RT Journal Article SR Electronic T1 Mechanism of action of novel lung oedema therapeutic AP301 by activation of ENaC JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP mol.113.089409 DO 10.1124/mol.113.089409 A1 Waheed Shabbir A1 Parastoo Scherbaum-Hazemi A1 Susan Tzotzos A1 Bernhard Fischer A1 Hendrik Fischer A1 Helmut Pietschmann A1 Rudolf Lucas A1 Rosa Lemmens-Gruber YR 2013 UL http://molpharm.aspetjournals.org/content/early/2013/09/27/mol.113.089409.abstract AB AP301, a cyclic peptide, comprising the human tumour necrosis factor lectin like domain (TIP domain) sequence, is currently being developed as a therapy for lung oedema and has been shown to reduce extravascular lung water and improve lung function in a mouse, rat and pig model. The current paradigm for liquid homeostasis in the adult mammalian lung is that passive apical uptake of sodium via the amiloride-sensitive epithelial sodium channel (ENaC) and non-selective cyclic-nucleotide-gated cation (CNG) channels, creates the major driving force for reabsorption of water through the alveolar epithelium besides other ion channels such as potassium and chloride channels. AP301 can increase amiloride-sensitive current in A549 cells as well as in freshly isolated type II alveolar epithelial cells from different species; in all these cell types ENaC is expressed endogenously. Consequently, the present study was undertaken to determine whether ENaC is the specific target of AP301. The effect of AP301 in A549 cells and in HEK and CHO cells heterologously expressing human ENaC subunits (α, β, γ and δ) was measured in patch clamp experiments. Also the congener TIP peptide AP318 activated ENaC by increasing single channel open probability. AP301 increased current in proteolytically activated (cleaved) but not near silent (uncleaved) ENaC in a reversible manner. For maximal activity αβγ- or δβγ-ENaC co-expression was required. After deglycosylation of extracellular domains of ENaC, no increase in current was observed. Thus, our data suggest that specific interaction of AP301 with both endogenously and heterologously expressed ENaC requires precedent binding to glycosylated extracellular loop(s).