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1 Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Vaud, Switzerland
2 The Hospital for Sick Children, Toronto, Ontario, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
3 Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Vaud, Switzerland; Service of Cardiology, University of Lausanne, CHUV, Lausanne, Vaud, Switzerland
* To whom correspondence should be addressed. E-mail: hugues.abriel{at}unil.ch.
The voltage-gated sodium channels (Nav) form a family composed of 10 genes. The C-termini of Navs contain a cluster of amino acids that are nearly identical among seven of the ten members. This C-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 the cardiac Nav1.5 sodium channel is regulated by Nedd4-2. In this study we further investigated the molecular determinants of regulation of Nav proteins. When expressed in HEK293 cells and studied by whole-cell voltage-clamp, the neuronal Nav1.2 and 1.3 were also down-regulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY-motif of Nav1.2, 1.3 and 1.5 indicated that mouse brain Nedd4-2 binds to the Nav PY-motif. Using intrinsic tryptophan fluorescence of WW domains, we found that Nav1.5 PY-motif binds preferentially to the fourth WW domain of Nedd4-2 with a Kd of about 55 µM. We tested the binding properties and the ability to ubiquitinate and down-regulate Nav1.5 of three Nedd4-like E3s, i.e. 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 down-regulated Nav1.5. Interestingly, co-expression 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 HEK293 cells. This study illustrates 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.
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