Molecular Mechanism of Lung Oedema Clearance by AP301: Dependence of ENaC Pore Forming Subunits

Abstract

Sodium absorption by epithial sodium channel (ENaC) is main driving force of lung liquid clearance from the alveolar space. Despite many years of intense research, the subunit composition of native ENaC in pulmonary epithelium is unknown. Different subunits (α, s , γ and δ) containing ENaC have been identified. It is not clear that α subunit containing ENaC channels are responsible for ENaC-mediated Na+ currents and subsequent fluid flux. AP301, a cyclic peptide, comprising the human tumour necrosis factor lectin like domain (TIP domain) sequence, has been shown to reduce extravascular lung water and improve lung function in a mouse, rat and pig model. Consequently, the present study was undertaken to determine: First, whether ENaC is the specific target of AP301. Second, what is the subunit combination of ENaC for maximum AP301-induced activation. Furthermore, this study explores the potential of AP301 to discriminate between different ENaC channel populations. The effect of AP301 in A549 cells and in HEK and CHO cells heterologously expressing human ENaC subunits (α, s, γ and δ) was measured in patch clamp experiments. For maximal AP301 activity αβγ- or δβγ-ENaC co-expression was required. Conclusively, AP301 increased current in proteolytically activated (cleaved) but not near silent (uncleaved) ENaC in a reversible manner. 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) and pore-forming α or δ delta subunits.