Figure 2.

IαI inhibits human epithelial sodium channel (ENaC) expressed in Xenopus oocytes. (A) Time course of inward Na⁺ currents (I_Na⁺) across Xenopus oocytes injected with cRNAs encoding wild-type α-, β-, and γ- human-ENaC (8.4 ng each). Addition of amiloride (10 μM) in the perfusion medium decreased I_Na⁺ from −40 to −5 μA. Perfusion with amiloride-free medium (ND96) restored I_Na⁺ to −40 μA. Perfusion with ND96 containing IαI (0.1 mg/ml) resulted in a time-dependent decrease of I_Na⁺. Addition of trypsin (2 μM) restored I_Na⁺ to its initial value. Results are from a typical experiment, which was repeated at least five times. (B) Oocytes were incubated with 0.1 mg/ml IαI or vehicle (ND96) for 60 minutes. Current voltage curves for amiloride-sensitive currents (I_ENaC) were calculated as described in Materials and Methods. Values are means ± SE; n (number of oocytes from 3 or 4 experiments) = 21 for vehicle and 19 for IαI. (C) ENaC-expressing oocytes were incubated with vehicle (ND96) or with equal concentrations of IαI and a specific blocking antibody (mAB) against IαI (0.1 mg/ml). Values means ± SE; n (number of oocytes from three or four experiments) = 8 for vehicle and 7 for IαI+mAB. Veh, vehicle.