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Molecular mechanism of lung oedema clearance by AP301: dependence of ENaC pore forming subunits (LB781)
Author(s) -
Shabbir Waheed,
Hasanovic Minela,
Jenn Soja,
Tzotzos Susan,
Fischer Bernhard,
Pietschmann Helmut,
Fischer Hendrik,
Lucas Rudolf,
LemmensGruber Rosa
Publication year - 2014
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.28.1_supplement.lb781
Subject(s) - epithelial sodium channel , protein subunit , chemistry , amiloride , mutant , asparagine , microbiology and biotechnology , a549 cell , transmembrane domain , biophysics , biochemistry , sodium , receptor , biology , cell , amino acid , gene , organic chemistry
Pulmonary oedema is a life threatening condition, caused by acute lung injury. Although failure of gas exchange in the alveolar spaces causes most of the morbidity and mortality, it remained uncertain how epithelial sodium channel lose their liquid clearance ability. Furthermore, the subunit composition of native ENaC in diseased pulmonary epithelium is still unknown. AP301, a lectin‐like domain derived peptide of TNF‐α, increases amiloride‐sensitive current in A549 cells as well as in heterologously expressed hENaC, an effect which is lost after deglycosylation. Consequently, the present study was undertaken to determine the interaction of AP301 with hENaC glycosylation sites and subunit compositions of hENaC. We substituted asparagine to glutamine in α‐hENaC, and studied the AP301 induced increase in sodium current in different subunit compositions. The effect of AP301 in HEK and CHO cells heterologously expressing human ENaC WT and mutants in addition to different subunit compositions (α, ß, γ and δ) was measured in patch clamp experiments. Indeed, AP301 weakly activated single as well as multiple asparagine mutants of α‐hENaC. For maximal AP301 activity αβγ‐ or δβγ‐hENaC co‐expression was required. Conclusively, αβγδ‐hENaC elicited a robust AP301 response at very low EC50 values. These findings suggest that specific interaction of AP301 ENaC requires precedent binding to glycosylated extracellular loop and pore‐forming α or δ delta subunits. Grant Funding Source : Supported by Austrian Research Promotion Agency (FFG).