Cleavage of endogenous γENaC and elevated abundance of αENaC are associated with increased Na+ transport in response to apical fluid volume expansion in human H441 airway epithelial cells

Using human H441 airway epithelial cells cultured at air–liquid interface (ALI), we have uniquely correlated the functional response to apical fluid volume expansion with the abundance and cleavage of endogenous α- and γENaC proteins in the apical membrane. Monolayers cultured at ALI rapidly elevated I sc when inserted into fluid-filled Ussing chambers. The increase in I sc was not significantly augmented by the apical addition of trypsin, and elevation was abolished by the protease inhibitor aprotinin and an inhibitor of the proprotein convertase, furin. These treatments also increased the IC 50 amiloride indicating that the effect was via inhibition of highly Na + -selective ENaC channels. Apical fluid, 5–500 μl for 1 h in culture, increased the spontaneous starting I sc in a dose-dependent manner, whilst maximal fluid-induced I sc in the Ussing chamber was unchanged. Apical fluid expansion increased the abundance of 63–65-kDa αENaC proteins in the apical membrane. However, this could not be attributed to increased cleavage as protease inhibitors had no effect on the ratio of cleaved to non-cleaved (90 kDa) αENaC proteins. Instead, fluid expansion increased αENaC abundance in the membrane. In contrast, function correlated well with γENaC cleavage at known sites by furin and extracellular proteases. Interestingly, cleavage of γENaC was associated with increased retrieval from the membrane via the proteosomal pathway. Thus, the response to apical fluid volume expansion in H441 airway epithelial cells involves cleavage of γENaC, and changes in α- and γENaC protein abundance at the apical membrane.