The role of individual Nedd4-2 (KIAA0439) WW domains in binding and regulating epithelial sodium channels

The amiloride-sensitive epithelial sodium channel (ENaC) is essential for fluid and electrolyte homeostasis. ENaC consists of α, β, and γ subunits, each of which contains a PPxY motif that interacts with the WW domains of the ubiquitin-protein ligases Nedd4 and Nedd4-2. Disruption of this interaction, as in Liddle's syndrome in which mutations delete or alter the PPxY motif of either the β or the γ subunits, results in increased ENaC activity. We report here that Nedd4-2 has two major isoforms that show tissue-specific expression; however, both isoforms can inhibit ENaC in Xenopus oocytes. Because there are four WW domains in Nedd4-2, we analyzed binding kinetics and affinity between individual WW domains and ENaC subunits. Using whole cell patch-clamp techniques, we studied the role of individual WW domains in the regulation of ENaC in mammalian cells. We report here that unlike Nedd4, only two of the Nedd4-2 WW domains, WW3 and WW4, are required for both the binding to ENaC subunits and the regulation of Na⁺ feedback control of ENaC. Although both WW3 and WW4 individually can interact with all three ENaC subunits in vitro, both domains together are essential for in vivo function of Nedd4-2 in ENaC regulation. These data suggest that Nedd4-2 WW3 and WW4 interact with distinct, noninterchangeable sites in ENaC and that to prevent Na⁺ feedback control of ENaC it is necessary to occlude both sites.Andrew B. Fotia, Anuwat Dinudom, Keith E. Shearwin, Jan-Peter Koch, Christoph Korbmacher, David I. Cook, and Sharad Kuma