TNF-α Lectin-Like Domain Derived Peptide Modulates Epithelial Sodium Channel Current

Abstract

Sodium absorption by epithial sodium channel (ENaC) is main driving force of lung liquid clearance at birth and lung edema clearance in adulthood. We investigated the molecular mechanism underlying the modulation of ENaC current by TNF-α and TNF-α lectin-like domain derived (TIP) peptides. With the help of the patch-clamp technique we show that TIP peptides caused a substantial increase in amiloride sensitive sodium current through ENaC in human alveolar adenocarcinoma cells (A549), in both whole cells as well as single channel configurations. ENaCs in A549 cells are proteolytically cleaved. This model cell line mimics the ENaC as in edema conditions. We next analyze the effect of TIP peptide in heterologous expression systems. To do so, we transiently transfect hENaC in CHO cells and studied the effect of TIP peptide. Our results show that TIP peptide has direct interaction with ENaC and can modulate the amiloride sensitive sodium current through these channels. In contrast, we barely observe an effect of TIP peptide when applied to the extracellular side of the HNEC cell line RPMI2650. It is widely accepted that two different populations of ENaC are expressed in cells. First, proteolytically cleaved with high open probability called active ENaCs and the second naive with low open probability silent/near silent ENaCs. In RPMI2650 cells ENaC are near silent. To activate these near silent ENaC in RPMI cells we applied Trypsin to the extracellular side and subsequently demonstrate that TIP peptide modulates the sodium current considerably. Our results strongly support a model where modulation of ENaC with TIP peptide AP301 happens in proteolytically active channels and that assaying proteolytic cleavage of ENaC could report on the benefit of therapeutic interventions.1. Hribar M, et al. (1999) Eur J Immunol 29: 3105-3111