Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins

The voltage-gated Na+ channels (Nav) form a family composed of 10 genes. The COOH termini of Nav 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 Nav1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Nav proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Nav1.2 and Nav1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Nav1.2, Nav1.3, and Nav1.5 indicated that mouse brain Nedd4-2 binds to the Nav PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Nav1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a K d of ∼55 μM. We tested the binding properties and the ability to ubiquitinate and downregulate Nav1.5 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 Nav1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Nav1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Nav1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Nav channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane.