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

Abstract

We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of ␣ENaC using the serine protease subtilisin. Trimeric channels formed with ␣FM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an ϳ10-fold decrease in amiloride-sensitive whole cell conductance as compared with ␣WT (2.2 versus 21.2 microsiemens ( S)). Both ␣WT and ␣FM were activated to similar levels by subtilisin cleavage. Channels formed with ␣FD, a construct that deleted the segment between the two furin sites (⌬175-204), exhibited an intermediate conductance of 13.2 S. More importantly, ␣FD retained the ability to be activated by subtilisin to 108.8 ؎ 20.9 S, a level not significantly different from that of subtilisin activated ␣WT (125.6 ؎ 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of ␣ was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with ␣LD, a construct that extended the deleted sequence of ␣FD by 17 amino acids (⌬175-221). Channels with ␣LD were uncleaved, exhibited low baseline activity (4.1 S), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.It is well established that serine proteases activate the epithelial sodium channel (ENaC). 2 Activation occurs by direct mechanisms that induce channel subunit cleavage (1, 2) as well as those that are cleavage-independent but may involve cleavage of protease-activated membrane receptors (3). Channel cleavage studies have established that cellular proteases such as furin endogenously cleave the channel ␣ and ␥ subunits. Mutation of identified endogenous cleavage sites on both of these subunits diminished baseline activity, demonstrating a role for cleavage in ENaC activation.The acute effects of ENaC cleavage have largely relied on examining the effects of the protease trypsin on the ␣ and ␥ subunits. These studies have examined the effects of cleavage on wild type and furin cleavage-deficient ENaC in oocytes and epithelial cells (1,4,5). Although these have markedly improved our understanding of channel activation by serine proteases, they suffer from the main shortcoming that trypsin is a non-selective serine protease that can cleave after a single arginine residue (6, 7), and therefore, it only offers a limited tool for examining the mechanisms of cleavage at specific sites. Consistent with the reduced specificity for trypsin is the observation that ENaC retains its cleavage by this protease after mutation of consensus cleavage sites for furin (1).Despite their limitations, these studies have ind...

The Cyp2c44 Epoxygenase Regulates Epithelial Sodium Channel Activity and the Blood Pressure Responses to Increased Dietary Salt

“…Multiple mechanisms are known to participate in the regulation of ENaC activity, including limited proteolysis (30,43,44), hormonal mediated effects on translocation and membrane assembly (9,13,45,46), a serum and glucocorticoid-regulated kinase (SGK-1)-mediated phosphorylation of Nedd4 -2 leading to reduced ubiquitination, retrieval and degradation (17,18,47,48), as well as negative and positive hormonal effects mediated by protein kinases including ERK1/2, protein kinases A and C, and phosphatidylinositol 3-kinase, among others (14 -19, 47-49). Added to the above is the present identification of the Cyp2c44 epoxygenase and its EET metabolites as a new class of in vivo regulators of ENaC activity.…”

Acute Cholesterol-induced Anti-natriuretic Effects

“…4 To circumvent these problems, we developed this assay using peptides with sequences specific to ENaC. Assuming that these sites are solution-accessible in the channel, a finding verified experimentally by our laboratory and by other investigators (21,22,29), it is likely that the reported activity is a reasonable representation of the activity of proteases that can target these sites. This approach sacrifices the ability to identify a specific proteases, probably a complicated issue because many proteases may act on these ENaC sequences, especially given the number of proteases that have been shown to cleave the ENaC subunits (see discussion in Ref.…”

“…This approach sacrifices the ability to identify a specific proteases, probably a complicated issue because many proteases may act on these ENaC sequences, especially given the number of proteases that have been shown to cleave the ENaC subunits (see discussion in Ref. 29). However, this approach results in a better understanding of the overall activity of proteases targeting these sequences and therefore provides a better representation or index of real-time channel activation.…”

“…In this case, the ensuing effect is probably due to either a combination of potential direct effects of cholesterol on the channel mediated by modification of the lipid bilayer or secondary effects of ECD on ENaC by modifying the activity of ENaCacting proteases. In this respect, both mechanisms have been shown to affect ENaC activity, where it was shown in cell systems that membrane cholesterol content modifies the activity of channels and transporters, including ENaC (5,13,15,37,38) and that ␣-and ␥-ENaC cleavage enhances channel activity (21,22,29). Interestingly, recent data further indicate that it is cleavage of ␥ in vivo that is more important to channel activation rather than ␣ (28, 30, 32), a result consistent with the current findings.…”

Proteolytic Cleavage of Human Acid-sensing Ion Channel 1 by the Serine Protease Matriptase

“…The exact mechanism of how cleavage activates ENaC is incompletely understood, but one hypothesis is that cleavage removes inhibitory peptides from ␣ENaC and ␥ENaC (12). An alternative mechanism of ENaC activation by cleavage, proposed by Hu et al (14), involves loss of the ␣ENaC N terminus (including the first transmembrane domain) from the channel complex. Cleavage by proteases does not change the number of ENaC channels at the surface but rather increases channel open probability and is a mechanism for regulating ENaC activity (13).…”