The epithelial Na ؉ channel (ENaC) mediates Na ؉ transport across high resistance epithelia. This channel is assembled from three homologous subunits with the majority of the protein's mass found in the extracellular domains. Acid-sensing ion channel 1 (ASIC1) is homologous to ENaC, but a key functional domain is highly divergent. Here we present molecular models of the extracellular region of ␣ ENaC based on a large data set of mutations that attenuate inhibitory peptide binding in combination with comparative modeling based on the resolved structure of ASIC1. The models successfully rationalized the data from the peptide binding screen. We engineered new mutants that had not been tested based on the models and successfully predict sites where mutations affected peptide binding. Thus, we were able to confirm the overall general fold of our structural models. Further analysis suggested that the ␣ subunit-derived inhibitory peptide affects channel gating by constraining motions within two major domains in the extracellular region, the thumb and finger domains.Epithelial Na ϩ channels (ENaCs) 3 are members of the ENaC/degenerin family of ion channels, of which the high resolution structure of acid-sensing ion channel 1 (ASIC1) has been reported. These channels are probably trimers (1, 2) with each subunit having two transmembrane helices, large extracellular regions, and short cytosolic amino and carboxyl termini (3). The resolved structure of the extracellular region of ASIC1 is composed of core ␤-sheet domains (termed palm and ␤-ball) surrounded by peripheral ␣-helical domains (termed finger, thumb, and knuckle) (1). Channels in the ENaC/degenerin family are Na ϩ -permeable and are gated by a diverse set of stimuli, including external ligands and mechanical forces (4). As such, ENaC/degenerin family members play diverse roles in biology. For ENaC, these include the regulation of extracellular volume and blood pressure by mediating Na ϩ transport in the distal nephron of the kidney, regulation of airway surface liquid volume and mucociliary clearance by facilitating Na ϩ transport in airways, and facilitation of salt taste by transporting Na ϩ in lingual epithelium (4). ENaC is assembled from homologous ␣, ␤, and ␥ subunits and is allosterically inhibited by extracellular Na ϩ by a phenomenon referred to as Na ϩ self-inhibition (5-7). Within the ENaC/degenerin family, sequence conservation is conspicuously lacking within the finger domains of the extracellular regions of these channels (1). This fact may partly account for the diversity in the regulation of channel gating observed among gene family members and is an obstacle in building comparative models of ENaC subunits based on the resolved ASIC1 structure.Among the panoply of ENaC properties is its activation by proteolytic cleavage, which is unusual for ion channels (8). Proteolytic activation of ENaC occurs through the cleavage of both the ␣ and ␥ subunits at multiple sites within their finger domains, leading to the release of inhibitory tracts (9 -12). Pe...

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Background: Proteases activate ENaC by releasing inhibitory tracts. Results: Inhibitory peptides cross-link to the finger and thumb domains of ENaC. Simply cross-linking these domains inhibits channel activity. Conclusion: Inhibitory peptides bind at a finger-thumb interface, inhibiting the channel by maintaining the interface in a tight conformation. Significance: These observations provide insights regarding the mechanisms of channel activation by proteases.

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Background: Laminar shear stress (LSS) regulates epithelial sodium channel (ENaC) activity, primarily by increasing single channel open probability. Results: Mutations introduced within the extracellular finger domain altered the LSS response. Conclusion:The finger domain participates in the LSS response. Significance: Our results enhance our understanding of the regulation of ENaC by mechanical forces.

Background: The epithelial Na + channel (ENaC) is intrinsically linked to fluid volume homeostasis and blood pressure. Specific rare mutations in SCNN1A , SCNN1B , and SCNN1G , genes encoding the α, β, and γ subunits of ENaC, respectively, are associated with extreme blood pressure phenotypes. No associations between blood pressure and SCNN1D , which encodes the δ subunit of ENaC, have been reported. A small number of sequence variants in ENaC subunits have been reported to affect functional transport in vitro or blood pressure. The effects of the vast majority of rare and low-frequency ENaC variants on blood pressure are not known. Methods: We explored the association of low frequency and rare variants in the genes encoding ENaC subunits, with systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure. Using whole-genome sequencing data from 14 studies participating in the Trans-Omics in Precision Medicine Whole-Genome Sequencing Program, and sequence kernel association tests. Results: We found that variants in SCNN1A and SCNN1B were associated with diastolic blood pressure and mean arterial pressure ( P <0.00625). Although SCNN1D is poorly expressed in human kidney tissue, SCNN1D variants were associated with systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure ( P <0.00625). ENaC variants in 2 of the 4 subunits ( SCNN1B and SCNN1D ) were also associated with estimated glomerular filtration rate ( P <0.00625), but not with stroke. Conclusions: Our results suggest that variants in extrarenal ENaCs, in addition to ENaCs expressed in kidneys, influence blood pressure and kidney function.

Epithelial Na channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, β-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αβγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na. The lack of N-linked glycans on the β-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the β-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type β-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.

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