Mineralocorticoid receptor degradation is promoted by Hsp90 inhibition and the ubiquitin-protein ligase CHIP.

The mineralocorticoid receptor (MR) plays a crucial role in the regulation of Na(+) balance and blood pressure, as evidenced by gain of function mutations in the MR of hypertensive families. In the kidney, aldosterone binds to the MR, induces its nuclear translocation, and promotes a transcriptional program leading to increased transepithelial Na(+) transport via the epithelial Na(+) channel. In the unliganded state, MR is localized in the cytosol and part of a multiprotein complex, including heat shock protein 90 (Hsp90), which keeps it ligand-binding competent. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a benzoquinone ansamycin antibiotic that binds to Hsp90 and alters its function. We investigated whether 17-AAG affects the stability and transcriptional activity of MR and consequently Na(+) reabsorption by renal cells. 17-AAG treatment lead to reduction of MR protein level in epithelial cells in vitro and in vivo, thereby interfering with aldosterone-dependent transcription. Moreover, 17-AAG inhibited aldosterone-induced Na(+) transport, possibly by interfering with MR availability for the ligand. Finally, we identified the ubiquitin-protein ligase, COOH terminus of Hsp70-interacting protein, as a novel partner of the cytosolic MR, which is responsible for its polyubiquitylation and proteasomal degradation in presence of 17-AAG. In conclusion, 17-AAG may represent a novel pharmacological tool to interfere with Na(+) reabsorption and hypertension.

BRAF mutation in 'sarcomas': a possible method to detect de-differentiated melanomas

AIMS: BRAF is mutated in 50-60% of melanomas, but BRAF mutation in sarcomas has not been systematically evaluated. Some melanomas are spindled and may show no immunohistochemical evidence of melanocytic differentiation. Similarly, many sarcomas are undifferentiated, i.e. undifferentiated pleomorphic sarcomas (UPS). Diagnosing melanoma versus sarcoma in an undifferentiated spindle cell malignancy can be challenging. Our aim was to evaluate the prevalence of BRAF mutation in sarcomas and the use of BRAF mutational status in the diagnosis of spindle cell malignancies. METHODS AND RESULTS: BRAF mutational analysis was performed on tissue from 104 patients: 90 with sarcoma only (50 UPS) and 14 with sarcoma and melanoma (seven UPS). In the sarcoma-only group, BRAF mutation was absent. In the sarcoma-melanoma group, three sarcomas showed BRAF mutation; all were UPS, occurred after the melanomas and did not stain for melanocytic markers. One melanoma-sarcoma pair showed identical BRAF V600E mutations. CONCLUSIONS: The presence of BRAF mutation in these tumours raises the possibility that poorly differentiated spindle cell malignancies with BRAF mutation may represent melanomas, and BRAF mutational analysis should be considered in a patient with a spindle cell malignancy and a history of melanoma, as a positive result may indicate de-differentiated melanoma.

Analyses of a novel SCN5A mutation (C1850S): conduction vs. repolarization disorder hypotheses in the Brugada syndrome.

AIMS: Brugada syndrome (BrS) is characterized by arrhythmias leading to sudden cardiac death. BrS is caused, in part, by mutations in the SCN5A gene, which encodes the sodium channel alpha-subunit Na(v)1.5. Here, we aimed to characterize the biophysical properties and consequences of a novel BrS SCN5A mutation. METHODS AND RESULTS: SCN5A was screened for mutations in a male patient with type-1 BrS pattern ECG. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in HEK293 cells. Sodium currents (I(Na)) were analysed using the whole-cell patch-clamp technique at 37 degrees C. The electrophysiological effects of the mutation were simulated using the Luo-Rudy model, into which the transient outward current (I(to)) was incorporated. A new mutation (C1850S) was identified in the Na(v)1.5 C-terminal domain. In HEK293 cells, mutant I(Na) density was decreased by 62% at -20 mV. Inactivation of mutant I(Na) was accelerated in a voltage-dependent manner and the steady-state inactivation curve was shifted by 11.6 mV towards negative potentials. No change was observed regarding activation characteristics. Altogether, these biophysical alterations decreased the availability of I(Na). In the simulations, the I(to) density necessary to precipitate repolarization differed minimally between the two genotypes. In contrast, the mutation greatly affected conduction across a structural heterogeneity and precipitated conduction block. CONCLUSION: Our data confirm that mutations of the C-terminal domain of Na(v)1.5 alter the inactivation of the channel and support the notion that conduction alterations may play a significant role in the pathogenesis of BrS.

