Figure 3. Mutating a putative prostasin site in the γ-subunit increases ENaC whole-oocyte currents, but decreases channel surface expression and reduces the stimulatory effect of extracellular proteases.

In A, the amino acid sequence alignment illustrates a highly conserved putative prostasin cleavage site (bold letters) in γ-ENaC of different species. In B and C, αβγ-ENaC (wt) or αβ-ENaC together with the mutant K181A γ-ENaC (γK181A) were expressed in Xenopus laevis oocytes to test the effect of the mutation on ΔI_ami baseline whole-oocyte currents and on the responsiveness to extracellular proteases. A representative whole-oocyte current (I) trace from an oocyte expressing γK181A is shown in B. In C, open columns represent the average ΔI_ami values before application of proteases. The black, grey and dotted columns represent the average ΔI_ami values 2 min after exposure to trypsin (2 μg ml⁻¹; n= 82 for wt, n= 83 for γK181A; N= 8), chymotrypsin (2 μg ml⁻¹; n= 43 for each group; N= 3) or human neutrophil elastase (hNE) (10 μg ml⁻¹; n= 32 for each group; N= 3), respectively. ΔI_ami values were normalized to the average ΔI_ami measured in the αβγ-ENaC expressing oocytes before the application of proteases. In D, surface expression (hatched columns) and ΔI_ami (open columns) were measured in matched oocytes expressing either wild-type ENaC or the γK181A mutant channel (N= 4). Non-injected oocytes (n.i.) served as control.