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

Abstract

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.

A rescuable folding defective Nav1.1 (SCN1A) sodium channel mutant causes GEFS+: Common mechanism in Nav1.1 related epilepsies?

“…In fact, Nedd-4 and FGF14-1A decreased the current density of Na v 1.1-R1916G but had no effect on the wild type, consistently with published data (Fotia et al, 2004;Lou et al, 2005;Rougier et al, 2005). Thus, the effects of R1916G depend on the contrasting action of rescuing and downregulating interactions.…”

“…The interaction with voltage gated Na + channel α subunits in general increases the internalization of the channel and possibly its degradation, but Nedd4-1 can interact with some Na + channel α subunits inducing just a moderate downregulation, or no downregulation at all (Fotia et al, 2004;Rougier et al, 2005). Na v 1.1 contains a conserved PY motif in the C-terminal cytoplasmic domain, downstream of R1916G (Fig.…”

The KCNQ1 potassium channel is down-regulated by ubiquitylating enzymes of the Nedd4/Nedd4-like family

“…Similarly, pcDNA3.1-hNedd4-2, pcDNA3.1-hNedd4-1, and pcDNA3.1-hWWP2-T7 were described earlier [16]. KCNQ1 PY motif, Nedd4-2CS, and Nedd4-1CS mutant constructs were generated using the QuickChange Mutagenesis Kit (Stratagene) and pIRES-CD8-hNedd4-2 plasmids (wt/CS) were generated from pIRES-CD8 and pcDNA3.1-hNedd4-2 (wt/CS).…”

The ubiquitin-proteasome system in cardiac physiology and pathology

“…Recent studies indicate that the Nedd4-like family of E3s, members of the HECT-domain group, play a major role in regulation of this channel. Nedd4-like E3s have two to four WW domains that can target proteins with specific PY motifs (PPXY), such as those found in the carboxy termini of all voltage-gated Na ϩ channels (50,64,183,184). In this regard, Nedd4 -2 downregulates Na v 1.5 by ubiquitination, leading to its internalization and decreased surface channel density (1,217).…”