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.