Abstract
Voltage-gated Ca(2+) (Ca(V)) channels are transmembrane proteins comprising the pore-forming subunit Ca(V)α(1) and the ancillary proteins Ca(V)α(2)δ and Ca(V)β. They are expressed in various tissues, including the nervous system, where they regulate Ca(2+) entry in response to membrane potential changes. The increase in intracellular Ca(2+) allows for regulating cell excitability and releasing neurotransmitters, among other cellular events. Leucine-rich repeat kinase 2 (LRRK2) is a serine-threonine kinase involved in vesicular mobilization. Previously, it has been shown that LRRK2 regulates neurotransmission by phosphorylating the Ca(V)β auxiliary subunit of the Ca(V)2.1 (P/Q-type) presynaptic channels. However, it is unknown whether the kinase can regulate the activity of other Ca(V) channel subtypes, such as Ca(V)1.3 (L-type), which play a significant role in the excitability of dopaminergic neurons in the substantia nigra pars compacta (SNc) and whose dysregulation contributes to neurodegeneration in Parkinson's disease (PD). Here, we found potential phosphorylation sites for LRRK2 in Ca(V)β(3) and examined how these molecules interact. We used immunoprecipitation and electrophysiology in HEK-293 cells expressing recombinant Ca(V)1.3 channels, both with and without wild-type LRRK2 or its LRRK2(G2019S) mutation, which plays a role in familial PD through a possible gain-of-toxic-function mechanism. Our results show that LRRK2(G2019S) significantly increases current density through Ca(V)1.3 channels, and this effect depends on the presence of Ca(V)β(3). Site-directed mutagenesis revealed that phosphorylation at S152 in the sequence of Ca(V)β(3) is necessary and sufficient to explain the abnormal regulation of the channels mediated by LRRK2(G2019S). These data provide new insights into the molecular regulation that mutant LRRK2 may exert on L-type Ca(V)1.3 channels, which determine pacemaker activity in dopaminergic neurons of the SNc and may, therefore, play a relevant role in the molecular pathophysiology of PD.