Functional Characterization of a Partial Loss-of-Function Mutation of the Epithelial Sodium Channel (ENaC) Associated with Atypical Cystic Fibrosis

Huber, Regina; Krueger, Bettina; Diakov, Alexei; Korbmacher, Judit; Einsiedel, Jürgen; Gmeiner, Peter; Azad, Abul Kalam; Cuppens, Harry; Cassiman, Jean‐Jacques; Korbmacher, Christoph; Rauh, R. D.

doi:10.1159/000272059

Cited by 27 publications

(27 citation statements)

References 55 publications

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“…Human, mouse, and rat are indicated as h, m, and r, respectively. severity in individuals with one or two mutant CFTR alleles as suggested by others (2,20,33).…”

Section: Discussionmentioning

confidence: 64%

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Gain-of-function variant of the human epithelial sodium channel

Chen

Kleyman

Sheng

2013

American Journal of Physiology-Renal Physiology

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ϩ channel (ENaC) mutations are associated with several human disorders, underscoring the importance of these channels in human health. Recent human genome sequencing projects have revealed a large number of ENaC gene variations, several of which have been found in individuals with salt-sensitive hypertension, cystic fibrosis, and other disorders. However, the functional consequences of most variants are unknown. In this study, we used the Xenopus oocyte expression system to examine the functional properties of a human ENaC variant. Oocytes expressing ␣␤␥L511Q human ENaCs showed 4.6-fold greater amiloridesensitive currents than cells expressing wild-type channels. The ␥L511Q variant did not significantly alter channel surface expression. Single channel recordings revealed that the variant had fourfold higher open probability than wild type. In addition, ␥L511Q largely eliminated the Na ϩ self-inhibition response, which reflects a downregulation of ENaC open probability by extracellular Na ϩ . Moreover, ␥L511Q diminished chymotrypsin-induced activation of the mutant channel. We conclude that ␥L511Q is a gain-of-function human ENaC variant. Our results suggest that ␥L511Q enhances ENaC activity by increasing channel open probability and dampens channel regulation by extracellular Na ϩ and proteases.

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“…Human, mouse, and rat are indicated as h, m, and r, respectively. severity in individuals with one or two mutant CFTR alleles as suggested by others (2,20,33).…”

Section: Discussionmentioning

confidence: 64%

“…These include SNPs, INDELs, and microsatellite repeats. Aside from a small number of variants (2,16,20,33,36,43), the functional properties of the vast majority of these ENaC variants are unknown.…”

Section: Epithelial Namentioning

confidence: 99%

Gain-of-function variant of the human epithelial sodium channel

Chen

Kleyman

Sheng

2013

American Journal of Physiology-Renal Physiology

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“…Corresponding I/V plots (Fig. 1D) indicate that the tripletagged channel has a single-channel conductance (5.2 pS) that is typical for WT human ENaC (17,26). We also investigated the ion selectivity of the triple-tagged channel.…”

Section: Resultsmentioning

confidence: 90%

Atomic Force Microscopy Reveals the Architecture of the Epithelial Sodium Channel (ENaC)

Stewart

Haerteis

Diakov

et al. 2011

Journal of Biological Chemistry

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The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (␣, ␤, and ␥); however, the subunit stoichiometry is still controversial. Here, we addressed this issue using atomic force microscopy imaging of complexes between isolated ENaC and antibodies/Fab fragments directed against specific epitope tags on the ␣-, ␤-and ␥-subunits. We show that for ␣-, ␤-and ␥-ENaC alone, pairs of antibodies decorate the channel at an angle of 120°, indicating that the individual subunits assemble as homotrimers. A similar approach demonstrates that ␣␤␥-ENaC assembles as a heterotrimer containing one copy of each subunit. Intriguingly, all four subunit combinations also produce higher-order structures containing two or three individual trimers. The trimer-of-trimers organization would account for earlier reports that ENaC contains eight to nine subunits.The epithelial sodium channel (ENaC) 5 is a member of the ENaC/degenerin superfamily, which also includes acid-sensing ion channels (ASICs; reviewed in Ref. 1). ENaC mediates Na ϩ entry across the apical membranes of many absorptive epithelia, including the alveolar epithelium, salivary duct, distal colon, and sweat glands. ENaC plays a particularly important role in Na ϩ transport across the aldosterone-sensitive distal nephron (reviewed in Ref. 2), which is responsible for the regulation of Na ϩ and K ϩ excretion, and thus plays an important role in the control of blood pressure. ENaC is the target of potassiumsparing diuretics such as amiloride and spironolactone used in the treatment of cardiovascular disease. Whereas amiloride is a direct blocker of the channel, the mineralocorticoid receptor antagonist spironolactone inhibits ENaC activity by preventing its stimulation by aldosterone.ENaC is a heteromultimer containing three homologous subunits (␣, ␤, and ␥), which have 30 -40% amino acid identity (3, 4). A fourth ENaC subunit, ␦-ENaC, has been cloned from a human kidney library (5). In heterologous expression systems, ␦-ENaC has functional similarities with ␣-ENaC, but its physiological role is still unclear (6). Each ENaC subunit contains two transmembrane domains, a large extracellular loop, and short intracellular N and C termini. Full ENaC activity requires coexpression of all three subunits (4). In the past, it has been proposed that ENaC is assembled from either four (7-9) or eight to nine subunits (10, 11). However, the recently published crystal structure of ASIC 1a (12) suggests that ENaC is likely to be a trimer (13), although this has not been demonstrated.We have developed a method, based on AFM imaging, for determining the stoichiometry and arrangement of subunits within multimeric proteins (reviewed in Ref. 14). The method involves engineering specific epitope tags onto each protein subunit and expressing the proteins exogenously in a suitable cell line (usually tsA 201). Membrane fractions from the transfected cells are solubilized in detergent, and the proteins are isola...

