Ivan Gautschi

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.

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.

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.

An external site controls closing of the epithelial Na+ channel ENaC.

Members of the ENaC/degenerin family of ion channels include the epithelial sodium channel (ENaC), acid‐sensing ion channels (ASICs) and the nematode Caenorhabditis elegans degenerins. These channels are activated by a variety of stimuli such as ligands (ASICs) and mechanical forces (degenerins), or otherwise are constitutively active (ENaC). Despite their functional heterogeneity, these channels might share common basic mechanisms for gating. Mutations of a conserved residue in the extracellular loop, namely the ‘degenerin site’ activate all members of the ENaC/degenerin family. Chemical modification of a cysteine introduced in the degenerin site of rat ENaC (βS518C) by the sulfhydryl reagents MTSET or MTSEA, results in a ∼3‐fold increase in the open probability. This effect is due to an 8‐fold shortening of channel closed times and an increase in the number of long openings. In contrast to the intracellular gating domain in the N‐terminus which is critical for channel opening, the intact extracellular degenerin site is necessary for normal channel closing, as illustrated by our observation that modification of βS518C destabilises the channel closed state. The modification by the sulfhydryl reagents is state‐ and size‐dependent consistent with a conformational change of the degenerin site during channel opening and closing. We propose that the intracellular and extracellular modulatory sites act on a common channel gate and control the activity of ENaC at the cell surface.

Common variants of the beta and gamma subunits of the epithelial sodium channel and their relation to plasma renin and aldosterone levels in essential hypertension

BackgroundRare mutations of the epithelial sodium channel (ENaC) result in the monogenic hypertension form of Liddles syndrome. We decided to screen for common variants in the ENaC βand γ subunits in patients with essential hypertension and to relate their occurrence to the activity of circulating renin-angiotensin-aldosterone system.MethodsInitially, DNA samples from 27 patients with low renin/low aldosterone hypertension were examined. The DNA variants were subsequently screened for in 347 patients with treatment-resistant hypertension, 175 male subjects with documented long-lasting normotension and 301 healthy Plasma renin and aldosterone levels were measured under baseline conditions and during postural and captopril challenge tests.ResultsTwo commonly occurring βENaC variants (G589S and a novel intronic i12-17CT substitution) and one novel γENaC variant (V546I) were detected. One of these variants occurred in a heterozygous form in 32 patients, a prevalence (9.2%) significantly higher than that in normotensive males (2.9%, p = 0.007) and blood donors (3.0%, p = 0.001). βENaC i12-17CT was significantly more prevalent in the hypertension group than in the two control groups combined (4.6% vs. 1.1%, p = 0.001). When expressed in Xenopus oocytes, neither of the two ENaC amino acid-changing variants showed a significant difference in activity compared with ENaC wild-type. No direct evidence for a mRNA splicing defect could be obtained for the βENaC intronic variant. The ratio of daily urinary potassium excretion to upright and mean (of supine and upright values) plasma renin activity was higher in variant allele carriers than in non-carriers (p = 0.034 and p = 0.048).ConclusionsAt least 9% of Finnish patients with hypertension admitted to a specialized center carry genetic variants of β and γENaC, a three times higher prevalence than in the normotensive individuals or in random healthy controls. Patients with the variant alleles showed an increased urinary potassium excretion rate in relation to their renin levels.

Identification of a highly conserved sequence at the N-terminus of the epithelial Na+ channel α subunit involved in gating

Abstract. The epithelial Na+ channel (ENaC) is responsible for Na+ reabsorption in aldosterone target tissues such as distal nephron and colon. ENaC is a heterotetramer composed of three homologous subunits, α, β, and γENaC. Mutations leading to loss of function or reduced channel activity have been identified in all three subunits in patients with pseudohypoaldosteronism type-1. One missense mutation substituting a glycine (G95S) which is completely conserved throughout the gene family reduced ENaC open probability, Po. In this study we have performed systematic alanine substitutions of 28 residues of αENaC encompassing the glycine (G95). This screen identified a stretch of ten consecutive amino acids (αT92–αC101) which, when mutated, lead to a decrease in Na+ current (INa) expressed with no significant changes in channel surface expression. This inhibitory effect was strongest for G95 and for two additional highly conserved amino acids – H94 and R98. The R98A mutant led to an important reduction in channel Po with no change in single-channel conductance, indicating that the segment encompassing H94, G95 and R98 is involved in modulation of channel gating kinetics.

A Gating Mutation in the Internal Pore of ASIC1a

Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd2+ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd2+ on R43C can be obtained with Cd2+ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore.

Congenital ataxia and hemiplegic migraine with cerebral edema associated with a novel gain of function mutation in the calcium channel CACNA1A

Mutations in the CACNA1A gene, encoding the α1 subunit of the voltage-gated calcium channel Ca(V)2.1 (P/Q-type), have been associated with three neurological phenotypes: familial and sporadic hemiplegic migraine type 1 (FHM1, SHM1), episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 (SCA6). We report a child with congenital ataxia, abnormal eye movements and developmental delay who presented severe attacks of hemiplegic migraine triggered by minor head traumas and associated with hemispheric swelling and seizures. Progressive cerebellar atrophy was also observed. Remission of the attacks was obtained with acetazolamide. A de novo 3 bp deletion was found in heterozygosity causing loss of a phenylalanine residue at position 1502, in one of the critical transmembrane domains of the protein contributing to the inner part of the pore. We characterized the electrophysiology of this mutant in a Xenopus oocyte in vitro system and showed that it causes gain of function of the channel. The mutant Ca(V)2.1 activates at lower voltage threshold than the wild type. These findings provide further evidence of this molecular mechanism as causative of FHM1 and expand the phenotypic spectrum of CACNA1A mutations with a child exhibiting severe SHM1 and non-episodic ataxia of congenital onset.