I. INTRODUCTION

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The epithelial sodium channel (ENaC)/degenerin gene family represents a new class of ion channels that was discovered at the beginning of the 1990s. The degenerin genes deg-1 and mec-4 were first identified from a genetic screen of the mechanosensory pathway of Caenorhabditis elegans. The name degenerin (DEG) comes from the cellular phenotype induced by mutations of the deg-1 gene and other related genes that result in selective degeneration of sensory neurons involved in touch sensitivity. At the time of the identification of the DEG gene family in C. elegans, a functional cloning strategy in Xenopus laevis oocytes allowed the isolation and sequencing of a cDNA encoding the -subunit of the amiloride-sensitive epithelial Na⁺ channel ENaC (found in databanks under SCA, "sodium channel, amiloride sensitive," and SCNN1, "sodium channel, nonneuronal"). This channel was already known to play a crucial role in Na⁺ absorption in the distal part of the kidney tubule and to be the target of aldosterone action. ENaC and degenerins were found to have substantial sequence homology. Additional members that formed a new subfamily within this emerging ion channel family were subsequently identified by sequence homology and characterized by functional expression. These related genes were found to be expressed mainly in the central and peripheral nervous system and were called mammalian degenerins (MDEG) or brain Na⁺ channels (BNaC, BNC). After the discovery of their activation by extracellular protons these channels were named acid-sensing ion channels (ASICs). At about the same time the FMRF-amide-gated ion channel (FaNaC) was cloned from the mollusk Helix aspersa. This channel forms its own subfamily within the ENaC/DEG family of ion channels.

In contrast to channels that appeared at an early stage of evolution such as potassium, chloride, or water channels, ENaC/DEG genes are present only in animals (metazoa) with specialized organ functions for reproduction, digestion, and coordination. The members of the ENaC/DEG gene family show a high degree of functional heterogeneity that is unusual among the known gene families of ion channels. Their wide tissue distribution that includes transporting epithelia as well as neuronal excitable tissues best reflects the functional heterogeneity of the ENaC/DEG family members. Depending on their function in the cell, these channels are either constitutively active like ENaC or activated by mechanical stimuli as postulated for C. elegans degenerins, or by ligands such as peptides or protons in the case of FaNaC and ASICs. The evolution of the ENaC/DEG gene family certainly followed quite divergent paths to finally achieve a variety of different functions in the cell.

This review summarizes our present knowledge of the fundamental roles of the ENaC/DEG proteins and the relationship between their biophysical properties and structural characteristics. Despite their different functions in the cell, the members of the ENaC/DEG family retain common functional characteristics. Other recent reviews cover relevant aspects of ENaC/DEG family members in more detail than this review: amiloride-sensitive channels in epithelia (87), molecular mechanisms of human hypertension (153, 209), taste reception (155, 156), nociception (131, 175, 269), touch sensation in C. elegans (82, 243), and mechanotransduction (70, 88, 102).

	II. PHYLOGENETIC AND SEQUENCE COMPARISON

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Figure 1 shows a phylogenetic tree of the most relevant ENaC/DEG sequences available to date. Seven different branches can presently be distinguished in this gene family: the three main subfamilies comprise the SCNN1 genes encoding the ENaC -, -, -, and -subunits; the C. elegans degenerins (UNC, MEC, DEG, DEL); and the ASICs. Smaller subfamilies found in invertebrates include the Drosophila proteins RPK/dGNaC1 and PPK/dmdNaC1, the peptide-gated Na⁺ channel FaNaC of mollusks, and FLR-1 in C. elegans that is clearly distinct from the degenerins (171). The mammalian BLINaC (brain-liver-intestine amiloride-sensitive Na⁺ channel) and hINaC (human intestine Na⁺ channel) genes encode Na⁺ channels that are clearly distinct from ENaC. The amino acid sequence identity between the different ENaC/DEG subfamilies is ~15-20%, whereas the identity within subfamilies is ~30% for the different ENaC subunits; ~30% for degenerins; 45-60% between the four ASIC genes ASIC1, ASIC2, ASIC3, and ASIC4; 38% for the two characterized Drosophila members; and ~65% for the three FaNaC orthologs. Within the ENaC subfamily two branches can be distinguished, one leading to the - and -subunits and the other to - and -subunits. The ENaC genes have been cloned from various species such as rat, human, cow, mouse, and Xenopus laevis, and for the sake of clarity, only human and rat sequences were used for the phylogenetic tree in Figure 1. The homology between human and rat orthologs of ENaC subunits is ~85%, and it is close to 100% between human and rat orthologs of ASIC1, ASIC2, and ASIC4 and ~83% for ASIC3. The hINaC and BLINaC genes share 79% sequence identity, and hINaC is probably the ortholog of the rodent BLINaC.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Phylogenetic tree of the epithelial sodium channel (ENaC)/degenerin (DEG) family showing the organization into subfamilies of related sequences. Sequences were aligned by using the ClustalW algorithm. The channels from vertebrates are divided into three groups: ENaC, acid-sensing ion channels (ASICs), and brain-liver-intestine sodium channel (BLINaC)/human intestine sodium channel (hINaC). ENaC/DEG proteins of invertebrates can be divided into four groups: 1) the degenerins from Caenorhabditis elegans; 2) the Drosophila channels RPK/dGNaC1 and PPK/dmdNaC1; 3) FMRFamide-gated sodium channel (FaNaC), which is expressed in mollusks; and 4) FLR-1, which is the only characterized member of a group of C. elegans ENaC/DEG family members that are different from the degenerins.

ENaC (47, 54, 142, 151, 247) and ASIC genes (see below) have different splice variants. For ASIC channels the nomenclature remains somewhat confusing in the literature. Initially ASICs were considered as the homologs of the degenerins because of their distribution in the central nervous system (CNS), and they were named mammalian degenerins (MDEG). The phylogenic tree however shows that the DEG and ENaC subfamilies are equally distant from the ASIC subfamily (Fig. 1); therefore, the name mammalian DEG for ASIC is phylogenetically incorrect. We use here a unified nomenclature adapted from Waldmann and Lazdunski (267). The ASIC subfamily comprises the following members: ASIC1a (also known as BNaC2, Refs. 83, 264), ASIC1b (or ASIC, Refs. 18, 40), and additional splice variants of ASIC1 (40, 252), ASIC2a (or MDEG, MDEG1, BNC1, BNaC1, Refs. 83, 201, 266, 268), ASIC2b (MDEG2, Ref. 158), ASIC3 (or DRASIC, hTNaC1, Refs. 13, 61, 116, 263), and ASIC4 (or SPASIC, Refs. 7, 97).

The degenerin subfamily comprises only 6 members out of the 23 predicted ENaC/DEG proteins identified in the C. elegans genome (171, 246). FLR-1 is the only characterized member of a subfamily of eight members that can be defined by the presence of a conserved region in the extracellular domain related to, but distinct from, degenerin CRDII (171). FLR-1 is expressed in the C. elegans intestine and appears to be also functionally distinct from the degenerins (126, 134, 241).

