ENaC Proteolytic Regulation by Channel-activating Protease 2

Garcı́a-Caballero, Agustı́n; Dang, Yan L.; He, Hong; Stutts, M. Jackson

doi:10.1085/jgp.200810030

Cited by 63 publications

(95 citation statements)

References 33 publications

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“…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).…”

Section: Discussionmentioning

confidence: 69%

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

Bengrine

Lis

et al. 2009

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 69%

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

Bengrine

Lis

et al. 2009

Journal of Biological Chemistry

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“…The reason for the discrepancy is not known but could potentially be because of an additional proteolytic cleavage by proteases, such as channel-activating protease 2, that induces cleavage near the second transmembrane domain in gENaC. 32 However, addition of the molecular mass of this C-terminal fragment to the putative N-terminal fragment detected by mAb inhibit adds up to 82 kD, which matches the full length rather precisely.…”

Section: Discussionmentioning

confidence: 98%

The Epithelial Sodium Channel γ-Subunit Is Processed Proteolytically in Human Kidney

Zachar¹,

Skjødt

Marcussen³

et al. 2015

Journal of the American Society of Nephrology

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The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the g-subunit and putative release of a 43-amino acid inhibitory tract from the g-subunit ectodomain. We hypothesized that proteolytic processing of gENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAb inhibit , and mAb4C11) and the neoepitope generated after proteolytic cleavage at the prostasin/kallikrein cleavage site (K181-V182 and mAb prostasin ) to examine human nephrectomy specimens. By immunoblotting, kidney cortex homogenate from patients treated with angiotensin II type 1 receptor antagonists (n=6) or angiotensin-converting enzyme inhibitors (n=6) exhibited no significant difference in the amount of full-length or furin-cleaved gENaC or the furin-cleaved-to-full-length ratio of gENaC compared with homogenate from patients on no medication (n=5). Patients treated with diuretics (n=4) displayed higher abundance of full-length and furincleaved gENaC, with no significant change in the furin-cleaved-to-full-length gENaC ratio. In patients with proteinuria (n=6), the inhibitory tract was detected only in full-length gENaC by mAb inhibit . Prostasin/ kallikrein-cleaved gENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney gENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin/kallikrein site and removal of the inhibitory tract within gENaC.

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“…Increasing evidence also indicates that modulation of ENaC activity by serine or metalloproteases (33)(34)(35) is linked to ASL volume regulation (36,37). Cysteine proteases, cathepsin B and S, have also been implicated in the regulation of ENaC activity in Xenopus oocytes (38,39) and, like many proteases, cathepsins are generally more active at lower pH (40).…”

Section: A Balance Between Enac and Cftr Activities Is Required For Ementioning

confidence: 99%

Glandular Proteome Identifies Antiprotease Cystatin C as a Critical Modulator of Airway Hydration and Clearance

Evans¹,

Joo

Keiser³

et al. 2016

Am J Respir Cell Mol Biol

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Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel lead to viscous secretions from submucosal glands that cannot be properly hydrated and cleared by beating cilia in cystic fibrosis (CF) airways. The mechanisms by which CFTR, and the predominant epithelial sodium channel (ENaC), control the hydration and clearance of glandular secretions remain unclear. We used a proteomics approach to characterize the proteins contained in CF and non-CF submucosal gland fluid droplets and found that differentially regulated proteases (cathepsin S and H) and their antiprotease (cystatin C) influenced the equilibration of fluid on the airway surface and tracheal mucociliary clearance (MCC). Contrary to prevailing models of airway hydration and clearance, cystatin C, or raising the airway surface liquid (ASL) pH, inhibited cathepsindependent ENaC-mediated fluid absorption and raised the height of ASL, and yet decreased MCC velocity. Importantly, coupling of both CFTR and ENaC activities were required for effective MCC and for effective ASL height equilibration after volume challenge. Cystatin C-inhibitable cathepsins controlled initial phases of ENaC-mediated fluid absorption, whereas CFTR activity was required to prevent ASL dehydration. Interestingly, CF airway epithelia absorbed fluid more slowly owing to reduced cysteine protease activity in the ASL but became abnormally dehydrated with time. Our findings demonstrate that, after volume challenge, pH-dependent protease-mediated coupling of CFTR and ENaC activities are required for rapid fluid equilibration at the airway surface and for effective MCC. These findings provide new insights into how glandular fluid secretions may be equilibrated at the airway surface and how this process may be impaired in CF.Keywords: cystic fibrosis; airway clearance; cystic fibrosis transmembrane conductance regulator; epithelial sodium channel; submucosal glands Clinical RelevanceAirway submucosal glands play an important role in cystic fibrosis (CF) lung disease. Using a proteomics approach to study pure glandular secretions from CF and non-CF human samples, we identified dysregulated proteases and antiproteases that impact airway clearance and airway surface liquid homeostasis. We demonstrate that cysteine proteases and the antiprotease cystatin C regulate pH-dependent protease coupling of CF transmembrane conductance regulator and epithelial sodium channel activities that allow for rapid fluid equilibration at the airway surface required for effective mucociliary clearance.

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ENaC Proteolytic Regulation by Channel-activating Protease 2

Cited by 63 publications

References 33 publications

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

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

The Epithelial Sodium Channel γ-Subunit Is Processed Proteolytically in Human Kidney

Glandular Proteome Identifies Antiprotease Cystatin C as a Critical Modulator of Airway Hydration and Clearance

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