Figure 3.
The γ subunit thumb domain is not required for inhibition by α-8. TEVC recordings of currents at −60 mV were performed 1 day after injection of 2 ng/subunit of cRNA encoding the ENaC subunits indicated. A, representative recordings of the effects of 0.3 and 3 μm α-8 are shown. Na+ self-inhibition and peptide inhibition were determined as described in the legend to Fig. 2. Baseline currents were −3.7 ± 2.3 μA for αβγ and −3.5 ± 2.0 μA for αβγΔThumb. B, dose-response curves were plotted (mean ± S.D., n = 9–14) for αβγ and αβγΔThumb, and data were analyzed by two-way ANOVA followed by Sidak's multiple comparison test. Differences were detected as the result of changing concentrations (p < 0.0001) and between groups (p < 0.0001), but not for the interaction between groups and concentrations (p = 0.11). Differences were also detected both groups at 3 μm α-8 (***, p < 0.001 versus WT). C, oocytes expressing the ENaC subunits indicated were perfused with 2 μg/ml of chymotrypsin for 2 min in 110 mm Na+ buffer to determine whether each channel could be proteolytically activated. Currents were measured by TEVC at −60 mV, and measurements were taken just prior to chymotrypsin addition, at the end of chymotrypsin addition, and at the end of 10 μm amiloride addition. The change in amiloride sensitive current in response to chymotrypsin addition is reported. ****, p < 0.0001 by Student's t test.