The C-Terminal of NaV1.7 Is Ubiquitinated by NEDD4L

Na(V)1.7, the neuronal voltage-gated sodium channel isoform, plays an important role in the human body's ability to feel pain. Mutations within Na(V)1.7 have been linked to pain-related syndromes, such as insensitivity to pain. To date, the regulation and internalization mechanisms of the Na(V)1.7 channel are not well known at a biochemical level. In this study, we perform biochemical and biophysical analyses that establish that the HECT-type E3 ligase, NEDD4L, ubiquitinates the cytoplasmic C-terminal (CT) region of Na(V)1.7. Through in vitro ubiquitination and mass spectrometry experiments, we identify, for the first time, the lysine residues of Na(V)1.7 within the CT region that get ubiquitinated. Furthermore, binding studies with an NEDD4L E3 ligase modulator (ubiquitin variant) highlight the dynamic partnership between NEDD4L and Na(V)1.7. These investigations provide a framework for understanding how NEDD4L-dependent regulation of the channel can influence the Na(V)1.7 function.