Pin1 promotes human Ca2.1 channel polyubiquitination by RNF138: pathophysiological implication for episodic ataxia type 2.

Loss-of-function mutations in the human gene encoding the neuron-specific Ca channel Ca2.1 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 Ca2.1 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 Ca2.1 at the ER. Herein we aimed to elucidate the ER proteostasis mechanism of Ca2.1. The peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1) was identified as a novel neuronal Ca2.1 binding partner that promoted polyubiquitination and proteasomal degradation of Ca2.1. Suppression of endogenous Pin1 level with either shRNA knockdown or the Pin1 inhibitor all-trans retinoic acid (ATRA) enhanced endogenous Ca2.1 protein level in neurons, and attenuated ER-associated degradation of Ca2.1 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 Ca2.1. We further generated Pin1-insensitive Ca2.1 constructs and demonstrated that, during ER quality control, Pin1 served as an upstream regulator of Ca2.1 polyubiquitination and degradation by RNF138. Pin1 regulation was required for the dominant-negative effect of EA2 missense mutants, but not nonsense mutants, on Ca2.1 WT protein expression. Our data are consistent with the idea that Ca2.1 proteostasis at the ER, as well as dominant-negative suppression of disease-causing loss-of-function mutants on Ca2.1 WT, entail both Pin1/RNF138-dependent and -independent mechanisms.