Using the yeast two-hybrid system, we identified a number of proteins that interacted with the carboxyl termini of murine epithelial sodium channel (ENaC) subunits. Initial screens indicated an interaction between the carboxyl terminus of β-ENaC and IκB kinase-β (IKKβ), the kinase that phosphorylates IκB and results in nuclear targeting of NF-κB. A true two-hybrid reaction employing full-length IKKβ and the carboxyl termini of all three subunits confirmed a strong interaction with β-ENaC, a weak interaction with γ-ENaC, and no interaction with α-ENaC. Co-immunoprecipitation studies for IKKβ were performed in a murine cortical collecting duct cell line that endogenously expresses ENaC. Immunoprecipitation with β-ENaC, but not γ-ENaC, resulted in co-immunoprecipitation of IKKβ. To examine the direct effects of IKKβ on ENaC activity, co-expression studies were performed using the two-electrode voltage clamp technique in Xenopus oocytes. Oocytes were injected with cRNAs for αβγ-ENaC with or without cRNA for IKKβ. Co-injection of IKKβ significantly increased the amiloride-sensitive current above controls. Using cell surface ENaC labeling, we determined that an increase of ENaC in the plasma membrane accounted for the increase in current. The injection of kinase-dead IKKβ (K44A) in ENaC-expressing oocytes resulted in a significant decrease in current. Treatment of mpkCCDc14 cells with aldosterone increased whole cell amounts of IKKβ. Because this result suggested that aldosterone might activate NF-κB, mpkCCDc14 cells were transiently transfected with a luciferase reporter gene responsive to NF-κβ activation. Both aldosterone and tumor necrosis factor-α (TNFα) stimulation caused a similar and significant increase in luciferase activity as compared with controls. We conclude that IKKβ interacts with ENaC by up-regulating ENaC at the plasma membrane and that the presence of IKKβ is at very least permissive to ENaC function. These studies also suggest a previously unexpected interaction between the NF-κβ transcription pathway and steroid regulatory pathways in epithelial cells.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology