Baldin JP et al. (2020), Epithelial Na+ Channel (ENaC) Formed by One or ...

XB-ART-56865

Front Physiol 2020 Jan 01;11:141. doi: 10.3389/fphys.2020.00141.

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Epithelial Na+ Channel (ENaC) Formed by One or Two Subunits Forms Functional Channels That Respond to Shear Force.

Baldin JP , Barth D , Fronius M .

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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.

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GO keywords: sodium channel activity

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	Figure 1. Homotrimeric ENaC forms functional channels except for Î³. Electrophysiological characterization of homotrimeric ENaC (Î±, Î´, Î², and Î³) vs. canonical ENaC (Î±Î²Î³ and Î´Î²Î³) expressed in oocytes. Representative current traces of Î±Î²Î³- (A), Î±- (B), Î²- (C), Î³- (D), Î´Î²Î³- (E), and Î´-injected oocytes (F) in response to the application of amiloride (10Î¼M). (C) Oocytes injected with ENaC encoding RNA revealed variable responses. Only a proportion of oocytes responded to amiloride, whereas the majority of oocytes did not show an amiloride response (Câ). (D) Oocytes injected with Î³ ENaC did not show a reaction in response to amiloride. (G) Amiloride dose response curve of Î±Î²Î³, Î±, and Î² ENaC and calculated IC50 values for amiloride (H) showing a decreased amiloride sensitivity for Î² when compared with Î±Î²Î³, whereas Î± is unchanged. (I) Amiloride dose response curves of Î´Î²Î³ and Î´. (J) Î´ ENaC displays a significantly reduced amiloride IC50 value compared with Î´Î²Î³. Paired t-test (AâF), one-way ANOVA with multiple comparison (H) and unpaired t-test (J); p < 0.05; p < 0.01; p < 0.001; **p < 0.0001.
	Figure 2. Biotinylation assay of Î² ENaC expressing oocytes. (A) Representative western blot showing Î² ENaC in presence (+) or absence (â) of biotin. Each group consists of the following three samples: whole cell (Wc), cytoplasmic (Cyto), and membrane bound (Mb). Exposure time for Wc and Cyto: 5 s and for Mb: 10 min N = 3. After an incubation of 24 h Î² ENaC was present in the Wc and Cyto fractions but absent in the Mb sample. It may also be noted that the Cyto and Mb fraction were treated with PNGase F, resulting in a lower molecular weight compared to the band detected in the Wc sample. (B) Fraction of Î² ENaC expressing oocytes exhibiting amiloride-sensitive currents (+ami) after 24 h incubation. (C) Increasing the incubation time of Î² ENaC to 48 h shows a clear band within the Mb fraction and increases the number of oocytes exhibiting amiloride-sensitive currents (D).
	Figure 3. Homotrimeric ENaC except for Î³ is activated by SF. Electrophysiological characterization of homotrimeric ENaC in response to 0.2 dyncmâ€“ 2 SF (colored bar). (A) H2O-injected control oocytes neither respond to SF nor amiloride (10 Î¼M). (B) SF response of Î±Î²Î³ ENaC is absent in presence of amiloride (10 Î¼M). Î±Î²Î³ (C) and Î´Î²Î³ (D) ENaC are significantly activated by SF. Left panels show representative traces. The right panels show the averages Â± SEM. Homotrimeric Î±, Î´, and Î² (Eâ€“G, respectively) respond to SF, whereas Î³ (H) does not. (I) The SF response (I0.2/I0) of homotrimeric ENaC subunits was normalized with respect to canonical Î±Î²Î³ and Î´Î²Î³ ENaC. The SF response of Î± ENaC was unchanged while Î² ENaC showed a reduced SF effect when compared with Î±Î²Î³. Î´ ENaC displayed an increased SF effect compared with canonical Î´Î²Î³ ENaC. Paired t-test (Câ€“H) and one-way ANOVA with multiple comparison (I); p < 0.05; p < 0.01; **p < 0.0001.
