title: ENaC activity and expression is decreased in the lungs of protein kinase C-α knockout mice.
abstract: We used a PKC-α knockout model to investigate the regulation of alveolar epithelial Na(+) channels (ENaC) by PKC. Primary alveolar type II (ATII) cells were subjected to cell-attached patch clamp. In the absence of PKC-α, the open probability (Po) of ENaC was decreased by half compared with wild-type mice. The channel density (N) was also reduced in the knockout mice. Using in vivo biotinylation, membrane localization of all three ENaC subunits (α, β, and γ) was decreased in the PKC-α knockout lung, compared with the wild-type. Confocal microscopy of lung slices showed elevated levels of reactive oxygen species (ROS) in the lungs of the PKC-α knockout mice vs. the wild-type. High levels of ROS in the knockout lung can be explained by a decrease in both cytosolic and mitochondrial superoxide dismutase activity. Elevated levels of ROS in the knockout lung activates PKC-δ and leads to reduced dephosphorylation of ERK1/2 by MAP kinase phosphatase, which in turn causes increased internalization of ENaC via ubiquitination by the ubiquitin-ligase Nedd4-2. In addition, in the knockout lung, PKC-δ activates ERK, causing a decrease in ENaC density at the apical alveolar membrane. PKC-δ also phosphorylates MARCKS, leading to a decrease in ENaC Po. The effects of ROS and PKC-δ were confirmed with patch-clamp experiments on isolated ATII cells in which the ROS scavenger, Tempol, or a PKC-δ-specific inhibitor added to patches reversed the observed decrease in ENaC apical channel density and Po. These results explain the decrease in ENaC activity in PKC-α knockout lung.
authors.affiliation: Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Amity F
authors.initials: AF
authors.lastname: Eaton
authors.affiliation: Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Yue
authors.affiliation: Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia deaton@emory.edu.
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Eaton
authors.affiliation: Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Hui-Fang
authors.initials: HF
authors.lastname: Bao
mesh_terms: D000818:Animals; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D005260:Female; D015151:Immunoblotting; D008168:Lung / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D051571:Protein Kinase C-alpha / Q000502:physiology*; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D053503:Epithelial Sodium Channels; D017382:Reactive Oxygen Species; C496630:Prkca protein, mouse; D051571:Protein Kinase C-alpha
keywords: alveoli; epithelial Na+ channels; knockout mice; lung fluid balance; protein kinase C-α; single channels