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. 2022 Apr 30;298(6):102004. doi: 10.1016/j.jbc.2022.102004

Figure 9.

Figure 9

TMPRSS2 cleaves γ-ENaC at sites proximal to the γ-inhibitory tract.A, representative whole-cell current traces recorded in individual oocytes from the same batch injected with 1 ng/subunit/oocyte of cRNA for wildtype (αβγ) or mutant ENaC (αβγR138A) either alone (−) or in combination with 5 ng/oocyte cRNA for TMPRSS2 (+). V5-tag epitope was attached to the N terminus of γ-ENaC. Amiloride (ami, 2 μM) was present in the bath solution as indicated by black bars. Dashed lines indicate zero current level. Summary data obtained in similar experiments are shown to the right of the representative traces. Mean ± SD and data points for individual oocytes are shown; ∗∗∗p < 0.001; two-tailed unpaired Student’s t test (27≤ n ≤ 31, N = 5). B, ΔIami values of individual oocytes obtained in the same experiments as shown in (A) were normalized as described for Figure 8B. Dashed line indicates a normalized ΔIami value of one (no effect). Mean ± SD and data points for individual oocytes are shown; ns; one-way ANOVA with Bonferroni post hoc test. C, schematic diagram showing γ-ENaC cleavage fragments, which can be detected using an antibody raised against a C-terminal γ-ENaC epitope (in blue) or an anti-V5 antibody (in green). The expected molecular weights of the corresponding C-terminal and N-terminal γ-ENaC cleavage fragments are given below in the respective color. D, representative western blots showing whole-cell expression of γ-ENaC detected using the C-terminal anti-γ-ENaC (upper panel) or N-terminal anti-V5 antibody (lower panel) in oocytes expressing wildtype (αβγ) or mutant ENaC (αβγR138A) either without (−) or with (+) TMPRSS2 coexpression. Noninjected oocytes served as control (n.i.). Uncleaved γ-ENaC (∼87 kDa), cleaved in the proximal (∼76 kDa and ∼15 kDa) or distal (∼67 kDa and ∼21 kDa) regions of the γ-inhibitory tract, are indicated by open, gray, and black-filled arrowheads, respectively. A putative N-terminal γ-ENaC degradation product (∼12 kDa) is indicated by an open arrowhead with dot pattern. Similar results were obtained in three additional repeats (n = 4). cRNA, complementary RNA; ENaC, epithelial sodium channel; ns, not significant; TMPRSS2, transmembrane serine protease 2.