γ subunit proteolytic fragment binds the epithelial Na+ channel at the finger‐thumb domain interface of the γ subunit

Epithelial Na + channel (ENaC) maturation and activation entail proteolysis of both the α and γ subunits. Cleavage at multiple sites in the finger domain of each subunit liberates the subunit's autoinhibitory peptide. Synthetic peptides derived from the proteolytically released fragments inhibit the channel, likely by reconstituting key interactions removed by proteolysis. We previously showed that a peptide derived from the α‐subunit's autoinhibitory sequence binds at the α subunit's finger‐thumb domain interface. Despite low sequence similarity between the α and γ subunit finger domains, we hypothesized that a peptide derived from γ subunit's autoinhibitory sequence inhibits the channel through an analogous mechanism. We tested our hypothesis using a crosslinking approach. We used bifunctional methane thiosulfonate (MTS) reagents to cross‐link experimentally‐introduced cysteines in the γ inhibitory peptide, and at select sites within the ENaC γ subunit. Our data show that the γ inhibitory peptide can be linked to both the finger and thumb domains of the γ subunit, consistent with our hypothesis. Our data also suggest that direct manipulation of the finger‐thumb interface using MTS reagents influences channel gating. Support or Funding Information This work is supported by NIH grant DK098204