A long isoform of the epithelial sodium channel alpha subunit forms a highly active channel

A long isoform of the human Epithelial Sodium Channel (ENaC) α subunit has been identified, but little data exist regarding the properties or regulation of channels formed by α₇₂₈. The baseline whole cell conductance of oocytes expressing trimeric α₇₂₈βγ channels was 898.1 ± 277.2 and 49.59 ± 13.2 µS in low and high sodium solutions, respectively, and was 11 and 2 fold higher than the conductances of α₆₆₉βγ in same solutions. α₇₂₈βγ channels were also 2 to 5 fold less sensitive to activation by the serine proteases subtilisin and trypsin than α₆₆₉βγ in low and high Na⁺ conditions. The long isoform exhibited lower levels of full length and cleaved protein at the plasma membrane and a rightward shifted sensitivity to inhibition by increases of [Na⁺]_i. Both channels displayed similar single channel conductances of 4 pS, and both were activated to a similar extent by reducing temperature, altogether indicating that activation of baseline conductance of α₇₂₈βγ was likely mediated by enhanced channel activity or open probability. Expression of α₇₂₈ in native kidneys was validated in human urinary exosomes. These data demonstrate that the long isoform of αENaC forms the structural basis of a channel with different activity and regulation, which may not be easily distinguishable in native tissue, but may underlie sodium hyperabsorption and salt sensitive differences in humans.     

Sensitivity of oocyte-expressed epithelial Na channel to glibenclamide

The effect of glibenclamide on heterologously expressed amiloride-sensitive sodium channels (ENaCs) was investigated in Xenopus oocytes. The ENaC is a heteromer and consists of α-, β- and γ-subunits and the α- and β-subunits have previously been shown to confer sensitivity to glibenclamide. We coexpressed either colonic rat α- (rα) or guinea-pig α-subunit (gpα) with Xenopus βγ-subunits. The gpαxβγ was significantly stimulated by glibenclamide (100 μM) (184±15%), whereas the rα-combination was slightly down-regulated by the sulfonylurea (79±4%). The stimulating effect did not interfere with Na⁺-self-inhibition resulting from intracellular accumulation of Na⁺-ions. We exchanged cytosolic termini between both orthologs but the gpα-chimera with the termini from rat retained sensitivity to glibenclamide. The effect of glibenclamide on Xenopus ENaC (xENaC) was inhibited by ADP-β-S but not by ATP-γ-S, when applied intracellularly. Intracellular loading with Na⁺-ions after inhibition of Na⁺/K⁺-ATPases with ouabain prevented an up-regulation of ENaC activity by glibenclamide. Pretreatment of oocytes expressing xENaC with edelfosine (ET-18-OCH₃) slightly reduced stimulation of I_ami (118±12%; control: 132±9%) while phosphatidylinositol-4,5-biphosphate (PIP₂) significantly reduced the effect of glibenclamide to 101±3%.     

Frog atrial natriuretic peptide and cGMP activate amiloride-sensitive Na<Superscript>+</Superscript> channels in urinary bladder cells of Japanese tree frog, <Emphasis Type="Italic">Hyla japonica</Emphasis>

In our previous study, it was suggested that ANP and cGMP may increase Na⁺ absorption in the urinary bladder of the Japanese tree frog, Hyla japonica. Thus, Na⁺ transport activated by ANP was investigated electrophysiologically by using a cell-attached patch-clamp technique in freshly isolated cells from the urinary bladder. A predominant channel expressed was a low conductance Na⁺ channel in the epithelial cells. The channel exhibited conductance for inward currents of 4.9 ± 0.2 pS, long open and closed times (c.a. 190 ms), and positive reversal potential. The channel activity was decreased under the pipette solution including 10⁻⁶ M amiloride. These characteristics were similar to those of amiloride-sensitive Na⁺ channels (ENaC). Addition of 10⁻⁹ M ANP activated and significantly increased the ENaC activity from 0.58 ± 0.09 to 1.47 ± 0.34. On the other hand, mean amplitudes and conductance of single channel did not change significantly after the addition of ANP. Addition of 10⁻⁵ M 8-Br-cGMP also activated the ENaC and significantly increased the channel activity from 0.56 ± 0.10 to 2.00 ± 0.33. The addition of ANP failed to activate the ENaC in the presence of 10⁻⁶ M amiloride. These results suggested that ANP and cGMP activate Na⁺ transport via ENaC in the epithelial cells of frog urinary bladder.     

Non-specific activation of the epithelial sodium channel by the CFTR chloride channel

The genetic disease cystic fibrosis is caused by mutation of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Controversial studies reported regulation of the epithelial sodium channel (ENaC) by CFTR. We found that uptake of ²²Na⁺ through ENaC is modulated by activation of CFTR in oocytes, coexpressing CFTR and ENaC, depending on extracellular chloride concentration. Furthermore we found that the effect of CFTR activation could be mimicked by other chloride channels. Voltage– and patch–clamp measurements, however, showed neither stimulation nor inhibition of ENaC-mediated conductance by activated CFTR. We conclude that the observed modulation of ²²Na⁺ uptake by activated CFTR is due to the effect of CFTR-mediated chloride conductance on the membrane potential. These findings argue against the notion of a specific influence of CFTR on ENaC and emphasize the chloride channel function of CFTR.     

Of cilia,titin, and neurosteroids

Missense mutations at arginine residues in the S4 voltage-sensor domains of Na_V1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K⁺. Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K⁺ during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K⁺, yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na⁺ current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type Na_V1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na⁺ was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼10 µS/cm² or 1% of the total resting conductance.Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na⁺ current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na⁺ current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of Na_V, Ca_V, and K_V channels associated with human disease.     

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

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

Associated Proteins and Renal Epithelial Na+ Channel Function

The hypothesis that amiloride-sensitive Na⁺ channel complexes immunopurified from bovine renal papillary collecting tubules contain, as their core conduction component, an ENaC subunit, was tested by functional and immunological criteria. Disulfide bond reduction with dithiothreitol (DTT) of renal Na⁺ channels incorporated into planar lipid bilayers caused a reduction of single channel conductance from 40 pS to 13 pS, and uncoupled PKA regulation of this channel. The cation permeability sequence, as assessed from bi-ionic reversal potential measurements, and apparent amiloride equilibrium dissociation constant (K ^amil _i ) of the Na⁺ channels were unaltered by DTT treatment. Like ENaC, the DTT treated renal channel became mechanosensitive, and displayed a substantial decrease in K ^amil _i following stretch (0.44 ± 0.12 μm versus 6.9 ± 1.0 μm). Moreover, stretch activation induced a loss in the channel's ability to discriminate between monovalent cations, and even allowed Ca²⁺ to permeate. Polyclonal antibodies generated against a fusion protein of αbENaC recognized a 70 kDa polypeptide component of the renal Na⁺ channel complex. These data suggest that ENaC is present in the immunopurified renal Na⁺ channel protein complex, and that PKA sensitivity is conferred by other associated proteins. Received: 5 June 1995/Revised: 29 September 1995     

Multiple conductance levels of the dihydropyridine-sensitive calcium channel in GH3 cells

Summary Calcium channels in GH₃ cells exhibit at least five conductance levels when examined in cell-attached or outside-out patches. These channels resemble the high threshold Ca²⁺ current in their range of activation and inactivation, and in their sensitivity to dihydropyridines (DHP). Mean open times for the five levels were brief (<1 msec) in control solutions but increased in the presence of BAY K 8644. In 100mm Ba²⁺ and BAY K 8644, the five predominant slope conductances were 8–9, 12–13, 16–18, 23–24, and 28 pS. The present study is the first report of multiple levels of the DHP-sensitive Ca²⁺ channel occurring with high frequency in native membranes. The range of conductance levels that we observed encompasses the range of conductances found for two other different types of Ca²⁺ channels and indicates that unit conductance should be used with caution as a distinguishing characteristic for identification of different channel types.     