Sequencing of the Drosophila genome identified 24 putative proteins related to the ENaC/DEG family (162), but so far only the two gene products Ripped Pocket (RPK/dGNaC1) (3, 55) and Pickpocket (PPK/dmdNaC1) (3, 56) have been characterized with regard to their function and/or tissue distribution.

The first draft sequence of the human genome reveals the presence of 11 proteins that can be assigned to the ENaC/DEG family: -, -, -, and -ENaC, ASIC1, -2, -3, and -4, and two or three proteins related to hINaC (256). Thus, at the present state of completion of the human genome, it seems unlikely that the number of mammalian genes encoding ENaC/ASIC proteins will greatly expand. It is interesting that no mammalian orthologs of the C. elegans DEG or FLR-1 genes, the Drosophila RPK/dGNaC1 and PPK/dmdNaC1 genes or of FaNaC have yet been identified in the human and the mouse genomes. This might indicate a divergence of the different ENaC/DEG subfamilies early in evolution.

	III. PHYSIOLOGICAL ROLE

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The ENaC is located in the apical membrane of polarized epithelial cells where it mediates Na⁺ transport across tight epithelia. In contrast to other Na⁺-selective channels involved in the generation of electrical signals in excitable cells, the basic function of ENaC in polarized epithelial cells is to allow vectorial transcellular transport of Na⁺. This transepithelial Na⁺ transport through a cell basically involves two steps as illustrated in Figure 2. The large electrochemical gradient for Na⁺ existing across the apical membrane provides the driving force for the entry of Na⁺ into the cell. Active Na⁺ transport across the basolateral membrane is accomplished by the Na⁺-K⁺-ATPase.

View larger version (100K): [in this window] [in a new window]   Fig. 2. Transepithelial ion transport in a principal cell of the cortical collecting duct (CCD). ENaC mediates Na+ entry from the tubule lumen at the apical membrane, and the Na+-K+-ATPase extrudes Na+ at the basolateral side. K+ channels are present on the basolateral and apical membranes. K+ channels at the apical membrane mediate K+ secretion into the tubular lumen. The diagram also illustrates the action of aldosterone (Aldo) which binds to intracellular receptors that are translocated to the nucleus and affect the expression and subcellular localization of ENaC and the Na+-K+-ATPase as well as other target proteins via aldosterone-induced transcripts (AITs) and aldosterone-repressed transcripts (ARTs).

The apical entry of Na⁺ is blocked by submicromolar concentrations of amiloride. The ENaC-mediated electrogenic Na⁺ absorption in the distal nephron creates a favorable electrochemical driving force for K⁺ secretion into the tubule lumen. This active transepithelial transport of Na⁺ is important for maintaining the composition and the volume of the fluid on either side of the epithelium. In the kidney and the colon, which are target tissues for aldosterone action, the transepithelial sodium transport is crucial for the maintenance of blood Na⁺ and K⁺ levels and their homeostasis. In the lung or in salivary glands, Na⁺ transport is certainly not important for the whole body Na⁺ homeostasis, but rather for keeping the composition and the volume of the luminal fluid, i.e., the saliva or the alveolar fluid constant.

In the kidney, ENaC is expressed in the distal nephron where sodium reabsorption is controlled by the mineralocorticoid hormone aldosterone. In the distal nephron, Na⁺ reabsorption can be measured in in vitro microperfused tubules as a large amiloride-sensitive Na⁺ flux in sodium-deprived or aldosterone-treated animals (41, 204, 221). The first descriptions of the amiloride-sensitive Na⁺ current at the single-channel level using the patch-clamp technique were obtained from principal cells of microdissected cortical collecting ducts (CCDs) (190) and from a cell line derived from the distal nephron of amphibians that responds to aldosterone (104). The presence of the amiloride-sensitive Na⁺ current correlates with the expression of ENaC both at the mRNA and protein levels in the distal convoluted tubule, the connecting tubule, the CCD, and the outer medullary collecting duct. There is a clear axial heterogeneity of ENaC expression along the distal nephron; expression of ENaC is higher in superficial cortical regions of the distal nephron such as the connecting tubule or the CCD than in the deeper medullary regions such as the inner medullary collecting duct (IMCD) (69, 167, 168, 173, 260). In the CCD ENaC colocalizes with the water channel aquaporin-2, both being stimulated by vasopressin in this nephron segment (166). An electrogenic Na⁺ transport sensitive to aldosterone is important in amphibian bladder and has also been found in the bladder of some mammals (150).

Under physiological conditions of hydration and salt balance, Na⁺ reabsorbed in the distal nephron represents only a small fraction (<5%) of the filtered load. However, Na⁺ reabsorption in the distal nephron can increase considerably in response to aldosterone or vasopressin secretion that is stimulated by dehydration and/or salt deprivation. This hormonal control is essential for the fine-tuning of Na⁺ reabsorption in the distal nephron and the maintenance of sodium and fluid balance (208). The physiological and pathophysiological role of ENaC in Na⁺ and K⁺ homeostasis has been clearly demonstrated in human genetic studies and later confirmed by disruption of ENaC genes in mouse models by homologous recombination. Mutations in the - and -ENaC genes causing hyperactive channels have been found in patients with Liddle's syndrome (105, 106, 114, 152, 218). Liddle's syndrome is a rare hereditary form of hypertension characterized by low plasma aldosterone levels and a low renin activity often associated with hypokalemia and/or metabolic alkalosis. These clinical features reflect an abnormally high Na⁺ reabsorption in the distal nephron leading to expansion of the extracellular fluid volume and to the development of arterial hypertension. The insertion of a mutation causing Liddle's syndrome in the mouse -ENaC gene locus by homologous recombination generated a mouse with a phenotype milder than the Liddle's syndrome in humans with extracellular volume expansion, high blood pressure associated with metabolic alkalosis, and hypokalemia that become evident only when animals are challenged with a high-salt diet (199). The elucidation of the genetic basis of Liddle's syndrome demonstrated the critical role of ENaC in maintaining a balance between the Na⁺ intake and Na⁺ excretion by the kidney (see Ref. 153 for review). The role of ENaC in Na⁺ homeostasis was further evidenced by the identification of mutations in ENaC causing reduced channel activity or complete loss of channel function associated with pseudohypoaldosteronism type 1 (PHA-I) (39). The renal symptoms of this heterogeneous syndrome include hyponatremia, hypotension, and hyperkalemia and are associated with elevated plasma aldosterone and renin levels. Mouse models for PHA-I were obtained by different gene targeting strategies leading to decreased expression of - or -ENaC genes (112, 198) or complete knockout of the -, -, or -ENaC gene (15, 111, 176). The renal symptoms of these PHA-I mice were similar to those of the PHA-I patients, with a renal loss of sodium associated with hyperkalemia despite elevated plasma aldosterone levels. The physiological consequences of ENaC gene targeting in these animal models confirmed the critical importance of the three genes -, -, and -ENaC for channel function and Na⁺ absorption in the distal nephron.

The airway epithelia absorb Na⁺ via an amiloride-sensitive electrogenic transport. This active Na⁺ absorption is important for the maintenance of the composition of the airway surface liquid. The expression of the ENaC subunits along the respiratory epithelium is complex and varies between species. In adult rats and humans, the -, -, and -ENaC subunits are highly expressed in small and medium-sized airways (30, 74, 242). The - and -subunits but not the -subunit are expressed more distally in the lung, which may well correspond to a localization in the type II alveolar cells. This heterogeneity of the expression of ENaC subunits along the airways suggests differential regulation of liquid absorption by channels of various subunit compositions.

At birth the amiloride-sensitive electrogenic Na⁺ transport is important to clear the liquid that fills the alveoli and the airways of the fetal mouse lung. mRNAs for -, -, and -ENaC can be detected in the fetal lung around days 15-17 of gestation, and expression of ENaC subunits (mainly - and -ENaC) sharply increases in the late fetal and early postnatal life when the lung turns from a secretory to an absorptive organ (242). The physiological role of ENaC in lung liquid balance was clearly demonstrated in mice in which the -ENaC gene was inactivated by homologous recombination (111). These -ENaC knock-out mice die soon after birth from respiratory failure due to a severe defect in the clearance of the fetal liquid that fills the lungs. These studies suggest that at birth -ENaC in the mouse fetal lung is essential for Na⁺ absorption. The disruption of the - and -ENaC gene loci results in a slower clearance of the fetal lung liquid at birth but does not severely affect the blood gas parameters. The - or -ENaC knockout mice die slightly later than the -knockout from severe electrolyte imbalance, namely, hyperkalemia due to deficient renal K⁺ secretion (15, 176). Thus, in contrast to the kidney, the Na⁺ transport in the lung can be maintained efficiently by only two functional ENaC genes, i.e., the pairs - or -.

In humans the contribution of -ENaC to the clearance of fetal lung liquid at birth is still unclear. Very premature infants with respiratory distress syndrome have a reduced sodium absorption across the respiratory epithelia, as demonstrated by a reduced nasal transepithelial potential difference, likely contributing to the pathogenesis of this syndrome (14). However, PHA-I patients with severe disruption of the -ENaC gene leading to near-complete channel loss of function have no report of respiratory distress syndrome at birth but show a more than twofold higher liquid volume in airway epithelia than normal individuals (139). Thus ENaC function in humans does not seem to be limiting at birth for the liquid clearance in the mature fetal lung. Differences between species in maturation of the lung, in mucociliary clearance, or in ENaC subunit expression in the respiratory epithelium may account for the phenotypic differences between human and mice.

In salivary glands, -, -, and -ENaC are detected at the RNA and protein levels in the apical membrane of the striated and interlobular ducts (69). An amiloride-sensitive Na⁺ conductance has been described in mandibular duct cells using the whole cell patch-clamp technique (63). The IC₅₀ of amiloride inhibition was ~5 µM, thus ~50 times higher than the IC₅₀ of ENaC. The functional characteristics of this amiloride-sensitive current still await more detailed characterization to ascertain the functional contribution of ENaC in mediating Na⁺ reabsorption in salivary glands.

The colon is a tight epithelium and an important site for Na⁺ absorption. Part of this Na⁺ transport is electrogenic, sensitive to amiloride and stimulated by aldosterone (71). Initially, the -, -, and -ENaC cDNAs were isolated from a rat distal colon cDNA library because of the high level of channel expression in this tissue (31, 33, 159).

The classical model of transepithelial Na⁺ transport in tight epithelia (144) was originally described by Koefoed-Johnson and Ussing in the skin of amphibians and postulated the presence of an apical Na⁺ channel and a basolateral Na⁺-K⁺-ATPase in series. The Na⁺ transport in the amphibian skin responds to aldosterone with simultaneous morphological changes (261). In mammals, the three ENaC subunits are expressed in keratinocytes of all epidermal layers as well as in epithelial cells of hair follicles and sweat glands (210). ENaC transcripts can also be found in the pluristratified epithelium of the esophagus where ENaC's role is unknown (74). In sweat glands ENaC likely controls Na⁺ excretion as in the kidney, but in the epidermis and hair follicles, the physiological role of ENaC is less clear. There is still no clear evidence for an ENaC-mediated transcellular Na⁺ transport in mammalian epidermal cells, despite the presence of a benzamil-sensitive current in cultured human keratinocytes consistent with ENaC activity (29). Interestingly, expression of the - and -ENaC subunits is upregulated in cultured keratinocytes during differentiation, suggesting that ENaC may play a role in epidermal differentiation and skin development. Alternatively, this upregulation might be indirectly linked to the regulation of cell volume during this process (29, 183).

Finally, salt taste is transduced by direct amiloride-sensitive influx of Na⁺ in the taste cells of the fungiform papillae of the anterior part of the tongue, suggesting the presence of an amiloride-sensitive Na⁺ channel (for review, see Refs. 155, 156). The three -, -, and -ENaC subunits are expressed in the taste receptor cells of the fungiform papillae (147, 154). The specific role of ENaC in salty taste transduction still remains to be clearly demonstrated, since the amiloride-sensitive inward currents recorded from whole taste receptor cells show lower affinity for amiloride and a lower single-channel conductance than expected from a typical ENaC current as estimated from noise analysis (9, 10).

The -ENaC subunit, closer to - than to - and -ENaC in its amino acid sequence, has been identified by sequence homology (Fig. 1). As expected from its close homology to -ENaC, the -ENaC subunit can substitute for -ENaC to form functional amiloride-sensitive Na⁺ channels. The -ENaC subunit is expressed in testis, ovary, pancreas, and to a lesser extent in brain and heart (265). In the pancreas as in the ducts of salivary glands, an amiloride-sensitive channel might be responsible for Na⁺ reabsorption. In the other tissues expressing -ENaC, amiloride-sensitive Na⁺ currents have not yet been detected, and therefore, the physiological role of -ENaC in these tissues remains unknown.

The membranous labyrinth of the cochlea is a complex sensorineural epithelium. The basolateral side is bathed with the perilymph of similar ionic composition as the plasma or as cerebrospinal fluid. The endolymph at the apical surface of the membranous labyrinth is a K⁺- rich, hyperosmotic fluid almost devoid of Na⁺. The endolymph bathes the hair bundles of the sensory cells, and its ionic composition is critical for mechanotransduction. The -, -, and -ENaC subunit mRNAs are detected in the rat cochlea in both epithelial and nonepithelial structures (53). Interestingly, expression of the three ENaC subunits was strongest in epithelial cells (Claudius cells in particular) lining the scala media. The Claudius cells might be responsible for Na⁺ reabsorption from the endolymph; the unusually high potential difference existing across the apical membrane of these cells (+80 mV in the endolymph and 100 mV intracellular) allows absorption of Na⁺ from the endolymph at lower than millimolar concentrations (99). Furthermore, the mRNA abundance of the -, -, and -ENaC subunits in the cochlea increases during the first 10 days of life when the high-K⁺, low-Na⁺ composition of the endolymph is established. This observation suggests that ENaC in the epithelial cells of the scala media is critical for Na⁺ extraction from the endolymph. However, no severe hearing problems have been reported for PHA-I patients with loss-of-function mutations of ENaC.

ENaC transcripts and proteins were detected in retina photoreceptors, but ENaC's role in phototransduction remains to be established (89). Finally, the -ENaC subunit has been localized in baroreceptor nerve termini innervating the carotid sinus (67). Structures specialized in mechanosensation in nerve endings of the rat footpad also express - and -ENaC, and it was proposed that ENaC subunits represent components of mechanoreceptors for touch or blood pressure sensing (66). An involvement of ENaC in mechanotransduction has not yet been directly demonstrated.

ASICs were cloned based on their homology to ENaC/DEG channels (for a recent review, see Ref. 267). Four ASIC genes (ASIC1-4) have been identified, and three of them exist in different splice variants (Fig. 1, see sect. II). The expression of ASIC mRNAs has been analyzed by Northern blotting and in situ hybridization (26, 267) and shows a high expression in the nervous system. ASIC1a, ASIC2a, and ASIC2b have a similar widespread distribution pattern in the brain, with the highest expression levels in the hippocampus, the cerebellum, the neo- and allocortical regions, the main olfactory bulb, the habenula, and the basolateral amygdaloid nuclei. Coexpression with ASIC4 is found in many areas of the brain (7, 97). In addition, strong expression of ASIC4 has been found in the pituitary gland (97).

In the peripheral nervous system, the ASIC channels are found predominantly in small-diameter sensory neurons involved in pain sensation (40). Rat ASIC3 is expressed almost exclusively in sensory neurons (12, 13, 40, 263). ASIC3 is expressed in sympathetic cardiac afferents that innervate the heart where they may play a role as mediators of cardiac pain (23, 240). Expression of ASIC2a in sensory neurons is lower compared with other ASICs (158, 200). ASIC2a is also found in the taste buds of the circumvallate papillae where it may have a function in sour taste perception (250). ASIC2b has to date only been found in the rat. As for ENaC subunits, ASIC4 expression was demonstrated in the inner ear, but its role in hearing function remains to be established (97).

ASIC channels are activated by a drop of the extracellular pH. Expression of homomultimers or heteromers of ASIC1-3 in heterologous cell systems generates proton-gated cation currents with functional properties resembling the native currents found in sensory and CNS neurons and in oligodendrocytes (25, 92, 148, 164, 255, 267). The magnitude of these depolarizing H⁺-gated currents is sufficient to initiate action potentials in neurons (72, 255). So far, no channel function has been found for ASIC4 (7, 97).

The physiological role of ASICs remains uncertain. Their expression in sensory neurons suggests a role in pain perception following tissue acidosis (203, 263, 267). For instance, good electrophysiological evidence supports the involvement of ASIC3 in cardiac ischemic pain sensation (23, 240). Myocardial extracellular pH drops to 6.7 during severe cardiac ischemia, a pH value that can evoke a high-amplitude current in the sympathetic cardiac afferents expressing ASIC3 (240). This current shows the same functional characteristics as the H⁺-gated current in COS-7 cells expressing ASIC3 (240). Amiloride that blocks ASICs with low affinity has recently been shown to have analgesic effects in a variety of animal pain models (75). This finding however has to be interpreted with some caution because at concentrations at which amiloride blocks ASICs, it also blocks other channels and proteins in the brain, as, e.g., voltage-gated Ca²⁺ channels (143). Finally, nonsteroid anti-inflammatory drugs inhibit ASIC currents (259). The potential involvement of ASICs and other ion channels in pain sensation has been reviewed (131, 175, 269).

In the CNS, tissue acidosis is a well-established feature of cerebral ischemia which aggravates cell damage (reviewed in Ref. 161). Transient global ischemia induces expression of ASIC2a protein in neurons that survive ischemia (129). ASICs may also be involved in controlling neuronal activity associated with external pH fluctuations (44). A significant acidification of the extracellular space has been found to be associated with repetitive stimulation and epileptiform activity (43, 44, 233, 253, 270). Recently, it has been shown that expression levels of ASIC1a and ASIC2b (but not ASIC2a) decrease in specific brain regions in the pilocarpine model of epilepsy (26). The question remains if in vivo extracellular pH changes are large enough to activate ASIC channels. It is possible that under physiological conditions endogenous ligands of ASICs such as Zn²⁺ or FMRFamide-related peptides may facilitate activation (8, 16).

ASIC2 knockout mice (200) had normal appearance, growth, size, temperature, fertility, and life span, with no obvious defects in pain sensation. The only phenotype that could be detected in an in vitro skin-nerve preparation from ASIC2^/ mice was a reduced sensitivity of a specific component of mechanosensation, involving the low-threshold rapidly adapting mechanoreceptors. In rodent hairy skin, these mechanoreceptors are excited by hair movement. Consistent with this function, ASIC2a/b was found in the lanceolate nerve endings that lie adjacent to and surround the hair follicle. In a recent immunocytochemical study, expression of ASIC2a was found in specialized cutaneous mechanosensory nerve terminals (86). The relatively discrete phenotype due to ASIC2 loss of function could be due to a redundancy of ASIC proteins in the formation of heteromultimeric channels.

Mechanosensory transduction refers to processes that convert mechanical forces into bioelectrical signals. Mechanotransduction is important for different physiological functions such as touch, hearing, or proprioception. The extensive genetic dissection of mechanosensory behavior in C. elegans led to the identification of a number of genes involved in the development, survival, function, and regulation of touch receptor neurons (37). Among these genes, the degenerins that form a subgroup of the ENaC/DEG family play a critical role in touch sensation and proprioception.

Specific mutations in MEC-4, MEC-10, UNC-8, UNC-105, or DEG-1 cause swelling and subsequent death of the cells in which the mutant proteins are expressed. These proteins were called degenerins because of the cell degenerative phenotype they cause when mutated (reviewed in Refs. 171, 243). Typical morphological features involved in this phenomenon include the production of membrane infoldings, membrane whorls, and vacuoles that resemble those found in excitotoxic cell death after ischemia, hypoxia, or epilepsy. The initial response of the cell is an enhanced membrane cycling that may reflect attempts to dilute or to sequester harmful components localized to various membranes and to translocate them to degradative vacuoles (101). On the basis of their homology with ENaC, it has been proposed that these degenerins function as mechanosensory channels which when mutated become hyperactive leading to increased cation influx into the cell and to cell death.

Mechanotransduction in C. elegans has been described in detail in several recent reviews (82, 171, 243). When a C. elegans moving over an agar plate is gently touched with an eyelash hair on the posterior part of the body, the animal will move forward, and when touched on the anterior body, it will move backward. This gentle touch is sensed by six touch receptor neurons. Their processes run longitudinally along the body wall on the ventral and both lateral sides and are embedded in the hypodermis (the "worm skin"), to which they appear to be glued by an extracellular material called the mantle (reviewed in Refs. 82, 243). The position of the processes along the body axis correlates with the sensory field of the touch cells.

Genetic analysis has identified 16 genes which, when mutated, specifically disrupt gentle body touch sensation; they represent, therefore, candidate mediators of touch sensitivity. These genes were named mec genes because when defective the animals become "mechanosensory abnormal." Many of the mec genes have been molecularly identified, and the encoded proteins were postulated to form a touch-transducing complex that converts the mechanical stimuli into an electrical signal. Only some of the mec genes belong to the ENaC/DEG family such as MEC-4 (243) or MEC-10 expressed in the touch receptor neurons (110).

The transduction of mechanical stimuli into bioelectrical signals requires activation of an ion channel to generate an ionic current. So far, functional expression experiments have failed to demonstrate mechanically inducible channel activity. Constitutively active channels were obtained after coexpression of MEC-4 and MEC-10 with the stomatin-related protein MEC-2 (Fig. 4) or upon expression of UNC-105 containing activating mutations (84, 90). However, the mechanosensitivity of these degenerins still remains to be demonstrated in heterologous expression systems.

UNC-8 (unc stands for uncoordinated) is expressed in several sensory neurons, interneurons, and the motorneurons VA and VB (245). This subclass of motorneurons shows the peculiar neuroanatomical feature of having their synapses with the muscle or the interneurons restricted to a limited region close to the cell body. Similar anatomical features are found in corresponding motorneurons in the nematode Ascaris suum that are known to be stretch responsive, suggesting that VA and VB motorneurons in C. elegans have similar functions (57). The degenerins UNC-8 and DEL-1 are coexpressed in the ventral cord VA and VB motorneurons. Functional evidence for involvement of UNC-8 in modulation of locomotion comes from observation of wild-type and UNC-8 mutant animals. When moving on agar plates, wild-type worms inscribe a sinusoidal wave. In unc-8 null mutants, the amplitude is reduced by ~3.5-fold (245). It has been proposed that the contribution of UNC-8 to modulation of nematode locomotion relies on feedback information on body posture, thus on an activity that is related to proprioception (243, 245). Alternatively UNC-8, which is also expressed in interneurons that regulate locomotion, might act within these neurons to modulate coordinated movement. The only evidence for the involvement of DEL-1 in modulation of C. elegans locomotion lies in its coexpression with UNC-8 in the VA and VB motorneurons and its homology to other ENaC/DEG family members (243, 245). Like MEC-4 and MEC-10, UNC-8 and DEL-1 are thought to form the ion channel core of the stretch-sensitive complex mediating modulation of C. elegans locomotion.

Yet another member of the C. elegans degenerin subfamily contributes to proprioception by monitoring muscle stretch. The UNC-105 gene is not expressed in neurons but in muscles. Mutations of the UNC-105 ENaC/DEG gene have been identified that create constitutively activated channels that cause muscle hypercontraction (163, 194), presumably because muscle cells are depolarized by an inappropriate cation influx through the UNC-105 channels. As mentioned above, expression of UNC-105 containing activating mutations in Xenopus oocytes results in ion channel activity, but mechanosensitivity of the UNC-105 channel has not been demonstrated (84).

Among the 24 predicted proteins of Drosophila related to the ENaC/DEG family (162), only Ripped Pocket (RPK/dGNaC1) (3, 55) and Pickpocket (PPK/dmdNaC1) (3, 56) have so far been characterized with regard to their function and/or tissue distribution. RPK/dGNaC1 transcripts are present exclusively in fly ovary and testis; they are maternally deposited into the embryo, where they persist until late gastrulation. The expression of RPK/dGNaC1 transcripts in Drosophila is restricted to oocytes in late vitellogenic stages and to early embryos, as well as to nurse and follicular cells. This suggests that RPK/dGNaC1 is involved in early development. When expressed in Xenopus oocytes RPK/dGNaC1 forms functional Na⁺-selective channels with low apparent affinity for amiloride (see Table 1). It was not possible to record any RPK/dGNaC1 activity in transfected mammalian COS cells. This observation was interpreted as RPK/dGNaC1 activity being modulated by specific factors that are present only during early development and might therefore be present in oocytes but not in COS cells (55). Amiloride-sensitive Na⁺ channels in mammalian embryos play an important role in fluid transport across the trophectoderm and in the formation of the blastocyst (3, 55). Like RPK/dGNaC1, these channels appear to have a low sensitivity to amiloride (K_i = 12 µM, Ref. 207). RPK/dGNaC1 and related members in Drosophila may play roles in fluid distribution and cell volume regulation during gametogenesis and early development.

The PPK/dmdNaC1 gene product appears at later developmental stages of Drosophila. It is expressed in sensory dendrites of a subset of peripheral neurons in late-stage embryos and early larvae. The late PPK/dmdNaC1 expression coincides with the end of elaboration of the nervous system, after the complete determination and differentiation of neuroprecursor cells. This led to the hypothesis that PPK/dmdNaC1 is not involved in the differentiation of the peripheral nervous system but rather in the function of PPK/dmdNaC1-expressing neurons (56). In insects, multiple dendritic neurons in which PPK/dmdNaC1 is expressed are thought to play a role in touch sensation and proprioception. For this reason, it has been proposed that in analogy to degenerins, PPK/dmdNaC1 may function as a mechanotransduction channel. PPK/dmdNaC1 does not produce functional ion channels when expressed in Xenopus oocytes (3, 56).

The FMRFamide (Phe-Met-Arg-Phe amide)-gated channel, or FaNaC, was originally cloned from a cDNA library from the snail Helix aspersa nervous system, based on its homology to ENaC/DEG family members. FaNaC mRNA is detected in the nervous system and in pedal muscle of this snail (157). The biophysical and pharmacological properties of the cloned FaNaC expressed in Xenopus oocytes are very similar to those of the native FMRFamide-gated current in Helix neurons (51, 93, 94). FMRFamide induces a fast excitatory depolarizing response due to direct activation of the channel in neurons of the snail Helix aspersa as well as in Aplysia motor neurons (19, 94, 211). This fast response is readily distinguished from other, slower neuronal FMRFamide responses in Helix aspersa (reviewed in Ref. 51) that depend on G proteins. Evidence that the peptide directly gates a channel was obtained using isolated membrane patches from the Helix C2 neuron and from Xenopus oocytes expressing the cloned FaNaC. Inward unitary currents could be generated by external application of FMRFamide to excised outside-out patches when G protein-mediated responses were inhibited (94, 157). This finding represented the first functional description of a peptide-gated ion channel. Recently, the cDNAs encoding FMRFamide-gated channels from two other mollusks, Helisoma trivolvis and Lymnaea stagnalis, were cloned (127, 196). These different clones share 60-65% identity of the amino acid sequence. So far, no mammalian homologs of FaNaC have been identified (8, 256), but FMRFamide and the mammalian neuropeptide FF as well as related peptides can modulate the activity of ASICs in heterologous expression systems (8).

