RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels

Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (Na_V) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which Na_V channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of Na_V channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary Na_Vβ subunits. Collectively, these results indicate that RNF121 participates in the quality control of Na_V channels during their synthesis and subsequent transport to the membrane.Voltage-gated sodium channels (Na_V) are large (∼230 kDa) multipass transmembrane proteins (1). The Na_V channel family is comprised of nine members (Na_V1.1–Na_V1.9), whose activity typically underlies the rising phase of action potentials in excitable cells. In excitable cells, Na_V channels form complexes with auxiliary β subunits (Na_Vβ_1–4) in the Golgi apparatus (2), a process that enhances the kinetics and membrane localization of Na_V channels (3, 4). In addition to these roles, several Na_Vβ subunits also function as cell adhesion molecules independent of Na_V channels (5). At the axon initial segment (AIS) and nodes of Ranvier of neurons, Na_V channels form clusters that facilitate the generation and propagation of action potentials. Although the molecular basis of Na_V clustering at these sites has been extensively studied (6), the transport of Na_V channels to these sites has been less explored. For instance, to date, only the annexin II light chain (p11) has been shown to associate with and facilitate the transport of Na_V1.8 to the plasma membrane (7). Furthermore, subsequent efforts revealed that p11 acts only on Na_V1.8 (8). Thus, the transport of other Na_V channels remains unclear.In zebrafish, several studies have explored the contribution of Na_V channels and their auxiliary Na_Vβ subunits through the use of forward and reverse genetics. In brief, impairments in Na_V1.1, Na_V1.6a, and Na_Vβ_1b have been shown to diminish touch-evoked escape responses and Na_V channel activity in Rohon–Beard (RB) sensory neurons (9–11). In addition, two other mutants identified in forward genetic screens have been shown to affect Na_V channel activity indirectly. The first, pigu, arises from a mutation in a GPI-transamidase necessary for the proper localization of Na_V channels (12). Although the genetic locus of the second mutation, macho (13, 14), has yet to be identified, rough mapping indicates that it lies within a region lacking both Na_V channels and auxiliary Na_Vβ subunits. Collectively, these results indicate that the characterization of touch-unresponsive zebrafish mutants is an efficient strategy to gain insight into the trafficking and function of Na_V channels.In this study, we identified a touch-unresponsive zebrafish mutant (mi500), which was found to be a new allele of the molecularly unidentified motor mutant alligator (13). Electrophysiological analysis revealed that Na_V channel activity was severely diminished throughout the sensorimotor circuit in mutants. Further characterization uncovered that Na_V channels were not localized at the AIS in mutant RBs, but instead seem to be accumulated within the endoplasmic reticulum (ER) and cis-Golgi compartments. Meiotic mapping and sequence analysis showed that the alligator locus encodes really interesting new gene (RING) finger protein 121 (RNF121), an ER- and cis-Golgi–resident E3-ubiquitin ligase that mediates the ubiquitination of Na_V1.6. We found that RNF121 promotes the degradation and membrane transport of Na_V1.6. Furthermore, overexpression of Na_V1.6 worsened the touch response in rnf121-knockdown larvae, suggesting that an excess amount of Na_V exerts proteotoxicity. These findings suggest that the proper transport of Na_V channels is attributable to RNF121-mediated quality control of Na_V channels within the ER and Golgi apparatus.