Modulation of distinct isoforms of L-type calcium channels by Gq-coupled receptors in Xenopus oocytes

L-type voltage dependent Ca²⁺ channels (L-VDCCs; Ca_v1.2) are crucial in cardiovascular physiology. In heart and smooth muscle, hormones and transmitters operating via G_q enhance L-VDCC currents via essential protein kinase C (PKC) involvement. Heterologous reconstitution studies in Xenopus oocytes suggested that PKC and G_q-coupled receptors increased L-VDCC currents only in cardiac long N-terminus (NT) isoforms of α_1C, whereas known smooth muscle short-NT isoforms were inhibited by PKC and G_q activators. We report a novel regulation of the long-NT α_1C isoform by Gβγ. Gβγ inhibited whereas a Gβγ scavenger protein augmented the G_q- but not phorbol ester-mediated enhancement of channel activity, suggesting that Gβγ acts upstream from PKC. In vitro binding experiments reveal binding of both Gβγ and PKC to α_1C-NT. However, PKC modulation was not altered by mutations of multiple potential phosphorylation sites in the NT, and was attenuated by a mutation of C-terminally located serine S1928. The insertion of exon 9a in intracellular loop 1 rendered the short-NT α_1C sensitive to PKC stimulation and to Gβγ scavenging. Our results suggest a complex antagonistic interplay between G_q-activated PKC and Gβγ in regulation of L-VDCC, in which multiple cytosolic segments of α_1C are involved.     

Feedback inhibition of ENaC: Acute and chronic mechanisms

Intracellular [Na⁺] ([Na⁺]_i) modulates the activity of the epithelial Na channel (ENaC) to help prevent cell swelling and regulate epithelial Na⁺ transport, but the underlying mechanisms remain unclear. We show here that short-term (60–80 min) incubation of ENaC-expressing oocytes in high Na⁺ results in a 75% decrease in channel activity. When the β subunit was truncated, corresponding to a gain-of-function mutation found in Liddle's syndrome, the same maneuver reduced activity by 45% despite a larger increase in [Na⁺]_i. In both cases the inhibition occurred with little to no change in cell-surface expression of γENaC. Long-term incubation (18 hours) in high Na⁺ reduced activity by 92% and 75% in wild-type channels and Liddle's mutant, respectively, with concomitant 70% and 52% decreases in cell-surface γENaC. In the presence of Brefeldin A to inhibit forward protein trafficking, high-Na⁺ incubation decreased wt ENaC activity by 52% and 88% after 4 and 8 hour incubations, respectively. Cleaved γENaC at the cell surface had lifetimes at the surface of 6 hrs in low Na⁺ and 4 hrs in high Na⁺, suggesting that [Na⁺]_i increased the rate of retrieval of cleaved γ ENaC by 50%. This implies that enhanced retrieval of ENaC channels at the cell surface accounts for part, but not all, of the downregulation of ENaC activity shown with chronic increases in [Na⁺]_i.     

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

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

Intracellular Na+ Regulates Epithelial Na+ Channel Maturation

Epithelial Na⁺ channel (ENaC) function is regulated by the intracellular Na⁺ concentration ([Na⁺]_i) through a process known as Na⁺ feedback inhibition. Although this process is known to decrease the expression of proteolytically processed active channels on the cell surface, it is unknown how [Na⁺]_i alters ENaC cleavage. We show here that [Na⁺]_i regulates the posttranslational processing of ENaC subunits during channel biogenesis. At times when [Na⁺]_i is low, ENaC subunits develop mature N-glycans and are processed by proteases. Conversely, glycan maturation and sensitivity to proteolysis are reduced when [Na⁺]_i is relatively high. Surface channels with immature N-glycans were not processed by endogenous channel activating proteases, nor were they sensitive to cleavage by exogenous trypsin. Biotin chase experiments revealed that the immature surface channels were not converted into mature cleaved channels following a reduction in [Na⁺]_i. The hypothesis that [Na⁺]_i regulates ENaC maturation within the biosynthetic pathways is further supported by the finding that Brefeldin A prevented the accumulation of processed surface channels following a reduction in [Na⁺]_i. Therefore, increased [Na⁺]_i interferes with ENaC N-glycan maturation and prevents the channel from entering a state that allows proteolytic processing.     

Intersubunit conformational changes mediate epithelial sodium channel gating

The epithelial Na⁺ channel (ENaC) functions as a pathway for Na⁺ absorption in the kidney and lung, where it is crucial for Na⁺ homeostasis and blood pressure regulation. However, the basic mechanisms that control ENaC gating are poorly understood. Here we define a role in gating for residues forming interfaces between the extracellular domains of the three ENaC subunits. Using cysteine substitution combined with chemical cross-linking, we determined that residues located at equivalent positions in the three subunits (α_K477, β_E446, and γ_E455) form interfaces with residues in adjacent subunits (β_V85, γ_V87, and α_L120, respectively). Cross-linking of these residues altered ENaC activity in a length-dependent manner; long cross-linkers increased ENaC current by increasing its open probability, whereas short cross-linkers reduced ENaC open probability. Cross-linking also disrupted ENaC gating responses to extracellular pH and Na⁺, signals which modulate ENaC activity during shifts in volume status. Introduction of charged side chains at the interfacing residues altered ENaC activity in a charge-dependent manner. Current increased when like charges were present at both interfacing residues, whereas opposing charges reduced current. Together, these data indicate that conformational changes at intersubunit interfaces participate in ENaC transitions between the open and closed states; movements that increase intersubunit distance favor the open state, whereas the closed state is favored when the distance is reduced. This provides a mechanism to modulate ENaC gating in response to changing extracellular conditions that threaten Na⁺ homeostasis.     

Feedback inhibition of ENaC: Acute and chronic mechanisms

Intracellular [Na⁺] ([Na⁺]_i) modulates the activity of the epithelial Na channel (ENaC) to help prevent cell swelling and regulate epithelial Na⁺ transport, but the underlying mechanisms remain unclear. We show here that short-term (60–80 min) incubation of ENaC-expressing oocytes in high Na⁺ results in a 75% decrease in channel activity. When the β subunit was truncated, corresponding to a gain-of-function mutation found in Liddle''s syndrome, the same maneuver reduced activity by 45% despite a larger increase in [Na⁺]_i. In both cases the inhibition occurred with little to no change in cell-surface expression of γENaC. Long-term incubation (18 hours) in high Na⁺ reduced activity by 92% and 75% in wild-type channels and Liddle''s mutant, respectively, with concomitant 70% and 52% decreases in cell-surface γENaC. In the presence of Brefeldin A to inhibit forward protein trafficking, high-Na⁺ incubation decreased wt ENaC activity by 52% and 88% after 4 and 8 hour incubations, respectively. Cleaved γENaC at the cell surface had lifetimes at the surface of 6 hrs in low Na⁺ and 4 hrs in high Na⁺, suggesting that [Na⁺]_i increased the rate of retrieval of cleaved γ ENaC by 50%. This implies that enhanced retrieval of ENaC channels at the cell surface accounts for part, but not all, of the downregulation of ENaC activity shown with chronic increases in [Na⁺]_i.     

