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. 2021 Feb 10;296:100404. doi: 10.1016/j.jbc.2021.100404

Figure 8.

Figure 8

Inhibition of ENaC currents by Cx30 is due to decreased ENaC expression at the cell surface.A, the following groups of oocytes were used: ENaC (injected with 0.6 ng/subunit/oocyte of ENaC cRNA); ENaC+Cx30 (injected with 0.6 ng/subunit/oocyte of ENaC cRNA and 2 ng/oocyte of Cx30 cRNA); 1/3 ENaC (injected with 0.2 ng/subunit/oocyte of ENaC cRNA). To detect channel expression at the cell surface, FLAG-tagged βENaC was coexpressed with wild-type α- and γENaC. ΔIAmi values of individual oocytes were normalized to the mean ΔIAmi measured in oocytes from the corresponding ENaC group. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; n.s. not significant; Kruskall–Wallis with Dunn’s post hoc test (n = 23, N = 3). B, in parallel with the ΔIAmi measurements shown in A, ENaC-βFLAG surface expression was detected as chemiluminescence signal in relative light units (RLU) using oocytes from the same batch. Control oocytes not expressing ENaC were used to determine the nonspecific background chemiluminescence. Mean ± SEM and data points for individual oocytes are shown; ∗∗∗p < 0.001; Kruskall–Wallis with Dunn’s post hoc test (n = 70, N = 3). C–E, representative western blots showing whole-cell expression of αENaC (C), βENaC (D), or γENaC (E) in oocytes expressing ENaC alone (ENaC) or coexpressing ENaC and Cx30. Specific bands for full-length α-, β-, and γENaC at ∼95 kDa (C–E) and for cleaved γENaC at ∼74 kDa (E) were not detected in control oocytes not expressing ENaC. F–H, densitometric evaluation of full-length bands for αENaC (F) and βENaC (G) and of full-length and cleaved bands for γENaC (H) from similar blots as shown in (C–E). Density values were normalized in each blot to the signal of αENaC (F), βENaC (G), or γENaC (H) bands obtained from oocytes expressing ENaC alone. Lines connect data points obtained in the same experiment, and mean ± SEM are shown; n.s. not significant; one sample Wilcoxon signed-rank test (N = 7). I, in parallel experiments to those shown in (C–H), ΔIAmi was measured to confirm the inhibitory effect of Cx30 on ENaC in these batches of oocytes. Each ΔIAmi value was normalized to the mean ΔIAmi obtained in oocytes expressing ENaC alone. Mean ± SEM and individual data points for each experiment are shown; ∗∗∗p < 0.001; Mann–Whitney test (n = 81, N = 7).