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

Figure 8.

Figure 8

Eliminating putative TMPRSS2 cleavage sites distal to the γ-inhibitory tract significantly reduces the stimulatory effect of TMPRSS2 coexpression on ENaC and prevents the appearance of fully cleaved γ-ENaC in whole-cell lysates.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 (αβγRKRK178AAAA, αβγK168A;K170A;R172A;K189A or αβγRKRK178AAAA;K168A;K170A;R172A;K189A) either alone (−) or in combination with 5 ng/oocyte cRNA for TMPRSS2 (+). 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 (22≤ n ≤ 61, 3 ≤ N ≤ 9). B, ΔIami values of individual oocytes obtained in the same experiments as shown in (A) were normalized to the corresponding mean ΔIami recorded in oocytes from the same batch expressing wildtype (αβγ) or mutant ENaC (αβγRKRK178AAAA, αβγK168A;K170A;R172A;K189A, or αβγRKRK178AAAA;K168A;K170A;R172A;K189A) without TMPRSS2 coexpression. Dashed line indicates a normalized ΔIami value of one (no effect). Mean ± SD and data points for individual oocytes are shown; ∗∗∗p < 0.001; ∗p < 0.05; ns; Kruskal–Wallis with Dunn’s post hoc test. C, representative western blots showing whole-cell expression of γ-ENaC detected using the same antibody as for Figure 4 in oocytes expressing wildtype (αβγ) or mutant ENaC (αβγRKRK178AAAA, αβγK168A;K170A;R172A;K189A, or αβγRKRK178AAAA;K168A;K170A;R172A;K189A) either without (−) or with (+) TMPRSS2 coexpression. Noninjected oocytes served as control (n.i.). Uncleaved (∼87 kDa), partially cleaved (∼76 kDa), and fully cleaved γ-ENaC (∼67 kDa) are indicated by open, light gray, and dark grayfilled arrowheads, respectively. Similar results were obtained in additional repeats shown in Fig. S3. cRNA, complementary RNA; ENaC, epithelial sodium channel; ns, not significant; TMPRSS2, transmembrane serine protease 2.