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

Knoepp, Fenja; Ashley, Zoe; Barth, Daniel; Kazantseva, Marina; Szczęśniak, Paweł; Clauss, Wolfgang; Althaus, Mike; Rosa, Diego Álvarez de la; Fronius, Martin

doi:10.1101/102756

Cited by 5 publications

(5 citation statements)

References 89 publications

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“…While the findings of Knoepp at al. suggested a link between ␣-subunit N-linked glycans and the channel's LSS response (35), our results seemingly rule out a role for N-linked glycans on a specific subunit in ENaC mechanosensing. Experimental differences may underlie these contrasting results.…”

Section: Discussionsupporting

confidence: 50%

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N-linked glycans are required on epithelial Na⁺channel subunits for maturation and surface expression

Kashlan

Kinlough

Myerburg

et al. 2018

American Journal of Physiology-Renal Physiology

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Epithelial Na channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, β-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αβγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na. The lack of N-linked glycans on the β-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the β-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type β-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.

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Section: Discussionsupporting

confidence: 50%

“…These data contrast with a recent report from Knoepp at al. that implicated links between N-linked glycans on the ␣-subunit and the extracellular matrix in the channel's LSS response (35) and suggest an alternate model for how LSS regulates ENaC function (see DISCUSSION). Fig.…”

Section: Enac Activity Is Dependent On N-glycosylation Of Each Subunitmentioning

confidence: 99%

N-linked glycans are required on epithelial Na⁺channel subunits for maturation and surface expression

Kashlan

Kinlough

Myerburg

et al. 2018

American Journal of Physiology-Renal Physiology

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“…Endothelial ENaC activation has been proposed to blunt nitric oxide release and prevent vasodilation (24). This response appears to be flow dependent, suggesting that endothelial ENaC may be functioning as a mechanosensor (110; 111). Vascular smooth muscle ENaC has also been suggested to have a role in the pressure-induced myogenic response (25).…”

Section: Extracellular Regulators Of Enacmentioning

confidence: 99%

“…At present, it is unclear whether tethering ENaC subunits to the extracellular matrix and/or intracellular cytoskeleton is required for mechanosensing. One group has suggested that the extracellular matrix has a role in shear stress activation of ENaC in Xenopus oocytes and endothelium, and that specific α subunit N-glycans facilitate interactions between extracellular matrix and ENaC that are required for flow sensing (111). In contrast, our unpublished work (Kashlan et al) indicates that ENaC N-glycans are not required for flow sensing.…”

Section: Extracellular Regulators Of Enacmentioning

confidence: 99%

Epithelial Na⁺ Channel Regulation by Extracellular and Intracellular Factors

Kleyman

Kashlan

Hughey

2018

Annu. Rev. Physiol.

102

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Epithelial Na channels (ENaCs) are members of the ENaC/degenerin family of ion channels that evolved to respond to extracellular factors. In addition to being expressed in the distal aspects of the nephron, where ENaCs couple the absorption of filtered Na to K secretion, these channels are found in other epithelia as well as nonepithelial tissues. This review addresses mechanisms by which ENaC activity is regulated by extracellular factors, including proteases, Na, and shear stress. It also addresses other factors, including acidic phospholipids and modification of ENaC cytoplasmic cysteine residues by palmitoylation, which enhance channel activity by altering interactions of the channel with the plasma membrane.

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“…Sound induced vibrations lead to motion between adjacent cilia and activation of the tethered channels. The family of DEG/ENaC/ASIC channels are MSCs present in both non-specialized and specialized mechanosensory cells [24, 25], and are gated by movement of their extracellular domains [26] that are associated with the ECM [27]. This connection to the ECM is disrupted during patch formation which is likely why these channels do not show mechanosensitivity in patches [25, 28].…”

Section: Msc Gating Mechanisms and Molecular Identitymentioning

confidence: 99%

Piezo channels and GsMTx4: Two milestones in our understanding of excitatory mechanosensitive channels and their role in pathology

Suchyna

2017

Progress in Biophysics and Molecular Biology

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Discovery of Piezo channels and the reporting of their sensitivity to the inhibitor GsMTx4 were important milestones in the study of non-selective cationic mechanosensitive channels (MSCs) in normal physiology and pathogenesis. GsMTx4 had been used for years to investigate the functional role of cationic MSCs, especially in muscle tissue, but with little understanding of its target or inhibitory mechanism. The sensitivity of Piezo channels to bilayer stress and its robust mechanosensitivity when expressed in heterologous systems were keys to determining GsMTx4’s mechanism of action. However, questions remain regarding Piezo’s role in muscle function due to the non-selective nature of GsMTx4 inhibition toward membrane mechanoenzymes and the implication of MCS channel types by genetic knockdown. Evidence supporting Piezo like activity, at least in the developmental stages of muscle, is presented. While the MSC targets of GsMTx4 in muscle pathology are unclear, its muscle protective effects are clearly demonstrated in two recent in situ studies on normal cardiomyocytes and dystrophic skeletal muscle. The muscle protective function may be due to the combined effect of GsMTx4’s inhibitory action on cationic MSCs like Piezo and TRP, and its potentiation of repolarizing K+ selective MSCs like K2P and SAKCa. Paradoxically, the potent in vitro action of GsMTx4 on many physiological functions seems to conflict with its lack of in situ side-effects on normal animal physiology. Future investigations into cytoskeletal control of sarcolemma mechanics and the suspected inclusion of MSCs in membrane micro/nano sized domains with distinct mechanical properties will aide our understanding of this dichotomy.

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

Abstract: Mechanotransduction describes how cells perceive their mechanical environment and mechanosensitive ion channels are important for this process. ENaC (epithelial Na

Cited by 5 publications

References 89 publications

N-linked glycans are required on epithelial Na⁺channel subunits for maturation and surface expression

N-linked glycans are required on epithelial Na⁺channel subunits for maturation and surface expression

Epithelial Na⁺ Channel Regulation by Extracellular and Intracellular Factors

Piezo channels and GsMTx4: Two milestones in our understanding of excitatory mechanosensitive channels and their role in pathology

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

Abstract: Mechanotransduction describes how cells perceive their mechanical environment and mechanosensitive ion channels are important for this process. ENaC (epithelial Na

Cited by 5 publications

References 89 publications

N-linked glycans are required on epithelial Na+channel subunits for maturation and surface expression

N-linked glycans are required on epithelial Na+channel subunits for maturation and surface expression

Epithelial Na+ Channel Regulation by Extracellular and Intracellular Factors

Piezo channels and GsMTx4: Two milestones in our understanding of excitatory mechanosensitive channels and their role in pathology

Contact Info

Product

Resources

About

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

N-linked glycans are required on epithelial Na⁺channel subunits for maturation and surface expression

N-linked glycans are required on epithelial Na⁺channel subunits for maturation and surface expression

Epithelial Na⁺ Channel Regulation by Extracellular and Intracellular Factors