Laurent Schild

Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination.

The epithelial Na+ channel (ENaC), composed of three subunits (αβγ), plays a critical role in salt and fluid homeostasis. Abnormalities in channel opening and numbers have been linked to several genetic disorders, including cystic fibrosis, pseudohypoaldosteronism type I and Liddle syndrome. We have recently identified the ubiquitin‐protein ligase Nedd4 as an interacting protein of ENaC. Here we show that ENaC is a short‐lived protein (t1/2 ∼1 h) that is ubiquitinated in vivo on the α and γ (but not β) subunits. Mutation of a cluster of Lys residues (to Arg) at the N‐terminus of γENaC leads to both inhibition of ubiquitination and increased channel activity, an effect augmented by N‐terminal Lys to Arg mutations in αENaC, but not in βENaC. This elevated channel activity is caused by an increase in the number of channels present at the plasma membrane; it represents increases in both cell‐surface retention or recycling of ENaC and incorporation of new channels at the plasma membrane, as determined by Brefeldin A treatment. In addition, we find that the rapid turnover of the total pool of cellular ENaC is attenuated by inhibitors of both the proteasome and the lysosomal/endosomal degradation systems, and propose that whereas the unassembled subunits are degraded by the proteasome, the assembled αβγENaC complex is targeted for lysosomal degradation. Our results suggest that ENaC function is regulated by ubiquitination, and propose a paradigm for ubiquitination‐mediated regulation of ion channels.

The heterotetrameric architecture of the epithelial sodium channel (ENaC)

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (α, β and γ) assemble to form a highly Na+‐selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC α, β and γ subunits shows that they assemble according to a fixed stoichiometry, with α ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each α, β and γ subunit are consistent with a four subunit stoichiometry composed of two α, one β and one γ. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two α subunits separated by β and γ subunits.

Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome

Liddle syndrome is an autosomal dominant form of hypertension, resulting from mutations in the cytoplasmic C‐terminus of either the beta or gamma subunits of the amiloride‐sensitive epithelial Na channel (ENaC) which lead to constitutively increased channel activity. Most mutations reported to date result in the elimination of 45–75 normal amino acids from these segments, leaving open the question of the identity of the precise amino acids in which mutation can lead to an enhanced channel activity. To address this question, we have performed a systematic mutagenesis study of the C‐termini of the alpha, beta and gamma ENaC subunits of the rat channel and have analyzed their function by expression in Xenopus oocytes. The results demonstrate that a short proline‐rich segment present in the cytoplasmic C‐terminus of each subunit is required for the normal regulation of channel activity. Missense mutations altering a consensus PPPXY sequence of the alpha, beta or gamma subunits reproduced the increase in channel activity found in mutants in which the entire cytoplasmic C‐termini are deleted. This proline‐rich sequence, referred to as the PY motif, is known to be a site of binding by proteins bearing a WW domain. These findings show that the three PY motifs in the C‐termini of ENaC are involved in the regulation of channel activity, probably via protein‐protein interactions. This new regulatory mechanism of channel function is critical for the maintenance of normal Na reabsorption in the kidney and of Na+ balance and blood pressure.

Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome

Liddles syndrome is an inherited form of hypertension linked to mutations in the epithelial Na+ channel (ENaC). ENaC is composed of three subunits (alpha, beta, gamma), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddles syndrome alter or delete the PY motifs of beta- or gamma-ENaC. We recently demonstrated that the ubiquitin-protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of beta-ENaC), as originally described in patients with Liddles syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddles syndrome may explain the increase in channel number at the cell surface, increased Na+ reabsorption by the distal nephron, and hence the hypertension.

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 β 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 α, β or γ 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 (Po), 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 α subunit (αG95S) directly demonstrate a striking reduction in Po. 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.

Epithelial sodium channels.

The highly selective amiloride-sensitive epithelial sodium channel is expressed in the distal part of the nephron, the distal colon, and the lung. It plays a critical role in the control of sodium balance, extracellular volume, blood pressure, and of fluid reabsorption in the lung. The primary structure of the rat epithelial sodium channel has recently been determined. It is a heteromultimeric protein made up of three homologous subunits (alpha, beta, and gamma). The biophysical properties, the cell distribution, and the regulation of this channel will be reviewed, with emphasis on its expression in the kidney, colon, and lung, where the clinical implications are most relevant. The epithelial sodium channel is a member of a novel gene superfamily that encodes cation channels involved in the control of cellular and extracellular volume and in the control of distinct functions such as taste transduction and mechanotransduction.

