Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca_v2.1 (P/Q-Type) Calcium Channels.

Voltage-gated Ca_V2.1 channels comprise a pore-forming α_1A subunit with auxiliary α₂δ and β subunits. Ca_V2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the Ca_V2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of Ca_V2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human Ca_V2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel Ca_V2.1-binding partner. In neurons, RNF138 and Ca_V2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of Ca_V2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the Ca_V2.1 protein level and enhances Ca_V2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of Ca_V2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on Ca_V2.1 WT functional expression, which can be attributed to defective membrane trafficking of Ca_V2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of Ca_V2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human Ca_V2.1 subunits.SIGNIFICANCE Loss-of-function mutations in the human Ca_V2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by paroxysmal attacks of ataxia and nystagmus. EA2-causing mutants may exert dominant-negative effects on the Ca_V2.1 wild-type subunit via aberrant proteasomal degradation. The molecular nature of the Ca_V2.1 ubiquitin-proteasome degradation pathway is currently unknown. The present study reports the first identification of an E3 ubiquitin ligase for Ca_V2.1, RNF138. Ca_V2.1 protein stability is dynamically regulated by RNF138 and auxiliary α₂δ and β subunits. We provide a proof of concept that protecting the human Ca_V2.1 subunit from excessive proteasomal degradation with specific interruption of endogenous RNF138 function may partially contribute to the future development of a novel therapeutic strategy for EA2 patients.