title: Interacting domains in the epithelial sodium channel that mediate proteolytic activation.
abstract: Epithelial Sodium Channel (ENaC) proteolysis at sites in the extracellular loop of the α and γ 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 α and γ 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 α and γ ENaC interact with the channel complex; with interactions between the N-terminal γ and the C-terminal α 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 γ exhibited the largest inhibition of channel function. This region was found to interact with the C-terminal α 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 α and γ subunit and indicate that the untethered N-terminal fragments of these subunits interact with the channel complex.
authors.affiliation: a Department of Physiology and Biophysics ; State University of New York at Buffalo ; Buffalo NY USA.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Berman
authors.affiliation: a Department of Physiology and Biophysics ; State University of New York at Buffalo ; Buffalo NY USA.
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Awayda
authors.affiliation: a Department of Physiology and Biophysics ; State University of New York at Buffalo ; Buffalo NY USA.
authors.forename: Mouhamed S
authors.initials: MS
authors.lastname: Awayda
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D003545:Cysteine / Q000235:genetics; D053503:Epithelial Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D014981:Xenopus
chemical_list: D053503:Epithelial Sodium Channels; D003545:Cysteine
keywords: ENaC; activity; furin; proteolysis; structure-function; subtilisin; trypsin