A novel dominant mutation of the Nav1.4 alpha-subunit domain I leading to sodium channel myotonia.

BACKGROUND: Mutations in SCN4A may lead to myotonia. METHODS: Presentation of a large family with myotonia, including molecular studies and patch clamp experiments using human embryonic kidney 293 cells expressing wild-type and mutated channels. RESULTS: In a large family with historic data on seven generations and a clear phenotype, including myotonia at movement onset, with worsening by cold temperature, pregnancy, mental stress, and especially after rest after intense physical activity, but without weakness, the phenotype was linked with the muscle sodium channel gene (SCN4A) locus, in which a novel p.I141V mutation was found. This modification is located within the first transmembrane segment of domain I of the Na(v)1.4 alpha subunit, a region where no mutation has been reported so far. Patch clamp experiments revealed a mutation-induced hyperpolarizing shift (-12.9 mV) of the voltage dependence of activation, leading to a significant increase (approximately twofold) of the window current amplitude. In addition, the mutation shifted the voltage dependence of slow inactivation by -8.7 mV and accelerated the entry to this state. CONCLUSIONS: We propose that the gain-of-function alteration in activation leads to the observed myotonic phenotype, whereas the enhanced slow inactivation may prevent depolarization-induced paralysis.

Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.

The amiloride-sensitive epithelial Na channel (ENaC) is a heteromultimeric channel made of three alpha beta gamma subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in beta and gamma subunits at position beta G525 and gamma G537 increased the apparent inhibitory constant (Ki) for amiloride by > 1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the alpha subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated alpha beta gamma subunits resulted in a non-conducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by external Zn2+ ions, in particular the alpha S583C mutant showing a Ki for Zn2+ of 29 microM. Mutations of residues alpha W582L, or beta G522D also increased amiloride Ki, the later mutation generating a Ca2+ blocking site located 15% within the membrane electric field. These experiments provide strong evidence that alpha beta gamma ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of alpha beta gamma subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ ions at an external Na+ binding site preventing ion permeation through the channel pore.

Voltage-gated sodium channel expression in mouse DRG after SNI leads to re-evaluation of projections of injured fibers.

BACKGROUND: Dysregulation of voltage-gated sodium channels (Na(v)s) is believed to play a major role in nerve fiber hyperexcitability associated with neuropathic pain. A complete transcriptional characterization of the different isoforms of Na(v)s under normal and pathological conditions had never been performed on mice, despite their widespread use in pain research. Na(v)s mRNA levels in mouse dorsal root ganglia (DRG) were studied in the spared nerve injury (SNI) and spinal nerve ligation (SNL) models of neuropathic pain. In the SNI model, injured and non-injured neurons were intermingled in lumbar DRG, which were pooled to increase the tissue available for experiments. RESULTS: A strong downregulation was observed for every Na(v)s isoform expressed except for Na(v)1.2; even Na(v)1.3, known to be upregulated in rat neuropathic pain models, was lower in the SNI mouse model. This suggests differences between these two species. In the SNL model, where the cell bodies of injured and non-injured fibers are anatomically separated between different DRG, most Na(v)s were observed to be downregulated in the L5 DRG receiving axotomized fibers. Transcription was then investigated independently in the L3, L4 and L5 DRG in the SNI model, and an important downregulation of many Na(v)s isoforms was observed in the L3 DRG, suggesting the presence of numerous injured neurons there after SNI. Consequently, the proportion of axotomized neurons in the L3, L4 and L5 DRG after SNI was characterized by studying the expression of activating transcription factor 3 (ATF3). Using this marker of nerve injury confirmed that most injured fibers find their cell bodies in the L3 and L4 DRG after SNI in C57BL/6 J mice; this contrasts with their L4 and L5 DRG localization in rats. The spared sural nerve, through which pain hypersensitivity is measured in behavioral studies, mostly projects into the L4 and L5 DRG. CONCLUSIONS: The complex regulation of Na(v)s, together with the anatomical rostral shift of the DRG harboring injured fibers in C57BL/6 J mice, emphasize that caution is necessary and preliminary anatomical experiments should be carried out for gene and protein expression studies after SNI in mouse strains.

