Mapping allosteric linkage to channel gating by extracellular domains in the human epithelial sodium channel

The epithelial sodium channel (ENaC) mediates sodium absorption in lung, kidney, and colon epithelia. Channels in the ENaC/degenerin family possess an extracellular region that senses physicochemical changes in the extracellular milieu and allosterically regulates the channel opening. Proteolytic cleavage activates the ENaC opening, by the removal of specific segments in the finger domains of the - and ENaC-subunits. Cleavage causes perturbations in the extracellular region that propagate to the channel gate. However, it is not known how the channel structure mediates the propagation of activation signals through the extracellular sensing domains. Here, to identify the structure–function determinants that mediate allosteric ENaC activation, we performed MD simulations, thiol modification of residues substituted by cysteine, and voltage-clamp electrophysiology recordings. Our simulations of an ENaC heterotetramer, ₁₂, in the proteolytically cleaved and uncleaved states revealed structural pathways in the -subunit that are responsible for ENaC proteolytic activation. To validate these findings, we performed site-directed mutagenesis to introduce cysteine substitutions in the extracellular domains of the -, -, and ENaC-subunits. Insertion of a cysteine at the -subunit Glu⁵⁵⁷ site, predicted to stabilize a closed state of ENaC, inhibited ENaC basal activity and retarded the kinetics of proteolytic activation by 2-fold. Our results suggest that the lower palm domain of ENaC is essential for ENaC activation. In conclusion, our integrated computational and experimental approach suggests key structure–function determinants for ENaC proteolytic activation and points toward a mechanistic model for the allosteric communication in the extracellular domains of the ENaC/degenerin family channels.