Targeted degradation of ENaC in response to PKC activation of the ERK1/2 cascade

Renal A6 epithelial cells were used to determine the mechanism by which protein kinase C ( PKC) decreases epithelial Na (+) channel ( ENaC) activity. Activation of PKC reduced relative Na (+) reabsorption to < 20% within 60 min. This decrease was sustained over the next 24 - 48 h. In response to PKC signaling, alpha-, beta-, and gamma- ENaC levels were 0.97, 0.36, and 0.39, respectively, after 24 h, with the levels of the latter two subunits being significantly decreased. The PKC- mediated decreases in beta- and gamma- ENaC were significantly reversed by simultaneous addition of the mitogen- activated protein kinase ( MAPK)/ extracellular signal- regulated kinase- 1/ 2 inhibitors U- 0126 and PD- 98059. These inhibitors, in addition, protected Na (+) reabsorption from PKC, demonstrating that the MAPK1/ 2 cascade, in some instances, plays a central role in downregulation of ENaC activity. The effects of PKC on beta- and gamma- ENaC levels were additive with those of inhibitors of transcription ( actinomycin D) and translation ( emetine and cycloheximide), suggesting that PKC promotes subunit degradation and does not affect subunit synthesis. The bulk of whole cell gamma- ENaC was degraded within 1 h after treatment with inhibitors of synthesis; however, a significant pool was " protected" from inhibitors for up to 12 h. PKC affected this protected pool of gamma- ENaC. Moreover, proteosome inhibitors ( MG- 132 and lactacystin) reversed PKC effects on this protected pool of gamma- ENaC. Thus PKC signaling via MAPK1/ 2 cascade activation in A6 cells promotes degradation of gamma- ENaC.