Pin1 promotes human Ca_V2.1 channel polyubiquitination by RNF138: pathophysiological implication for episodic ataxia...

Area of Science:

• Neuroscience
• Molecular Biology
• Cell Biology

Background:

• Loss-of-function mutations in Ca_V2.1 channels cause episodic ataxia type 2 (EA2) and other neurological disorders.
• Ca_V2.1 mutants can disrupt proteostasis and lead to ER-associated degradation of wild-type (WT) Ca_V2.1.
• RNF138, an E3 ubiquitin ligase, mediates Ca_V2.1 degradation at the ER.

Purpose of the Study:

• To investigate the ER proteostasis mechanism of the Ca_V2.1 channel.
• To identify novel binding partners of Ca_V2.1 involved in its ER quality control.
• To understand the role of Pin1 in Ca_V2.1 degradation and the dominant-negative effects of EA2 mutants.

Main Methods:

• Identified Pin1 as a Ca_V2.1 binding partner using neuronal cell models.
• Utilized shRNA knockdown and a Pin1 inhibitor (ATRA) to modulate Pin1 levels.
• Performed mutation analyses to map Pin1 interaction sites on Ca_V2.1.
• Generated Pin1-insensitive Ca_V2.1 constructs to assess Pin1's regulatory role.

Main Results:

• Pin1 binds to Ca_V2.1 and promotes its polyubiquitination and proteasomal degradation.
• Suppression of Pin1 increases endogenous Ca_V2.1 levels and reduces ER-associated degradation of WT and mutant channels.
• Pin1 interacts with specific phosphorylated motifs in the Ca_V2.1 II-III loop and C-terminus.
• Pin1 acts upstream of RNF138 in Ca_V2.1 ubiquitination and degradation during ER quality control.
• Pin1 is essential for the dominant-negative impact of EA2 missense mutants on WT Ca_V2.1.

Conclusions:

• Pin1 is a key regulator of Ca_V2.1 proteostasis at the ER.
• Pin1-dependent degradation influences the severity of EA2-associated neurological disorders.
• Both Pin1/RNF138-dependent and -independent pathways govern Ca_V2.1 proteostasis and mutant effects.