	IV. STRUCTURAL ASPECTS

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The size of the ENaC/DEG proteins ranges from ~530 to ~740 amino acids. Regions of conserved amino acid sequences in ENaC/DEG family members are likely to represent structural elements important for channel function. These conserved domains shown in Fig. 3, A and B, are located essentially in the transmembrane segments and their proximity and in the extracellular loop.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Conserved domains and their localization in ENaC/DEG family members. A: linear representation of the primary structure shows the conserved regions found in each of the main subfamilies. The length of the domains in the diagram reflects their relative size in the protein. M1, M2, transmembrane segments; HG, conserved His-Gly motif within a conserved NH2-terminal domain; pM1, post-M1, conserved domain directly downstream of M1; CRD, cysteine-rich domain; pre-M2, hydrophobic domain directly preceding M2; ERD, extracellular regulatory domain unique to C. elegans degenerins; NTD, neurotoxin domain (244); PY, PPPxY domain of -, -, and -ENaC. B: membrane topological organization of an ENaC/DEG subunit. The part that is unique to degenerins (containing CRDI and ERD) is shown on a gray background encircled by an interrupted line. Abbreviations are as in A. The residue in the pre-M2 domain whose mutation induces degeneration of the cells expressing these mutant degenerins is indicated as "deg."

A few sequences are completely conserved among the ENaC/DEG family members; they include a HG motif (His-Gly) located in the NH₂-terminal cytoplasmic domain in close proximity to the M1 segment, a FPxxTxC sequence following M1 (post-M1) and completely conserved residues in the M2 region. The extracellular loop contains cysteine-rich domains (CRD) II and III. The conserved Cys residues might be involved in the formation of disulfide bonds to maintain the tertiary structure of the large extracellular loop. Candidate cysteine residues in the extracellular loop of -ENaC involved in disulfide bridges have been identified by tandem Cys mutations and functional analysis (77).

Other domains are conserved only within particular ENaC/DEG subfamilies. Two domains in the extracellular loop are conserved exclusively within the degenerin subfamily, the cysteine-rich domain CRDI and the extracellular regulatory domain ERD (85). ENaC subunits contain conserved proline-rich motifs in the cytoplasmic COOH terminus that are unique to the ENaC subfamily. These motifs have the consensus sequence PPPXYXXL and are involved in protein-protein interactions.

The membrane topology has been investigated essentially for ENaC, but we can assume from the overall sequence homology that this topology is shared by the other ENaC/DEG family members. Analysis of the membrane topology of ENaC was performed by protease digestion analysis of -ENaC translated in vitro and reconstituted in microsomes (205). This analysis was consistent with M1 and M2 being separated by a large extracellular loop of ~50 kDa, the NH₂ and COOH termini being cytoplasmic. This large extracellular loop makes up more than one-half of the channel protein and represents a structural feature unique to the ENaC/DEG family members that is not found in other ion channel families. Several putative glycosylation sites are present in the extracellular loop of ENaC subunits (6 for , 12 for , and 5 for ). Site-directed mutagenesis in -ENaC showed that the six potential N-glycosylation sites between M1 and M2 but not the two putative N-glycosylation sites preceding M1 are used, demonstrating the extracellular topology of the loop between M1 and M2 (32, 230). In addition, with the use of truncated -ENaC fused with the Na⁺-K⁺-ATPase -subunit that is known to be glycosylated, it was demonstrated that M1 and M2 serve as start-transfer and stop-transfer signals, respectively, consistent with both M1 and M2 crossing the membrane and leaving the COOH terminus cytoplasmic (32).

The hydrophobic profile of ENaC subunits reveals a stretch of hydrophobic amino acid residues preceding M1 and M2. The protease digestion pattern of -ENaC is consistent with the presence of a short segment preceding M2 that is accessible for the protease from the cytoplasmic side (205). In all known Na⁺- or K⁺-selective cation channels, the short hydrophobic segment preceding the most COOH-terminal transmembrane domain forms the outer pore of the channel with the receptor sites for external blockers. This analogy led to the suggestion that the pre-M2 segment of ENaC subunits may contribute to the formation of the channel pore.

B.  Multimeric Channels and Subunit Stoichiometry

1.  ENaC/FaNaC

The initial cloning experiments demonstrated that the three homologous ENaC subunits , , and  are required for maximal expression of ENaC activity (33). Thus it became clear that the structure of ENaC is a heteromultimeric channel similar to the nicotinic acetylcholine receptor. There is presently good evidence that the three ENaC subunits contribute to the formation of the channel pore and line a unique ion permeation pathway (137, 138, 220, 223, 231) (see sect. V). It should be mentioned that other models for the subunit arrangement around the channel pore have been proposed based on functional experiments with channels reconstituted in lipid bilayers. According to these models, ENaC shows a triple barrel organization to account for the different subconductance states of the channel reconstituted in lipid bilayers (22). Such models for ENaC have not gained any experimental support from functional studies in cellular expression systems such as the Xenopus oocyte.

The relative abundance of the three ENaC subunits at the cell surface in Xenopus oocytes expressing amiloride-sensitive Na⁺ current was determined by quantitative analysis of the binding of radiolabeled antibodies directed against a FLAG epitope introduced into the extracellular domain of -, -, and -subunits (78). The low current expression after expression of -ENaC alone is correlated with a low level of -ENaC subunit expression at the cell surface, suggesting a retention of -ENaC in the endoplasmic reticulum due to the absence of a molecular signal essential for targeting the channel to the cell surface. In oocytes expressing the three ENaC subunits , , and , the -subunit is approximately two times more abundant in active ENaC channels at the cell surface compared with - or -subunits (76). This 2:1:1 ratio of channel subunits remains constant even under conditions where one subunit is overexpressed relative to the two other homologous subunits (76), consistent with a preferred fixed subunit stoichiometry when the three ENaC subunits are available for channel assembly.

