Alternative mechanism of activation of the epithelial Na⁺ channel by cleavage

We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of αENaC using the serine protease subtilisin. Trimeric channels formed with αFM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an ∼10-fold decrease in amiloride-sensitive whole cell conductance as compared with αWT (2.2 versus 21.2 microsiemens (μS)). Both αWT and αFM were activated to similar levels by subtilisin cleavage. Channels formed with αFD, a construct that deleted the segment between the two furin sites (Δ175-204), exhibited an intermediate conductance of 13.2 μS. More importantly, αFD retained the ability to be activated by subtilisin to 108.8 ± 20.9 μS, a level not significantly different from that of subtilisin activated αWT (125.6 ± 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of α was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with αLD, a construct that extended the deleted sequence of αFD by 17 amino acids (Δ175- 221). Channels with αLD were uncleaved, exhibited low baseline activity (4.1 μS), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.