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Glycosylated extracellular asparagines of the α‐subunit provide a connection to the extracellular matrix and facilitate the shear force‐dependent activation of the human epithelial Na + ‐channel (ENaC)
Author(s) -
Knoepp Fenja,
Szczesniak Pawel,
Althaus Mike,
Fronius Martin
Publication year - 2015
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.29.1_supplement.845.1
Subject(s) - epithelial sodium channel , xenopus , mutant , chemistry , extracellular , protein subunit , microbiology and biotechnology , glycosylation , amiloride , transmembrane domain , biology , biochemistry , receptor , gene , organic chemistry , sodium
Epithelial Na + ‐channels (ENaCs) are activated by shear force (SF) but the underlying mechanism is yet unknown. In this study, we investigated if a linkage to the extracellular matrix (ECM) is crucial for SF‐sensing of ENaC. Glycosylated asparagines (N) are putative connection sites to the ECM. Five different N in the extracellular domain (ED) of αENaC were mutated by site‐directed mutagenesis. N‐mutants were expressed with wildtype (Wt) β and γENaC in Xenopus oocytes. To characterize the SF effect (0 → 0.2 dyn/cm 2 ), amiloride‐sensitive current (I ami ) was measured by two‐electrode voltage‐clamp technique. The localization of N was explored via homology modeling. Basic channel function was characterized by single channel recordings and glycosylation determined by western blot. SF increased I ami of WtENaC by 340 ± 40 %. Mutation of N232, N293 and N397 within the ED of αENaC failed to affect this SF‐induced increase of I ami . By contrast, mutation of N312 ( palm‐domain ) and N511 ( knuckle ‐ domain ) resulted in a decreased SF‐effect (220 ± 30 % and 236 ± 40 %, respectively). A combined mutation of N312 and N511 further reduced SF‐effect to 54 ± 10 %. Membrane expression, single channel amplitudes and open‐probability of these N‐mutants were equal to those of Wt. As glycosylation‐deficient mutants differ in molecular weight from Wt, band‐shifts were detected in western blots. SF activates human ENaC in the Xenopus expression system via a connection to the ECM. This connection relies on glycosylation of N312 and N511 within the ED of αENaC, thus providing a new mechanism of how SF regulates ENaC activity.

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