Question: 

Epithelial sodium transport mediated by the epithelial sodium channel (ENaC) critically depends on regulated expression of the channel at the cell surface. Phosphorylation of rat b- and gENaC at bT613 and gT623 is thought to facilitate the interaction with the ubiquitin ligase Nedd4-2 thereby promoting channel internalization^1,2.3. However, the role of bENaC phosphorylation in regulating channel surface expression is still unclear.

Methodology: 

Rat wild-type (wt) abg- or mutant abT613AgT623A-ENaC was expressed in Xenopus laevisoocytes alone or together with Nedd4-2. Channel surface expression was detected with biotinylation and Western blot analysis. A phospho-specific antibody against phospho-bT613 was used to demonstrate phosphorylation of bT613. Protein interaction of wt and mutant ENaC with Nedd4-2 was analyzed by co-immunoprecipitation. ENaC activity was determined by measuring amiloride-sensitive whole-cell currents (DI_ami) with two-electrode voltage-clamp.

Results: 

As previously shown³, DI_ami was higher in oocytes expressing mutant ENaC than in oocytes expressing wt ENaC. Western blot analysis of biotinylated cell surface protein revealed two specific bands for both wt and mutant b-ENaC at about 110 kDa and 96 kDa. The 110 kDa band was the predominant band in particular for mutant b-ENaC. Intracellular b-ENaC mainly migrated at 96 kDa. Pretreatment of oocytes with kifunensine, an ER mannosidase I inhibitor, shifted the size of cell surface b-ENaC from 110 kDa to 96 kDa. However, pretreatment with kifunensine did not affect average DI_ami. These results indicate that glycosylated b-ENaC predominates at the cell surface but that glycosylation is not required for channel function. In oocytes expressing wt ENaC the phosphospecific antibody demonstrated phosphorylation of bT613 at 110 kDa. Importantly, no phosphorylation signal could be detected in mutant ENaC. Inhibition of DI_ami by co-expression of Nedd4-2 was more pronounced in wt ENaC expressing oocytes than in mutant ENaC expressing oocytes and was associated with a decrease of the110 kDa band of b-ENaC at the cell surface. Interestingly, Nedd4-2 co-immunoprecipitated with both wt and mutant ENaC.

Conclusion: 

Using a phosphospecific antibody we provide direct evidence for phosphorylation of b-ENaC at residue bT613. Mutating this site prevents its phosphorylation and stabilizes the mature 100 kDa glycosylated form of b-ENaC in the plasma membrane probably by reducing Nedd4-2 mediated channel retrieval. However, the mutation does not abolish Nedd4-2 interaction with the channel.

1. Shi et al., J Biol Chem 2002, 277:13539

2. Michlig et al., J Biol Chem 2005, 280:38264

3. Yang et al., J Biol Chem 2006, 281:9859