ENaC open probability (Po) requires the association of ENaC with anionic phospholipids like PIP2. Protein kinase C is also alters ENaC activity, but the mechanism is unclear. Recently we showed that PKC can affect ENaC through phosphorylation of myristolyated‐alanine‐rich‐C‐kinase‐substrate protein (MARCKS). MARCKS acts to sequester PIP2 in close proximity to ENaC in the membrane and phosphorylation of MARCKS by protein kinase C (PKC) causes MARCKS to dissociate from the membrane. Without MARCKS in the membrane, PIP2 no longer associates with ENaC and ENaC Po decreases. To test whether PKC‐α influences ENaC activity in the lung, we examined ENaC regulation in a PKCα‐/‐ murine model. Primary cultures of alveolar type II cells were prepared from wild‐type and knockout animals, and were subjected to cell‐attached patch clamp. In PKC‐α KO, ENaC Po was significantly less (0.100 ± 0.0241, n = 18) compared to wildtype SV129 mice (0.200 ± 0.0310, n = 28, p = 0.025). The number of channels per patch (N) also decreased. Evan’s blue assays showed a two‐fold decrease in ENaC‐mediated fluid uptake in the KO mice, while wet‐dry assays also showed a decrease in ENaC activity in the KO animals. To confirm this decrease, a lung from each animal was perfused with biotin and membrane protein pulled down with streptavidin. While total ENaC protein did not change in PKCα‐/‐ mice compared with wildtype, membrane localization of all three ENaC subunits decreased. Using confocal microscopy, we observed an increase in reactive oxygen species (ROS) in the alveolar type II cells of PKCα‐/‐ mice. The increased ROS stimulated PKCδ which phosphorylatyed MARCKS in lieu of PKCα and decreased ENaC in Po in the knockout animals.Grant Funding Source: Supported by NIH