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

Figure 5.

Figure 5

Mutant Cx30 (M34A) with no detectable ion channel activity did not inhibit ENaC.A, representative whole-cell current traces recorded in oocytes expressing ENaC only (upper trace), coexpressing ENaC with C-terminally V5-tagged wild-type Cx30 (ENaC + Cx30, middle trace) or Cx30 with single point mutation M34A (ENaC + Cx30M34A, lower trace). Application of amiloride (Ami, 2 μM) and removal of divalent cations from the bath solution for 60 s (Ca2+ Mg2+-Removal) are indicated by corresponding bars. Dashed lines indicate zero current level. B, ΔICa2+Mg2+-Removal values were obtained from similar experiments as shown in A and calculated as described in Figure 1. Mean ± SEM and individual data points for each experiment are shown; ∗∗∗p < 0.001; n.s., not significant; one-way ANOVA with Bonferroni post hoc test (n = 36, N = 3). C, ΔIAmi were calculated from similar experiments as shown in A as described in Figure 3. 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. D, relative inhibitory effect of Cx30 on ENaC calculated from the data shown in C essentially as described in Figure 3. 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. E and F, representative western blots showing cell surface (E) or intracellular (F) expression of C-terminally V5-tagged wild-type Cx30 and mutant Cx30M34A in oocytes from the same batch as used in A. No specific signal was detected with the anti-V5 antibody in oocytes expressing ENaC alone. To validate separation of cell surface proteins from intracellular proteins, blots were stripped and reprobed using an antibody against β-actin. G and H, densitometric evaluation of Cx30 and Cx30M34A expression from three similar blots as shown in E and F. In each blot the density value of the Cx30M34A band was normalized to that of the Cx30 band. 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 = 3).