The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes.

Cellular directional migration in an electric field (galvanotaxis) is one of the mechanisms guiding cell movement in embryogenesis and in skin epidermal repair. The epithelial sodium channel (ENaC), in addition to its function of regulating sodium transport in kidney, has recently been found to modulate cell locomotory speed. Here we tested whether ENaC has an additional function of mediating the directional migration of galvanotaxis in keratinocytes. Genetic depletion of ENaC completely blocks only galvanotaxis and does not decrease migration speed. Overexpression of ENaC is sufficient to drive galvanotaxis in otherwise unresponsive cells. Pharmacologic blockade or maintenance of the open state of ENaC also decreases or increases, respectively, galvanotaxis, suggesting that the channel open state is responsible for the response. Stable lamellipodial extensions formed at the cathodal sides of wild-type cells at the start of galvanotaxis; these were absent in the ENaC knockout keratinocytes, suggesting that ENaC mediates galvanotaxis by generating stable lamellipodia that steer cell migration. We provide evidence that ENaC is required for directional migration of keratinocytes in an electric field, supporting a role for ENaC in skin wound healing.

A Heterozygous Deletion Mutation in the Cardiac Sodium Channel Gene SCN5A with Loss- and Gain-of-Function Characteristics Manifests as Isolated Conduction Disease, without Signs of Brugada or Long QT Syndrome.

BACKGROUND: The SCN5A gene encodes for the α-subunit of the cardiac sodium channel NaV1.5, which is responsible for the rapid upstroke of the cardiac action potential. Mutations in this gene may lead to multiple life-threatening disorders of cardiac rhythm or are linked to structural cardiac defects. Here, we characterized a large family with a mutation in SCN5A presenting with an atrioventricular conduction disease and absence of Brugada syndrome. METHOD AND RESULTS: In a large family with a high incidence of sudden cardiac deaths, a heterozygous SCN5A mutation (p.1493delK) with an autosomal dominant inheritance has been identified. Mutation carriers were devoid of any cardiac structural changes. Typical ECG findings were an increased P-wave duration, an AV-block I° and a prolonged QRS duration with an intraventricular conduction delay and no signs for Brugada syndrome. HEK293 cells transfected with 1493delK showed strongly (5-fold) reduced Na(+) currents with altered inactivation kinetics compared to wild-type channels. Immunocytochemical staining demonstrated strongly decreased expression of SCN5A 1493delK in the sarcolemma consistent with an intracellular trafficking defect and thereby a loss-of-function. In addition, SCN5A 1493delK channels that reached cell membrane showed gain-of-function aspects (slowing of the fast inactivation, reduction in the relative fraction of channels that fast inactivate, hastening of the recovery from inactivation). CONCLUSION: In a large family, congregation of a heterozygous SCN5A gene mutation (p.1493delK) predisposes for conduction slowing without evidence for Brugada syndrome due to a predominantly trafficking defect that reduces Na(+) current and depolarization force.

Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system.

Liddle syndrome is an autosomal dominant form of hypertension resulting from deletion or missense mutations of a PPPxY motif in the cytoplasmic COOH terminus of either the beta or gamma subunit of the epithelial Na channel (ENaC). These mutations lead to increased channel activity. In this study we show that wild-type ENaC is downregulated by intracellular Na+, and that Liddle mutants decrease the channel sensitivity to inhibition by intracellular Na+. This event results at high intracellular Na+ activity in 1.2-2.4-fold higher cell surface expression, and 2.8-3.5-fold higher average current per channel in Liddle mutants compared with the wild type. In addition, we show that a rapid increase in the intracellular Na+ activity induced downregulation of the activity of wild-type ENaC, but not Liddle mutants, on a time scale of minutes, which was directly correlated to the magnitude of the Na+ influx into the oocytes. Feedback inhibition of ENaC by intracellular Na+ likely represents an important cellular mechanism for controlling Na+ reabsorption in the distal nephron that has important implications for the pathogenesis of hypertension.

Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure.

The recently discovered epithelial sodium channel (ENaC)/degenerin (DEG) gene family encodes sodium channels involved in various cell functions in metazoans. Subfamilies found in invertebrates or mammals are functionally distinct. The degenerins in Caenorhabditis elegans participate in mechanotransduction in neuronal cells, FaNaC in snails is a ligand-gated channel activated by neuropeptides, and the Drosophila subfamily is expressed in gonads and neurons. In mammals, ENaC mediates Na+ transport in epithelia and is essential for sodium homeostasis. The ASIC genes encode proton-gated cation channels in both the central and peripheral nervous system that could be involved in pain transduction. This review summarizes the physiological roles of the different channels belonging to this family, their biophysical and pharmacological characteristics, and the emerging knowledge of their molecular structure. Although functionally different, the ENaC/DEG family members share functional domains that are involved in the control of channel activity and in the formation of the pore. The functional heterogeneity among the members of the ENaC/DEG channel family provides a unique opportunity to address the molecular basis of basic channel functions such as activation by ligands, mechanotransduction, ionic selectivity, or block by pharmacological ligands.

Role of the epithelial sodium channel (ENaC) and its regulator CAP1/Prss8 in colon

Dans les cellules épithéliales sensibles à l'aldostérone, le canal sodique épithélial (ENaC) joue un rôle critique dans le contrôle de l'équilibre sodique, le volume sanguin, et la pression sanguine. Le rôle d'ENaC est bien caractérisé dans le rein et les poumons, cependant le rôle d'ENaC et son régulateur positif la protéase activatrice de canal 1 (CAP1 /Prss8) sur le transport sodique dans le côlon reste en grande partie inconnu. Nous avons étudié l'importance d'ENaC et de CAPMPrss8 dans le côlon. Les souris déficientes pour la sous- unité aENaC (souris ScnnlaKO) dans les cellules superficielles intestinales étaient viables et ne montraient pas de létalité embryonnaire ou postnatale. Sous diète normale (RS) ou pauvre en sodium (LS), la différence de potentiel rectale sensible à l'amiloride (APDamii) était drastiquement diminuée et son rythme circadien atténué. Sous diète normale (RS) ou diète riche en sodium (HS) ou fort chargement de potassium, le sodium et le potassium plasmatique et urinaire n'étaient pas significativement changé. Cependant, sous LS, les souris Senni aK0 perdaient des quantités significativement augmentées de sodium dans leurs fèces, accompagnées par de très hauts taux d'aldostérone plasmatique et une rétention urinaire en sodium augmentée. Les souris déficientes en CAPl/PmS (Prss8K0) dans les cellules superficielles intestinales étaient viables et ne montraient pas de létalité embryonnaire ou postnatale. Sous diètes RS et HS cependant, les souris Prss8KO montraient une diminution significative du APDamil dans l'après-midi, mais le rythme circadien était maintenu. Sous diète LS, la perte de sodium par les fèces était accompagnée par des niveaux d'aldostérone plasmatiques plus élevés. Par conséquent, nous avons identifié la protéase activatrice de canal CAP 1 IPrss8 comme un régulateur important d'ENaC dans le côlon in vivo. De plus, nous étudions l'importance d'ENaC et de CAPIIPrss8 dans les conditions pathologiques comme les maladies inflammatoires chroniques de l'intestin (MICI). Le résultat préliminaire out montre qu'une déficience d'Prss8 mènait à la détérioration de la colite induite par le DSS comparé aux modèles contrôles respectifs. En résumé, l'étude a montré que sous restriction de sel, l'absence d'ENaC dans Pépithélium de surface du côlon était compensée par 1'activation du système rénine-angiotensine- aldostérone (RAAS) dans le rein. Ceci a mené à un pseudohypoaldostéronisme de type I spécifique au côlon avec résistance aux minéralocorticoïdes sans signe d'altération de rétention de potassium. - In aldosterone-responsive epithelial cells of kidney and colon, the epithelial sodium channel (ENaC) plays a critical role in the control of sodium balance, blood volume, and blood pressure. The role of ENaC is well characterized in kidney and lung, whereas role of ENaC and its positive regulator channel-activating protease 1 (CAPl/PrasS) on sodium transport in colon is largely unknown. We have investigated the importance of ENaC and CAPI/Prss8 in colon for sodium and potassium balance. Mice lacking the aENaC subunit (Scnnla mice) in intestinal superficial cells were viable and did not show any fetal or perinatal lethality. Under regular (RS) or low salt (LS) diet, the amiloride sensitive rectal potential difference (APDamii) was drastically decreased and its circadian rhythm blunted. Under regular salt (RS) or high salt (HS) diets or under potassium loading, plasma and urinary sodium and potassium were not significantly changed. However, upon LS, the ScnnlaK0 mice lost significant amounts of sodium in their feces, accompanied by very high plasma aldosterone and increased urinary sodium retention. Mice lacking the CAPl/PrasS (Prss8K0) in intestinal superficial cells were viable and did not show any fetal or perinatal lethality. Upon RS and HS diets, however, Prss8K0 exhibited a significantly reduced APDamii in the afternoon, but its circadian rhythm was maintained. Upon LS diet, sodium loss through feces was accompanied by higher plasma aldosterone levels. Thus, we have identified the channel-activating protease CAPl/Prss8 as an important in vivo regulator of ENaC in colon. Furthermore, we are investigating the importance of ENaC and CAPI/Prss8 in pathological conditions like inflammatory bowel disease (IBD). Preliminary data showed that PmS-deficiency led to worsening of DSS-induced colitis as compared to their respective controls. Overall, the present study has shown that under salt restriction, the absence of ENaC in colonic surface epithelium was compensated by the activation of renin-angiotensin- aldosterone (RAAS) system in the kidney. This led to a colon specific pseudohypoaldosteroni sm type 1 with mineralocorticoid resistance without evidence of impaired potassium retention.

