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.
Canonical epithelial sodium channels (ENaCs) are heterotrimers formed by α, β, and γ ENaC subunits in vertebrates and belong to the Degenerin/ENaC family of proteins. Proteins from this family form mechanosensitive channels throughout the animal kingdom. Activity of canonical ENaC is regulated by shear force (SF) mediating Na + absorption in the kidney and vascular tone of arteries. Expression analysis suggests that non-canonical ENaC, formed by single or only two subunits, exist in certain tissues, but it is unknown if these channels respond to SF. α, β, γ, and δ ENaC subunits were expressed either alone or in combinations of two subunits in Xenopus oocytes. Amiloride-sensitive currents and the responses to SF were assessed using two-electrode voltage clamp recordings. With the exception of γ ENaC, all homomeric channels provided amiloride-sensitive currents and responded to SF applied via a fluid stream directed onto the oocytes. Channels containing two subunits were also activated by SF. Here, the presence of the γ ENaC subunit when co-expressed with α or δ augmented the SF response in comparison to the αβγ/δβγ ENaC. Overall, we provide evidence that non-canonical ENaC can form channels that respond to SF. This supports a potential function of non-canonical ENaC as mechanosensors in epithelial, vascular, and sensory cells.
-In pulmonary epithelia, -adrenergic agonists regulate the membrane abundance of the epithelial sodium channel (ENaC) and, thereby, control the rate of transepithelial electrolyte absorption. This is a crucial regulatory mechanism for lung liquid clearance at birth and thereafter. This study investigated the influence of the gaseous signaling molecule hydrogen sulfide (H 2S) on -adrenergic agonistregulated pulmonary sodium and liquid absorption. Application of the H2S-liberating molecule Na2S (50 M) to the alveolar compartment of rat lungs in situ decreased baseline liquid absorption and abrogated the stimulation of liquid absorption by the -adrenergic agonist terbutaline. There was no additional effect of Na2S over that of the ENaC inhibitor amiloride. In electrophysiological Ussing chamber experiments with native lung epithelia (Xenopus laevis), Na 2S inhibited the stimulation of amiloride-sensitive current by terbutaline. -adrenergic agonists generally increase ENaC abundance by cAMP formation and activation of PKA. Activation of this pathway by forskolin and 3-isobutyl-1-methylxanthine increased amiloride-sensitive currents in H441 pulmonary epithelial cells. This effect was inhibited by Na 2S in a dose-dependent manner (5-50 M). Na2S had no effect on cellular ATP concentration, cAMP formation, and activation of PKA. By contrast, Na 2S prevented the cAMP-induced increase in ENaC activity in the apical membrane of H441 cells. H441 cells expressed the H 2S-generating enzymes cystathionine--synthase, cystathionine-␥-lyase, and 3-mercaptopyruvate sulfurtransferase, and they produced H 2S amounts within the employed concentration range. These data demonstrate that H 2S prevents the stimulation of ENaC by cAMP/PKA and, thereby, inhibits the proabsorptive effect of -adrenergic agonists on lung liquid clearance.
The epithelial sodium channel (ENaC) is expressed in the kidney and vasculature and is important for blood pressure regulation. In the kidney and vasculature, ENaC is exposed to mechanical force such as shear force (SF) caused by the urine or blood flow. Canonical ENaC consists of three homologous subunits: α, β, γ and studies have shown that α‐ENaC plays an important role for the ability of the channel to respond to SF. Further evidence suggests that β‐and γ‐ENaC alone may be involved in mechanotransduction processes in arteries. Since little is known about the function of individual ENaC subunits and their regulation by SF, this project aims to characterize the role of β‐ and γ‐ENaC for SF sensation.Human ENaCs were expressed as homotrimers (α, β or γ alone) or heterotrimers (αβ, αγ or βγ) in Xenopus oocytes. Two‐electrode voltage‐clamp experiments were performed to measure ENaC activity in response to a fluid stream used for the application of SF. SF responses were normalized and expressed as percentage.Homotrimeric α‐ENaC showed an activation of 46 ± 7.4 (Mean ± SEM, p < 0.0001, n = 19) in response to SF, whereas homotrimeric β‐ or γ‐ENaCs did not respond to SF. In heterotrimeric αβ‐ENaC channels the activation by SF was 17 ± 3 (p < 0.0001, n = 19) and in heterotrimeric αγ‐ENaC 222 ± 35 (p < 0.0001, n = 17). Heterotrimeric βγ‐ENaC did not respond to SF.Homotrimeric α‐ENaC form a functional channel that responds to SF whereas β‐ and γ‐ENaC did not. Heterotrimeric αβ‐ENaC showed a lower SF responds, whereas αγ‐ENaC showed a higher responds to SF. This experiments identify a modulatory role of β‐ and γ‐ENaC for the activation in response to SF. Therefore, ENaC dysfunction is observed in hypertension may be due to changes in β‐ and γ‐ENaC expression that could influence its activity in response to SF caused by the urine or blood flow.Support or Funding InformationUniversity of Otago Doctoral ScholarshipDepartment of PhysiologyRoyal Society Te Aparangi ‐ Marsden FundThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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