Figure 7.

Three aspartates in the δ-ENaC acidic cleft distinctly affect ENaC sensitivity to pH and Na⁺. a, representative I_M recordings of Xenopus oocytes expressing WT δβγ-ENaC or channels containing a single aspartate to asparagine (δ_D296Nβγ) mutation. Channel sensitivity to pH was assessed by a stepwise reduction of extracellular pH from 8.0 to 6.0 (pH 0.2 increments). b, pH-dependent activation of δβγ-ENaC containing none (wt), one (δ_D105Kβγ, δ_D293Nβγ, and δ_D296Nβγ), two (δ_D293N,D296Nβγ and δ_D105K,D296Nβγ) or three (δ_D3βγ: δ_{D105K,D293N,D296N}βγ) mutations leading to substitution of single aspartates. Maximum acid-induced channel activation is reduced in ENaC-containing single mutations (δ_D105Kβγ, δ_D293Nβγ, and δ_D296Nβγ) when compared with WT δβγ-ENaC. There is no cumulative effect in channels containing two mutated aspartates, but substitution of three aspartates (δ_D3βγ) further decreases maximum acid-induced activation as well as alkaline channel inhibition (Kruskal-Wallis test, p < 0.0001; Dunn's multiple comparisons test). c, assessment of SSI at [Na⁺] from 3 to 120 mm in oocytes expressing WT (δβγ) or mutant (δ_D296Nβγ) ENaC. d, Na⁺ self-inhibition ((I_{M, peak} − I_{M, 3 min})/I_{M, peak}) of WT, single, double, and triple mutant channels. Introduction of δ_D293N or δ_D105K moderately decreases maximal SSI when compared with WT δβγ-ENaC, whereas ENaC containing δ_D296N has a profoundly reduced SSI irrespective of the extracellular [Na⁺]. Substitution of additional aspartates in double or triple mutant channels does not further decrease ENaC SSI (one-way ANOVA, F = 10.26, p = 0.0003; Tukey's multiple comparisons test). e, proton-sensitivity of δβγ ENaC mutants was assessed by determining fractional channel activation ((I_{M, x} − I_{M, min})/Δ_max I_M) resulting from a stepwise reduction of the extracellular pH (pH 8.5–6.0; pH 0.5 increments) in the presence of 90 or 3 mm extracellular Na⁺. f, reduction of extracellular [Na⁺] evokes an alkaline-shift of the pH EC₅₀ in WT δβγ-ENaC but not in channels containing the δ_D296Nβγ or δ_D3βγ mutations. However, in the presence of 90 mm Na⁺, introduction of these mutations shifts the pH EC₅₀ to more alkaline values, when compared with WT ENaC (one-way ANOVA, F = 48.02, p < 0.0001; Tukey's multiple comparisons test). g, pH-sensitivity of Xenopus αβγ-ENaC (n = 11) is not enhanced by partial (α_K105Dβγ; n = 13) or full (α_{K105D,E296D,Q297L}βγ; n = 12) reconstitution of the δ-ENaC acidic cleft in this subunit. Individual values for pH-mediated regulation and SSI of WT and mutant ENaC (a–d), including statistical analyses, are listed in Table 1.