Constraint-based, Homology Model of the Extracellular Domain of the Epithelial Na+ Channel α Subunit Reveals a Mechanism of Channel Activation by Proteases

Abstract

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...

Inhibitory Tract Traps the Epithelial Na+ Channel in a Low Activity Conformation

“…1A). We recently showed that N-Cys can be cross-linked to E174C but not E173C (11). We mutated each of the remaining sites to Cys and attempted to cross-link these mutants to both N-Cys and C-Cys using bifunctional MTS compounds with alkanediyl linkers of various lengths.…”

“…Except for Y474C, successful cross-linking depended on the length of the alkanediyl linker of the MTS compound. Of the sites 170 -175 at the N terminus of helix ␣1, we previously examined MTS-4-MTS cross-linking of N-Cys and C-Cys to Cys introduced at Glu-173 and Glu-174 (11). These data provided evidence for cross-linking between E174C and N-Cys but not cross-linking involving E173C.…”

“…Our model placed the bound peptide at the finger-thumb domain interface, suggesting that this domain interface changes conformation upon gating. Motions around this interface were correlated with twisting motions in the pore in normal mode analysis calculations performed on both ASIC1 (14) and our ␣ ENaC model (11).…”

“…We recently reported that introduction of Trp mutations at a large number of sites in the ␣ subunit reduced the inhibition of channel activity by the ␣ subunit-derived 8-mer peptide, Ac-LPHPLQRL-amide (10). A structural model of the ␣ subunit extracellular region was constructed based on these data and on homology to ASIC1 (11), the only member of the ENaC/degenerin family whose structure has been resolved (12,13). ASIC1 is homotrimeric, and the extracellular region of each subunit has three peripheral helical domains (termed the thumb, finger, and knuckle) surrounding two central ␤ sheet domains (termed palm and ␤-ball) that form the core of the structure.…”

Activation of the Caenorhabditis elegans Degenerin Channel by Shear Stress Requires the MEC-10 Subunit

“…Sequencing the two tm1552 transcripts revealed that the large transcript introduced a pre- mature stop codon after Ile 189 . The small but more abundant transcript encoded a MEC-10 protein with an in-frame deletion of 150 residues (Ala 162 -Lys 311 ) from a peripheral part of the extracellular region that is poorly conserved among members of the ENaC/degenerin family, referred to as the finger domain (33,34). We generated both gene products, MEC-10 L190X and MEC-10 ⌬162-311, and examined the LSS response of channels containing these MEC-10 mutants.…”