Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.
Voltage-gated CaV2.1 channels comprise a pore-forming α1Asubunit with auxiliary α2δ and β subunits. CaV2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the CaV2.1 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 CaV2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human CaV2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel CaV2.1-binding partner. In neurons, RNF138 and CaV2.1 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 CaV2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the CaV2.1 protein level and enhances CaV2.1 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 CaV2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on CaV2.1 WT functional expression, which can be attributed to defective membrane trafficking of CaV2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of CaV2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human CaV2.1 subunits.SIGNIFICANCE STATEMENTLoss-of-function mutations in the human CaV2.1 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 CaV2.1 wild-type subunit via aberrant proteasomal degradation. The molecular nature of the CaV2.1 ubiquitin-proteasome degradation pathway is currently unknown. The present study reports the first identification of an E3 ubiquitin ligase for CaV2.1, RNF138. CaV2.1 protein stability is dynamically regulated by RNF138 and auxiliary α2δ and β subunits. We provide a proof of concept that protecting the human CaV2.1 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.
Department of Physiology and.
Ssu-Ju
SJ
Fu
Institute of Anatomy and Cell Biology, School of Medicine and.|Brain Research Center, National Yang-Ming University, Taipei, 12212 Taiwan.
Chung-Jiuan
CJ
Jeng
Department of Psychiatry and.
Chia-Hao
0000-0001-9721-1047
CH
Ma
Department of Physiology and.
Yi-Jheng
YJ
Peng
Department of Physiology and.
Chi-Ming
CM
Lee
Department of Physiology and.
Ya-Ching
YC
Fang
Department of Physiology and.
Yi-Ching
YC
Lee
Department of Neurology, National Taiwan University Hospital, Taipei, 10002 Taiwan.
Sung-Chun
SC
Tang
Department of Physiology and.
Meng-Chun
MC
Hu
Department of Physiology and tang@ntu.edu.tw.|Graduate Institute of Brain and Mind Sciences, College of Medicine, National Taiwan University, Taipei, 10051 Taiwan.
Chih-Yung
CY
Tang
D000818:Animals; D001259:Ataxia / Q000235:genetics; D001921:Brain / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D003513:Cycloheximide / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009447:Neuroblastoma / Q000473:pathology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D009759:Nystagmus, Pathologic / Q000235:genetics; D009865:Oocytes; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D059748:Proteolysis / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects / Q000235:genetics; D014981:Xenopus
C500545:CACNA1B protein, human; D020864:Calcium Channels, N-Type; D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide; C577594:RNF138 protein, human; D044767:Ubiquitin-Protein Ligases
E3 ubiquitin ligase; calcium channel; episodic ataxia; membrane trafficking; protein degradation; ubiquitin–proteasome system