Insight toward epithelial Na<sup>+</sup> channel mechanism revealed by the acid‐sensing ion channel 1 structure

Stockand, James D.; Staruschenko, Alexander; Pochynyuk, Oleh; Booth, Rachell E.; Silverthorn, Dee U.

doi:10.1002/iub.89

Cited by 90 publications

(77 citation statements)

References 27 publications

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“…This segment between Arg-178 and Arg-204 maps at or near the exposed finger domain of the crystallized ASIC1 (25). Therefore, it is expected to be solution-exposed and amenable to extracellular cleavage.…”

Section: Mutation Of ␣ Enac Cleavage Sites Andmentioning

confidence: 99%

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

Bengrine

Lis

et al. 2009

Journal of Biological Chemistry

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We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of ␣ENaC using the serine protease subtilisin. Trimeric channels formed with ␣FM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an ϳ10-fold decrease in amiloride-sensitive whole cell conductance as compared with ␣WT (2.2 versus 21.2 microsiemens ( S)). Both ␣WT and ␣FM were activated to similar levels by subtilisin cleavage. Channels formed with ␣FD, a construct that deleted the segment between the two furin sites (⌬175-204), exhibited an intermediate conductance of 13.2 S. More importantly, ␣FD retained the ability to be activated by subtilisin to 108.8 ؎ 20.9 S, a level not significantly different from that of subtilisin activated ␣WT (125.6 ؎ 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of ␣ was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with ␣LD, a construct that extended the deleted sequence of ␣FD by 17 amino acids (⌬175-221). Channels with ␣LD were uncleaved, exhibited low baseline activity (4.1 S), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.It is well established that serine proteases activate the epithelial sodium channel (ENaC). 2 Activation occurs by direct mechanisms that induce channel subunit cleavage (1, 2) as well as those that are cleavage-independent but may involve cleavage of protease-activated membrane receptors (3). Channel cleavage studies have established that cellular proteases such as furin endogenously cleave the channel ␣ and ␥ subunits. Mutation of identified endogenous cleavage sites on both of these subunits diminished baseline activity, demonstrating a role for cleavage in ENaC activation.The acute effects of ENaC cleavage have largely relied on examining the effects of the protease trypsin on the ␣ and ␥ subunits. These studies have examined the effects of cleavage on wild type and furin cleavage-deficient ENaC in oocytes and epithelial cells (1,4,5). Although these have markedly improved our understanding of channel activation by serine proteases, they suffer from the main shortcoming that trypsin is a non-selective serine protease that can cleave after a single arginine residue (6, 7), and therefore, it only offers a limited tool for examining the mechanisms of cleavage at specific sites. Consistent with the reduced specificity for trypsin is the observation that ENaC retains its cleavage by this protease after mutation of consensus cleavage sites for furin (1).Despite their limitations, these studies have ind...

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Section: Mutation Of ␣ Enac Cleavage Sites Andmentioning

confidence: 99%

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

Bengrine

Lis

et al. 2009

Journal of Biological Chemistry

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“…The ENaC is a heterotrimer formed by the three subunits, a, b, and g (7,8,9,10). All subunits are thought to have a comparable three-dimensional structure containing two transmembrane segments, intracellular N-and C-termini, and a large extracellular loop (11).…”

Section: Introductionmentioning

confidence: 99%

Five novel mutations in the SCNN1A gene causing autosomal recessive pseudohypoaldosteronism type 1

Welzel

Akın

Büscher³

et al. 2013

European Journal of Endocrinology

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Background: Pseudohypoaldosteronism type 1 (PHA1) is a monogenic disease caused by mutations in the genes encoding the human mineralocorticoid receptor (MR) or the a (SCNN1A), b (SCNN1B) or g (SCNN1G) subunit of the epithelial Na C channel (ENaC). While autosomal dominant mutation of the MR cause renal PHA1, autosomal recessive mutations of the ENaC lead to systemic PHA1. In the latter, affected children suffer from neonatal onset of multi-organ salt loss and often exhibit cystic fibrosis-like pulmonary symptoms. Objective: We searched for underlying mutations in seven unrelated children with systemic PHA1, all offsprings of healthy consanguineous parents. Methods and results: Amplification of the SCNN1A gene and sequencing of all 13 coding exons unraveled mutations in all of our patients. We found five novel homozygous mutations (c.587_588insC in two patients, c.1342_1343insTACA, c.742delG, c.189COA, c.1361-2AOG) and one known mutation (c.1474COT) leading to truncation of the aENaC protein. All parents were asymptomatic heterozygous carriers of the respective mutations, confirming the autosomal recessive mode of inheritance. Five out of seven patients exhibited pulmonary symptoms in the neonatal period. Conclusion: The a subunit is essential for ENaC function and mutations truncating the pore-forming part of the protein leading to systemic PHA1. Based on current knowledge, the pulmonary phenotype cannot be satisfactorily predicted.

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“…In the past, it has been proposed that ENaC is assembled from either four (7)(8)(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.…”

mentioning

confidence: 99%

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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Insight toward epithelial Na⁺ channel mechanism revealed by the acid‐sensing ion channel 1 structure

Cited by 90 publications

References 27 publications

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

Five novel mutations in the SCNN1A gene causing autosomal recessive pseudohypoaldosteronism type 1

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

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Insight toward epithelial Na+ channel mechanism revealed by the acid‐sensing ion channel 1 structure

Cited by 90 publications

References 27 publications

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

Five novel mutations in the SCNN1A gene causing autosomal recessive pseudohypoaldosteronism type 1

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

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Insight toward epithelial Na⁺ channel mechanism revealed by the acid‐sensing ion channel 1 structure