Ubiquitin ligase RNF138 promotes episodic ataxia type 2-associated aberrant degradation of human Cav2.1 (P/Q-type) calcium channels
Journal Volume
37
Journal Issue
9
Pages
2485-2503
Date Issued
2017
Author(s)
Fu S.-J.
Jeng C.-J.
Ma C.-H.
Peng Y.-J.
Lee C.-M.
Fang Y.-C.
Lee Y.-C.
Abstract
Voltage-gated CaV2.1 channels comprise a pore-formingα1A subunit with auxiliaryα2δ 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 CaV2.1WTsubunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on CaV2.1WTfunctional 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. ? 2017 the authors.
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Other Subjects
benzylsulfonyl fluoride; DNA binding protein; glutathione transferase alpha; lentivirus vector; lipofectamine; muscle RING finger 1 protein; ubiquitin protein ligase; ubiquitin protein ligase E3; voltage gated calcium channel; CACNA1B protein, human; calcium channel N type; cycloheximide; protein synthesis inhibitor; RNF138 protein, human; ubiquitin protein ligase; adult; agar gel electrophoresis; animal experiment; animal model; animal tissue; Article; biotinylation; brain cell culture; brain electrophysiology; cell fractionation; cerebellar ataxia; confocal laser scanning microscopy; controlled study; DNA transfection; female; fluorescence microscopy; forebrain; HEK293 cell line; human; immunoblotting; immunofluorescence; immunohistochemistry; immunoprecipitation; loss of function mutation; nonhuman; nystagmus; priority journal; protein degradation; protein expression; protein stability; rat; reverse transcription polymerase chain reaction; RNA interference; sequence analysis; signal transduction; transient transfection; Xenopus laevis; animal; ataxia; brain; brain cortex; cell culture; cell line; cytology; drug effects; genetics; membrane potential; metabolism; mutation; nerve cell; neuroblastoma; oocyte; pathology; ubiquitination; ultrastructure; Xenopus; Animals; Ataxia; Brain; Calcium Channels, N-Type; Cell Line; Cells, Cultured; Cerebral Cortex; Cycloheximide; HEK293 Cells; Humans; Membrane Potentials; Mutation; Neuroblastoma; Neurons; Nystagmus, Pathologic; Oocytes; Protein Synthesis Inhibitors; Proteolysis; Subcellular Fractions; Ubiquitin-Protein Ligases; Ubiquitination; Xenopus
Type
journal article