Lawong RY et al. (2023), Recording Sodium Self-Inhibition of Epithelial ...

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Membranes (Basel) 2023 May 19;135:. doi: 10.3390/membranes13050529.

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Recording Sodium Self-Inhibition of Epithelial Sodium Channels Using Automated Electrophysiology in Xenopus Oocytes.

Lawong RY , May F , Etang EC , Vorrat P , George J , Weder J , Kockler D , Preller M , Althaus M .

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The epithelial sodium channel (ENaC) is a key regulator of sodium homeostasis that contributes to blood pressure control. ENaC open probability is adjusted by extracellular sodium ions, a mechanism referred to as sodium self-inhibition (SSI). With a growing number of identified ENaC gene variants associated with hypertension, there is an increasing demand for medium- to high-throughput assays allowing the detection of alterations in ENaC activity and SSI. We evaluated a commercially available automated two-electrode voltage-clamp (TEVC) system that records transmembrane currents of ENaC-expressing Xenopus oocytes in 96-well microtiter plates. We employed guinea pig, human and Xenopus laevis ENaC orthologs that display specific magnitudes of SSI. While demonstrating some limitations over traditional TEVC systems with customized perfusion chambers, the automated TEVC system was able to detect the established SSI characteristics of the employed ENaC orthologs. We were able to confirm a reduced SSI in a gene variant, leading to C479R substitution in the human α-ENaC subunit that has been reported in Liddle syndrome. In conclusion, automated TEVC in Xenopus oocytes can detect SSI of ENaC orthologs and variants associated with hypertension. For precise mechanistic and kinetic analyses of SSI, optimization for faster solution exchange rates is recommended.

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	Figure 1. Sodium self-inhibition (SSI) of guinea pig αβγ- or δβγ-ENaCs heterologously expressed in Xenopus oocytes. (a) Left panel: representative transmembrane (IM) recording of an oocyte expressing αβγ-ENaC at 19 °C; right panel: mean ± SD plotted from experiments as shown in the left panel (n = 12). (b) Left panel: representative transmembrane (IM) recording of an oocyte expressing δβγ-ENaC at 19 °C; right panel: mean ± SD plotted from experiments as shown in the left panel (n = 9). (c) SSI was calculated as the percent decrease in amiloride-baseline subtracted IM within 3 min after switching the extracellular solution from low-sodium (1) to high-sodium (90) ORS. For αβγ-ENaC, n = 12; for δβγ-ENaC, n = 9. Student’s unpaired t-test (two-tailed). (d,e) Similar to panels (a,b) with recordings performed at 24 °C, for αβγ-ENaC, n = 17; for δβγ-ENaC, n = 11. (f) Similar to panel (c) with data from recordings performed at 24 °C, for αβγ-ENaC, n = 17; for δβγ-ENaC, n = 11. Student’s unpaired t-test (two tailed). Notes: 1 = low-sodium ORS containing 1 mM NaCl; 90 = high-sodium ORS containing 90 mM NaCl; a = amiloride.
	Figure 2. Sodium self-inhibition (SSI) of Xenopus laevis αβγ- or δβγ-ENaCs heterologously expressed in Xenopus oocytes. (a) Left panel: representative transmembrane (IM) recording of an oocyte expressing αβγ-ENaC at 19 °C; right panel: mean ± SD plotted from experiments as shown in the left panel (n = 11). (b) Left panel: representative transmembrane (IM) recording of an oocyte expressing δβγ-ENaC at 19 °C; right panel: mean ± SD plotted from experiments as shown in the left panel (n = 19). (c) SSI was calculated as the percent decrease in amiloride-baseline subtracted IM within 3 min after switching the extracellular solution from low-sodium (1) to high-sodium (90) ORS. For αβγ-ENaC, n = 11; for δβγ-ENaC, n = 19. Student’s unpaired t-test (two-tailed). (d,e) Similar to panels (a,b) with recordings performed at 24 °C, for αβγ-ENaC, n = 8; for δβγ-ENaC, n = 9. (f) Similar to panel (c) with data from recordings performed at 24 °C, for αβγ-ENaC, n = 8; for δβγ-ENaC, n = 9. Student’s unpaired t-test (two-tailed). Notes: 1 = low-sodium ORS containing 1 mM NaCl; 90 = high-sodium ORS containing 90 mM NaCl; a = amiloride.
	Figure 3. Structural model of the human α-ENaC subunit after energy minimization. (a) Superposition of the energy-minimized wildtype α-ENaC subunit (dark blue cartoon representation) and the C479R mutation (light blue cartoon and red sticks representation) indicates local rearrangements of the loop region between beta-strand β9 and the α4 helix. This region is suggested as a fulcrum for conformational changes during the open–closed transition of the ENaC channel. Subunits β and γ are shown in grey. (b) Close-up view on the wildtype α subunit of ENaC (dark blue) with the conserved disulfide bridge between C394 and C479. This disulfide bridge appears to stabilize the adjacent loop region. (c) Close-up view on the mutated α-ENaC subunit (light blue) shows that the inserted, positively charged arginine at position 479 leads to the repulsion of surrounding, positively charged amino acids of the loop, thereby potentially affecting intrinsic conformational changes and interactions with the lipid membrane.
	Figure 4. Sodium self-inhibition (SSI) of human αβγ-, δβγ-, or αC479Rβγ-ENaCs heterologously expressed in Xenopus oocytes. (a) Left panel: representative transmembrane (IM) recording of an oocyte expressing αβγ-ENaC at 19 °C; right panel: mean ± SD plotted from experiments as shown in the left panel (n = 18). (b) Left panel: representative transmembrane (IM) recording of an oocyte expressing δβγ-ENaC at 19 °C; right panel: mean ± SD plotted from experiments as shown in the left panel (n = 17). (c) Left panel: representative transmembrane (IM) recording of an oocyte expressing αC479Rβγ-ENaC at 19 °C; right panel: mean ± SD plotted from experiments as shown in the left panel (n = 17). (d) SSI was calculated as the percent decrease in amiloride-baseline subtracted IM within 3 min after switching the extracellular solution from low-sodium (1) to high-sodium (90) ORS. For αβγ-ENaC, n = 18; for δβγ-ENaC, n = 17; for αC479Rβγ-ENaC, n = 17. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (e–g) Similar to panels (a–c) with recordings performed at 24 °C, for αβγ-ENaC, n = 14; for δβγ-ENaC, n = 11; for αC479Rβγ-ENaC, n = 13. (h) Similar to panel (d) with data from recordings performed at 24 °C, for αβγ-ENaC, n = 14; for δβγ-ENaC, n = 11; for αC479Rβγ-ENaC, n = 13. One-way ANOVA followed by Tukey’s multiple comparisons test. Notes: 1 = low-sodium ORS containing 1 mM NaCl; 90 = high-sodium ORS containing 90 mM NaCl; a = amiloride.

References [+] :

Babini, A new subunit of the epithelial Na+ channel identifies regions involved in Na+ self-inhibition. 2003, Pubmed, Xenbase