Regulation of voltage-gated calcium channels in PC12 cells by Leucine Rich Repeat Kinase 2

Abstract

Leucine rich repeat kinase two (LRRK2) is a widely expressed protein belonging to the Roco family of proteins, mutations in which have recently been discovered as a cause of familial Parkinson’s disease (PD). Despite an array of interacting proteins having been identified across multiple cellular systems, LRRK2’s functional role remains to be determined. Manipulation of LRRK2 expression disrupts many Ca2+ dependent cellular processes. It, therefore, may act as an upstream regulator of initial Ca2+ signalling events, which could explain LRRK2’s widespread effects. The central aim of this study was to determine whether LRRK2 alters endogenous voltage-gated Ca2+ (CaV) channel function in PC12 cells using whole cell patch clamp electrophysiology. Additionally, transiently transfected PC12 cells underwent epifluorescence imaging to identify morphological changes and identify any effects of L-type Ca2+ blockers on morphology.

Peak CaV channel currents in LRRK2 transfected cells showed a significantly (p=0.0025, n≥7, one way ANOVA with Tukeys post-hoc test) higher current density across a number of holding voltages relative to untransfected and EGFP transfected controls. These results indicate that LRRK2 up regulates endogenous CaV channel function. Morphological assessment, however showed no significant effect of LRRK2 transfection on morphological parameters relative to EGFP transfected and non-transfected controls (N≥63, Kruskal-Wallis test). Furthermore, addition of the L-type Ca2+ channel blocker nifedipine had no significant effect relative to untransfected and ethanol vehicle controls (N≥43,Kruskal-Wallis test). These results suggest that LRRK2 dependent modulation of CaV channel function does not affect neurite differentiation. Overall, this study has identified a novel effect of LRRK2 on CaV channels, which may explain how LRRK2 has such widespread cellular effects and advances our understanding of LRRK2s functional role. If the effect of LRRK2 on CaV channels is responsible for pathology, CaV channel blockers currently being investigated for Parkinson’s therapy may be particularly effective in Parkinson’s patients harbouring LRRK2 mutations.