Mechanism of action of novel lung edema therapeutic AP301 by activation of the epithelial sodium channel.
AP301 [Cyclo(CGQRETPEGAEAKPWYC)], a cyclic peptide comprising the human tumor necrosis factor lectin-like domain (TIP domain) sequence, is currently being developed as a treatment for lung edema and has been shown to reduce extravascular lung water and improve lung function in mouse, rat, and pig models. The current paradigm for liquid homeostasis in the adult mammalian lung is that passive apical uptake of sodium via the amiloride-sensitive epithelial Na⁺ channel (ENaC) and nonselective cyclic-nucleotide-gated cation channels creates the major driving force for reabsorption of water through the alveolar epithelium in addition to 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. ENaC is expressed endogenously in all of these cell types. Consequently, this study was undertaken to determine whether ENaC is the specific target of AP301. The effect of AP301 in A549 cells as well as in human embryonic kidney cells and Chinese hamster ovary cells heterologously expressing human ENaC subunits (α, β, γ, and δ) was measured in patch clamp experiments. The congener TIP peptide AP318 [Cyclo(4-aminobutanoic acid-GQRETPEGAEAKPWYD)] 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. αβγ- or δβγ-ENaC coexpression was required for maximal activity. No increase in current was observed after deglycosylation of extracellular domains of ENaC. Thus, our data suggest that the specific interaction of AP301 with both endogenously and heterologously expressed ENaC requires precedent binding to glycosylated extracellular loop(s).
Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria (W.S., P.S.-H., R.L.-G.); APEPTICO Forschung und Entwicklung GmbH, Vienna, Austria (S.T., B.F., H.F., H.P.); and Division of Pulmonary Medicine, Department of Pharmacology and Toxicology, Vascular Biology Center, Medical College of Georgia, Georgia Regents University, Augusta, Georgia (R.L.).
Waheed
W
Shabbir
Parastoo
P
Scherbaum-Hazemi
Susan
S
Tzotzos
Bernhard
B
Fischer
Hendrik
H
Fischer
Helmut
H
Pietschmann
Rudolf
R
Lucas
Rosa
R
Lemmens-Gruber
D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D018408:Patch-Clamp Techniques; D010456:Peptides, Cyclic / Q000494:pharmacology*; D021122:Protein Subunits / Q000378:metabolism; D011654:Pulmonary Edema / Q000188:drug therapy*
D053503:Epithelial Sodium Channels; D010456:Peptides, Cyclic; D021122:Protein Subunits; C583752:AP301 peptide