Ca²⁺-activated potassium channel.

The majority of upper airway epithelial cells are cuboidal cells with cilia (219). These ciliated epithelial cells coordinate the beating of their cilia and govern ion transfer, and the efficiencies and fidelity of these two processes are necessary for proper mucociliary clearance (MCC). Efficient MCC requires that the airway surface liquid (ASL), which is composed of the mucous layer above and the periciliary liquid (PCL) below, is appropriately hydrated and of the right viscosity (mucus layer) to allow for the cilia to beat properly and move the mucus layer at an appropriate rate (142). MCC is a part of the innate immune system that is responsible for trapping and clearing the airways from inhaled pathogens and other noxious particles (142). Although the trapped pathogens and particles are transported within the mucus layer, the composition and thickness, pH, and viscosity of the PCL determines the optimal ciliary beat and thus the effectiveness of mucociliary clearance (156, 158, 217). The PCL characteristics are dependent on the active coordination between the cystic fibrosis transmembrane conductance regulator (CFTR), Ca²⁺-activated chloride channels (CaCC), chloride channel (CLC) 2, and the epithelial sodium channel (ENaC) (246). The fluid follows passively and results in the osmotic gradient that moves fluid from the airspace to interstitium [alveolar fluid clearance (AFC)] (156), and this regulates the ASL height (43, 217).

The apical cyclic nucleotide-gated channels (CNG) may support ENaC function, whereas CLC2 chloride channel and CaCC, including the transmembrane domain 16a (TMEM16) channels, contribute to apical chloride secretion (79, 191). Chloride secretion is sustained by the basolateral Na⁺-K⁺-2Cl⁻ cotransporter and $\mathrm{HC}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$/Cl⁻ anion exchanger, whereas basolateral Na⁺/H⁺ exchanger (NHE) maintains intracellular pH (74). Additionally, the basolateral membrane contains chloride channels [basolateral outward rectifying channel (BORC), basolateral inward rectifying channel (BIRC), and basolateral CFTR-like channel (BCFTR)] that along with basolateral- and apical membrane-located potassium channels {basolateral α-subunit of a voltage-dependent potassium channel (Kv7.1) to K7.5 and Ca²⁺-dependent potassium channels [small-conductance Ca²⁺-activated potassium channels (SK4), large-conductance Ca²⁺-activated, voltage-dependent potassium channel (BK_Ca), and Ca²⁺-activated potassium channel (K_Ca3.1)]} may modulate apical chloride secretion (9, 42, 68, 103, 139).

Finally, the epithelial cells that form the alveolar epithelium (AE) are crucial for efficient gas exchange and are separated from the gas phase by a thin layer of alveolar lining fluid (ALF). The amount, volume, and composition of ALF are crucial for gas diffusion, and therefore the electrolyte composition of ALF is strictly controlled by AE-mediated ion transport. For example, amiloride inhibition of sodium reabsorption in AE results in a significant reduction of basal fluid clearance in human lungs (159, 160). The AE consist of two types of epithelial cells, alveolar type I (ATI) and alveolar type II (ATII) cells, that mediate vectorial ion processes (49, 58, 239) despite their differences in function and morphology (49, 70). Both ATI and ATII express ENaC, amiloride-insensitive CNG cation channels (239), potassium channels, CFTR, γ-aminobutyric acid type A (GABA_A) channels, and voltage-gated chloride channels (CLC2 and CLC5) (103). Furthermore, ATI cells express aquaporin 5 (AQP5) and exhibit the highest water permeability of any mammalian cell type (58). As in ciliated epithelial cells, fluid removal out of alveoli in the interstitium uses a transepithelial osmotic gradient created in AE cells by sodium uptake through the coordinated action of apical ENaC (117, 155, 172), CNG cation channels (239), and the basal ouabain-sensitive Na⁺-K⁺-ATPase (197). However, the mechanism for chloride and potassium transport by AE cells is less clear (170). AT II cells are also important for enriching the ALF with surfactant (83, 105, 199, 225) and immunoreactive proteins (27, 48, 128, 211) which facilitate innate immune responses.