The evolution of XY recombination: sexually antagonistic selection versus deleterious mutation load.

Recombination arrest between X and Y chromosomes, driven by sexually antagonistic genes, is expected to induce their progressive differentiation. However, in contrast to birds and mammals (which display the predicted pattern), most cold-blooded vertebrates have homomorphic sex chromosomes. Two main hypotheses have been proposed to account for this, namely high turnover rates of sex-determining systems and occasional XY recombination. Using individual-based simulations, we formalize the evolution of XY recombination (here mediated by sex reversal; the "fountain-of-youth" model) under the contrasting forces of sexually antagonistic selection and deleterious mutations. The shift between the domains of elimination and accumulation occurs at much lower selection coefficients for the Y than for the X. In the absence of dosage compensation, mildly deleterious mutations accumulating on the Y depress male fitness, thereby providing incentives for XY recombination. Under our settings, this occurs via "demasculinization" of the Y, allowing recombination in XY (sex-reversed) females. As we also show, this generates a conflict with the X, which coevolves to oppose sex reversal. The resulting rare events of XY sex reversal are enough to purge the Y from its load of deleterious mutations. Our results support the "fountain of youth" as a plausible mechanism to account for the maintenance of sex-chromosome homomorphy.

Patient with syncope and LQTS carrying a mutation in the PAS domain of the hERG1 channel.

We report the case of a woman with syncope and persistently prolonged QTc interval. Screening of congenital long QT syndrome (LQTS) genes revealed that she was a heterozygous carrier of a novel KCNH2 mutation, c.G238C. Electrophysiological and biochemical characterizations unveiled the pathogenicity of this new mutation, displaying a 2-fold reduction in protein expression and current density due to a maturation/trafficking-deficient mechanism. The patient's phenotype can be fully explained by this observation. This study illustrates the importance of performing genetic analyses and mutation characterization when there is a suspicion of congenital LQTS. Identifying mutations in the PAS domain or other domains of the hERG1 channel and understanding their effect may provide more focused and mutation-specific risk assessment in this population.

Lung adenocarcinoma with BRAF G469L mutation refractory to vemurafenib.

BRAF V600E is an emerging drug target in lung cancer, but the clinical significance of non-V600 BRAF mutations in lung cancer and other malignancies is less clear. Here, we report the case of a patient with metastatic lung adenocarcinoma with BRAF G469L mutation refractory to vemurafenib. We calculated a structure model of this very rare type of mutated BRAF kinase to explain the molecular mechanism of drug resistance. This information may help to develop effective targeted therapies for cancers with non-V600 BRAF mutations.

Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays.

Malignant melanoma, the deadliest form of skin cancer, is characterized by a predominant mutation in the BRAF gene. Drugs that target tumours carrying this mutation have recently entered the clinic. Accordingly, patients are routinely screened for mutations in this gene to determine whether they can benefit from this type of treatment. The current gold standard for mutation screening uses real-time polymerase chain reaction and sequencing methods. Here we show that an assay based on microcantilever arrays can detect the mutation nanomechanically without amplification in total RNA samples isolated from melanoma cells. The assay is based on a BRAF-specific oligonucleotide probe. We detected mutant BRAF at a concentration of 500 pM in a 50-fold excess of the wild-type sequence. The method was able to distinguish melanoma cells carrying the mutation from wild-type cells using as little as 20 ng µl(-1) of RNA material, without prior PCR amplification and use of labels.

