The ubiquitin E3 ligase Parkin regulates neuronal CaV1.3 channel functional expression

Abstract

Neuronal L-type Ca²⁺ channels of the Ca_V1.3 subclass are transmembrane protein complexes that contribute to the pacemaker activity in the adult substantia nigra dopaminergic neurons. The altered function of these channels may play a role in the development and progress of neurodegenerative mechanisms implicated in Parkinson's disease (PD). Although L-type channel expression is precisely regulated, an increased functional expression has been observed in PD. Previously, we showed that Parkin, an E3 enzyme of the ubiquitin-proteasome system (UPS) interacts with neuronal Ca_V2.2 channels promoting their ubiquitin-mediated degradation. In addition, previous studies show an increase in Ca_V1.3 channel activity in dopaminergic neurons of the SNc and that Parkin expression is reduced in PD. These findings suggest that the decrease in Parkin may affect the proteasomal degradation of Ca_V1.3, which helps explain the increase in channel activity. Therefore, the present report aims to gain insight into the degradation mechanisms of the neuronal Ca_V1.3 channel by the UPS. Immunoprecipitation assays showed the interaction between Parkin and the Ca_V1.3 channels expressed in HEK-293 cells and neural tissues. Likewise, Parkin overexpression reduced the total and membrane channel levels and decreased the current density. Consistent with this, patch-clamp recordings in the presence of an inhibitor of the UPS, MG132, prevented the effects of Parkin, suggesting enhanced channel proteasomal degradation. In addition, the half-life of the pore-forming Ca_V1.3α₁ protein was significantly reduced by Parkin overexpression. Finally, electrophysiological recordings using a PRKN knockout HEK-293 cell line generated by CRISPR/Cas9 showed increased current density. These results suggest that Parkin promotes the proteasomal degradation of Ca_V1.3, which may be a relevant aspect for the pathophysiology of PD.NEW & NOTEWORTHY The increased expression of Ca_V1.3 calcium channels is a crucial feature of Parkinson's disease (PD) pathophysiology. However, the mechanisms that determine this increase are not yet defined. Parkin, an enzyme of the ubiquitin-proteasome system, is known to interact with neuronal channels promoting their ubiquitin-mediated degradation. Interestingly, Parkin mutations also play a role in PD. Here, the degradation mechanisms of Ca_V1.3 channels and their relationship with the pathophysiology of PD are studied in detail.

Keywords: CaV1.3; L-type Ca2+ channel; Parkin; Parkinson’s disease; ubiquitination.