Pin1 promotes human CaV2.1 channel polyubiquitination by RNF138: pathophysiological implication for episodic ataxia type 2.

Abstract

Loss-of-function mutations in the human gene encoding the neuron-specific Ca²⁺ channel Ca_V2.1 are linked to the neurological disease episodic ataxia type 2 (EA2), as well as neurodevelopmental disorders such as developmental delay and developmental epileptic encephalopathy. Disease-associated Ca_V2.1 mutants may exhibit defective proteostasis and promote endoplasmic reticulum (ER)-associated degradation of their wild-type (WT) counterpart in a dominant-negative manner. The E3 ubiquitin ligase RNF138 was previously shown to mediate EA2-related aberrant degradation of Ca_V2.1 at the ER. Herein we aimed to elucidate the ER proteostasis mechanism of Ca_V2.1. The peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1) was identified as a novel neuronal Ca_V2.1 binding partner that promoted polyubiquitination and proteasomal degradation of Ca_V2.1. Suppression of endogenous Pin1 level with either shRNA knockdown or the Pin1 inhibitor all-trans retinoic acid (ATRA) enhanced endogenous Ca_V2.1 protein level in neurons, and attenuated ER-associated degradation of Ca_V2.1 WT and EA2-causing mutants. Detailed mutation analyses suggested that Pin1 interacted with specific phosphorylated serine/threonine-proline motifs in the intracellular II-III loop and the distal carboxy-terminal region of human Ca_V2.1. We further generated Pin1-insensitive Ca_V2.1 constructs and demonstrated that, during ER quality control, Pin1 served as an upstream regulator of Ca_V2.1 polyubiquitination and degradation by RNF138. Pin1 regulation was required for the dominant-negative effect of EA2 missense mutants, but not nonsense mutants, on Ca_V2.1 WT protein expression. Our data are consistent with the idea that Ca_V2.1 proteostasis at the ER, as well as dominant-negative suppression of disease-causing loss-of-function mutants on Ca_V2.1 WT, entail both Pin1/RNF138-dependent and -independent mechanisms.