Inhibitory tract traps the epithelial Na+ channel in a low activity conformation

Proteolysis plays an important role in the maturation and activation of epithelial Na(+) channels (ENaCs). Non-cleaved channels are inactive at high extracellular Na(+) concentrations and fully cleaved channels are constitutively active. Cleavage of the α and γ subunits at multiple sites activates the channel through the release of imbedded inhibitory tracts. Peptides derived from these released tracts are also inhibitory, likely through binding at the inhibitory tract sites. We recently reported a model of the α subunit. We have now cross-linked Cys derivatives of the inhibitory peptide to the channel, using our model to predict sites at a domain interface of the α subunit that is in proximity to the N terminus of the peptide. Furthermore, peptide inhibition was mimicked in the absence of peptide by cross-linking the channel across the domain interface. Our results suggest a dynamic domain interface that can be exploited by inhibitory peptides and provides a mechanism for peptide inhibition and proteolytic activation.     

Atomic force microscopy reveals the architecture of the epithelial sodium channel (ENaC)

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (α, β, and γ); 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 α-, β- and γ-subunits. We show that for α-, β- and γ-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 αβγ-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.     

Independent Contribution of Extracellular Proton Binding Sites to ASIC1a Activation

Acid-sensing ion channels (ASICs) are a group of trimeric cation permeable channels gated by extracellular protons that are mainly expressed in the nervous system. Despite the structural information available for ASIC1, there is limited understanding of the molecular mechanism that allows these channels to sense and respond to drops in extracellular pH. In this report, we employed the substituted cysteine accessibility method and site-directed mutagenesis to examine the mechanism of activation of ASIC1a by extracellular protons. We found that the modification of E238C and D345C channels by MTSET reduced proton apparent affinity for activation. Furthermore, the introduction of positively charged residues at position 345 rendered shifted biphasic proton activation curves. Likewise, channels bearing mutations at positions 79 and 416 in the palm domain of the channel showed reduced proton apparent affinity and biphasic proton activation curves. Of significance, the effect of the mutations at positions 79 and 345 on channel activation was additive. E79K-D345K required a change to a pH lower than 2 for maximal activation. In summary, this study provides direct evidence for the presence of two distinct proton coordination sites in the extracellular region of ASIC1a, which jointly facilitate pore opening in response to extracellular acidification.