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

Abstract

Following their synthesis in the endoplasmic reticulum (ER), voltagegated 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 V β 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.oltage-gated sodium channels (Na V ) are large (∼230 kDa) multipass transmembrane proteins (1). The Na V channel family is comprised of nine members (Na V 1.1-Na V 1.9), 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 V β 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 V β 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 V 1.8 to the plasma membrane (7). Furthermore, subsequent efforts revealed that p11 acts only on Na V 1.8 (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 V β subunits through the use of forward and reverse genetics. In brief, impairments in Na V 1.1, Na V 1.6a, and Na V β 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 GPItransamidase 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 with...

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