Role of Membrane Potential

The membrane potential of the apical membrane (V_a) is governed by its major conductances, which are the apical sodium and chloride conductances, whereas the basolateral membrane potential (V_b) is largely governed by its potassium conductance. The primary ion transport function of lung epithelia is sodium absorption across apical sodium channels driven by the basolaterally located Na⁺-K⁺-ATPase. Recycling of potassium across the basolateral membrane results in a hyperpolarization of the cell, and chloride distributes passively across the apical membrane according to its electrochemical driving forces. Although there are a number of other ion channels expressed in lung epithelium, including the apical hydrogen ion channel, their contributions to the membrane potentials can be safely neglected by the high sodium and chloride conductances (67, 155).

The differential expression of the major ion conductances in the two membranes and the selective ion gradients toward the mucosal and the serosal compartment result in distinct electromotive forces across the two membranes. However, the difference in the membrane potentials of the apical and basolateral membrane is solely caused by the junctional resistance, R_j, where the voltage difference between V_a and V_b drops. Thus, the tightness of the epithelial monolayer determines the depolarization of V_a with respect to V_b. In leaky epithelia, where R_j is small compared with R_a + R_b, there is little difference between V_a and V_b, and as a result the transepithelial potential, V_t, is small (67).

In alveolar cells, in comparison, membrane potentials have been little studied because of their complicated anatomy in vivo and the lack of good cell culture models for alveolar epithelia. Stimulation of type II monolayers with a cAMP agonist activates both apical CFTR chloride channels and ENaC sodium channels and results in a transepithelial hyperpolarization. Indeed, Lazrak and Matalon in their study in human lung ATI-like cells (H441) observed that stimulation with cAMP leads to sodium entry and thus the membrane depolarization (137). Hence, it can be concluded that maintaining proper membrane potential requires the coordinated modulation of potassium and choride ion channel activities.

Sodium Transport

The driving force for apical sodium reabsorption in airway epithelial cells is provided by the Na⁺-K⁺-ATPase located along the basolateral membrane (50). The Na⁺-K⁺-ATPase is composed of a heterodimer of two subunits (α and β) and small accessory transmembrane proteins of the FXYD family (66). The conversion of one ATP to ADP results in transport of three sodium ions out of the cell in exchange for two potassium ions pumped into the cell (213). The Na⁺-K⁺-ATPase activity is stimulated by sodium and potassium ions from the cytoplasmic and extracellular side, respectively (66). However, because potassium and sodium compete at the cytoplasmic site, an acute increase in intracellular sodium stimulates Na⁺-K⁺-ATPase activity and leads to a rapid export of intracellular sodium (66). Furthermore, the sodium activation of the Na⁺-K⁺-ATPase is strongly cooperative, and small changes in intracellular sodium levels lead to changes in Na⁺-K⁺-ATPase activity. Thus, under physiological conditions, intracellular sodium is rate limiting for Na⁺-K⁺-ATPase activity, whereas the intracellular ATP concentration is not. ATP may become rate limiting, however, when its synthesis is decreased under pathological conditions such as during periods of hypoxia (121, 132, 181) or oxidative stress (230). Furthermore, during acute lung injury, the accumulation of a protein-rich edema impairs gas exchange and leads to hypoxemia that is accompanied by a decrease of Na⁺-K⁺-ATPase function (234). Hypoxia reduces alveolar fluid clearance and the nasal potential difference (35, 252). In alveolar epithelial cells exposed to severe hypoxia, an increased production of mitochondrial reactive oxygen species (ROS) leads to Na⁺-K⁺-ATPase endocytosis and ubiquitin-dependent degradation (138). Additionally, the basal membrane of ciliated cells contains ATP-dependent amiloride-sensitive electroneutral NHE channels (176, 177) that are involved in maintaining intracellular pH balance (74).

Alveolar edema clearance requires active sodium transport that is mediated by Na⁺-K⁺-ATPases (161, 224). Under normal conditions, this pump is therefore required for maintenance of a dry alveolar space. Furthermore, downregulation of alveolar Na⁺-K⁺-ATPase promotes pulmonary edema in models of lung injury, and therefore activation of dopaminergic or adrenergic receptors that increase Na⁺-K⁺-ATPase activity provides a therapeutic potential for increasing fluid clearance from the alveolar space (161, 166, 224). The obvious importance of this pump is clearly reflected in its ability to resolve alveolar edema under pathological conditions (159).

The main mediator of transcellular sodium reabsorbtion is ENaC that is found at the apical membrane of human airway epithelia. Although it is well established that ENaC forms a trimer consisting of α-, β- and γ-subunits (33), a novel δ-subunit has been identified in both epithelial and nonepithelial tissues (including human lung) (86, 110). Because the δ-subunit is able to form an amiloride-sensitive functional channel along with β- and γ-subunits (236), at least two different channel compositions are possible (110). The δ-subunit-containing ENaC channels have been shown to be responsible for up to one-half of the amiloride-sensitive transport in human nasal epithelial cells (8). The ENaC complexes consisting of other combinations besides the usual α-β-γ complex, however, exhibit lower selectivity of sodium over potassium and are less sensitive to amiloride (38). Although deregulation of the δ-subunit is associated with a predisposition to respiratory diseases (110), the determination of the exact role of this subunit in maintaining ion lung homeostasis is limited because of the absence of δ-subunit expression in mice (87). Because ENaC is often considered the rate-limiting step of lung sodium homeostasis, it is not surprising that changes in sodium channel function that result from epigenetic or pathological factors (reviewed in Ref. 156) often contribute to lung disease. To date, numerous physiological (7, 41, 62, 73, 76, 78, 82, 89, 90, 108, 122, 192, 226), environmental (54, 60, 98, 111, 162, 231), and genetic [mutations in ENaC subunits (188)] factors have been shown to modulate ENaC activity in lung epithelium and thus affect ASL balance (see Table 1) (156).

Table 1.

Factors that elevate or inhibit ENaC channel expression and/or function in alveolar cells and thus affect ASL homeostasis

Factor	Impact on ENaC Function and ASL	Ref. No.
SPLUNC1	SPLUNC1 is highly expressed in the airways and secreted on mucosal surfaces as a volume sensor that regulates ENaC activity and mucosal volumes, including that of the airway surface liquid. SPLUNC1 binds to ENaC, preventing ENaC cleavage and activation by serine proteases.	82, 101, 125, 126, 242
Female sex hormones	Estradiol and progesterone directly regulate expression and activity of alveolar ENaC and ASL volume. Estradiol increases ENaC channel activity and apical plasma membrane abundance of the αENaC through G protein-coupled estrogen receptors.	90, 108
β-Agonists and neurotransmitters	β-Adrenergic stimulation enhances fluid absorption mediated by ENaC stimulation. In the submucosal glands, however, increased cAMP from β-adrenergic receptor stimulation increases ion and fluid secretion and facilitates mucociliary clearance. β2-Agonists and dopamine activate ENaC channels in ATII cells. The muscarinic agonists, carbachol and oxotremorine, activate epithelial ENaC via a RhoA/ROCK signaling pathway in ATII cells.	7, 89, 226
Cytokines	IL-4 may favor the hydration of the airway surface by decreasing Na+ absorption and increasing Cl− secretion to hydrate the mucus, which is hypersecreted during inflammatory conditions.	53, 54, 73, 76–78, 122, 146, 180, 192
	IL-1β increases lung epithelial and endothelial permeability. IL-1β reduces αENaC expression and activity through the activation of p38 MAPK in ATII cells. p38 MAPK-dependent inhibition of αENaC promoter and an alteration in ENaC trafficking to the apical membrane of ATII cells reduces ion and water transport across the lung.
	TGF-β1 has an inhibitory effect on Na+ uptake and αENaC expression in ATII cells that is mediated by ERK1/2-dependent inhibition of the αENaC promoter activity. The recent studies proposed a mechanism in which TGF-β rapidly and sequentially activates phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NOX4 to produce ROS, driving internalization of βENaC, the subunit responsible for cell surface stability of the αβγENaC complex. TNF-α, a cytokine present during lung infection, has a profound influence on the capacity of alveolar epithelial cells to transport Na+. TNF-α decreased the expression of the α-, β-, and γ-subunits of epithelial ENaC mRNA and reduced αENaC protein and ENaC activity. Furthermore, the lectin-like domain of TNF (mimicked by the TIP peptide) was shown to improve ALC in models of both hydrostatic and permeability edema. The TIP peptide directly binds to the α-subunit of ENaC and stabilizes the channel’s complex formation with myristoylated alanine-rich C kinase substrate and phosphatidylinositol 4,5-bisphosphate, which are essential for the open conformation, in the presence of pneumococcal pneumolysin, an important mediator of permeability edema.
PKC	Activation of PKC has been shown to reduce ENaC activity, and, conversely, inhibition of PKC has been observed to increase ENaC function in ATII cells.	47, 48
uPA	Urokinase upregulates ENaC activity and fluid reabsorption in vivo and in vitro. The proposed mechanisms for the regulation of ENaC by uPA include augmentation of Na+-K+-ATPase, proteolysis, and inhibition of ERK1/2 phosphorylation.	41
ROS	GSH serves as a main antioxidant in the lungs, and changes in GSH redox potential have an effect on ENaC activity. ROS GSSG impairs ENaC function and alveolar fluid clearance in vivo. Hence, ROS through increases in GSSG reduce ALF.	60
Hypoxia	Although the results of several studies suggest that severe alveolar hypoxia results in decreased AFC and Na+ transport, the molecular mechanisms underlying this observation require further studies. Furthermore, chronic hypoxia leads to increased ROS formation that may contribute to observed impaired ENaC activity as well.	98, 173, 231
Hypercapnia	Hyperercapnia (elevated CO2 levels) has been shown to impair AFC, thereby causing retention of pulmonary edema and may lead to worse outcomes; hypercapnia affects ENaC cell surface stability by stimulation of polyubiquitination of βENaC and subsequent endocytosis of the α/βENaC complex in AECs. Hypercapnia induced ubiquitination and cell surface retrieval of ENaC and was critically dependent on phosphorylation of the Thr615 residue of βENaC, which was mediated by ERK1/2. The hypercapnia-induced ENaC dysfunction may contribute to impaired alveolar edema clearance.	92, 94, 95
LPS	LPS, a glycolipid abundant in the outermost membrane of Gram-negative bacteria, is a potent proinflammatory molecule. LPS has been reported to decrease ENaC activity and expression of αENAC in AEC. It has been postulated that LPS attenuates αENaC mRNA expression via decreased transcription.	162

SPLUNC1, short-palate lung and nasal epithelial clone 1; ENaC, epithelial sodium channel; ASL, airway surface liquid; ROCK, Rho kinase; ATII, alveolar type II; IL, interleukin; MAPK, mitogen-activated protein kinase; TGF-β1, tumor growth factor-β1; NOX4, NADPH oxidase 4; TNF-α, tumor necrosis factor-α; TIP, TIP peptide of the lectin-like domain of TNF-α; PKC, protein kinase C; uPA, urokinase-like plasminogen activator; ROS, reactive oxygen species; GSH, glutathione; GSSG, oxidized glutathione; ALF, alveolar lining fluid; AFC, alveolar fluid clearance; AEC, alveolar epithelial cells; ERK, extracellular signal-regulated kinase; LPS, liposaccharide. Na⁺/H⁺ exchanger is responsible for preserving intracellular pH. The apical chloride secretion is mediated mainly by cystic fibrosis transmembrane conductance regulator (CFTR); however, Ca²⁺-dependent apical channels like transmembrane domain 16a contribute as well. Chloride secretion is maintained by the basolateral Na⁺-K⁺-2Cl⁻ cotransporter and $\mathrm{HC}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$/Cl⁻ anion exchanger. Furthermore, other basolateral chloride channels, including basolateral outward rectifying channel, basolateral inward rectifying channel, and basolateral CFTR-like channel, have been proposed to modulate apical chloride secretion. Basolateral voltage-dependent potassium channels (Kv7.1–Kv7.5) as well as basolateral and apical Ca²⁺-dependent potassium channels (small-conductance Ca²⁺-activated potassium channels; large-conductance Ca²⁺-activated, voltage-dependent potassium channels; Ca²⁺-activated potassium channels) have also been proposed to modulate apical chloride secretion.

Many of these ENaC-modulating factors, especially those using general signaling pathways such as protein kinase A, mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), or PKC, are ubiquitous and regulate a wide range of cellular activities (157, 243). Until recently, it has proven to be particularly difficult to use ENaC modulation as a potential therapy for lung diseases such as CF. Nevertheless, a deeper understanding of the molecular and physiological mechanisms that modulate airway sodium homeostasis remains a viable target for potential therapies. For example, the short palate lung and nasal epithelial clone 1 (SPLUNC1), a peptide inhibitor of ENaC that is endogenously secreted in ASL, functions as ENaC activity modulator in response to changes in ASL volume (101). Following any increase in ASL volume, this soluble ENaC inhibitor concentration decreases, and this allows for ENaC activation and increased sodium resorption, whereas, with a reduction in ASL volume, the SPLUNC1 peptide is concentrated and inhibits ENaC activation (101, 126).

The recent identification of the SPLUNC1 ENaC-inhibitory domain has contributed to the design of novel specific ENaC peptide inhibitors that have been shown to prevent sodium hyperabsorption in CF lungs (101, 136, 141). The therapeutic potential of ENaC inhibition is further supported by a recent study showing that use of a specific ENaC blocker could lead to ASL rehydration and increased ciliary function and mucociliary clearance (5). These data imply that ENaC inhibition may also be an effective strategy to treat other lung diseases such as COPD. Finally, because ENaC requires proteolytic cleavage of its extracellular loops by specific extracellular proteases for activity (127), it is possible that, in conditions of chronic neutrophilia present in CF and COPD lungs, the increased levels of human neutrophil elastase (HNE) may nonspecifically activate ENaC and contribute to enhanced sodium absorption and ASL dehydration (32). Nonantibiotic macrolides that prevent HNE activity could be used for limiting ENaC activity and thus may be useful in CF and COPD therapies (227). Inter-α-inhibitor (IαI), a serum protease inhibitor consisting of three polypeptides and known to be present in the alveolar spaces after lung injury and in CF, also inhibits ENaC by preventing its cleavage by serine proteases but does not affect CFTR function. A single intranasal instillation of IαI in ∆F508 mice decreased ENaC-nasal potential difference 24 h later by 25% (136).

AE cells contain numerous antioxidants, such as glutathione and ascorbate, that protect cells from oxidants (34, 59). Oxidative stress is a concern because increased levels of ROS that can affect lung redox potential are observed in numerous lung pathologies, including COPD, asthma, lung cancer, and acute lung injury (ALI), and are often seen during influenza infections (39, 69, 133, 178, 185, 229). Recent studies have also shown that exposure to high levels of ROS results in increased amounts of oxidized glutathione in the ALF, and this reduces the ENaC activity in primary AE cells and impairs the lung fluid balance (60). This last observation suggests that antioxidant-based therapies should be considered.

Besides ENaC, the apical membrane-located CNG cation channels may provide another sodium entry pathway in lung epithelial cells (93, 239). These nonselective amiloride-insensitive CNG channels were detected in both ATI and ATII cells, show no preference for sodium over potassium, and can be activated by micromolar concentrations of cGMP (123, 239). Because exogenous cGMP was shown to stimulate liquid absorption, the CNG channels have been proposed to play a role in the regulation of ALF volume (93, 239). However, the exact role of these channels in the regulation of lung fluid homeostasis remains poorly understood (93, 119, 120).

Chloride Transport

The apically located cAMP-dependent CFTR chloride (3) and bicarbonate channel (183) is responsible for a large part of the airway chloride secretion in humans (47). The importance of this channel for lung homeostasis is clearly illustrated by its role in CF, where loss of CFTR function leads to dehydrated mucus, airway obstruction, chronic bacterial infections, inflammatory responses, and eventually bronchiectasis and respiratory failure (174, 194). Furthermore, the decreases in CFTR function have been proposed to play an important role in the pathology of COPD as well (61, 184, 187, 193).

Given that the CFTR-driven apical chloride secretion and ENaC-mediated sodium reabsorption are critical for maintaining PCL homeostasis, it is not surprising that these two activities need to be balanced relative to each other and that one activity may regulate the activity of the other. This hypothesis is supported by the observation that airway epithelia of CF patients have elevated amiloride-sensitive sodium reabsorption, illustrating that CFTR regulates ENaC activity (23, 107, 129, 131). Furthermore, studies in CFTR knockout mice demonstrate the same elevated ENaC activity (135). Interestingly, in contrast to this hypothesis, in the CF pig model, loss of CFTR does not result in increased transepithelial sodium absorption (37). Additionally, the question of whether ENaC regulates CFTR activity remains unclear (46). Furthermore, understanding the mechanism of the ENaC-CFTR cross talk in airway epithelia is complicated by the fact that CFTR activity and ENaC function are often affected by the same physiological [β-agonists (7, 179)] and environmental factors that include oxidative stress (19, 20, 175, 203, 218, 248), hypoxia (36, 244), inflammatory responses (28, 30, 169, 202, 241), and influenza infections (102, 109, 144, 189, 235).

The apical side of airway epithelia contains other channels besides CFTR that contribute to chloride secretion. These include the chloride voltage-gated channel 2 (CLCN2) and Ca²⁺-activated ion channels (CaCCs), including TMEM16 family members that have been shown to contribute to apical chloride secretion (79, 191, 198). Although the extent of TMEM16A channel contribution to Ca²⁺-induced chloride secretion in airways is limited (167, 200), the mechanism of Ca²⁺-activated chloride secretion requires further analysis. Recent studies, however, have shown that the inhibition of these channels along with the modulation of Na⁺-K⁺-Cl⁻ cotransporter (NKCC) activity leads to smooth muscle relaxation and thus, along with β-agonists, provides a novel basis for therapy in asthma (55, 56). Furthermore, gaining control over CLCN2 or CaCC activity may provide an alternative approach to CFTR loss and thus provide a potential therapeutic benefit for CF patients (99, 198). On the other hand, activation of TMEM16A following exposure of airway smooth muscle cells to oxidant gases results in membrane depolarization that may contribute to the activation of RhoA and airway smooth muscle contraction (134).

During development, ClCN2 is highly expressed in lung tissue and has been proposed as a potential pathway to replace the missing chloride conductance in patients suffering from CF (204), However, Clcn2^−/− mice do not suffer from lung disease, and additional ClCN-2 knockout in CFTR^−/− mice did not deteriorate the phenotype of CF (245).

TMEM16A epithelial expression is increased in asthmatic patients, particularly in secretory cells, and inhibition of TMEM16A-CaCC activity significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human airway smooth muscle (ASM) contraction in response to cholinergic agonists (104). Furthermore, increased expression of TMEM16A was observed in the airways of CF patients, suggesting that TMEM16A expression/function is upregulated in CF lung disease, possibly as a response to the presence of bacteria in the airways (31).

NKCC mediates chloride entry across the basolateral membranes of secretory epithelia. The driving force for cellular chloride entry and accumulation is supplied primarily by the inward chemical gradient of sodium established by the pump; the basolateral membrane potential does not influence this electrically neutral process. Ultimately, the rate of basolateral chloride entry via NKCC determines the overall rate of chloride secretion. The timing of NKCC activation generally follows that of CFTR and the potassium channels, resulting in some loss of cellular chloride and potassium and a decrease in the cell volume (74). NKCC is upregulated in response to Gram-negative bacterial toxins like lipopolysaccharide (LPS) in the lung. Recently, basolateral NKCC1 and apical-expressed CFTR were shown to be critical for the reversal of fluid uptake in a secretory alveolar epithelium by driving chloride secretion. It was reported that an acute increase in left atrial pressure decreases amiloride-sensitive sodium uptake across the alveolar epithelium and concomitantly stimulates sodium and chloride uptake via basolateral NKCC and chloride secretion in the alveolar space via apical CFTR, thus effectively reversing sodium-driven AFC in chloride-driven alveolar fluid secretion (AFS). Importantly, inhibition of CFTR and NKCC1 improved AFC and attenuated edema formation (238). This was also the case in influenza infection: Wolk et al. reported that inhibition of CFTR decreased alveolar edema in influenza infection (240). Others, however, have reported that influenza viruses and its M2 protein decrease CFTR activity and levels in the apical membranes of epithelial cells (91, 143, 145).

Despite the continuous development of novel therapies for CF (17, 36, 45, 47, 51, 52, 75, 84, 163, 196, 215), the vast majority treats the symptoms rather than correcting molecular defects in the channel. Correcting the CFTR channel has been difficult because, besides the most common mutation, ∆F508 CFTR, >2,000 mutations in the CFTR gene have been identified (11, 195). Translating this complex genetic CF background into CFTR protein structure and function has not proven to be an easy task. In that regard, a recent high-resolution structure of the human CFTR protein (3.9-Å as determined by electron cryomicroscopy) was presented that may provide new insights into channel function (140). The structure revealed a previously unresolved helix belonging to the R domain that docked inside the intracellular vestibule that blocks channel opening (140). Furthermore, a structure of the purified G551D CFTR, a channel-gating mutant, has also been determined by electron cryomicroscopy (72), suggesting that this type of methodology is being used more and more. And, finally, another recent study identified extracellular loop 1 (ECL1) of CFTR as critical for proper channel function (106). These studies increase our understanding of structural basis of chloride channel function (71, 140, 247) in CFTR and will likely aid in understanding the effects of mutations in CFTR (44) and hopefully allow for the development of more effective therapies.

