H202 regulates lung ENaC via ubiquitin‐like protein Nedd8 in alveolar type 1 and type 2 cells.

Abstract

Proteolytic degradation of epithelial sodium channels (ENaC) assists in regulating net salt and water balance in lung epithelia. Classical ubiquitination of ENaC involves Nedd 4–2; however, less attention has been given to the role of ubiquitin‐like protein Nedd8. We tested the hypothesis that Nedd8 and its associated E2 carrier protein (Ubc12) and E3‐SCF ligase, plays a key role in regulating the expression of ENaC in alveolar type 1 (T1) and type 2 (T2) cells. Using single channel patch clamp analysis in the cell attached configuration, we exposed alveolar T2 cells to 250 μM H2O2 and observed that channel open probability increased by 35 min exposure (p <0.05) with a significant increase in α‐ENaC protein at 60 and 120 minutes (p<0.05). Patch clamp analysis of T1 and T2 cells treated with MLN 4924, a specific Nedd8 activating enzyme inhibitor, showed a significant increase in ENaC activity. Cycloheximide chase assays performed +/− MLN 4924 showed a significant increase in α‐ENaC in MLN4924 treated cells compared to cycloheximide alone at 60 and 120 minutes (p<0.05). MLN 4924 treated mice showed accelerated rates of alveolar fluid clearance, confirming our electrophysiological measurements and supporting a significant role for Nedd8 in the ubiquitination of α‐ENaC. Research was supported by R00HL09226 awarded to MNH; Children's Center for Development in Lung Biology Grant awarded to MNH