Regulation of the NaCI cotransporter by the SGK1-Nedd4-2 pathway

SummaryRegulation of renal Na+ transport is essential for controlling blood pressure, as well as Na+ and K+ homeostasis. Aldosterone stimulates Na+ reabsorption in the aldosterone-sensitive distal nephron (ASDN), via the Na+-CI" cotransporter (NCC) in the distal convoluted tubule (DCT), and the epithelial Na+ channel (ENaC) in the late DCT, connecting tubule and collecting duct. Importantly, aldosterone increases NCC protein expression by an unknown post-translational mechanism. The ubiquitin-protein ligase Nedd4-2 is expressed along the ASDN and regulates ENaC: under aldosterone induction, the serum/glucocorticoid-regulated kinase SGK1 phosphorylates Nedd4-2 on S328, thus preventing the Nedd4-2/ENaC interaction, ubiquitylation and degradation of the channel. Here, we present evidence that Nedd4-2 regulates NCC. In transfected HEK293 cells, Nedd4-2 co-immunoprecipitates with NCC and stimulates NCC ubiquitylation at the cell surface. In Xenopus laevis oocytes, co- expression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreases NCC activity and surface expression. This inhibition is prevented by SGK1 in a kinase-dependent manner. Moreover, we show that NCC expression is up-regulated in inducible renal tubule-specific Nedd4-2 knockout mice and in mDCT15 cells silenced for Nedd4-2. On the other hand, in inducible renal tubule-specific SGK1 knockout mice, NCC expression is down-regulated.Interestingly, in contrast to ENaC, Nedd4-2-mediated NCC inhibition is independent of a PY motif in NCC. Moreover, whereas single mutations of Nedd4-2 S328 or S222 to alanine do not interfere with SGK1 action, the double mutation enhances Nedd4-2 activity and abolishes SGK1-dependent inhibition. These results indicate that NCC expression and activity is controlled by a regulatory pathway involving SGK1 and Nedd4-2, and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.RésuméLa régulation du transport de sodium est cruciale dans le maintien de la pression artérielle. L'aldostérone stimule la réabsorption de Na+ dans la partie du néphron sensible à l'aldostérone (ASDN), via le co-transporteur Na+-CI" (NCC) au niveau du tubule contourné distale et via le canal à sodium (Epithelial Na+ Channel ; ENaC) dans la deuxième partie du tubule contourné distale, dans le tube connecteur et le tube collecteur. L'aldostérone augmente l'expression de NCC au niveau protéique par un mécanisme non élucidé. La protéine ubiquitine ligase Nedd4-2 est exprimée tout le long du néphron sensible à l'aldostérone. ENaC est connu pour être régulé par Nedd4-2. Suite à une stimulation par l'aldostérone, la kinase Ser/Thr SGK1 phosphoryle Nedd4-2, ce qui empêche l'interaction entre Nedd4-2 et ENaC. Dans des cellules HEK293 transfectées, nous avons montré que Nedd4-2 interagit avec le co-transporteur NCC et stimule l'ubiquitylation de NCC à la surface. Nous avons montré dans les oocytes de Xenopus laevis que l'expression de NCC avec Nedd4-2 diminue l'activité du co-transporteur. Cette diminution n'est pas observée lorsqu'on exprime NCC avec le mutant inactif de Nedd4-2. Cette inhibition de NCC est contrée par SGK1. L'effet de SGK1 sur NCC dépend de son activité kinase. Nous avons montré dans des souris knock-out pour Nedd4-2, dans le néphron et de manière inductible, que l'expression de NCC est augmentée. Nous avons également montré que la suppression de la protéine Nedd4-2 dans les cellules mDCT15 provoque l'augmentation de NCC. Au contraire dans les souris knock-out pour la kinase SGK1, dans le néphron et de manière inductible, nous observons une diminution de la protéine NCC. Contrairement à ce qui a été montré pour le canal ENaC l'inhibition de NCC par Nedd4-2 est indépendante des motifs PY. De plus, La mutation des sérines 328 ou 222 sur Nedd4-2 en alanine n'interfère pas avec l'action de SGK1 pour prévenir l'inhibition. Par contre, la double mutation, les sérines 222 et 328 mutées en alanine, augmente l'action de Nedd4-2 sur l'activité de NCC et prévient l'effet de SGK1. Ces résultats montrent que l'expression et l'activité de NCC sont contrôlées par une voie de régulation impliquant Nedd4-2-SGK1 et nous fournissent une explication pour l'augmentation de NCC observé après une induction avec l'aldostérone.Résumé large publicOn estime que des millions de personnes seraient hypertendues. L'hypertension artérielle est responsable d'environ 8 millions de décès par ans dans le monde. L'hypertension est responsable de la moitié environs des accidents cardiaques, mais aussi des accidents vasculaires cérébraux. Il est très important de comprendre les mécanismes qui se trouvent derrière cette pathologie.Le co-transporteur NCC joue un grand rôle dans le maintien de la balance sodique. Il a été montré que des perturbations dans l'expression de NCC pouvaient engendrer de l'hypertension.Le co-transporteur NCC est exprimé dans la partie distale du néphron, l'unité fonctionnelle du rein. Plusieurs études ont montrées que NCC était sous le contrôle de l'hormone aldostérone.Le travail de cette thèse consiste à étudier les mécanismes impliqués dans la régulation de NCC. On a ainsi pu montrer que NCC interagit avec la protéine ubiquitine ligase Nedd4-2. La protéine Nedd4-2 diminue l'expression de NCC à la surface cellulaire et aussi son activité Nous avons également montré que la kinase SGK1 pouvait prévenir l'interaction entre Nedd4-2 et NCC par phosphorylation de Nedd4-2. Nous avons montré dans des souris deletée pour Nedd4-2, dans le néphron, que l'expression de NCC est augmentée. Nous avons également montré que la suppression de la protéine Nedd4-2 dans les cellules mDCT15 provoque l'augmentation de NCC. Au contraire, dans les souris deletée pour la kinase SGK1, dans le néphron, nous observons une diminution de la protéine NCC. La connaissance des processus impliqués dans la régulation du co-transporteur NCC pourrait amener au développement de nouveau médicaments pour soigner l'hypertension.

