The Epithelial Na+ Channel Is Inhibited by a Peptide Derived from Proteolytic Processing of Its α Subunit

Abstract

Epithelial sodium channels (ENaCs) mediate Na؉ entry across the apical membrane of high resistance epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels are composed of three homologous subunits, termed ␣, ␤, and ␥, which have intracellular amino and carboxyl termini and two membranespanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the ␣ subunit and at a single site within the ␥ subunit. Epithelial sodium channels (ENaCs) 2 mediate Na ϩ entry across the apical membrane of high resistance epithelia, including the distal nephron, airway and alveolar epithelia, and distal colon. These channels are composed of three homologous subunits, termed ␣, ␤, and ␥, which have intracellular amino and carboxyl termini and two membranespanning domains connected by large extracellular loops (1). Residues preceding and within the second membrane-spanning domain constitute the channel pore (2-5). ENaCs have a key role in the regulation of urinary Na ϩ reabsorption, extracellular fluid volume homeostasis, and control of blood pressure (6 -9). Epithelial Na ϩ channel gain-of-function mutations have been identified in patients with Liddle syndrome, a disorder characterized by volume expansion and hypertension (10 -15).In airway epithelia, ENaC has an important role in regulating the volume of airway surface liquids and mucociliary clearance. Increased ENaC activity is thought to contribute to poor mucociliary clearance observed in cystic fibrosis (16). Maturation of ENaC subunits in Xenopus oocytes, Chinese hamster ovarycells,andMadin-Darbycaninekidney(MDCK)cellsinvolvesfurindependent proteolysis at two sites within the ␣ subunit (after Arg-205 and Arg-231) and at a single site within the ␥ subunit (after Arg-143) (17, 18). Interestingly, both processed (i.e. cleaved) and unprocessed ENaCs are expressed on the cell surface (19). Proteolytic processing of ENaC likely occurs within the biosynthetic pathway as well as at the cell surface. Furin, a serine protease that resides primarily in the trans-Golgi network, is required both for cleavage and activation of ENaC in oocytes, Chinese hamster ovary, and MDCK cells (18). Other proteases, such as prostasin (also known as CAP-1) (20 -23), CAP-2 and CAP-3 (24), trypsin (18,20,25), and elastase (26), are also thought to have a role in the proteolytic processing and activation of ENaC. Proteolysis activates the channel by increasing channel open probability (25,27,28).We recently reported that ␣ subunits lacking either one or both cleavage sites exhibited a marked enhancement of the Na ϩ self-inhibition response, suggesting that ␣ subunits must be cleaved at both furin consensus sites in order to activate the channel (28). We now report that ENaCs with mutant ␣ subunits lacking either one or both furin cleavage sites exhibited markedly reduced activity, confirming that cleavage at a single furin site in the ␣ subunit was not sufficient to a...

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

“…In this case the ability of the peptide with sequences originating from this segment to inhibit ENaC may be due to other unrelated mechanisms such as interaction with other parts of the channel that are different than those that aa 175-204 interacted with when they were part of the native channel. In support of such an alternative indirect inhibitory mechanism is the observation that the efficiency of inhibition by this peptide differs by nearly 100-fold between oocytes and mammalian cells expressing ENaC (10), indicating that this is not likely because of inhibition of an intrinsic channel property. These possibilities remain to be tested and represent future experimental challenges to a better understanding of channel regulation.…”

“…Removal of a Short Inhibitory Domain-As mentioned above, it has been proposed that channel activation requires obligatory removal of the tract in ␣ between the two furin cleavage sites (10). To directly test this hypothesis we removed the equivalent tract in human ␣.…”

“…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

“…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).…”