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Conformational trapping of the closed state of ENaC using sites at the finger –thumb domain interface of the alpha subunit
Author(s) -
Kashlan Ossama B.,
Blobner Brandon M.,
Zuzek Zachary,
Kleyman Thomas R.
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.1039.10
Subject(s) - epithelial sodium channel , protein subunit , peptide , chemistry , biophysics , gating , biochemistry , biology , sodium , organic chemistry , gene
The epithelial Na+ channel (ENaC) is assembled from three homologous subunits, two of which are cleaved resulting in the release of inhibitory tracts and channel activation. Peptides corresponding to these tracts are inhibitory. Mutagenesis and double mutant cycle data implicated sites in the finger‐thumb domain interface in the periphery of the α subunit in inhibitory peptide binding. We built a model of the α subunit of ENaC based on these data and on homology to ASIC1, which predicts specific sites that are in close proximity to the bound inhibitory peptide. We examined whether introducing Cys at specific sites in the channel and peptide would allow for crosslinking with bifunctional Cys reactive crosslinkers resulting in an inhibition of channel activity. Peptide‐channel crosslinking was dependent on the sites of the introduced Cys, and in most cases on the length of the crosslinker. We also found that a crosslink between specific sites in the finger and thumb domains of the α subunit reduced channel activity, suggesting that these sites move relative to one another during gating. The observed effect was dependent on crosslinker length.