Fig. 2.

Stimulation of ENaC-mediated whole-cell currents by recombinant serine protease domain of FSAP. a–c Representative whole-cell current traces recorded in oocytes expressing human αβγENaC before (left panels) and after (middle panels) 30-min incubation in ND96 containing FSAP-SPD-WT (50 µg mL⁻¹ or 1.67 µM; a) or FSAP-SPD-MI (50 µg mL⁻¹; b) or in protease-free ND96 (control; c). Data obtained from similar experiments as shown in left and middle panels are summarized in corresponding right panels. Amiloride-sensitive currents (ΔI_ami) were determined before ( −) and after ( +) incubation with FSAP-SPD-WT (a), FSAP-SPD-MI (b), or control solution (c). Measurements performed in the same oocyte are connected by a line (n = 4–7). d Summary of data from the same experiments as shown in a–c and from additional experiments in which different protease concentrations were used as indicated. Incubation time (30 min) was the same in all experiments. The relative effect on ΔI_ami was calculated for each oocyte as the ratio of ΔI_ami measured after and before the incubation period (n = 4–7). Each data point corresponds to one individual oocyte. e Effect of 1 (0.03 µM) or 10 µg mL⁻¹ (0.33 µM) FSAP-SPD-WT on ΔI_ami for different incubation times (30, 120, or 240 min). Significance is indicated for comparison with baseline ΔI_ami measured before incubation with the protease (n = 5–7). f Time course of proteolytic activity in the indicated incubation solutions detected using the fluorogenic substrate Boc-QAR-AMC (RFU = relative fluorescence unit; n = 10–11). *p < 0.05; †p < 0.01; ns non-significant; paired t-test (a–c, e) or one-way ANOVA with Bonferroni post hoc test (d). Error bars, S.E