Role of the epithelial sodium channel (ENaC) in the epidermal barrier function of the skin

Abstract In humans, the skin is the largest organ of the body, covering up to 2m2 and weighing up to 4kg in an average adult. Its function is to preserve the body from external insults and also to retain water inside. This barrier function termed epidermal permeability barrier (EPB) is localized in the functional part of the skin: the epidermis. For this, evolution has built a complex structure of cells and lipids sealing the surface, the stratum corneum. The formation of this structure is finely tuned since it is not only formed once at birth, but renewed all life long. This active process gives a high plasticity and reactivity to skin, but also leads to various pathologies. ENaC is a sodium channel extensively studied in organs like kidney and lung due to its importance in regulating sodium homeostasis and fluid volume. It is composed of three subunits α, ß and r which are forming sodium selective channel through the cell membrane. Its presence in the skin has been demonstrated, but little is known about its physiological role. Previous work has shown that αENaC knockout mice displayed an abnormal epidermis, suggesting a role in differentiation processes that might be implicated in the EPB. The principal aim of this thesis has been to study the consequences for EPB function in mice deficient for αENaC by molecular and physiological means and to investigate the underlying molecular mechanisms. Here, the barrier function of αENaC knockout pups is impaired. Apparently not immediately after birth (permeability test) but 24h later, when evident water loss differences appeared compared to wildtypes. Neither the structural proteins of the epithelium nor the tights junctions showed any obvious alterations. In contrary, stratum corneum lipid disorders are most likely responsible for the barrier defect, accompanied by an impairment of skin surface acidification. To analyze in details this EPB defect, several hypotheses have been proposed: reduced sensibility to calcium which is the key activator far epidermal formation, or modification of ENaC-mediated ion fluxes/currents inside the epidermis. The cellular localization of ENaC and the action in the skin of CAPl, a positive regulator of ENaC, have been also studied in details. In summary, this study clearly demonstrates that ENaC is a key player in the EPB maintenance, because αENaC knockout pups are not able to adapt to the new environment (ex utero) as efficiently as the wildtypes, most likely due to impaired of sodium handling inside the epidermis. Résumé Chez l'homme, la peau est le plus grand organe, couvrant presque 2m2 et pesant près de 4kg chez l'adulte. Sa fonction principale est de protéger l'organisme des agressions extérieures mais également de conserver l'eau à l'intérieur du corps. Cette fonction nommée barrière épithéliale est localisée dans la partie fonctionnelle de la peau : l'épiderme. A cette fin, l'évolution s'est dotée d'une structure complexe composée de cellules et de lipides recouvrant la surface, la couche cornée. Sa formation est finement régulée, car elle n'est pas seulement produite à la naissance mais constamment renouvelée tout au long de la vie, ce qui lui confère une grande plasticité mais ce qui est également la cause de nombreuses pathologies. ENaC est un canal sodique très étudié dans le rein et le poumon pour son importance dans la régulation de l'homéostasie sodique et la régulation du volume du milieu intérieur. Il est composé de 3 sous unités, α, ß et y qui forment un pore sélectif pour le sodium dans les membranes. Ce canal est présent dans la peau mais sa fonction n'y est pas connue. Des travaux précédents ont pu montrer que les souris dont le gène codant pour αENaC a été invalidé présentent un épiderme pathologique, suggérant un rôle dans la différentiation et pourrait même être impliqué dans la barrière épithéliale. Le but de cette thèse fut l'étude de la barrière dans ces souris knockouts avec des méthodes moléculaires et physiologiques et la caractérisation des mécanismes moléculaire impliqués. Dans ce travail, il a été montré que les souris mutantes présentaient un défaut de la barrière. Ce défaut n'est pas visible immédiatement à la naissance (test de perméabilité), mais 24h plus tard, lorsque les tests de perte d'eau transépithéliale montrent une différence évidente avec les animaux contrôles. Ni les protéines de structures ni les jonctions serrées de l'épiderme ne présentaient d'imperfections majeures. A l'inverse, les lipides de la couche cornée présentaient un problème de maturation (expliquant le phénotype de la barrière), certainement consécutif au défaut d'acidification à la surface de la peau que nous avons observé. D'autres mécanismes ont été explorées afin d'investiguer cette anomalie de la barrière, comme la réduction de sensibilité au calcium qui est le principal activateur de la formation de l'épiderme, ou la modification des flux d'ions entre les couches de l'épiderme. La localisation cellulaire d'ENaC, et l'action de son activateur CAPl ont également été étudiés en détails. En résumé, cette étude démontre clairement qu'ENaC est un acteur important dans la formation de la barrière épithéliale, car la peau des knockouts ne s'adapte pas aussi bien que celle des sauvages au nouvel environnement ex utero à cause de la fonction d'ENaC dans les mouvements de sodium au sein même de l'épiderme. Résumé tout public Chez l'homme, la peau est le plus grand organe, couvrant presque 2m2 et pesant près de 4kg chez l'adulte. Sa fonction principale est de protéger l'organisme des agressions extérieures mais également de conserver l'eau à l'intérieur du corps. Cette fonction nommée barrière épithéliale est localisée dans la partie fonctionnelle de la peau : l'épiderme. A cette fin, l'évolution s'est dotée d'une structure complexe composée de cellules et de lipides recouvrant la surface, la couche cornée. Sa formation est finement régulée, car elle n'est pas seulement produite à la naissance mais constamment renouvelée tout au long de la vie, ce qui lui confère une grande plasticité mais ce qui est également la cause de nombreuses maladies. ENaC est une protéine formant un canal qui permet le passage sélectif de l'ion sodium à travers la paroi des cellules. Il est très étudié dans le rein pour son importance dans la récupération du sel lors de la concentration de l'urine. Ce canal est présent dans la peau mais sa fonction n'y est pas connue. Des travaux précédents ont pu montrer que les souris où le gène codant pour αENaC a été invalidé présentent un épiderme pathologique, suggérant un rôle dans la peau et plus particulièrement la fonction de barrière de l'épiderme. Le but de cette thèse fut l'étude de la fonction de barrière dans ces souris mutantes, au niveau tissulaire et cellulaire. Dans ce travail, il a été montré que les souris mutantes présentaient une peau plus perméable que celle des animaux contrôles, grâce à une machine mesurant la perte d'eau à travers la peau. Ce défaut n'est visible que 24h après la naissance, mais nous avons pu montrer que les animaux mutants perdaient quasiment 2 fois plus d'eau que les contrôles. Au niveau moléculaire, nous avons pu montrer que ce défaut provenait d'un problème de maturation des lipides qui composent la barrière de la peau. Cette maturation est incomplète vraisemblablement à cause d'un défaut de mouvement des ions dans les couches les plus superficielles de l'épiderme, et cela à cause de l'absence du canal ENaC. En résumé, cette étude démontre clairement qu'ENaC est un acteur important dans la formation de la barrière épithéliale, car la peau des mutants ne s'adapte pas aussi bien que celle des sauvages au nouvel environnement ex utero à cause de la fonction d'ENaC dans les mouvements de sodium au sein même de l'épiderme.

Scnn1 sodium channel gene family in genetically engineered mice.

The amiloride-sensitive epithelial sodium channel is the limiting step in salt absorption. In mice, this channel is composed of three subunits (alpha, beta, and gamma), which are encoded by different genes (Scnn1a, Scnn1b, and Scnn1c, respectively). The functions of these genes were recently investigated in transgenic (knockout) experiments, and the absence of any subunit led to perinatal lethality. More defined phenotypes have been obtained by introducing specific mutations or using transgenic rescue experiments. In this report, these approaches are summarized and a current gene-targeting strategy to obtain conditional inactivation of the channel is illustrated. This latter approach will be indispensable for the investigation of channel function in a wide variety of organ systems.

Rufinamide Attenuates Mechanical Allodynia in a Model of Neuropathic Pain in the Mouse and Stabilizes Voltage-gated Sodium Channel Inactivated State.

BACKGROUND:: Voltage-gated sodium channels dysregulation is important for hyperexcitability leading to pain persistence. Sodium channel blockers currently used to treat neuropathic pain are poorly tolerated. Getting new molecules to clinical use is laborious. We here propose a drug already marketed as anticonvulsant, rufinamide. METHODS:: We compared the behavioral effect of rufinamide to amitriptyline using the Spared Nerve Injury neuropathic pain model in mice. We compared the effect of rufinamide on sodium currents using in vitro patch clamp in cells expressing the voltage-gated sodium channel Nav1.7 isoform and on dissociated dorsal root ganglion neurons to amitriptyline and mexiletine. RESULTS:: In naive mice, amitriptyline (20 mg/kg) increased withdrawal threshold to mechanical stimulation from 1.3 (0.6-1.9) (median [95% CI]) to 2.3 g (2.2-2.5) and latency of withdrawal to heat stimulation from 13.1 (10.4-15.5) to 30.0 s (21.8-31.9), whereas rufinamide had no effect. Rufinamide and amitriptyline alleviated injury-induced mechanical allodynia for 4 h (maximal effect: 0.10 ± 0.03 g (mean ± SD) to 1.99 ± 0.26 g for rufinamide and 0.25 ± 0.22 g to 1.92 ± 0.85 g for amitriptyline). All drugs reduced peak current and stabilized the inactivated state of voltage-gated sodium channel Nav1.7, with similar effects in dorsal root ganglion neurons. CONCLUSIONS:: At doses alleviating neuropathic pain, amitriptyline showed alteration of behavioral response possibly related to either alteration of basal pain sensitivity or sedative effect or both. Side-effects and drug tolerance/compliance are major problems with drugs such as amitriptyline. Rufinamide seems to have a better tolerability profile and could be a new alternative to explore for the treatment of neuropathic pain.

Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes.