Role of intracellular cysteines in ENaC function

The epithelial sodium channel (ENaC) regulates the sodium reabsorption in the collecting duct principal cells of the nephron. ENaC is mainly regulated by hormones such as aldosterone and vasopressin, but also by serine proteases, Na+ and divalent cations. The crystallization of an ENaC/Deg member, the Acid Sensing Ion Channel, has been recently published but the pore-lining residues constitution of ENaC internal pore remains unclear. It has been reported that mutation aS589C of the selectivity filter on the aENaC subunit, a three residues G/SxS sequence, renders the channel permeant to divalent cations and sensitive to extracellular Cd2+. We have shown in the first part of my work that the side chain of aSer589 residue is not pointing toward the pore lumen, permitting the Cd2+ to permeate through the ion pore and to coordinate with a native cysteine, gCys546, located in the second transmembrane domain of the gENaC subunit. In a second part, we were interested in the sulfhydryl-reagent intracellular inhibition of ENaC-mediated Na+ current. Kellenberger et al. have shown that ENaC is rapidly and reversibly inhibited by internal sulfhydryl reagents underlying the involvement of intracellular cysteines in the internal regulation of ENaC. We set up a new approach comprising a Substituted Cysteine Analysis Method (SCAM) using intracellular MTSEA-biotin perfusion coupled to functional and biochemical assays. We were thus able to correlate the cysteine-modification of ENaC by methanethiosulfonate (MTS) and its effect on sodium current. This allowed us to determine the amino acids that are accessible to intracellular MTS and the one important for the inhibition of the channel. RESUME : Le canal épithélial sodique ENaC est responsable de la réabsorption du sodium dans les cellules principales du tubule collecteur rénal. Ce canal est essentiellement régulé par voie hormonale via l'aldostérone et la vasopressine mais également par des sérines protéases, le Na+ lui-même et certains cations divalents. La cristallisation du canal sodique sensible au pH acide, ASIC, un autre membre de la famille ENaC/Deg, a été publiée mais les acides aminés constituant le pore interne d'ENaC restent indéterminés. Il a été montré que la mutation aS589C du filtre de sélectivité de la sous-unité aENaC permet le passage de cations divalents et l'inhibition du canal par le Cd2+ extracellulaire. Dans un premier temps, nous avons montré que la chaîne latérale de la aSer589 n'est pas orientée vers l'intérieur du pore, permettant au Cd2+ de traverser le canal et d'interagir avec une cysteine native du second domaine transrnembranaire de la sous-unité γENaC, γCys546. Dans un second temps, nous nous sommes intéressés au mécanisme d'inhibition d'ENaC par les réactifs sulfhydryl internes. Kellenberger et al. ont montré l'implication de cystéines intracellulaires dans la régulation interne d'ENaC par les réactifs sulfhydryl. Nous avons mis en place une nouvelle approche couplant la méthode d'analyse par substitution de cystéines (SCAM) avec des perfusions intracellulaires de MTSEAbiotine. Ainsi, nous pouvons meure en corrélation les modifications des cystéines d'ENaC par les réactifs methanethiosulfonates (MTS) avec leur effet sur le courant sodique, et donc mettre en évidence les acides aminés accessibles aux MTS intracellulaires et ceux qui sont importants dans la fonction du canal.