Multiple residues in the distal C terminus of the α-subunit have roles in modulating human epithelial sodium channel activity

Epithelial sodium channels (ENaC) are critically important in the regulation of ion and fluid balance in both renal and respiratory epithelia. ENaC functional polymorphisms may contribute to alterations in blood pressure in the general population. We previously reported that the A663T polymorphism in the C terminus of the α-subunit altered ENaC functional and surface expression in Xenopus laevis oocytes (Samaha FF, Rubenstein RC, Yan W, Ramkumar M, Levy DI, Ahn YJ, Sheng S, Kleyman TR. J Biol Chem 279: 23900-23907, 2004). We examined whether sites in the vicinity of 663 influenced channel activity by performing scanning Ala mutagenesis. Interestingly, only αT663/G667Aβγ channels exhibited increased currents compared with αT663βγ. This increase in channel activity reflected an increase in channel open probability and not an increase in channel surface expression. In contrast, decreases in channel activity were observed with both αT663/C664Aβγ and αT663/C664Mβγ channels. The decrease in functional expression of αT663/C664Mβγ channels correlated with decreased surface expression, suggesting that the αC664M mutation altered the intracellular trafficking of the channel. While cytoplasmic Cys residues may be modified by the addition of palmitate, we did not observe palmitoylation of αC664. Our results suggest that multiple residues in the distal part of the cytoplasmic C terminus have roles in modulating channel activity.     

Calreticulin positively regulates the expression and function of epithelial sodium channel

Epithelial sodium channel (ENaC) is a heteromultimeric Na⁺ channel at the apical membrane in the kidney, colon, and lung. Because ENaC plays a crucial role in regulating Na⁺ absorption and extracellular fluid volume, its dysregulation causes severe phenotypes including hypertension, hypokalemia, and airway obstruction. Despite the importance of ENaC, its protein quality control mechanism remains less established. Here we firstly show the role of calreticulin (CRT), a lectin-like molecular chaperone in the endoplasmic reticulum (ER), on the regulation of ENaC. Overexpression and knockdown analyses clearly indicated that CRT positively affects the expression of each ENaC subunit (α, β and γ). CRT overexpression also up-regulated the cell surface expression of α-, β- and γ-ENaC. Moreover, we found that CRT directly interacts with each ENaC subunit. Although CRT knockdown did not affect the de novo synthesis of ENaC subunits, CRT overexpression decreased α-, β- and γ-ENaC expression in the detergent (RIPA)-insoluble fraction, suggesting that CRT enhanced the solubility of ENaC subunits. Consistent with the increased intracellular and cell surface expression of ENaC subunits, increased channel activity of ENaC was also observed upon overexpression of CRT. Our study thus identifies CRT as an ER chaperone that regulates ENaC expression and function.     

ENaC at the Cutting Edge: Regulation of Epithelial Sodium Channels by Proteases

Epithelial Na⁺ channels facilitate the transport of Na⁺ across high resistance epithelia. Proteolytic cleavage has an important role in regulating the activity of these channels by increasing their open probability. Specific proteases have been shown to activate epithelial Na⁺ channels by cleaving channel subunits at defined sites within their extracellular domains. This minireview addresses the mechanisms by which proteases activate this channel and the question of why proteolysis has evolved as a mechanism of channel activation.Many ion channels are silent at rest and are activated in response to a variety of factors, including membrane potential, external ligands, and intracellular signaling processes. The ENaC² has evolved as a channel that is thought to reside primarily in an active state, facilitating the bulk movement of Na⁺ out of renal tubular or airway lumens. The regulated insertion and retrieval of channels at the plasma membrane have important roles in modulating ENaC-dependent Na⁺ transport (1). A number of factors also have a role in regulating ENaC activity via changes in channel P_o or gating. In this regard, it has become increasingly apparent that proteolysis of ENaC subunits has a key role in this process (2). This minireview addresses several questions regarding the role of ENaC subunit proteolysis in regulating channel gating. (i) Where are ENaC subunits cleaved? (ii) Which proteases mediate ENaC cleavage? (iii) Why are channels activated by proteolysis? (iv) Is proteolysis responsible, in part, for the highly variable channel P_o that has been noted for ENaC? (v) Why have ENaCs evolved as channels that require proteolysis for activation?     

