Background: Ubiquitination controls trafficking of the epithelial Na ϩ channel (ENaC) in the endocytic pathway.

Residues forming interfaces between the three ENaC subunits participate in conformational changes required for transition between open and closed states.

The epithelial sodium channel (ENaC) plays a critical role in blood pressure control. Defects in ENaC regulation by the E3 ubiquitin ligase Nedd4‐2 cause Liddle’s syndrome, an inherited form of hypertension. Nedd4‐2 catalyzes ubiquitination of ENaC lysines, which enhances ENaC endocytosis and degradation. To identify the lysines required for Nedd4‐2 regulation, we mutated them to arginine and tested the effect of Nedd4‐2 on ENaC cell surface expression. Mutation of all cytoplasmic lysines in ENaC reduced the ability of Nedd4‐2 to decrease ENaC surface expression (detected by biotinylation) and ENaC current. In contrast, mutation of all cytoplasmic lysines in ‐ and ENaC had no effect. By mutating ENaC lysines individually and in combination, we identified differential roles for each. K8, K10, and K26 were partially required for Nedd4‐2 regulation, whereas K6, K12, and K13 were not. Mutation of lysines in ‐ and ENaC also revealed differential roles. In ENaC, mutation of K22 and K25 to arginine reduced ENaC inhibition by Nedd4‐2. In contrast, mutation of K83 enhanced Nedd4‐2 inhibition. In ENaC, mutation of K16 and K23 produced a small decrease in Nedd4‐2 regulation, but mutation of other lysines did not. Combination of mutations in ‐, , and ENaC produced an additive reduction in Nedd4‐2 regulation, but did not abolish regulation. Thus, Nedd4‐2 regulates ENaC in part through a subset of ENaC lysines, as well as through a second mechanism that is independent of ENaC lysines. Grant Funding Source: supported by NIH HL072256 (PMS)