Recessive multiple epiphyseal dysplasia (rMED) with homozygosity for C653S mutation in the DTDST gene - phenotype, molecular diagnosis and surgical treatment of habitual dislocation of multilayered patella: case report.

BACKGROUND: Multiple epiphyseal dysplasia (MED) is one of the more common generalised skeletal dysplasias. Due to its clinical heterogeneity diagnosis may be difficult. Mutations of at least six separate genes can cause MED. Joint deformities, joint pain and gait disorders are common symptoms. CASE PRESENTATION: We report on a 27-year-old male patient suffering from clinical symptoms of autosomal recessive MED with habitual dislocation of a multilayered patella on both sides, on the surgical treatment and on short-term clinical outcome. Clinical findings were: bilateral hip and knee pain, instability of femorotibial and patellofemoral joints with habitual patella dislocation on both sides, contractures of hip, elbow and second metacarpophalangeal joints. Main radiographic findings were: bilateral dislocated multilayered patella, dysplastic medial tibial plateaus, deformity of both femoral heads and osteoarthritis of the hip joints, and deformity of both radial heads. In the molecular genetic analysis, the DTDST mutation g.1984T > A (p.C653S) was found at the homozygote state. Carrier status was confirmed in the DNA of the patient's parents. The mutation could be considered to be the reason for the patient's disease. Surgical treatment of habitual patella dislocation with medialisation of the tibial tuberosity led to an excellent clinical outcome. CONCLUSIONS: The knowledge of different phenotypes of skeletal dysplasias helps to select genes for genetic analysis. Compared to other DTDST mutations, this is a rather mild phenotype. Molecular diagnosis is important for genetic counselling and for an accurate prognosis. Even in case of a multilayered patella in MED, habitual patella dislocation could be managed successfully by medialisation of the tibial tuberosity.

Concomitant calcium entry blockade and inhibition of the renin-angiotensin system: a rational and effective means for treating hypertension

Pharmacological treatment of hypertension is effective in preventing cardiovascular and renal complications. Calcium antagonists (CAs) and blockers of the renin-angiotensin system [angiotensin-converting enzyme (ACE) inhibitors and angiotensin II antagonists (ARBs)] are widely used today to initiate antihypertensive treatment but, when given as monotherapy, do not suffice in most patients to normalise blood pressure (BP). Combining a CA and either an ACE-inhibitor or an ARB considerably increases the antihypertensive efficacy, but not at the expense of a deterioration of tolerability. Several fixed-dose combinations are available (CA + ACE-inhibitors: amlodipine + benazepril, felodipine + ramipril, verapamil + trandolapril; CA + ARB: amlodipine + valsartan). They are expected not only to improve BP control, but also to facilitate long-term adherence with antihypertensive therapy, thereby providing maximal protection against the cardiovascular and renal damage caused by high BP.

Identification of an RP1 Prevalent Founder Mutation and Related Phenotype in Spanish Patients with Early-Onset Autosomal Recessive Retinitis.