Epithelial sodium channel modulates platelet collagen activation

Activated platelets adhere to the exposed subendothelial extracellular matrix and undergo a rapid cytoskeletal rearrangement resulting in shape change and release of their intracellular dense and alpha granule contents to avoid hemorrhage. A central step in this process is the elevation of the intracellular Ca²⁺ concentration through its release from intracellular stores and on throughout its influx from the extracellular space. The Epithelial sodium channel (ENaC) is a highly selective Na⁺ channel involved in mechanosensation, nociception, fluid volume homeostasis, and control of arterial blood pressure. The present study describes the expression, distribution, and participation of ENaC in platelet migration and granule secretion using pharmacological inhibition with amiloride. Our biochemical and confocal analysis in suspended and adhered platelets suggests that ENaC is associated with Intermediate filaments (IF) and with Dystrophin-associated proteins (DAP) via α-syntrophin and β-dystroglycan. Migration assays, quantification of soluble P-selectin, and serotonin release suggest that ENaC is dispensable for migration and alpha and dense granule secretion, whereas Na⁺ influx through this channel is fundamental for platelet collagen activation.     

The epithelial sodium channel and the control of sodium balance

Studies aiming at the elucidation of the genetic basis of rare monogenic forms of hypertension have identified mutations in genes coding for the epithelial sodium channel ENaC, for the mineralocorticoid receptor, or for enzymes crucial for the synthesis of aldosterone. These genetic studies clearly demonstrate the importance of the regulation of Na⁺ absorption in the aldosterone-sensitive distal nephron (ASDN), for the maintenance of the extracellular fluid volume and blood pressure.Recent studies aiming at a better understanding of the cellular and molecular basis of ENaC-mediated Na⁺ absorption in the distal part of nephron, have essentially focused on the regulation ENaC activity and on the aldosterone-signaling cascade. ENaC is a constitutively open channel, and factors controlling the number of active channels at the cell surface are likely to have profound effects on Na⁺ absorption in the ASDN, and in the amount of Na⁺ that is excreted in the final urine.A number of membrane-bound proteases, kinases, have recently been identified that increase ENaC activity at the cell surface in heterologous expressions systems. Ubiquitylation is a general process that regulates the stability of a variety of target proteins that include ENaC. Recently, deubiquitylating enzymes have been shown to increase ENaC activity in heterologous expressions systems.These regulatory mechanisms are likely to be nephron specific, since in vivo studies indicate that the adaptation of the renal excretion of Na⁺ in response to Na⁺ diet occurs predominantly in the early part (the connecting tubule) of the ASDN.An important work is presently done to determine in vivo the physiological relevance of these cellular and molecular mechanisms in regulation of ENaC activity. The contribution of the protease-dependent ENaC regulation in mediating Na⁺ absorption in the ASDN is still not clearly understood. The signaling pathway that involves ubiquitylation of ENaC does not seem to be absolutely required for the aldosterone-mediated control of ENaC. These in vivo physiological studies presently constitute a major challenge for our understanding of the regulation of ENaC to maintain the Na⁺ balance.     

Phylogenetic characterization of the epithelial Na+ channel (ENaC) family

Summary

Twenty-one sequenced protein members of the epithelial Na⁺ channel (ENaC) family have been identified and characterized in terms of their sizes, hydropathy profiles, sequence similarities and phylogenies. These proteins derive from mammals, the frog Xenopus laevis and the worm Caenorhabditis elegans. The eleven sequenced vertebrate proteins fall into four subfamilies designated α, β, γ, and δ. The 10 C. elegans proteins do not cluster with the vertebrate proteins, and they all proved to be distantly related to each other. Nonetheless, the 21 ENaC proteins exhibit the same apparent topology, each with two transmembrane spanning segments separated by a large extracellular loop. All but two ENaC proteins possess highly conserved extracellular domains containing numerous conserved cysteine residues as well as adjacent C-terminal amphipathic transmembrane spanning segments, postulated to contribute to the formation of the hydrophilic pores of these oligomeric channel protein complexes. It is proposed that the well-conserved extracellular domains serve as receptors to control the activities of the channels. A topological model for the ENaC family proteins is presented.