Cardiac voltage-gated sodium channel Nav1.5 is regulated by Nedd4-2 mediated ubiquitination.
Na(v)1.5, the cardiac isoform of the voltage-gated Na+ channel, is critical to heart excitability and conduction. However, the mechanisms regulating its expression at the cell membrane are poorly understood. The Na(v)1.5 C-terminus contains a PY-motif (xPPxY) that is known to act as binding site for Nedd4/Nedd4-like ubiquitin-protein ligases. Because Nedd4-2 is well expressed in the heart, we investigated its role in the ubiquitination and regulation of Na(v)1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Na(v)1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Na(v)1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2-dependent ubiquitination of Na(v)1.5 was observed. To test for a functional role of Nedd4-2, patch-clamp experiments were performed on HEK cells expressing wild-type and mutant forms of both Na(v)1.5 and Nedd4-2. Na(v)1.5 current density was decreased by 65% upon Nedd4-2 cotransfection, whereas the PY-motif mutant channels were not affected. In contrast, a catalytically inactive Nedd4-2 had no effect, indicating that ubiquitination mediates this downregulation. However, Nedd4-2 did not alter the whole-cell or the single channel biophysical properties of Na(v)1.5. Consistent with the functional findings, localization at the cell periphery of Na(v)1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Na(v)1.5, suggesting that Nedd4-2 is a specific regulator of Na(v)1.5. These results demonstrate that Na(v)1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Na(v)1.5 by decreasing the channel density at the cell surface.
van Bemmelen
Miguel X
MX
Department of Pharmacology and Toxicology, University of Lausanne, Switzerland.
Rougier
Jean-Sébastien
JS
Gavillet
Bruno
B
Apothéloz
Florine
F
Daidié
Dorothée
D
Tateyama
Michihiro
M
Rivolta
Ilaria
I
Thomas
Marc A
MA
Kass
Robert S
RS
Staub
Olivier
O
Abriel
Hugues
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002460:Cell Line / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D005786:Gene Expression Regulation; D006801:Humans; D015640:Ion Channel Gating; D017136:Ion Transport; D007668:Kidney; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000235:genetics; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism; D062554:NAV1.5 Voltage-Gated Sodium Channel; D000075702:Nedd4 Ubiquitin Protein Ligases; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000502:physiology; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000502:physiology*
D056827:Endosomal Sorting Complexes Required for Transport; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C568320:SCN5A protein, human; C568321:Scn5a protein, mouse; D015222:Sodium Channels; D025801:Ubiquitin; D012964:Sodium; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases