Interacting Domains in the Epithelial Sodium Channel that Mediate Proteolytic Activation

ABSTRACTEpithelial Sodium Channel (ENaC) proteolysis at sites in the extracellular loop of the alpha and gamma subunits leads to marked activation. The mechanism of this effect remains debated, as well as the role of the N- and C-terminal fragments of these subunits created by cleavage. We introduced cysteines at sites bracketing upstream and downstream the cleavage regions in alpha and gamma ENaC to examine the role of these fragments in the activated channel. Using thiol modifying reagents, as well as examining the effects of cleavage by exogenous proteases we constructed a functional model that determines the potential interactions of the termini near the cleavage regions. We report that the N-terminal fragments of both alpha and gamma ENaC interact with the channel complex; with interactions between the N-terminal gamma and the C-terminal alpha fragments being the most critical to channel function and activation by exogenous cleavage by subtilisin. Positive charge modification at a.a.135 in the N-terminal fragment of gamma exhibited the largest inhibition of channel function. This region was found to interact with the C-terminal alpha fragment between a.a. 205 and 221; a tract which was previously identified to be the site of subtilisin's action. These data provide the first evidence for the functional channel rearrangement caused by proteolysis of the alpha and gamma subunit and indicate that the untethered N-terminal fragments of these subunits interact with the channel complex.