Question: 

Proteolytic processing of the epithelial sodium channel (ENaC) is thought to contribute to its activation. In nephrotic syndrome filtered plasminogen may be converted to plasmin by urokinase-type plasminogen activator. We and others demonstrated that plasmin can proteolytically activate ENaC and therefore may contribute to renal sodium retention in nephrotic syndrome (Svenningsen et al., 2009). In this study we further characterized cleavage sites in human gENaC that are relevant for channel activation by plasmin.

Methodology: 

Mutant human gENaC constructs were generated by site directed mutagenesis. Human wild-type or mutant abgENaC were expressed in Xenopus laevis oocytes. Amiloride-sensitive whole-cell currents (DI_ami) were determined by two-electrode voltage-clamp before and after 30 min incubation of the oocytes in human plasmin (10mg/ml).

Results: 

Mutating a previously described putative plasmin site in human gENaC (gK189A) significantly reduced the stimulatory effect of plasmin on ENaC activity but did not abolish it. In contrast, the stimulatory effect of plasmin was largely preserved in oocytes expressing ENaC with a mutated putative prostasin site (gRKRK178AAAA). Importantly, the combination of both mutations (gRKRK178AAAA;K189A) abolished the stimulatory effect of plasmin.

Conclusion: 

The reduced stimulatory effect of plasmin on abg_K189AENaC indicates that gK189 is a relevant plasmin cleavage site in human gENaC. However, the lack of a complete inhibition is in contrast to previously reported findings in mouse ENaC and suggests that an additional cleavage site is involved in the activation of human ENaC by plasmin. This is supported by our finding that a combination of the K189A and the RKRK178AAAA mutation abolished the stimulatory effect of plasmin. We conclude that in addition to the putative plasmin site (gK189A) a putative prostasin cleavage site (gRKRK178AAAA) is involved in mediating the stimulatory effect of plasmin on human ENaC.