APPS November 2002 Meeting Abstract 239

The epithelial Na⁺ channel (ENaC) is expressed in the apical membrane of many epithelia and plays a critical role in Na⁺ homeostasis and the maintenance of plasma Na⁺ concentration, blood volume and blood pressure. Its activity is controlled by intracellular Na⁺ concentration in a process called Na⁺ feedback regulation¹. We have used the whole-cell patch-clamp technique to investigate the mechanisms that underlie Na⁺ feedback regulation of ENaC in isolated mouse mandibular duct cells and have found that this mechanism is mediated via a G_o-dependent pathway¹. The pathway involves an ubiquitin protein-ligase that interacts with proline-rich motifs at the C-termini of the subunits of ENaC, which in turn induces ubiquitination and internalization of ENaC². Interaction between ENaC and the ubiquitin protein-ligase is essential for maintaining normal ENaC function. Mutations that delete of the PY motifs in the ľ- or γ-subunits of ENaC, as seen in Liddle's syndrome, increase ENaC activity and induce hypertension³. We have identified the ubiquitin protein-ligases Nedd4 and Nedd4-2 as possible candidate mediators of Na⁺ feedback^2,4. mNedd4 has three WW-domains and all three of these WW-domains are involved in mediating Na⁺ feedback regulation⁵. Interestingly, only WW2 and WW3 of mNedd4 interact with ENaC suggesting that WW1 of mNedd4 may interact with another regulatory protein outside the channel. In the case of mNedd4-2, only WW3 and WW4, bind to ENaC in vitro, whereas WW1 and WW2 do not interact with ENaC. Moreover, patch-clamp experiments indicated that only action of WW3 and WW4 of mNedd4-2 mediate Na⁺ feedback regulation.