Role of the actin cytoskeleton on epithelial Na+ channel regulation

Tumor necrosis factor (TNF) is known to activate the epithelial Na+ channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na+ current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na+ current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.

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In vitro phosphorylation of COOH termini of the epithelial Na+ channel and its effects on channel activity inXenopus oocytes

AJP Renal Physiology ◽

10.1152/ajprenal.2001.280.6.f1030 ◽

2001 ◽

Vol 280 (6) ◽

pp. F1030-F1036 ◽

Cited By ~ 23

Author(s):

Alexander Chigaev ◽

Gang Lu ◽

Haikun Shi ◽

Carol Asher ◽

Rong Xu ◽

...

Keyword(s):

Rat Colon ◽

Na Channel ◽

Xenopus Laevis Oocytes ◽

Channel Activity ◽

Glutathione S Transferase ◽

Single Well ◽

Py Motif ◽

Epithelial Na Channel

Recent findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na+ channel (ENaC). This study reports the in vitro phosphorylation of the COOH termini of ENaC subunits expressed as glutathione S-transferase fusion proteins. Channel subunits were specifically phosphorylated by kinase-enriched cytosolic fractions derived from rat colon. The phosphorylation observed was not mediated by the serum- and glucocorticoid-regulated kinase sgk. For the γ-subunit, phosphorylation occurred on a single, well-conserved threonine residue located in the immediate vicinity of the PY motif (T630). The analogous residue on β(S620) was phosphorylated as well. The possible role of γT630 and βS620 in channel function was studied in Xenopus laevis oocytes. Mutating these residues to alanine had no effect on the basal channel-mediated current. They do, however, inhibit the sgk-induced increase in channel activity but only in oocytes that were preincubated in low Na+ and had a high basal Na+ current. Thus mutating γT630 or βS620 may limit the maximal channel activity achieved by a combination of sgk and low Na+.

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Epithelial Na+ channel regulation by cytoplasmic and extracellular factors

Experimental Cell Research ◽

10.1016/j.yexcr.2012.02.024 ◽

2012 ◽

Vol 318 (9) ◽

pp. 1011-1019 ◽

Cited By ~ 48

Author(s):

Ossama B. Kashlan ◽

Thomas R. Kleyman

Keyword(s):

Na Channel ◽

Channel Regulation ◽

Epithelial Na Channel

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Enhanced Shear Force Responsiveness of Epithelial Na+ Channel’s (ENaC) δ Subunit Following the Insertion of N-Glycosylation Motifs Relies on the Extracellular Matrix

International Journal of Molecular Sciences ◽

10.3390/ijms22052500 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2500

Author(s):

Daniel Barth ◽

Fenja Knoepp ◽

Martin Fronius

Keyword(s):

Shear Force ◽

Xenopus Oocytes ◽

Site Directed Mutagenesis ◽

Na Channel ◽

Cell Matrix ◽

Core Feature ◽

Electrode Voltage ◽

Ecm Degradation ◽

Epithelial Na Channel

Members of the Degenerin/epithelial Na+ channel (ENaC) protein family and the extracellular cell matrix (ECM) form a mechanosensitive complex. A core feature of this complex are tethers, which connect the channel with the ECM, however, knowledge about the nature of these tethers is scarce. N-glycans of α ENaC were recently identified as potential tethers but whether N-glycans serve as a ubiquitous feature for mechanosensation processes remains unresolved. The purpose of this study was to reveal whether the addition of N-glycans to δ ENaC—which is less responsive to shear force (SF)—increases its SF-responsiveness and whether this relies on a linkage to the ECM. Therefore, N-glycosylation motifs were introduced via site-directed mutagenesis, the resulting proteins expressed with β and γ ENaC in Xenopus oocytes, and SF-activated currents measured by two-electrode voltage-clamp. The insertion of N-glycosylation motifs increases δ ENaC’s SF responsiveness. The inclusion of a glycosylated asparagine (N) at position 487 did increase the molecular mass and provided a channel whose SF response was abolished following ECM degradation via hyaluronidase. This indicates that the addition of N-glycans improves SF-responsiveness and that this effect relies on an intact ECM. These findings further support the role of N-glycans as tethers for mechanotransduction.

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Novel role of Rac1/WAVE signaling mechanism in regulation of the epithelial Na + channel (ENaC)

The FASEB Journal ◽

10.1096/fasebj.25.1_supplement.1039.1 ◽

2011 ◽

Vol 25 (S1) ◽

Author(s):

Alexander Staruschenko ◽

Alexey Karpushev ◽

Vladislav Levchenko ◽

Daria V. Ilatovskaya ◽

Tengis S. Pavlov

Keyword(s):

Na Channel ◽

Signaling Mechanism ◽

Epithelial Na Channel

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Mechanical activation of epithelial Na+ channel relies on an interdependent activity of the extracellular matrix and extracellular N-glycans of αENaC

10.1101/102756 ◽

2017 ◽

Cited By ~ 3

Author(s):

Fenja Knoepp ◽

Zoe Ashley ◽

Daniel Barth ◽

Marina Kazantseva ◽

Pawel P. Szczesniak ◽

...

