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

2017 ◽  
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.

scholarly journals CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1858
Author(s):  
Waheed Shabbir ◽  
Nermina Topcagic ◽  
Mohammed Aufy ◽  
Murat Oz
Keyword(s):  
A549 Cells ◽  
Na Channel ◽  
Na Current ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Knock Down ◽  
Whole Cell Patch Clamp ◽  
Glycosylation Sites ◽  
Epithelial Na Channel

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.

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

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.

scholarly journals Shear force sensing of epithelial Na+channel (ENaC) relies onN-glycosylated asparagines in the palm and knuckle domains of αENaC

2019 ◽  
Vol 117 (1) ◽  
pp. 717-726 ◽  
Author(s):  
Fenja Knoepp ◽  
Zoe Ashley ◽  
Daniel Barth ◽  
Jan-Peter Baldin ◽  
Michael Jennings ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Shear Force ◽  
Molecular Mechanisms ◽  
Cell Function ◽  
Physiological Function ◽  
Blood Pressure Regulation ◽  
Na Channel ◽  
Pressure Regulation ◽  
Vascular Responsiveness ◽  
Epithelial Na Channel

Mechanosensitive ion channels are crucial for normal cell function and facilitate physiological function, such as blood pressure regulation. So far little is known about the molecular mechanisms of how channels sense mechanical force. Canonical vertebrate epithelial Na+channel (ENaC) formed by α-, β-, and γ-subunits is a shear force (SF) sensor and a member of the ENaC/degenerin protein family. ENaC activity in epithelial cells contributes to electrolyte/fluid-homeostasis and blood pressure regulation. Furthermore, ENaC in endothelial cells mediates vascular responsiveness to regulate blood pressure. Here, we provide evidence that ENaC’s ability to mediate SF responsiveness relies on the “force-from-filament” principle involving extracellular tethers and the extracellular matrix (ECM). Two glycosylated asparagines, respectively theirN-glycans localized in the palm and knuckle domains of αENaC, were identified as potential tethers. Decreased SF-induced ENaC currents were observed following removal of the ECM/glycocalyx, replacement of these glycosylated asparagines, or removal ofN-glycans. Endothelial-specific overexpression of αENaC in mice induced hypertension. In contrast, expression of αENaC lacking these glycosylated asparagines blunted this effect. In summary, glycosylated asparagines in the palm and knuckle domains of αENaC are important for SF sensing. In accordance with the force-from-filament principle, they may provide a connection to the ECM that facilitates vascular responsiveness contributing to blood pressure regulation.

scholarly journals The function and regulation of acid-sensing ion channels (ASICs) and the epithelial Na+channel (ENaC): IUPHAR Review 19

2016 ◽  
Vol 173 (18) ◽  
pp. 2671-2701 ◽  
Author(s):  
Emilie Boscardin ◽  
Omar Alijevic ◽  
Edith Hummler ◽  
Simona Frateschi ◽  
Stephan Kellenberger
Keyword(s):  
Ion Channels ◽  
Na Channel ◽  
Acid Sensing Ion Channels ◽  
Epithelial Na Channel

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

2001 ◽  
Vol 280 (6) ◽  
pp. F1030-F1036 ◽  
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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