Epithelial Na+ Channels Are Fully Activated by Furin- and Prostasin-dependent Release of an Inhibitory Peptide from the γ-Subunit

Abstract

Epithelial sodium channels (ENaC) are expressed in the apical membrane of high resistance Na ؉ transporting epithelia and have a key role in regulating extracellular fluid volume and the volume of airway surface liquids. Maturation and activation of ENaC subunits involves furin-dependent cleavage of the ectodomain at two sites in the ␣ subunit and at a single site within the ␥ subunit. We now report that the serine protease prostasin further activates ENaC by inducing cleavage of the ␥ subunit at a site distal to the furin cleavage site. Dual cleavage of the ␥ subunit is predicted to release a 43-amino acid peptide. Channels with a ␥ subunit lacking this 43-residue tract have increased activity due to a high open probability. A synthetic peptide corresponding to the fragment cleaved from the ␥ subunit is a reversible inhibitor of endogenous ENaCs in mouse corticalcollecting duct cells and in primary cultures of human airway epithelial cells. Our results suggest that multiple proteases cleave ENaC ␥ subunits to fully activate the channel.Epithelial sodium channels (ENaC) 3 are expressed at the apical plasma membrane of cells lining the distal nephron, airway and alveoli, and distal colon, where they play a key role in the regulation of extracellular fluid volume, blood pressure, and airway surface liquid volume. These channels are composed of three homologous subunits, termed ␣, ␤, and ␥. Each subunit has cytosolic amino and carboxyl termini and two membranespanning domains separated by a large ectodomain (1-3). The second membrane-spanning domain and the preceding region of each subunit are predicted to form the channel pore (4 -7). Proteolysis of ENaC subunit extracellular domains at specific sites has a key role in modulating channel gating (8 -10). Maturation of ENaC subunits in Xenopus oocytes, Madin-Darby canine kidney (MDCK) cells, and Chinese hamster ovary cells involves furin-dependent cleavage at two sites in the extracellular loop of the ␣ subunit and at a single site within the extracellular loop of the ␥ subunit (8). Channels that lack proteolytic processing exhibit markedly reduced activity and enhanced inhibition by external Na ϩ , a process referred to as Na ϩ selfinhibition (9). ENaC subunit cleavage by furin or exogenous trypsin relieves channels from inhibition by external Na ϩ (9, 11). We previously proposed that furin-dependent proteolysis of the ␣ subunit activates ENaC by disassociating an inhibitory domain (␣Asp-206 -Arg-231) from its effector site within the channel complex (10).Endogenous proteases other than furin likely have a role in the processing and activation of ENaC. A number of serine proteases, referred to as "channel activating proteases," have been identified that increase ENaC activity when co-expressed with ENaC in heterologous expression systems (12-14). Furthermore, selective serine protease inhibitors that do not block furin, such as aprotinin and bikunin, reduce ENaC activity (14 -20). Prostasin is an aprotinin-sensitive "channel activating (serine) protease" that inc...

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

“…Thus, in both cases the loss of the segment between the two furin cleavage sites is not likely to be an obligatory mechanism of ENaC activation by ␣ cleavage. Such built-in redundancy into channel cleavage would certainly be consistent with the ability of multiple diverse proteases such as trypsin, subtilisin, elastase, prostasin, furin, and plasmin to activate ENaC in different systems with a similar final outcome (9,24,(31)(32)(33)(34)(35).…”

“…Kleyman and co-workers (9,10) have proposed that the mechanism of the P o increase involves the release of small inhibitory fragments from the ␣ and ␥ subunits and that cleavage at two points in the extracellular loop is necessary for ENaC activation. They also proposed that the resulting N-and C-terminal fragments of subunit cleavage remain associated with the active channel complex, although a direct comparison of fragment levels at the membrane was not feasible given the presence of two different tags on these fragments.…”

Inhibitory Tract Traps the Epithelial Na+ Channel in a Low Activity Conformation

“…ENaC proteolysis has been suggested to play a role in disorders such as nephrotic syndrome and cystic fibrosis, where concentrations of specific protease are elevated in the extracellular milieu (4,5). Activation requires cleavage at multiple sites within the ␣ and/or ␥ subunits with the liberation of imbedded inhibitory tracts (3,6,7). Crucially, it is the release of inhibitory tracts and not cleavage itself that activates ENaC.…”

Conserved cysteines in the finger domain of the epithelial Na+ channel α and γ subunits are proximal to the dynamic finger–thumb domain interface

“…1A) are divergent and may underlie functional differences between the channels (10,11). The sites of channel activating proteolytic cleavage in the a and g subunits map to their respective finger domains (6,12,13), underlining the notion that the divergent domains are functionally important. We previously reported a model of the a subunit extracellular domains using ASIC1 homology complemented by constraints for the divergent finger domain derived from functional experiments (10).…”

“…Double cleavage of the ENaC a and g subunits liberates autoinhibitory tracts from each subunit (13,16). Synthetic peptides corresponding to each of the liberated tracts inhibit the channel (17,18).…”