Protein modification by ubiquitination regulates protein abundance, function and localisation. Specificity of ubiquitination is largely determined by ubiquitinprotein ligases (E3s). The Nedd4–family proteins are a group of E3s containing a conserved domain structure of a C2 domain, multiple WW domains and a carboxyl terminal HECT domain, which is responsible for E3 activity. The prototypical member of this family, Nedd4, is known to down-regulate the epithelial Na+ channel (ENaC) by ubiquitination. This process requires interactions between ENaC and specific WW domains of Nedd4. Mutation or deletion of WW domain binding sites in ENaC leads to Liddle's syndrome, an autosomal dominant form of hypertension. At the beginning of this study there was evidence to suggest that Nedd4–2, a Nedd4–family protein closely related to Nedd4, could also regulate ENaC. The focus of this study was to characterise the ability of Nedd4–2 to regulate ENaC and other potential substrates. Two major splice variants of Nedd4–2 were identified, which were both found to down–regulate ENaC in Xenopus oocytes. In vitro binding studies and whole cell functional analysis showed that interactions between ENaC and Nedd4– 2 occur via two of the four Nedd4–2 WW domains. The E3 activity of Nedd4–2 was further examined, revealing that it can use the same ubiquitin–conjugating enzymes as Nedd4 and exhibits strongest activity in the presence of UbcH5b. An in vitro ubiquitination assay and whole cell functional analysis provided evidence that Nedd4–2 down-regulates ENaC via ubiquitination. The possibility that Nedd4 and Nedd4–2 could down-regulate a number of voltage–gated Na+ channels (Navs) by a similar mechanism to regulation of ENaC was investigated. Not only were Nedd4 and Nedd4–2 found to interact with seven Navs, but these channels and ENaC have conserved WW domain binding specificity. Ubiquitination studies indicated that these channels can be ubiquitinated by Nedd4 and Nedd4–2. Co–expression of Nedd4 or Nedd4–2 with neuronal Navs in Xenopus oocytes reduced channel activity to varying degrees. These data indicate that Nedd4 and Nedd4–2 are likely to be key regulators of neuronal Nav channels in vivo. / Thesis (Ph.D.)--Department of Medicine, 2004.