Less effort, however, has been placed on studying chloride movement across the basolateral membrane. Basolateral chloride transport appears to be mediated by the outwardly rectifying chloride channel (68), the inwardly rectifying chloride channel (68), and a cAMP-dependent low-conductance linear CFTR-like channel (42, 68). Furthermore, the action of these channels is facilitated by electroneutral channels such as the NKCC cotransporter (228) and the $\mathrm{HC}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$/Cl⁻ exchanger (14) that provide a sufficient intracellular supply of chloride ions (68).

Although the CFTR function in ciliated epithelial cells is well established, the role of this channel in AE is less clear. Despite the fact that numerous studies identified CFTR activity in AE [both in ATI (116) and ATII (65) cells] and proposed this channel to be involved in ASL regulation (65, 116), there is no consensus as to whether CFTR is responsible for chloride absorption (64, 65, 112) or secretion (25, 214). Furthermore, a recent study indicated that CFTR was at the basolateral membrane of AE cells (15). These controversies may partially be explained from limited availability of these cells and various culturing conditions that were used to isolate and culture these cells (57). There are other channels in AE besides CFTR that include electroneutral cotransporters like NKCC (96, 212), K⁺-Cl⁻ (KCC) (212) and the $\mathrm{HC}{\mathrm{O}}_{\mathrm{3}}^{\mathrm{-}}$/Cl⁻ exchanger (14), as well as the GABA_A chloride channel (113, 114), and the voltage-gated CLC2 and CLC5 (115). All of these channels and transporters have been proposed to be involved in transepithelial chloride transport.

Potassium Transport

The wide variety of diverse potassium channels [over 30 (9)] that are apically and basolaterally located in airway epithelia maintain the electrochemical gradient and thus support lung ion and fluid homeostasis (9, 139). Changes in activity of airway epithelial basolateraly located potassium channels (including KCNQ voltage-activated Na⁺–Kv7 channels) were shown to impact apical cAMP-dependent chloride secretion (26, 149, 150) and Ca²⁺-dependent chloride secretion (16). To date, the presence of all three classes of Ca²⁺-dependent potassium channels [big-conductance potassium (BK), intermediate-conductance potassium (IK)/K_Ca3.1, and small-conductance potassium (SK)] was confirmed at the basolateral membrane of airway epithelial cells (130, 149, 232). Furthermore, some apically located Ca²⁺-dependent potassium channels, including SK4-like channels (16) and BK_Ca (152, 153), have a significant influence on chloride secretion and thus are important for MCC and ASL volume regulation.

Importantly, the role of the potassium channels in AE cells is not limited to sodium reabsorption maintenance. The activity of ATP-sensitive potassium channels in alveolar cells strongly relies on intracellular ATP levels and thus links the membrane potential with the metabolic state of the cells (168, 233). Additionally, a recent study reported that the two-pore-domain potassium channel Trek-1 regulates release of proinflammatory interleukin-6 (IL-6) from AE cells (18, 206–208). This suggests that AE cells may use potential potassium channels to adapt the membrane potential in response to stress factors and thus are crucial for airway epithelial repair (29, 233). Hence, understanding their physiological role in airway regeneration and in inflammatory responses may be important for lung pathologies such as CF, ALI, and acute respiratory distress syndrome (208, 209).

Additionally, potassium channels act in alveolar epithelium as oxygen sensors and thus adjust lung function to environmental changes in O₂ levels (9, 124, 170, 171, 209). Alveolar potassium channels, in particular BK_Ca, that have been shown to reduce their opening time during hypoxia (118) are able to detect O₂ variation and modulate their activity to adjust ion transport and fluid clearance (9, 124, 170, 171). Although the molecular mechanism(s) governing oxygen sensing by alveolar potassium channels remains unclear, their properties make them potential therapeutic targets for lung diseases associated with hypoxia that include pulmonary hypertension (147, 209).