	Figure 4. Heterotrimeric ENaC composed of two subunits form functional channels. (AâG) Effect of amiloride (10 Î¼M) application on heterotrimeric ENaC currents of various subunit combinations (Î±Î²Î³, Î±Î², Î±Î³, Î²Î³, Î´Î²Î³, Î´Î², and Î´Î³). (H) Dose response curves for amiloride were fitted and derived IC50 values were used for comparison (I). Amiloride sensitivity of Î±Î² and Î±Î³ ENaC was statistically not different when compared with Î±Î²Î³, whereas Î²Î³ ENaC displays a significantly increased IC50 value with respect to Î±Î²Î³ and Î±Î³ ENaC. (J) Dose-response curves for channels containing Î´ ENaC. (K) Î´Î³ displayed a significantly reduced amiloride IC50 value compared with Î´Î²Î³, whereas Î´Î² remains unchanged. Paired t-test (AâG) and one-way ANOVA with multiple comparison (I,K); p < 0.01; p < 0.001; ***p < 0.0001.
	Figure 5. Heterotrimeric ENaC composed of two subunits responds to SF. SF response of heterotrimeric ENaC composed of two different subunits was assessed. Left panels show representative current traces whereas the right panels show the averages Â± SEM. All combinations of two different ENaC subunits, Î±Î² (A), Î±Î³ (B), Î´Î² (C), Î´Î³ (D), and Î²Î³ (E), were significantly activated by the application of SF (indicated as colored bar). (F) The SF response (I0.2/I0) of those ENaC subunit compositions was normalized with respect to canonical Î±Î²Î³ and Î´Î²Î³ ENaC. The SF effect of Î±Î² and Î²Î³ was unchanged when compared with Î±Î²Î³. A significantly increased SF response was observed for Î±Î³ when compared with Î±Î²Î³. Î´Î³ ENaC shows also a significantly increased SF effect when compared with Î´Î²Î³, whereas Î´Î² was unchanged. Paired t-test (Aâ€“E) and one-way ANOVA with multiple comparison (F); p < 0.01; p < 0.001; ***p < 0.0001.
	Supplementary Figure 1- Epithelial Na+ Channel (ENaC) Formed by One or Two Subunits Forms Functional Channels That Respond to Shear Force
	Supplementary Figure 2- Epithelial Na+ Channel (ENaC) Formed by One or Two Subunits Forms Functional Channels That Respond to Shear Force
	FIGURE 1. Homotrimeric ENaC forms functional channels except for Î³. Electrophysiological characterization of homotrimeric ENaC (Î±, Î´, Î², and Î³) vs. canonical ENaC (Î±Î²Î³ and Î´Î²Î³) expressed in oocytes. Representative current traces of Î±Î²Î³- (A), Î±- (B), Î²- (C), Î³- (D), Î´Î²Î³- (E), and Î´-injected oocytes (F) in response to the application of amiloride (10Î¼M). (C) Oocytes injected with ENaC encoding RNA revealed variable responses. Only a proportion of oocytes responded to amiloride, whereas the majority of oocytes did not show an amiloride response (Câ). (D) Oocytes injected with Î³ ENaC did not show a reaction in response to amiloride. (G) Amiloride dose response curve of Î±Î²Î³, Î±, and Î² ENaC and calculated IC50 values for amiloride (H) showing a decreased amiloride sensitivity for Î² when compared with Î±Î²Î³, whereas Î± is unchanged. (I) Amiloride dose response curves of Î´Î²Î³ and Î´. (J) Î´ ENaC displays a significantly reduced amiloride IC50 value compared with Î´Î²Î³. Paired t-test (AâF), one-way ANOVA with multiple comparison (H) and unpaired t-test (J); p < 0.05; p < 0.01; p < 0.001; **p < 0.0001.
	FIGURE 2. Biotinylation assay of Î² ENaC expressing oocytes. (A) Representative western blot showing Î² ENaC in presence (+) or absence (â) of biotin. Each group consists of the following three samples: whole cell (Wc), cytoplasmic (Cyto), and membrane bound (Mb). Exposure time for Wc and Cyto: 5 s and for Mb: 10 min N = 3. After an incubation of 24 h Î² ENaC was present in the Wc and Cyto fractions but absent in the Mb sample. It may also be noted that the Cyto and Mb fraction were treated with PNGase F, resulting in a lower molecular weight compared to the band detected in the Wc sample. (B) Fraction of Î² ENaC expressing oocytes exhibiting amiloride-sensitive currents (+ami) after 24 h incubation. (C) Increasing the incubation time of Î² ENaC to 48 h shows a clear band within the Mb fraction and increases the number of oocytes exhibiting amiloride-sensitive currents (D).
	FIGURE 3. Homotrimeric ENaC except for Î³ is activated by SF. Electrophysiological characterization of homotrimeric ENaC in response to 0.2 dyncmâ€“ 2 SF (colored bar). (A) H2O-injected control oocytes neither respond to SF nor amiloride (10 Î¼M). (B) SF response of Î±Î²Î³ ENaC is absent in presence of amiloride (10 Î¼M). Î±Î²Î³ (C) and Î´Î²Î³ (D) ENaC are significantly activated by SF. Left panels show representative traces. The right panels show the averages Â± SEM. Homotrimeric Î±, Î´, and Î² (Eâ€“G, respectively) respond to SF, whereas Î³ (H) does not. (I) The SF response (I0.2/I0) of homotrimeric ENaC subunits was normalized with respect to canonical Î±Î²Î³ and Î´Î²Î³ ENaC. The SF response of Î± ENaC was unchanged while Î² ENaC showed a reduced SF effect when compared with Î±Î²Î³. Î´ ENaC displayed an increased SF effect compared with canonical Î´Î²Î³ ENaC. Paired t-test (Câ€“H) and one-way ANOVA with multiple comparison (I); p < 0.05; p < 0.01; **p < 0.0001.
	FIGURE 4. Heterotrimeric ENaC composed of two subunits form functional channels. (AâG) Effect of amiloride (10 Î¼M) application on heterotrimeric ENaC currents of various subunit combinations (Î±Î²Î³, Î±Î², Î±Î³, Î²Î³, Î´Î²Î³, Î´Î², and Î´Î³). (H) Dose response curves for amiloride were fitted and derived IC50 values were used for comparison (I). Amiloride sensitivity of Î±Î² and Î±Î³ ENaC was statistically not different when compared with Î±Î²Î³, whereas Î²Î³ ENaC displays a significantly increased IC50 value with respect to Î±Î²Î³ and Î±Î³ ENaC. (J) Dose-response curves for channels containing Î´ ENaC. (K) Î´Î³ displayed a significantly reduced amiloride IC50 value compared with Î´Î²Î³, whereas Î´Î² remains unchanged. Paired t-test (AâG) and one-way ANOVA with multiple comparison (I,K); p < 0.01; p < 0.001; ***p < 0.0001.
	FIGURE 5. Heterotrimeric ENaC composed of two subunits responds to SF. SF response of heterotrimeric ENaC composed of two different subunits was assessed. Left panels show representative current traces whereas the right panels show the averages Â± SEM. All combinations of two different ENaC subunits, Î±Î² (A), Î±Î³ (B), Î´Î² (C), Î´Î³ (D), and Î²Î³ (E), were significantly activated by the application of SF (indicated as colored bar). (F) The SF response (I0.2/I0) of those ENaC subunit compositions was normalized with respect to canonical Î±Î²Î³ and Î´Î²Î³ ENaC. The SF effect of Î±Î² and Î²Î³ was unchanged when compared with Î±Î²Î³. A significantly increased SF response was observed for Î±Î³ when compared with Î±Î²Î³. Î´Î³ ENaC shows also a significantly increased SF effect when compared with Î´Î²Î³, whereas Î´Î² was unchanged. Paired t-test (Aâ€“E) and one-way ANOVA with multiple comparison (F); p < 0.01; p < 0.001; ***p < 0.0001.

