Cathepsin B Is Secreted Apically from Xenopus 2F3 Cells and Cleaves the Epithelial Sodium Channel (ENaC) to Increase Its Activity

Alli, Abdel A.; Song, John Z.; Al‐Khalili, Otor; Bao, Hui‐Fang; Ma, Heping; Alli, Alia A; Eaton, Douglas C.

doi:10.1074/jbc.m111.338574

Cited by 40 publications

(34 citation statements)

References 46 publications

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“…GST Pulldown Assay-Biotinylated TIP peptide, biotinylated rhTNF (0.5 nmol of peptide/recombinant protein) were incubated with 0.3 nmol each of GST, GST-wild type, and GST-3M C-terminal domain of ENaC-␣ fusion proteins, prepared as described (52), coupled to glutathione-Sepharose beads (corresponding to 1/10 of Microspin column, GE Healthcare, ϳ60 l of glutathione-Sepharose slurry) for 1 h at 4°C. Afterward, the beads were washed and incubated with streptavidin Alexa Fluor 594 conjugate for another 1 h at room temperature in dark.…”

Section: Methodsmentioning

confidence: 99%

“…Subunit Association Studies-GST-ENaC-␣ wild type, GSTENaC-␣ triple mutant, GST-ENaC-␤, and GST-ENaC-␥ fusion proteins were expressed and purified as previously described (52,53). In separate tubes, 40 g of each fusion protein was incubated with 50 l of prewashed glutathione-Sepharose 4B beads and 100 l of PBS at 4°C for 4 h with end over end mixing.…”

Section: Methodsmentioning

confidence: 99%

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The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit

Lucas

Yue

Alli

et al. 2016

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit

Lucas

Yue

Alli

et al. 2016

Journal of Biological Chemistry

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“…The membranes were incubated with SuperSignal Dura Chemiluminescent Substrate for 5 min before being developed using a Kodak Gel Logic 2200 Imager and Molecular Imaging software (Carestream Health, Rochester, NY). This method was used to detect ENaC subunits (with in-house antibodies) (1,4,8,40,46), ERK1/2 (9102, Cell Signaling) and phosphoERK1/2 (9101a, Cell Signaling). PKC isoforms were detected with antibodies obtained from Cell Signaling, (In particular, PKC␣ was detected with Cell Signaling no.…”

Section: Sds-page and Immunoblottingmentioning

confidence: 99%

ENaC activity is increased in isolated, split-open cortical collecting ducts from protein kinase Cα knockout mice

Bao

Thai

Yue

et al. 2014

American Journal of Physiology-Renal Physiology

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The epithelial Na channel (ENaC) is negatively regulated by protein kinase C (PKC) as shown using PKC activators in a cell culture model. To determine whether PKC␣ influences ENaC activity in vivo, we examined the regulation of ENaC in renal tubules from PKC␣ Ϫ/Ϫ mice. Cortical collecting ducts were dissected and split open, and the exposed principal cells were subjected to cell-attached patch clamp. In the absence of PKC␣, the open probability (P o) of ENaC was increased three-fold vs. wild-type SV129 mice (0.52 Ϯ 0.04 vs. 0.17 Ϯ 0.02). The number of channels per patch was also increased. Using confocal microscopy, we observed an increase in membrane localization of ␣-, ␤-, and ␥-subunits of ENaC in principal cells in the cortical collecting ducts of PKC␣ Ϫ/Ϫ mice compared with wild-type mice. To confirm this increase, one kidney from each animal was perfused with biotin, and membrane protein was pulled down with streptavidin. The nonbiotinylated kidney was used to assess total protein. While total ENaC protein did not change in PKC␣ Ϫ/Ϫ mice, membrane localization of all the ENaC subunits was increased. The increase in membrane ENaC could be explained by the observation that ERK1/2 phosphorylation was decreased in the knockout mice. These results imply a reduction in ENaC membrane accumulation and P o by PKC␣ in vivo. The PKC-mediated increase in ENaC activity was associated with an increase in blood pressure in knockout mice fed a high-salt diet.protein kinase C␣; ENaC; renal tubules; single channels; knockout mice; hypertension EPITHELIAL NA CHANNELS (ENaC) are sodium-permeable ion channels located in the apical membrane of polarized epithelial cells primarily in the distal nephron, lung, and distal colon. In the distal nephron, ENaC activity is the rate-limiting step for Na ϩ reabsorption (16, 34); therefore, ENaC activity is critical for the physiological maintenance of systemic Na ϩ homeostasis and long-term control of blood pressure. Because of its central role in responding to changes in Na ϩ uptake, ENaC activity is tightly regulated; dysregulation of this channel has been linked to abnormal blood pressure in several genetic disorders including Liddle's syndrome (18, 37) and pseudohypoaldosteronism type 1 (9, 33, 41).ENaC can be regulated either by altering the amount of time the channel spends open (open probability or P o ) or by altering the density of functional channels (N) in the apical membrane of distal nephron epithelial cells. One signaling molecule that appears to alter ENaC activity is protein kinase C (PKC). Activation of PKC with phorbol esters reduces ENaC activity in the apical membrane of A6 cells, an amphibian renal cell line, and in rat principal cells (15). In contrast to the inhibitory effect on ENaC due to activating PKC, pharmacologically inhibiting PKC increases ENaC P o (23, 49). A6 cells, on which many of the experiments described above were performed, contain several different PKC isoforms; so that it is difficult to determine which isoform is responsible for the changes in E...

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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). Thus, we hypothesized that increased cystatin C, a potent cysteine protease inhibitor, might influence MCC through one or more of the pathways described previously here.…”

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

confidence: 78%

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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Cathepsin B Is Secreted Apically from Xenopus 2F3 Cells and Cleaves the Epithelial Sodium Channel (ENaC) to Increase Its Activity

Cited by 40 publications

References 46 publications

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit

ENaC activity is increased in isolated, split-open cortical collecting ducts from protein kinase Cα knockout mice

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

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