Figure 2.

The γ subunit thumb domain is required for inhibition by γ-11. Oocytes were injected with 2 ng/subunit of cRNA encoding the ENaC subunits indicated, and whole cell recordings of currents were performed 1 day later by TEVC at −100 mV. A, representative recordings are shown; the response to two γ-11 concentrations were measured in each cell. Na⁺ self-inhibition was elicited by increasing bath [Na⁺] from 1 (1 Na⁺) to 110 mm (110 Na⁺). Indicated concentrations of γ-11 and 10 μm amiloride were diluted into 110 Na⁺ buffer. Current measurements were taken at the peak elicited by 110 Na⁺ (I_peak), and at the end of each bath condition, giving I_110Na, I_γ-11,1, I_γ-11,2, and I_amiloride. Na⁺ self-inhibition was determined as (1 − (I_peak − I_Na110)/(I_peak − I_amiloride)) × 100%. Peptide Inhibition was determined as (1 − (I_110Na − I_γ-11,X)/(I_110Na − I_amiloride)) × 100%. Baseline currents were −1.9 ± 1.2 μA for α^Fβγ^ΔI and −2.1 ± 0.9 μA for α^Fβγ^ΔI,ΔThumb. B, dose-response curves were plotted (mean ± S.D., n = 7–13) for α^Fβγ^ΔI and α^Fβγ^ΔI,ΔThumb, and fit to the Hill equation with the Hill coefficient fixed to 1. IC₅₀ values were 1.7 ± 0.9 μm for α^Fβγ^ΔI and >100 μm for α^Fβγ^ΔI,ΔThumb.