👤 Olivier Staub

The voltage-gated Na(+) channels (Na(v)) form a family composed of 10 genes. The COOH termini of Na(v) contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na(v)1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na(v) proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na(v)1.2 and Na(v)1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Na(v)1.2, Na(v)1.3, and Na(v)1.5 indicated that mouse brain Nedd4-2 binds to the Na(v) PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Na(v)1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a K(d) of approximately 55 muM. We tested the binding properties and the ability to ubiquitinate and downregulate Na(v)1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Na(v)1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Na(v)1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Na(v)1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Na(v) channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane. Show less

Ubiquitylation has emerged as an important mechanism for controlling surface expression of membrane proteins. This post-translational modification involves the sequential action of several enzymes including a ubiquitin-activating enzyme E1, a ubiquitin-conjugating enzyme E2 and a ubiquitin-protein ligase E3. E3s are responsible for substrate recognition. Here we describe the role of the Nedd4/Nedd4-like family of ubiquitin-protein ligases in the regulation of proteins involved in epithelial transport. The Nedd4/Nedd4-like proteins are composed of a N-terminal C2 domain, several WW domains and a catalytic HECT domain. The epithelial Na(+) channel ENaC is the best studied example of a Nedd4/Nedd4-like substrate. Its cell surface expression is regulated by the ubiquitin-protein ligase Nedd4-2 via direct PY motif/WW domain interaction. This regulatory mechanism is impaired in Liddle's disease, an inherited form of human hypertension, and is controlled by Sgk1, an aldosterone-inducible kinase which phosphorylates Nedd4-2. The regulation of ENaC by Nedd4-2 is a paradigm for the control of epithelial membrane proteins, as evidenced by the regulation of the ClC-5 chloride channel by the ubiquitin-protein ligase WWP2 or the tight junction protein Occludin by Itch. Show less