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“…It has also been demonstrated that manipulations that result in a maximum increment in opening kinetics were capable of abolishing selfinhibition, for example, serine protease proteolysis and locking all channels in the open state (b S520C , a degenerin mutant). A mutation (a F61L ) at the amino terminal of the protein, adjacent to the gating tract in the M1 domain, enhanced self-inhibition (25). Our current understanding of the nature of ENaC gating mechanisms provides no unequivocal answers to the question of how the aforementioned domains affect self-inhibition.…”

Section: Epithelial Namentioning

confidence: 99%

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na⁺ Self-Inhibition of Epithelial Na⁺ Channels

Han

Nie²,

et al. 2011

Am J Respir Cell Mol Biol

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Salt absorption via alveolar epithelial Na 1 channels (ENaC) is a critical step for maintaining an airspace free of flooding. Previously, we found that 8-(4-chlorophenylthio)-guanosine-39,59-cyclic monophosphate-Na (CPT-cGMP) activated native and heterologous ENaC. To investigate the potential pharmacological relevance, we applied this compound intratracheally to human lungs and found that ex vivo alveolar fluid clearance was increased significantly. Furthermore, this compound eliminated self-inhibition in human lung H441 cells and in oocytes expressing human abg but not dbg channels. To further elucidate this novel mechanism, we constructed mutants abolishing (b DV348 and g H233R ) or augmenting (a Y458A and g M432G ) self-inhibition. The mutants eliminating self-inhibition lost their responses to CPT-cGMP, whereas those enhancing self-inhibition facilitated the stimulatory effects of this compound. CPT-cGMP was unable to activate a high P o mutant (b S520C ) and plasmin proteolytically cleaved channels. Our data suggest that elimination of self-inhibition of abg ENaC may be a novel mechanism for CPT-cGMP to stimulate salt reabsorption in human lungs.Keywords: lung fluid reabsorption; amiloride-sensitive sodium channel; CPT-cGMP; ENaC self-inhibition Epithelial Na1 channels (ENaC) in polarized absorptive tight epithelium are responsible for the transapical salt influx. Together with basolaterally located Na 1 /K 1 -ATPase, the vectorial solute/fluid transport pathway is crucial for maintaining luminal and body fluid volume (1-3). Five subunits have been cloned: a, b, g, d, and e ENaC. Luminal impermeable reagents and hormones have been confirmed to regulate salt reabsorption in lung, kidney, and colon.ENaC has long been proposed to function as a ligand-gated channel on the basis of observations of other ENaC/DEG family members (1, 4). Acid-sensing Na 1 channels, a branch of the ENaC/DEG super gene family, are gated by external protons. Another branch of channels, termed FaNaC, are manipulated by FMRFamide and related tetrapeptides (5). With respect to ENaC channels, a number of luminal reagents, including parachloromercuribenzoate; benzimidazoly-2-guanidinium; bumetanide; and H 1 , Zn 21 , and Ni 21 ions, have been reported to alter external Na 1 self-inhibition, a temperature-sensitive intrinsic phenomenon of ENaC (1, 4, 6-10). Recently, a small molecule (S6939), cpt-cAMP, halothane, glibenclamide, and extracellular chloride ions (Cl 2 ) have been shown to act as ligands to regulate heterologous ENaC (11-16). Among these, halothane and Cl 2 ions may activate ENaC by eliminating Na 1 self-inhibition (17, 18). We recently found that 8-(4-chlorophenylthio)-guanosine-39,59-cyclic monophosphate-Na (CPT-cGMP) stimulated human abg ENaC in oocytes in a PKG-independent and domain-specific manner (19). The underlying mechanisms are unknown.Several ectodomains have been reported to modify self-inhibition by unknown mechanisms. Given that self-inhibition is governed by the extracellular Na 1 concentrations, the extracellu...

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Functional Characterization of a Partial Loss-of-Function Mutation of the Epithelial Sodium Channel (ENaC) Associated with Atypical Cystic Fibrosis

Cited by 27 publications

References 55 publications

Gain-of-function variant of the human epithelial sodium channel

Gain-of-function variant of the human epithelial sodium channel

Atomic Force Microscopy Reveals the Architecture of the Epithelial Sodium Channel (ENaC)

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na⁺ Self-Inhibition of Epithelial Na⁺ Channels

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Functional Characterization of a Partial Loss-of-Function Mutation of the Epithelial Sodium Channel (ENaC) Associated with Atypical Cystic Fibrosis

Cited by 27 publications

References 55 publications

Gain-of-function variant of the human epithelial sodium channel

Gain-of-function variant of the human epithelial sodium channel

Atomic Force Microscopy Reveals the Architecture of the Epithelial Sodium Channel (ENaC)

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na+ Self-Inhibition of Epithelial Na+ Channels

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8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na⁺ Self-Inhibition of Epithelial Na⁺ Channels