Despite the preferred subunit stoichiometry of ENaC, the -, -, and -subunits are to some extent interchangeable. Expression of - or -ENaC alone in Xenopus oocytes has been shown to generate small amiloride-sensitive currents, suggesting that homomeric -ENaC and -ENaC channels are functional. Expression of the ENaC subunit combinations - or - generates a similar level of amiloride-sensitive current that is higher than after expression of -ENaC alone, suggesting that - and -subunits can replace each other in the channel complex (33, 78, 177). Early expression experiments in Xenopus oocytes indicated that - channels were not expressed at the cell surface (33, 78). However, it has been later shown that 6 days after injection of cRNA encoding - and -ENaC subunits a small but significant amiloride-sensitive current could be detected, suggesting that functional channels can be formed with - and/or -subunits (27).

In the absence of high-resolution images, the determination of the number of subunits in the functional channel relies on a biochemical analysis of the channel complex at the cell surface. This approach was first performed on FaNaC because this channel is functional as a homomultimer. In addition, the mature FaNaC at the cell surface is fully glycosylated and thus biochemically identifiable. Following the maturation process and the oligomerization of the channel complex, Coscoy et al. (50) showed that the tetrameric assembly of the channel occurs already in the endoplasmic reticulum where this immature oligomeric complex is core glycosylated. Chemical cross-linking experiments were consistent with a minimal stoichiometry of four subunits, and the hydrodynamic characterization of the channel complex revealed a maximal number of five subunits (50).

Alternatively, the number of homologous subunits forming a channel can be assessed by the quantitative analysis of the contribution of each subunit to channel function. The identification of mutations in -, -, and -subunits that confer to the channel a resistance to block by amiloride or a sensitivity to block by Zn²⁺ allowed the design of experiments to assess for each of the three ENaC subunits the number of subunits per channel complex that contribute to a particular channel function (76, 220). These experiments followed the basic principles originally described to determine the subunit stoichiometry of the Shaker K⁺ channel by studying the interaction of a toxin with channels made of different fractions of wild-type and toxin-resistant mutant subunits (170). The Zn²⁺ sensitivity of ENaC was conferred to the channel when the channel complex contained two -subunits carrying a cysteine substitution at the amiloride binding site (76). Inversely, block by the sulfhydryl reagent 2-aminoethyl methanethiosulfonate (MTSEA) was conferred to the channel when at least one of two -subunits carried the same cysteine substitution (146). These experiments were consistent with the presence of two -ENaC subunits in the functional channel. The same approach using point mutations on - or -ENaC that confer amiloride resistance did not provide any information about the absolute number of - and -subunits, since the magnitude of the amiloride-resistant current was directly proportional to the fraction of mutant - or -subunit expressed (76, 146).

In contrast to FaNaC, ENaC at the cell surface of Xenopus oocytes is not fully glycosylated, making a biochemical approach difficult for assessing the channel subunit stoichiometry. Subunit stoichiometry of ENaC can be assessed by expression of trimeric or tetrameric constructs made of -, -, and -ENaC subunits linked in a head-to-tail fashion. The rationale of this approach relies on the assumption that the correct concatameric construct does not need to be complemented by monomeric subunits for channel function (145). The analysis revealed that the --- construct is sufficient for the expression of functional channels and does not require complementation by additional monomeric subunits (76). Thus, taken together, the following observations support a tetrameric subunit organization around the channel pore, as illustrated in Figure 4A: 1) the surface expression of -ENaC per active channel is approximately two times higher than for , 2) there are consistently two -subunits per active channel, 3) the number of - and -subunits are equal, and 4) --- is the minimal concatameric construct for functional channel expression. This tetrameric subunit composition of ENaC is consistent with the subunit stoichiometry of other Na⁺- or K⁺-selective ion channels and in particular with the stoichiometry of channels made of subunits with two transmembrane domains.

View larger version (54K): [in this window] [in a new window]   Fig. 4. Model of ENaC/DEG channel complexes. A: model of the tetrameric assembly of an ENaC channel. The model shows the tetrameric assembly of ENaC subunits around the central channel pore. The model is based on functional analyses that showed that all ENaC subunits participate in the formation of the channel pore and established the subunit stoichiometry (76). B: model of a touch-transducing complex in C. elegans. This model is based on genetic analysis. Touch transduction is thought to be mediated by a degenerin ion channel made of at least two MEC-4 (light gray) and two MEC-10 (dark gray) subunits. The large extracellular domains of the subunits project into a specialized extracellular matrix called the mantle, which includes the MEC-5 collagen and the MEC-9 protein. Inside the touch receptor neuron are special microtubules made up of the MEC-12 -tubulin and the MEC-7 -tubulin. The microtubules are proposed to be linked to the channel by means of stomatin-related MEC-2, a peripheral membrane protein. Proteins tethered to the extracellular and intracellular sides are thought to exert pressure on the channel, conferring gating tension when the channel is stretched between these two contact points. [Adapted from Mano and Driscoll (171).]

With a similar approach looking at interactions of sulfhydryl reagents with ENaC wild-type and mutants together with the biochemical determination of the molecular mass of ENaC and FaNaCh, Snyder et al. (229) reached the conclusion that ENaC as well as other members of the ENaC/DEG gene family are made of nine subunits: three -, three -, and three -subunits in the case of ENaC. This nonameric channel stoichiometry was further supported by freeze-fracture electron microscopy in oocytes expressing ENaC (73). Such high-magnification images revealed particles of 8 nm in diameter compatible with ENaC channels. Based on the assumption that the cross-sectional area of membrane proteins in freeze-fracture microscopy is proportional to the number of the membrane-spanning domains, ENaC was found to have 17 membrane-spanning segments corresponding to 8 or 9 subunits. These conclusions should be taken with caution because the relationship between the cross-sectional area and the number of transmembrane segments is certainly not constant for all membrane proteins, and the functional ENaC channel complex may well include additional helical segments embedded in the membrane or associated proteins.

C. elegans degenerins are implicated in touch sensation and proprioception. Based on the genetic analysis of C. elegans mutants with impaired touch and stretch sensation, a model of the touch-transducing complex was proposed in which the degenerins MEC-4 and MEC-10 constitute the channel core of the mechanosensory transduction complex (see Fig. 4B) (70). MEC-4 and MEC-10 subunits are coexpressed in touch receptor neurons, and genetic interactions could be demonstrated between mec-4 and mec-10 (100, 110, 243). In Xenopus oocytes expressing MEC-4 and MEC-10, both proteins could be immunoprecipitated, suggesting the presence of functional heteromeric channels (90). The number of MEC subunits that form the channel pore has not yet been established, but genetic evidence is compatible with at least two MEC-4 and two MEC-10 subunits per channel (108, 110).

Because mechanosensation could not be reconstituted in heterologous expression systems with MEC-4 and MEC-10 alone, additional associated proteins likely participate in the functional complex at the cell surface. Degeneration caused by mutations of a number of degenerins (deg-1, mec-4, mec-10, and unc-8) requires an additional gene, MEC-6, essential for activation of the mechanosensitive channel (reviewed in Ref. 243). MEC-6 has not been cloned yet and might encode a protein that mediates localization or posttranslational modification of the putative channel-forming subunits. In addition, MEC-2, a stomatin-related protein essential for touch sensation, is associated with the channel complex and increases channel activity in oocytes expressing MEC-4 and MEC-10 (90).

