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H 2 0 2 regulates lung ENaC via ubiquitin‐like protein Nedd8 in alveolar type 1 and type 2 cells.
Author(s) -
Johnson Nicholle M.,
Downs Charles A.,
Kreiner Lisa H.,
Helms My N.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.725.3
Subject(s) - epithelial sodium channel , cycloheximide , nedd8 , ubiquitin , chemistry , microbiology and biotechnology , lung , patch clamp , ubiquitin ligase , biology , biochemistry , medicine , sodium , protein biosynthesis , receptor , organic chemistry , gene
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 H 2 O 2 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