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

Abstract

Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (NaV) 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 NaV 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 NaV channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary NaVβ subunits. Collectively, these results indicate that RNF121 participates in the quality control of NaV channels during their synthesis and subsequent transport to the membrane.

Keywords: escape; touch response; ubiquitin; voltage-gated sodium channel; zebrafish.

Fig. 1.

mi500 is a new allele of the touch-unresponsive mutant alligator. Tactile stimuli delivered to the tail of WT (A), alligator allele mi500 (B), or alligator allele tm342 (C). Of note, the images are superimposed video stills of the first 100 ms following a stimulus. (D) Histogram representing the percentage of larvae that were touch-unresponsive or exhibited a normal touch response (Materials and Methods) from incrosses and complementation crosses of alligator alleles mi500 and tm342.

Fig. 2.

Na_V channel activity and membrane localization are diminished in mutants. (A) Schematic of the sensorimotor circuit in zebrafish. (B) Whole-cell current-clamp recordings showed that action potentials are elicited in WT RBs, motor neurons, and fast-twitch skeletal muscle. Current injection failed to initiate an action potential in mutant RB cells and most motor neurons and fast-twitch skeletal muscle. (C) Whole-cell voltage-clamp recordings made from indicated cells showing that voltage-dependent inward currents are missing in mutant RBs and motor neurons and significantly diminished in skeletal muscle (Table S3). (D–M) Immunohistochemical labeling of the following proteins in WT and mutant RBs: pan-Na_V, the neuronal RNA-binding protein HuC, acetylated α-tubulin common to axons, ER and cis-Golgi proteins containing KDEL tetrapeptides at the C terminus, and neurofascin common to the AIS. Arrows indicate Na_V and neurofascin found in the proximal tubulin-positive processes. Note that Na_V proteins in the proximal tubulin-positive processes is observed in WT RB cells but not in mutants, whereas neurofascin accumulates at the AIS in WT and mutant RBs.

Fig. 3.

RNF121 facilitates ubiquitination and membrane localization of Nav1.6 in HEK293T cells. Protein extracts from cells transfected with RNF121_WT or RNF121_V228A, Na_V1.6-FLAG, and/or Na_Vβ₁-V5-His expression vectors. Proteasome activity was inhibited by MG132. Whole-cell extracts probed with anti-RNF121 (A), anti-FLAG (B), anti-His (C), or anti-GAPDH (D). Assessing the ubiquitination of Na_V1.6 from whole-cell extracts was achieved by immunoprecipitation with anti-FLAG, followed by probing with anti-FLAG (E) or anti-ubiquitin (F), which represents total and ubiquitinated Na_V1.6-FLAG, respectively. Membrane localization of Na_V1.6-FLAG and Na_Vβ₁-V5-His assayed through incubation of cells in biotin, followed by purification of biotinylated proteins and probing with anti-FLAG (G) or anti-His (H), respectively. Note that ubiquitination of Na_V1.6-FLAG was enhanced when coexpressed with RNF121_WT, and that membrane localization of Na_V1.6-FLAG increased when RNF121_WT and Na_Vβ₁-V5-His were coexpressed.

Fig. 4.

Overexpression of Na_Vβ₁ partially compensates for the loss of RNF121. (A–L) Overexpression of Na_Vβ₁ restores Na_V localization at the proximal tubulin-positive processes in some RBs. Immunohistochemical labeling of RBs for the following proteins in mutants injected with control RNA (luciferase; A–F) or RNA encoding Na_Vβ₁ (G–L): pan-Na_V, HuC enriched in RB cell bodies, and acetylated α-tubulin common to axons. Arrows highlight Na_V localization in the proximal tubulin-positive processes. (M) Control, Na_V1.6, or Na_Vβ₁ RNA was injected into WT embryos with or without RNF121 antisense MO (1 or 5 ng). Histograms represents the percentage of larvae displaying a touch response. Note that overexpression of Na_V1.6 diminished touch responsiveness, whereas overexpression of Na_Vβ₁ partially restored the touch responsiveness in morpholino injected larvae. Touch responses were classified as described in Materials and Methods.

Fig. 5.

A model of RNF121-mediated quality control of Nav channels. (A) A WT neuron wherein RNF121 mediates ubiquitination of misfolded Na_V channels marking them for proteasome-mediated degradation. Properly folded Na_V channels (green) associate with Na_Vβ subunits (magenta) in the Golgi apparatus and are transported to the AIS. Of note, some Na_Vβ subunits are transported to the membrane independent of Na_V channels. (B) An rnf121 mutant neuron wherein misfolded Na_V channels (red) accumulate in the ER and cis-Golgi compartments, which, over time, depletes Na_Vβ subunits, preventing them from forming complexes with properly folded Na_V channels, causing an impairment of Na_V transport.