Question: 

The epithelial sodium channel (ENaC) is the rate limiting step for transepithelial sodium transport in several sodium absorbing tissues including the distal colon, distal nephron and respiratory tract. Proteolytic cleavage at specific sites in the extracellular domain of the a- and g-subunit of ENaC plays an important role in its activation. Cleavage of the g-subunit of ENaC seems to be essential for proteolytic channel activation which may involve the release of inhibitory peptides. However, the (patho-)physiologically relevant proteases for ENaC activation are not yet known. The serine protease trypsin IV (also called mesotrypsin) is expressed in several epithelial tissues. Trypsin IV has been shown to be up-regulated in inflammatory disease and may be co-expressed with ENaC. Therefore, we hypothesized that trypsin IV may activate ENaC.

Methodology: 

Human wild-type or mutant abg_K189AENaC 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 with trypsin IV. To identify a putative cleavage site for trypsin IV, a 23-mer peptide (TGRKRKVGGSIIHKASNVMHIES) was synthesized that corresponds to a region in the extracellular domain of gENaC thought to be critical for proteolytic channel activation. The 23-mer peptide was incubated with trypsin IV and cleavage products were identified using high-performance liquid chromatography (HPLC) and mass spectrometry.

Results: 

In an initial set of experiments we demonstrated that trypsin IV stimulates ENaC currents in oocytes expressing wild-type human ENaC in a concentration dependent manner. In vitro cleavage analysis of the 23-mer peptide suggested that trypsin IV may cleave human gENaC at a lysine residue in position 189 (K189) that has previously been described as a putative plasmin cleavage site in mouse gENaC. Mutating this lysine residue to alanine (K189A) significantly reduced the stimulatory effect of trypsin IV on ENaC currents in oocytes expressing the mutant channel.

Conclusion: 

Our results indicate that trypsin IV can stimulate ENaC and that this stimulation involves a critical cleavage site (K189) in the extracellular domain of the g-subunit. These findings suggest that trypsin IV is a candidate protease that may activate ENaC under (patho-)physiological conditions.