The epithelial sodium channel (ENaC) is responsible for Na+ and fluid absorption across colon, kidney, and airway epithelia. We have previously identified SPLUNC1 as an autocrine inhibitor of ENaC. We have now located the ENaC inhibitory domain of SPLUNC1 to SPLUNC1's N terminus, and a peptide corresponding to this domain, G22-A39, inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold). However, G22-A39 had no effect on the structurally related acid-sensing ion channels, indicating specificity for ENaC. G22-A39 preferentially bound to the β-ENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Addition of G22-A39 to CF human bronchial epithelial cultures (HBECs) resulted in an increase in airway surface liquid height from 4.2±0.6 to 7.9±0.6 μm, comparable to heights seen in normal HBECs, even in the presence of neutrophil elastase. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, which suggests that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the G22-A39 peptide suggests that this peptide may be suitable for treating CF lung disease.
Nedd4‐2 has been proposed to play a critical role in regulating epithelial Na+ channel (ENaC) activity through ubiquitination mediated degradation, and blood pressure (BP) regulation. To further understand the in vivo role of Nedd4‐2, we generated mice with global and kidney collecting duct (CD)‐specific inactivation of Nedd4‐2. The global knockout mice had higher BP on a normal diet, and a further increase in BP when on a high‐salt (HS) diet. The hypertension was mediated through ENaC, as both expression level and activity were increased in Nedd4‐2 null mice, and hypertension was substantially reduced in the presence of amiloride. However, unlike mice with global inactivation of Nedd4‐2, mice with CD‐specific inactivation did not have spontaneously elevated BP. When the mice were placed on HS diet, mice with CD‐specific inactivation of Nedd4‐2 had increased BP, although it was significantly lower than that of mice with global inactivation of Nedd4‐2. Overall, our results demonstrate that in vivo Nedd4‐2 is critical regulator of ENaC activity and BP, and suggest that kidney (CD) expression of Nedd4‐2 is necessary but not sufficient for the general increase in BP. Supported by NIH DK52617 to BY and JBS.
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