👤 Hugues Abriel

Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) proteins are ubiquitin ligases, which attach ubiquitin moieties to their target proteins, a post-translational modification that is most commonly associated with protein degradation. Nedd4 ubiquitin ligases have been shown to down-regulate both potassium and sodium channels. In this study, we investigated whether Nedd4 ubiquitin ligases also regulate Ca(v) calcium channels. We expressed three Nedd4 family members, Nedd4-1, Nedd4-2, and WWP2, together with Ca(v)1.2 channels in tsA-201 cells. We found that Nedd4-1 dramatically decreased Ca(v) whole-cell currents, whereas Nedd4-2 and WWP2 failed to regulate the current. Surface biotinylation assays revealed that Nedd4-1 decreased the number of channels inserted at the plasma membrane. Western blots also showed a concomitant decrease in the total expression of the channels. Surprisingly, however, neither the Ca(v) pore-forming α1 subunit nor the associated Ca(v)β and Ca(v)α(2)δ subunits were ubiquitylated by Nedd4-1. The proteasome inhibitor MG132 prevented the degradation of Ca(v) channels, whereas monodansylcadaverine and chloroquine partially antagonized the Nedd4-1-induced regulation of Ca(v) currents. Remarkably, the effect of Nedd4-1 was fully prevented by brefeldin A. These data suggest that Nedd4-1 promotes the sorting of newly synthesized Ca(v) channels for degradation by both the proteasome and the lysosome. Most importantly, Nedd4-1-induced regulation required the co-expression of Ca(v)β subunits, known to antagonize the retention of the channels in the endoplasmic reticulum. Altogether, our results suggest that Nedd4-1 interferes with the chaperon role of Ca(v)β at the endoplasmic reticulum/Golgi level to prevent the delivery of Ca(v) channels at the plasma membrane. Show less

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