| Title | Alternative mechanism of activation of the epithelial Na+ channel by cleavage. | | Author(s) | Hu JC, Bengrine A, Lis A, Awayda MS | | Institution | Univ. Buffalo, United States; | | Source | J Biol Chem 2009 Oct 26. | | Abstract | We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of ENaC using the serine protease subtilisin. Trimeric channels formed with FM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and ~10 fold decrease in amiloride sensitive whole cell conductance as compared to alphaWT (2.2 vs. 21.2 microS). Both alphaWT and alphaFM were activated to similar levels by subtilisin cleavage. Channels formed with alphaFD, a construct that deleted the segment between the two furin sites (175-204), exhibited an intermediate conductance of 13.2 microS. More importantly, alphaFD retained the ability to be activated by subtilisin to 108.8+/-20.9 microS, a level not significantly different from that of subtilisin activated alphaWT (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 alpha 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 1st transmembrane domain. This was confirmed in channels formed with alphaLD, a construct that extended the deleted sequence of alphaFD by 17 a.a. (175-221). Channels with alphaLD were uncleaved, exhibited low baseline activity (4.1 microS), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the 1st transmembrane domain from the channel complex. | | Language | ENG | | Pub Type(s) | JOURNAL ARTICLE
| | PubMed ID | 19858199 |
|