OBJECTIVE: To identify the genetic causes underlying early-onset autosomal recessive retinitis pigmentosa (arRP) in the Spanish population and describe the associated phenotype. DESIGN: Case series. PARTICIPANTS: A total of 244 unrelated families affected by early-onset arRP. METHODS: Homozygosity mapping or exome sequencing analysis was performed in 3 families segregating arRP. A mutational screening was performed in 241 additional unrelated families for the p.Ser452Stop mutation. Haplotype analysis also was conducted. Individuals who were homozygotes, double heterozygotes, or carriers of mutations in RP1 underwent an ophthalmic evaluation to establish a genotype-phenotype correlation. MAIN OUTCOME MEASURES: DNA sequence variants, homozygous regions, haplotypes, best-corrected visual acuity, visual field assessments, electroretinogram responses, and optical coherence tomography images. RESULTS: Four novel mutations in RP1 were identified. The new mutation p.Ser542Stop was present in 11 of 244 (4.5%) of the studied families. All chromosomes harboring this mutation shared the same haplotype. All patients presented a common phenotype with an early age of onset and a prompt macular degeneration, whereas the heterozygote carriers did not show any signs of retinitis pigmentosa (RP). CONCLUSIONS: p.Ser542Stop is a single founder mutation and the most prevalent described mutation in the Spanish population. It causes early-onset RP with a rapid macular degeneration and is responsible for 4.5% of all cases. Our data suggest that the implication of RP1 in arRP may be underestimated. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

HIV-1 protease mutation 82M contributes to phenotypic resistance to protease inhibitors in subtype G.

OBJECTIVES: The purpose of this study was the qualitative and quantitative assessment of the in vitro effect of HIV-1 protease (PR) mutation 82M on replication capacity and susceptibility to the eight clinically available PR inhibitors (PIs).¦METHODS: The 82M substitution was introduced by site-directed mutagenesis in wild-type subtype B and G strains, as well as reverted back to wild-type in a therapy-failing strain. The recombinant viruses were evaluated for their replication capacity and susceptibility to PIs.¦RESULTS: The single 82M mutation within a wild-type subtype B or G background did not result in drug resistance. However, the in vitro effect of single PR mutations on PI susceptibility is not always distinguishable from wild-type virus, and particular background mutations and polymorphisms are required to detect significant differences in the drug susceptibility profile. Consequently, reverting the 82M mutation back to wild-type (82I) in a subtype G isolate from a patient that failed therapy with multiple other PR mutations did result in significant increases in susceptibility towards indinavir and lopinavir and minor increases in susceptibility towards amprenavir and atazanavir. The presence of the 82M mutation also slightly decreased viral replication, whether it was in the genetic background of subtype B or subtype G.¦CONCLUSIONS: Our results suggest that 82M has an impact on PI susceptibility and that this effect is not due to a compensatory effect on the replication capacity. Because 82M is not observed as a polymorphism in any subtype, these observations support the inclusion of 82M in drug resistance interpretation systems and PI mutation lists.

Deleterious mutation accumulation in asexual Timema stick insects.

Sexual reproduction is extremely widespread in spite of its presumed costs relative to asexual reproduction, indicating that it must provide significant advantages. One postulated benefit of sex and recombination is that they facilitate the purging of mildly deleterious mutations, which would accumulate in asexual lineages and contribute to their short evolutionary life span. To test this prediction, we estimated the accumulation rate of coding (nonsynonymous) mutations, which are expected to be deleterious, in parts of one mitochondrial (COI) and two nuclear (Actin and Hsp70) genes in six independently derived asexual lineages and related sexual species of Timema stick insects. We found signatures of increased coding mutation accumulation in all six asexual Timema and for each of the three analyzed genes, with 3.6- to 13.4-fold higher rates in the asexuals as compared with the sexuals. In addition, because coding mutations in the asexuals often resulted in considerable hydrophobicity changes at the concerned amino acid positions, coding mutations in the asexuals are likely associated with more strongly deleterious effects than in the sexuals. Our results demonstrate that deleterious mutation accumulation can differentially affect sexual and asexual lineages and support the idea that deleterious mutation accumulation plays an important role in limiting the long-term persistence of all-female lineages.

A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.

Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild-type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P(o)), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant alpha subunit (alphaG95S) directly demonstrate a striking reduction in P(o). We propose that this mutation favors a gating mode characterized by short-open and long-closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.