Figure 1.

Stimulatory effect of TMPRSS2 on ENaC requires proteolytic activity of TMPRSS2.A, representative whole-cell current traces are shown for oocytes expressing human wildtype αβγ-ENaC alone (left trace, ENaC) or coexpressing αβγ-ENaC with human wildtype TMPRSS2 (middle trace, ENaC + TMPRSS2) or catalytically inactive TMPRSS2 (right trace, ENaC + TMPRSS2^S441A). HA tag epitope was attached to the C terminus of TMPRSS2. HA tag neither disturbed the proteolytic activity of TMPRSS2 nor affected the stimulatory effect of TMPRSS2 on ENaC (Fig. S1). Amiloride (ami, 2 μM) and chymotrypsin (chym, 2 μg/ml) were present in the bath solution as indicated by black and gray bars, respectively. Dashed lines indicate zero current level. B, ENaC-mediated ami-sensitive whole-cell currents (ΔI_ami) were determined from similar experiments as shown in (A) by subtracting the baseline current in the presence of ami from the current level reached in its absence before (−) or after (+) chym application. Lines connect data points obtained in an individual oocyte. Mean ± SD and data points for individual oocytes are shown; ∗∗∗p < 0.001; ns, Kruskal–Wallis with Dunn’s post hoc test (51 ≤ n ≤ 52, N = 5). C, relative stimulatory effect of chym on ΔI_ami summarized from data shown in (B). Dashed line indicates normalized ΔI_ami value of one (no effect). Mean ± SD and data points for individual oocytes are shown; ∗∗∗p < 0.001; ns, one-way ANOVA with Bonferroni post hoc test. D, in parallel experiments to those shown in (A–C), trypsin-like proteolytic activity at the cell surface was detected in the same batches of oocytes. Progress curves of proteolytic activity (RFU = relative fluorescent unit; mean ± SD) are shown. In each individual recording RFU values were normalized to the initial RFU value at the beginning of the measurement. ∗∗∗p < 0.001; Kruskal–Wallis with Dunn’s post hoc test (at the time point 190 min; 41≤ n ≤ 46, N = 5). E, representative western blots showing intracellular (left upper panel) or cell surface (right upper panel) expression of HA-tagged wildtype TMPRSS2 or mutant TMPRSS2^S441A in oocytes from one batch. No specific signal was detected with the anti-HA antibody in oocytes expressing ENaC alone. TMPRSS2 zymogen (∼65 kDa) and TMPRSS2 in its activated cleaved form (catalytic chain, ∼27 kDa) are indicated by open and filled arrowheads, respectively. To validate separation of cell surface proteins from intracellular proteins, blots were stripped and reprobed using an antibody against β-actin (lower panels). Similar results were obtained in three additional repeats (n = 4). ENaC, epithelial sodium channel; HA, hemagglutinin; ns, not significant; TMPRSS2, transmembrane serine protease 2.