References [+] :

Althaus, Mechano-sensitivity of epithelial sodium channels (ENaCs): laminar shear stress increases ion channel open probability. 2007, Pubmed, Xenbase

	Figure 1. Homotrimeric ENaC forms functional channels except for Î³. Electrophysiological characterization of homotrimeric ENaC (Î±, Î´, Î², and Î³) vs. canonical ENaC (Î±Î²Î³ and Î´Î²Î³) expressed in oocytes. Representative current traces of Î±Î²Î³- (A), Î±- (B), Î²- (C), Î³- (D), Î´Î²Î³- (E), and Î´-injected oocytes (F) in response to the application of amiloride (10Î¼M). (C) Oocytes injected with ENaC encoding RNA revealed variable responses. Only a proportion of oocytes responded to amiloride, whereas the majority of oocytes did not show an amiloride response (Câ). (D) Oocytes injected with Î³ ENaC did not show a reaction in response to amiloride. (G) Amiloride dose response curve of Î±Î²Î³, Î±, and Î² ENaC and calculated IC50 values for amiloride (H) showing a decreased amiloride sensitivity for Î² when compared with Î±Î²Î³, whereas Î± is unchanged. (I) Amiloride dose response curves of Î´Î²Î³ and Î´. (J) Î´ ENaC displays a significantly reduced amiloride IC50 value compared with Î´Î²Î³. Paired t-test (AâF), one-way ANOVA with multiple comparison (H) and unpaired t-test (J); p < 0.05; p < 0.01; p < 0.001; **p < 0.0001.
	Figure 2. Biotinylation assay of Î² ENaC expressing oocytes. (A) Representative western blot showing Î² ENaC in presence (+) or absence (â) of biotin. Each group consists of the following three samples: whole cell (Wc), cytoplasmic (Cyto), and membrane bound (Mb). Exposure time for Wc and Cyto: 5 s and for Mb: 10 min N = 3. After an incubation of 24 h Î² ENaC was present in the Wc and Cyto fractions but absent in the Mb sample. It may also be noted that the Cyto and Mb fraction were treated with PNGase F, resulting in a lower molecular weight compared to the band detected in the Wc sample. (B) Fraction of Î² ENaC expressing oocytes exhibiting amiloride-sensitive currents (+ami) after 24 h incubation. (C) Increasing the incubation time of Î² ENaC to 48 h shows a clear band within the Mb fraction and increases the number of oocytes exhibiting amiloride-sensitive currents (D).
	Figure 3. Homotrimeric ENaC except for Î³ is activated by SF. Electrophysiological characterization of homotrimeric ENaC in response to 0.2 dyncmâ€“ 2 SF (colored bar). (A) H2O-injected control oocytes neither respond to SF nor amiloride (10 Î¼M). (B) SF response of Î±Î²Î³ ENaC is absent in presence of amiloride (10 Î¼M). Î±Î²Î³ (C) and Î´Î²Î³ (D) ENaC are significantly activated by SF. Left panels show representative traces. The right panels show the averages Â± SEM. Homotrimeric Î±, Î´, and Î² (Eâ€“G, respectively) respond to SF, whereas Î³ (H) does not. (I) The SF response (I0.2/I0) of homotrimeric ENaC subunits was normalized with respect to canonical Î±Î²Î³ and Î´Î²Î³ ENaC. The SF response of Î± ENaC was unchanged while Î² ENaC showed a reduced SF effect when compared with Î±Î²Î³. Î´ ENaC displayed an increased SF effect compared with canonical Î´Î²Î³ ENaC. Paired t-test (Câ€“H) and one-way ANOVA with multiple comparison (I); p < 0.05; p < 0.01; **p < 0.0001.