We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits alpha, beta, and gamma of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 microg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (approximately 20-fold) with the channel obtained by coexpression of the alpha subunit of Xenopus ENaC with the beta and gamma subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

Revealing a subclinical salt-losing phenotype in heterozygous carriers of the novel S562P mutation in the alpha subunit of the epithelial sodium channel.

OBJECTIVE: Pseudohypoaldosteronism type I (PHA1) is a rare inborn disease causing severe salt loss. Mutations in the three coding genes of the epithelial sodium channel (ENaC) are responsible for the systemic autosomal recessive form. So far, no phenotype has been reported in heterozygous carriers. PATIENTS: A consanguineous family from Somalia giving birth to a neonate suffering from PHA1 was studied including clinical and hormonal characteristics of the family, mutational analysis of the SCNN1A, SCNN1B, SCNN1G and CFTR genes and in vitro analysis of the functional consequences of a mutant ENaC channel. RESULTS: CFTR mutations have been excluded. SCNN1A gene analysis revealed a novel homozygous c.1684T > C mutation resulting in a S562P substitution in the alphaENaC protein of the patient. Functional analysis showed a significantly reduced S562P channel function compared to ENaC wild type. Protein synthesis and channel subunit assembly were not altered by the S562P mutation. Co-expression of mutant and wild-type channels revealed a dominant negative effect. In heterozygote carriers, sweat sodium and chloride concentrations were increased without additional hormonal or clinical phenotypes. CONCLUSION: Hence, the novel mutation S562P is causing systemic PHA1 in the homozygous state. A thorough clinical investigation of the heterozygote SCNN1A mutation carriers revealed increased sweat sodium and chloride levels consistent with a dominant effect of the mutant S562P allele. Whether this subclinical phenotype is of any consequence for the otherwise asymptomatic heterozygous carriers has to be elucidated.

The epidthelial sodium channel ENaC and its regulators in the epidermal permeability barrier function

The highly amiloride-sensitive epithelial sodium channel ENaC is well known to be involved in controlling whole body sodium homeostasis and lung liquid clearance. ENaC expression has also been detected in the skin of amphibians and mammals. Mice lacking ENaC expression lose rapidly weight associated with an epidermal barrier defect that develops following birth. This dehydration is accompanied with a highly abnormal lipid matrix composition and an impaired skin surface acidification. This strongly suggests a role of ENaC in the maturation of barrier function rather than in the prenatal generation of the barrier, and may be as such an important modulator for skin hydration. In parallel, gene targeting experiments of regulators of ENaC activity, membrane serine proteases, also termed channel activating proteases, like CAP1/Prss8 and matriptase/MT-SP1 by themselves have been shown to be crucial for the epidermal barrier function. In our review, we mainly focus on the role of ENaC and its regulators in the skin and discuss their importance in the epidermal permeability barrier function.

Intracellular thiol-mediated modulation of epithelial sodium channel activity.

The epithelial sodium channel ENaC is physiologically important in the kidney for the regulation of the extracellular fluid volume, and in the lungs for the maintenance of the appropriate airway surface liquid volume that lines the pulmonary epithelium. Besides the regulation of ENaC by hormones, intracellular factors such as Na(+) ions, pH, or Ca(2+) are responsible for fast adaptive responses of ENaC activity to changes in the intracellular milieu. In this study, we show that ENaC is rapidly and reversibly inhibited by internal sulfhydryl-reactive molecules such as methanethiosulfonate derivatives of different sizes, the metal cations Cd(2+) and Zn(2+), or copper(II) phenanthroline, a mild oxidizing agent that promotes the formation of disulfide bonds. At the single channel level, these agents applied intracellularly induce the appearance of long channel closures, suggesting an effect on ENaC gating. The intracellular reducing agent dithiothreitol fully reverses the rundown of ENaC activity in inside-out patches. Our observations suggest that changes in intracellular redox potential modulate ENaC activity and may regulate ENaC-mediated Na(+) transport in epithelia. Finally, substitution experiments reveal that multiple cysteine residues in the amino and carboxyl termini of ENaC subunits are responsible for this thiol-mediated inhibition of ENaC.