Moreover, the involvement of potassium channels has been proposed in respiratory conditions such as asthma and chronic COPD (148). Pulmonary hypertension can be caused by a defect in the function of potassium channels or by alveolar hypoxia. Importantly, hypoxia selectively inhibits the function and expression of voltage-gated potassium (KV) channels in pulmonary arterial smooth muscle cells (SMCs). The activity of potassium channels regulates the membrane potential (E_m) of SMCs, which in turn regulates the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_cyt). Depolarization of the E_m leads to an elevated [Ca²⁺]_cyt by opening voltage-dependent Ca²⁺ channels. Elevated [Ca²⁺]_cyt is implicated in stimulating vascular SMC proliferation and inducing vasomotor tone and, hence, vasoconstriction. Vasoconstriction causes elevation of intravascular pressure and elastic stretch of the SMCs, both of which have been shown to play a role in pulmonary arterial cellular growth and synthetic activity, creating a vicious cycle of cellular hypertrophy, proliferation, and vascular remodeling. Dysfunction of potassium channels has also been linked to decreased apoptosis in pulmonary arterial SMCs, a condition that contributes further to the medial hypertrophy of the arterial walls and vascular remodeling (21, 151, 165, 190). Furthermore, KCNRG, a putative potassium channel-regulating protein expressed in bronchial epithelial cells, was identified as an autoantigen in autoimmune polyendocrine syndrome type 1 (APS-1) associated with pulmonary manifestations (1).

Role of Ca²⁺ Transport

As discussed above, both the chloride and potassium ion transport in airway epithelial cells rely to some extent on Ca²⁺-dependent channel activity (e.g., CaCCs and BK_Ca). Some support for this idea comes from the fact that the proper folding and membrane expression of crucial transmembrane ion channels like CFTR (12, 13, 154) relies on the endoplasmic reticulum being the main cellular Ca²⁺ reservoir (4, 10, 201). Furthermore, a recent study showed that the extracellular Ca²⁺-sensing receptor regulates human fetal lung development via CFTR (24).

A variety of environmental insults such as hypoxia (18, 85, 88, 182, 237, 251), oxidative stress (134, 220, 221, 223), and inflammation (2, 40, 63, 100, 250) that affect cellular Ca²⁺ homeostasis were shown to affect Ca²⁺ channel activity in airway epithelium as well. Because changes in airway Ca²⁺ transport can be reflected in Ca²⁺-dependent potassium and chloride channel activity, Ca²⁺ channels provide an additional mechanism that governs lung ion and fluid homeostasis. They are therefore emerging as a potential novel drug target for lung pathologies, including asthma (80, 216) and chronic inflammation (2, 40, 250), oxidative lung injury (81, 134, 164, 223), and CF (100). Importantly, the transient receptor potential vanilloid-4 channel (TVRPV4), a nonselective Ca²⁺ channel that responds to variety of stimuli (164, 186, 221, 222), governs the production of a number of proinflammatory mediators in airway epithelia. Thus, anti-inflammatory inhibitors of TRPV4 have been proposed as a therapeutic target for lung injury, CF (100), and acute respiratory distress syndrome (6, 221). Because of the concomitant pulmonary or systemic infections that accompany patients with these lung pathologies, the use of TRPV4 inhibitors, unfortunately, carries the risk of compromising immune responses (164). Maintaining the proper intracellular Ca²⁺ levels and Ca²⁺ transport by airway myocytes that control the extent of airway opening (97, 210) is often underappreciated and critical for normal lung function.

Current Limitations and Challenges

The proper maintenance of airway epithelium ion balance is imperative for healthy lung homeostasis. However, the complete characterization of biochemical and biophysical properties and the coordinated function of ion channels that govern lung function still remain a challenge. The apical and basal membranes have different sets of ion transporters, and different cell types have different channels as well, which adds to the cellular/tissue complexity. Hence, even understanding channel function by itself and its relation to ion exchange makes picking specific molecular targets for therapeutic approaches difficult at best and problematic at worst. Although the recent reports have increased our knowledge of lung epithelial ion transport, they have also exposed how limited our understanding is on the relationships between ion channels, intracellular signaling networks, and environmental factors as well (205). That being said, recent studies on the role of airway epithelial channels have provided promising new drug targets.

Concluding Remarks

The mechanisms that control ion homeostasis in airways rely on the coordinated action of an entire network of channels, and this is reflected in the complex pathological features of the majority of lung diseases. Hence, understanding the rules that govern these channel interactions within this network of airway ion regulation remains an important challenge to overcome for achieving successful interventions in various lung airway diseases.

GRANTS

This research was supported by the CounterACT Program, National Institutes of Health Grants 5U01-ES-026458 02 (S. Matalon), 1 U01-ES-027697 01 (S. Matalon), and P30-DK-072482 (J. F. Collawn), and by the National Science Center OPUS Program under contract 2015/17/B/NZ3/01485 (R. Bartoszewski).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.B. drafted manuscript; R.B., S.M., and J.F.C. edited and revised manuscript; R.B., S.M., and J.F.C. approved final version of manuscript.