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Glycosylated Asparagines Are Important Constituents For The Shear Force Dependent Activation Of The Epithelial Sodium Channel (ENaC)
Author(s) -
Barth Daniel,
Knoepp Fenja,
Fronius Martin
Publication year - 2016
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.30.1_supplement.1223.7
Subject(s) - epithelial sodium channel , chemistry , glycosylation , protein subunit , mutant , biochemistry , microbiology and biotechnology , sodium , biology , gene , organic chemistry
The epithelial sodium channel (ENaC) contributes to electrolyte/fluid‐homeostasis and blood pressure regulation and its activity is regulated by shear force (SF). Previous studies have shown that SF sensation of α‐ENaC depends on an interaction with the extracellular matrix (ECM) via N‐linked glycans of two glycosylated asparagines (N312 and N511). To determine whether the asparagines themselves or the attached N‐glycans are required for SF sensation, site‐directed mutagenesis combined with two‐electrode voltage clamp (TEVC) experiments were performed. α‐ENaC subunits were stripped of their N‐glycans by individually disrupting the glycosylation consensus sequence (NXS/T) N312NS to N312NA and N511YT to N511YA. When co‐expressed with β and γ ENaC, a reduced SF‐response was observed compared with wildtype α ENaC. Furthermore, the role of glycosylated asparagines for SF sensation was confirmed in experiments with the δ‐subunit. While functionally related to the α‐subunit (35% of sequence identity), δ ENaC (when co‐expressed with β and γ) is less SF sensitive and lacking the glycosylated asparagines responsible for proper SF sensation in α‐ENaC. Insertion of asparagines corresponding to α N312 and N511, including their glycosylation consensus sequence, at the corresponding positions in the δ‐subunit, generated two δ‐subunit mutants that were more sensitive to SF. These experiments further confirm that glycosylated asparagines and the attached N‐glycans in particular are crucial for SF sensation of ENaC. Support or Funding Information The work was supported by University of Otago and Justus‐Liebig‐University Giessen PhD Scholarships and research grants.