Keyword(s):

Extracellular Matrix ◽

Ion Channels ◽

Shear Force ◽

Na Channel ◽

Pressure Regulation ◽

Mechanical Environment ◽

Mechanosensitive Ion Channels ◽

Glycosylation Sites ◽

Epithelial Na Channel

AbstractMechanotransduction describes how cells perceive their mechanical environment and mechanosensitive ion channels are important for this process. ENaC (epithelial Na+ channel)/DEG (degenerin) proteins form mechanosensitive ion channels and it is hypothesized their interaction with the extracellular matrix (ECM) via ‘tethers’ is required for mechanotransduction. Channels formed by vertebrate α, β and γ ENaC proteins are activated by shear force (SF) and mediate electrolyte/fluid-homeostasis and blood pressure regulation. Here, we report an interdependent activity of ENaC and the ECM that mediates SF effects in murine arteries and heterologously expressed channels. Furthermore, replacement of conserved extracellular N-glycosylated asparagines of αENaC decreased the SF response indicating that the attached N-glycans provide a connection to the ECM. Insertion of N-glycosylation sites into a channel subunit, innately lacking these motifs, increased its SF response. These experiments confirm an interdependent channel/ECM activity of mechanosensitive ENaC channel and highlight the role of channel N-glycans as new constituents for the translation of mechanical force into cellular signals.

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Novel Role of Rac1/WAVE Signaling Mechanism in Regulation of the Epithelial Na + Channel

Hypertension ◽

10.1161/hypertensionaha.110.157784 ◽

2011 ◽

Vol 57 (5) ◽

pp. 996-1002 ◽

Cited By ~ 23

Author(s):

Alexey V. Karpushev ◽

Vladislav Levchenko ◽

Daria V. Ilatovskaya ◽

Tengis S. Pavlov ◽

Alexander Staruschenko

Keyword(s):

Na Channel ◽

Signaling Mechanism ◽

Epithelial Na Channel

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Participation of the Ubiquitin-Conjugating Enzyme UBE2E3 in Nedd4-2-Dependent Regulation of the Epithelial Na+ Channel

Molecular and Cellular Biology ◽

10.1128/mcb.24.6.2397-2409.2004 ◽

2004 ◽

Vol 24 (6) ◽

pp. 2397-2409 ◽

Cited By ~ 29

Author(s):

Christophe Debonneville ◽

Olivier Staub

Keyword(s):

Binding Sites ◽

Regulatory Mechanism ◽

Surface Expression ◽

Cell Surface Expression ◽

Na Channel ◽

Limiting Factor ◽

Ubiquitin Conjugating Enzyme ◽

Rate Limiting ◽

Epithelial Na Channel

ABSTRACT The epithelial Na+ channel (ENaC) is a heteromeric protein complex playing a fundamental role in Na+ homeostasis and blood pressure regulation. Specific mutations inactivating PY motifs in ENaC C termini cause Liddle's syndrome, an inherited form of hypertension. Previously we showed that these PY motifs serve as binding sites for the E3 enzyme Nedd4-2, implying ubiquitination as a regulatory mechanism of ENaC. Ubiquitination involves the sequential action of E1, E2, and E3 enzymes. Here we identify the E2 enzyme UBE2E3, which acts in concert with Nedd4-2, and show by coimmunoprecipitation that UBE2E3 and Nedd4-2 interact together. In Xenopus laevis oocytes, UBE2E3 reduces ENaC activity marginally, consistent with Nedd4-2 being the rate-limiting factor in this process, whereas a catalytically inactive mutant of UBE2E3 (UBE2E3-CS) causes elevated ENaC activity by increasing cell surface expression. No additive effect is observed when UBE2E3-CS is coexpressed with an inactive Nedd4-2 mutant, and the stimulatory role of UBE2E3-CS depends on the integrity of the PY motifs (Nedd4-2 binding sites) and the ubiquitination sites on ENaC. In renal mpkCCDcl4 cells, displaying ENaC-dependent transepithelial Na+ transport, Nedd4-2 and UBE2E3 can be coimmunoprecipitated and overexpression of UBE2E3 affects Na+ transport, corroborating the concept of a concerted action of UBE2E3 and Nedd4-2 in ENaC regulation.

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