	Figure 4. Heterotrimeric ENaC composed of two subunits form functional channels. (AâG) Effect of amiloride (10 Î¼M) application on heterotrimeric ENaC currents of various subunit combinations (Î±Î²Î³, Î±Î², Î±Î³, Î²Î³, Î´Î²Î³, Î´Î², and Î´Î³). (H) Dose response curves for amiloride were fitted and derived IC50 values were used for comparison (I). Amiloride sensitivity of Î±Î² and Î±Î³ ENaC was statistically not different when compared with Î±Î²Î³, whereas Î²Î³ ENaC displays a significantly increased IC50 value with respect to Î±Î²Î³ and Î±Î³ ENaC. (J) Dose-response curves for channels containing Î´ ENaC. (K) Î´Î³ displayed a significantly reduced amiloride IC50 value compared with Î´Î²Î³, whereas Î´Î² remains unchanged. Paired t-test (AâG) and one-way ANOVA with multiple comparison (I,K); p < 0.01; p < 0.001; ***p < 0.0001.
	Figure 5. Heterotrimeric ENaC composed of two subunits responds to SF. SF response of heterotrimeric ENaC composed of two different subunits was assessed. Left panels show representative current traces whereas the right panels show the averages Â± SEM. All combinations of two different ENaC subunits, Î±Î² (A), Î±Î³ (B), Î´Î² (C), Î´Î³ (D), and Î²Î³ (E), were significantly activated by the application of SF (indicated as colored bar). (F) The SF response (I0.2/I0) of those ENaC subunit compositions was normalized with respect to canonical Î±Î²Î³ and Î´Î²Î³ ENaC. The SF effect of Î±Î² and Î²Î³ was unchanged when compared with Î±Î²Î³. A significantly increased SF response was observed for Î±Î³ when compared with Î±Î²Î³. Î´Î³ ENaC shows also a significantly increased SF effect when compared with Î´Î²Î³, whereas Î´Î² was unchanged. Paired t-test (Aâ€“E) and one-way ANOVA with multiple comparison (F); p < 0.01; p < 0.001; ***p < 0.0001.
	Supplementary Figure 1- Epithelial Na+ Channel (ENaC) Formed by One or Two Subunits Forms Functional Channels That Respond to Shear Force
	Supplementary Figure 2- Epithelial Na+ Channel (ENaC) Formed by One or Two Subunits Forms Functional Channels That Respond to Shear Force
	FIGURE 1. Homotrimeric ENaC forms functional channels except for Î³. Electrophysiological characterization of homotrimeric ENaC (Î±, Î´, Î², and Î³) vs. canonical ENaC (Î±Î²Î³ and Î´Î²Î³) expressed in oocytes. Representative current traces of Î±Î²Î³- (A), Î±- (B), Î²- (C), Î³- (D), Î´Î²Î³- (E), and Î´-injected oocytes (F) in response to the application of amiloride (10Î¼M). (C) Oocytes injected with ENaC encoding RNA revealed variable responses. Only a proportion of oocytes responded to amiloride, whereas the majority of oocytes did not show an amiloride response (Câ). (D) Oocytes injected with Î³ ENaC did not show a reaction in response to amiloride. (G) Amiloride dose response curve of Î±Î²Î³, Î±, and Î² ENaC and calculated IC50 values for amiloride (H) showing a decreased amiloride sensitivity for Î² when compared with Î±Î²Î³, whereas Î± is unchanged. (I) Amiloride dose response curves of Î´Î²Î³ and Î´. (J) Î´ ENaC displays a significantly reduced amiloride IC50 value compared with Î´Î²Î³. Paired t-test (AâF), one-way ANOVA with multiple comparison (H) and unpaired t-test (J); p < 0.05; p < 0.01; p < 0.001; **p < 0.0001.
	FIGURE 2. Biotinylation assay of Î² ENaC expressing oocytes. (A) Representative western blot showing Î² ENaC in presence (+) or absence (â) of biotin. Each group consists of the following three samples: whole cell (Wc), cytoplasmic (Cyto), and membrane bound (Mb). Exposure time for Wc and Cyto: 5 s and for Mb: 10 min N = 3. After an incubation of 24 h Î² ENaC was present in the Wc and Cyto fractions but absent in the Mb sample. It may also be noted that the Cyto and Mb fraction were treated with PNGase F, resulting in a lower molecular weight compared to the band detected in the Wc sample. (B) Fraction of Î² ENaC expressing oocytes exhibiting amiloride-sensitive currents (+ami) after 24 h incubation. (C) Increasing the incubation time of Î² ENaC to 48 h shows a clear band within the Mb fraction and increases the number of oocytes exhibiting amiloride-sensitive currents (D).
	FIGURE 3. Homotrimeric ENaC except for Î³ is activated by SF. Electrophysiological characterization of homotrimeric ENaC in response to 0.2 dyncmâ€“ 2 SF (colored bar). (A) H2O-injected control oocytes neither respond to SF nor amiloride (10 Î¼M). (B) SF response of Î±Î²Î³ ENaC is absent in presence of amiloride (10 Î¼M). Î±Î²Î³ (C) and Î´Î²Î³ (D) ENaC are significantly activated by SF. Left panels show representative traces. The right panels show the averages Â± SEM. Homotrimeric Î±, Î´, and Î² (Eâ€“G, respectively) respond to SF, whereas Î³ (H) does not. (I) The SF response (I0.2/I0) of homotrimeric ENaC subunits was normalized with respect to canonical Î±Î²Î³ and Î´Î²Î³ ENaC. The SF response of Î± ENaC was unchanged while Î² ENaC showed a reduced SF effect when compared with Î±Î²Î³. Î´ ENaC displayed an increased SF effect compared with canonical Î´Î²Î³ ENaC. Paired t-test (Câ€“H) and one-way ANOVA with multiple comparison (I); p < 0.05; p < 0.01; **p < 0.0001.
	FIGURE 4. Heterotrimeric ENaC composed of two subunits form functional channels. (AâG) Effect of amiloride (10 Î¼M) application on heterotrimeric ENaC currents of various subunit combinations (Î±Î²Î³, Î±Î², Î±Î³, Î²Î³, Î´Î²Î³, Î´Î², and Î´Î³). (H) Dose response curves for amiloride were fitted and derived IC50 values were used for comparison (I). Amiloride sensitivity of Î±Î² and Î±Î³ ENaC was statistically not different when compared with Î±Î²Î³, whereas Î²Î³ ENaC displays a significantly increased IC50 value with respect to Î±Î²Î³ and Î±Î³ ENaC. (J) Dose-response curves for channels containing Î´ ENaC. (K) Î´Î³ displayed a significantly reduced amiloride IC50 value compared with Î´Î²Î³, whereas Î´Î² remains unchanged. Paired t-test (AâG) and one-way ANOVA with multiple comparison (I,K); p < 0.01; p < 0.001; ***p < 0.0001.
	FIGURE 5. Heterotrimeric ENaC composed of two subunits responds to SF. SF response of heterotrimeric ENaC composed of two different subunits was assessed. Left panels show representative current traces whereas the right panels show the averages Â± SEM. All combinations of two different ENaC subunits, Î±Î² (A), Î±Î³ (B), Î´Î² (C), Î´Î³ (D), and Î²Î³ (E), were significantly activated by the application of SF (indicated as colored bar). (F) The SF response (I0.2/I0) of those ENaC subunit compositions was normalized with respect to canonical Î±Î²Î³ and Î´Î²Î³ ENaC. The SF effect of Î±Î² and Î²Î³ was unchanged when compared with Î±Î²Î³. A significantly increased SF response was observed for Î±Î³ when compared with Î±Î²Î³. Î´Î³ ENaC shows also a significantly increased SF effect when compared with Î´Î²Î³, whereas Î´Î² was unchanged. Paired t-test (Aâ€“E) and one-way ANOVA with multiple comparison (F); p < 0.01; p < 0.001; ***p < 0.0001.