Dysfunction of the Epithelial Sodium Channel Expressed in the Kidney of a Mouse Model for Liddle Syndrome

The Liddle syndrome is a dominant form of salt-sensitive hypertension resulting from mutations in the beta or gamma subunit of ENaC. A previous study established a mouse model carrying a premature Stop codon corresponding to the R(566stop) mutation (L) found in the original pedigree that recapitulates to a large extent the human disease. This study investigated the renal Na(+) transport in vivo, ex vivo (intact perfused tubules), and in vitro (primary cultured cortical collecting ducts [CCD]). In vivo, upon 6 to 12 h of salt repletion, after 1 week of low-salt diet, the L/L mice showed a delayed urinary sodium excretion, despite a lower aldosterone secretion as compared with controls. After 6 h salt of repletion, ENaC gamma subunit is rapidly removed from the apical plasma membrane in wild-type mice, whereas it is retained at the apical membrane in L/L mice. Ex vivo, isolated perfused CCD from L/L mice exhibited higher transepithelial potential differences than perfused CCD isolated from +/+ mice. In vitro, confluent primary cultures of CCD microdissected from L/L kidneys grown on permeable filters exhibited significant lower transepithelial electrical resistance and higher negative potential differences than their cultured L/+ and +/+ CCD counterparts. The equivalent short-circuit current (I(eq)) and the amiloride-sensitive I(eq) was approximately twofold higher in cultured L/L CCD than in +/+ CCD. Aldosterone (5 x 10(-7)M for 3 h) further increased I(eq) from cultured L/L CCD. Thus, this study brings three independent lines of evidence for the constitutive hyperactivity of ENaC in CCD from mice harboring the Liddle mutation.

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.

International Union of Basic and Clinical Pharmacology. XCI. Structure, Function, and Pharmacology of Acid-Sensing Ion Channels and the Epithelial Na+ Channel

The epithelial Na+ channel (ENaC) and the acid-sensing ion channels (ASICs) form subfamilies within the ENaC/degenerin family of Na+ channels. ENaC mediates transepithelial Na+ transport, thereby contributing to Na+ homeostasis and the maintenance of blood pressure and the airway surface liquid level. ASICs are H+-activated channels found in central and peripheral neurons, where their activation induces neuronal depolarization. ASICs are involved in pain sensation, the expression of fear, and neurodegeneration after ischemia, making them potentially interesting drug targets. This review summarizes the biophysical properties, cellular functions, and physiologic and pathologic roles of the ASIC and ENaC subfamilies. The analysis of the homologies between ENaC and ASICs and the relation between functional and structural information shows many parallels between these channels, suggesting that some mechanisms that control channel activity are shared between ASICs and ENaC. The available crystal structures and the discovery of animal toxins acting on ASICs provide a unique opportunity to address the molecular mechanisms of ENaC and ASIC function to identify novel strategies for the modulation of these channels by pharmacologic ligands.

Mutational Analysis of Cysteine-rich Domains of the Epithelium Sodium Channel (ENaC) IDENTIFICATION OF CYSTEINES ESSENTIAL FOR CHANNEL EXPRESSION AT THE CELL SURFACE

One of the characteristic features of the structure of the epithelial sodium channel family (ENaC) is the presence of two highly conserved cysteine-rich domains (CRD1 and CRD2) in the large extracellular loops of the proteins. We have studied the role of CRDs in the functional expression of rat αβγ ENaC subunits by systematically mutating cysteine residues (singly or in combinations) into either serine or alanine. In the Xenopusoocyte expression system, mutations of two cysteines in CRD1 of α, β, or γ ENaC subunits led to a temperature-dependent inactivation of the channel. In CRD1, one of the cysteines of the rat αENaC subunit (Cys158) is homologous to Cys133 of the corresponding human subunit causing, when mutated to tyrosine (C133Y), pseudohypoaldosteronism type 1, a severe salt-loosing syndrome in neonates. In CRD2, mutation of two cysteines in α and β but not in the γ subunit also produced a temperature-dependent inactivation of the channel. The main features of the mutant cysteine channels are: (i) a decrease in cell surface expression of channel molecules that parallels the decrease in channel activity and (ii) a normal assembly or rate of degradation as assessed by nondenaturing co-immunoprecipitation of [35S]methionine-labeled channel protein. These data indicate that the two cysteines in CRD1 and CRD2 are not a prerequisite for subunit assembly and/or intrinsic channel activity. We propose that they play an essential role in the efficient transport of assembled channels to the plasma membrane.