Figure 2.

αS589 mutants are permeable to K⁺. Two-electrode voltage-clamp recordings in Xenopus oocytes expressing ENaC wt or the αS589A, C, or D mutant are shown. (A) Current traces of ENaC wt and the αS589D mutant in 120 mM K⁺ solution. Currents were elicited by 500-ms voltage steps from a holding potential of −20 mV to test potentials of −140 to +40 mV in 20-mV increments. Currents measured in the absence of amiloride were subtracted from currents measured in the presence of 5 μM amiloride, and these subtracted currents are shown. The dotted line indicates zero level of the amiloride-sensitive current. (B) Inhibition curves of I_K (▴) and I_Na (Δ) in the αS589D mutant by amiloride in one experiment including nine oocytes per condition. The fit of the data to Langmuir isotherm gives an apparent inhibitory constant (K_i) of 0.24 μM in K⁺ solution (dotted line) and 0.22 μM in Na⁺ solution (dashed line). (C) I/V relationship of amiloride-sensitive Na⁺, Li⁺, and K⁺ inward currents. Currents were measured as described under A in extracellular 40-mM Na⁺, 40-mM Li⁺, or 120-mM K⁺ solution. For comparison, current values were normalized to the I_Na at −100 mV. I_Na at −100 mV was 9.5 ± 2.3 μA for wt, 2.9 ± 0.5 μA for αS589A, 3.1 ± 0.5 μA for αS589C, and 1.4 ± 0.4 μA for the αS589D mutant. Shown are mean ± SEM from at least eight oocytes per condition.