The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel

Journal of Biological Chemistry (Impact Factor: 4.57). 06/2014; 289(33). DOI: 10.1074/jbc.M114.570952
Source: PubMed


The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular
domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed
or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by
intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental
techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction
with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly
conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We
also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of
allosteric control of ENaC proteolysis is dependent on Tyr370 in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on
a potential general mechanism of channel and receptor activation.

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