To sense mechanical stimuli, the large extracellular loops of MEC-4 /MEC-10 channels are postulated to be linked to the touch cell-specific extracellular matrix, by interaction with the MEC-5 collagen and/or MEC-9, a large protein rich in interaction domains such as epidermal growth factor (EGF)-like repeats (2 of them of the Ca²⁺-binding type) and Kunitz protease inhibitor domains (68) (see Fig. 4B). The intracellular domains of MEC-4 and MEC-10 are hypothesized to be tied to the unique 15-protofilament microtubules (made of the MEC-12 -tubulin and the MEC-7 -tubulin) by the linker protein MEC-2, which is related to stomatin (reviewed in Ref. 243). By anchoring the channel on both sides of the membrane, displacement of the extracellular matrix with respect to the cytoskeleton is supposed to open the channel in response to mechanical stimuli. A model for the stretch-sensitive complex mediating proprioception similar to that of the touch-transducing complex has been proposed, containing UNC-8 and DEL-1 as subunits of the channel core in the mechanotransducing complex (243).

In summary, there is good evidence that C. elegans DEG subfamily members represent the channel subunits of a large mechanotransduction complex in the plasma membrane. The channel core of this complex is heteromultimeric and made of at least four DEG channel subunits belonging to the ENaC/DEG family. Other components might be involved in this mechanotransduction complex to anchor the channel to cell constituents on both sides of the membrane.

The human -ENaC gene (SCNN1A) covers 17 kb on chromosome 12p13. The human - and -ENaC genes (SCNN1B and SCNN1G) are located on chromosome 16p in very close proximity, suggesting that the two genes arise from gene duplication. The three ENaC genes , , and  share a remarkable degree of conservation in their genomic organization (45, 169, 213, 249). The human ENaC genes are divided into 13 exons; the 2 transmembrane regions of ENaC proteins are encoded by parts of exon 2 and exon 13. The -ENaC gene is located on the chromosome 1.

The promoters of ENaC genes remain to be precisely identified. In human and rat, the region within 75 bp upstream of the transcription start site of the -ENaC gene contains two GC-rich boxes that are sufficient for promoter activity (248). Analysis of the nucleotide sequence in the region farther upstream between 289 and 142 shows two imperfect glucocorticoid response elements (GRE) that represent potential transcriptional regulatory elements necessary for ENaC regulation by glucocorticoids (214). Sequential deletions in this region showed that the downstream GRE is sufficient to confer glucocorticoid stimulation and is also able to bind glucocorticoids specifically. An additional third GRE motif further upstream (between 300 and 2,400) has been identified in the 5'-flanking region of human -ENaC. Reporter constructs containing this GRE motif also exhibit glucocorticoid-inducible expression (45). So far no specific mineralocorticoid response element has been identified, and the elements that determine the tissue specificity of the glucocorticoid versus mineralocorticoid regulation of ENaC genes remain to be elucidated.

ASIC1, ASIC2, ASIC3, and ASIC4 are located on human chromosomes 12, 17, 7, and 2, respectively (61, 256).

	V. ION CONDUCTANCE AND THE CHANNEL PORE

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The functional properties of the ENaC/DEG channels have been investigated in native tissues and in cell expression systems using electrophysiological techniques. Long before its cloning by functional expression, the highly selective amiloride-sensitive ENaC was functionally characterized in tight epithelia such as frog skin and toad urinary bladder. More recently, ENaC function at the single-channel level could be resolved in the rat cortical collecting tubule using the patch-clamp technique (87). Some members of the ENaC/DEG family exhibit macroscopic ionic currents when expressed in Xenopus oocytes, but the single-channel characteristics have not yet been determined (Table 1). Electrophysiological investigations of native C. elegans cells are possible but remain difficult because of their small size (91, 165). In cell expression systems, hINaC, BLINaC, and UNC-105 channels simply do not express ionic currents unless an activating mutation is introduced in their sequence. Thus much more functional data on ion permeation are available for the amiloride-sensitive ENaC channel compared with other family members.

The macroscopic amiloride-sensitive conductance measured in toad bladder or frog skin was found to be selective for the small inorganic Na⁺ and Li⁺. In these tissues, the Na⁺/K⁺ selectivity ratio was estimated to be >500 (20, 185). This high selectivity of ENaC was confirmed at the single-channel level in the mammalian CCD (190) and later in oocytes expressing the -, -, and -ENaC subunits (138). In the toad bladder an amiloride-sensitive current carried by protons could be measured when the mucosal milieu was acidified below pH 5, indicating that protons can permeate the epithelial Na⁺ channel (185, 186). The proton permeability of ENaC expressed in Xenopus oocytes has not been addressed yet. Thus ENaC seems to discriminate among cations based on their size, small cations such as Na⁺, Li⁺, or protons being permeant whereas larger cations such as K⁺ or organic NH cannot pass through the channel. This simple rule for the ionic permeability of ENaC applies only to monovalent cations, because ENaC is not permeable to divalent cations. In the CCD and in Xenopus oocytes expressing -, -, and -ENaC, the unitary conductance of the amiloride-sensitive epithelial Na⁺ channel measured in the presence of 140 mM Na⁺ at room temperature is 5 pS (33, 138, 190). A typical trace of ENaC single-channel activity is shown in Figure 5A. The single-channel conductance of ENaC saturates at external Na⁺ concentrations above 100 mM with a concentration for half-maximal conductance (K_m) around 20-50 mM extracellular Na⁺ (138, 184, 192). In the presence of 100-150 mM external Li⁺, the single-channel conductance measured at room temperature is 9-10 pS in the cortical collecting tubule and in Xenopus oocytes expressing -, -, and -ENaC. Similar values were measured in a canine kidney cell line (Madin-Darby canine kidney cells) stably transfected with -, -, and -ENaC (117). The apparent affinity for Li⁺ is lower than that for Na⁺, with a K_m for Li⁺ around 90 mM in cortical collecting tubule and 120 mM in Xenopus oocytes (138, 192). A higher dissociation rate of Li⁺ from its binding site in the channel pore toward the cytoplasmic side can account for the lower affinity for Li⁺ compared with Na⁺ and for the faster movement of Li⁺ through the channel, i.e., the higher channel conductance for Li⁺. These data obtained in the native tissue and in heterologous expression systems set unambiguously the functional conductance signature of ENaC: 4-5 pS and 9-10 pS conductance with Na⁺ and Li⁺ at concentrations on the order of 100-150 mM and a high Na⁺/K⁺ selectivity.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Functional fingerprints of some ENaC/DEG proteins. A: ENaC single-channel trace from an outside-out patch of an oocyte expressing rat