Lower Affinity of Isradipine for L-Type Ca Channels during Substantia Nigra Dopamine Neuron-Like Activity: Implications for Neuroprotection in Parkinson's Disease.

Ca-influx through L-type Ca-channels (LTCCs) is associated with activity-related stressful oscillations of Ca levels within dopaminergic (DA) neurons in the substantia nigra (SN), which may contribute to their selective degeneration in Parkinson's disease (PD). LTCC blockers were neuroprotective in mouse neurotoxin models of PD, and isradipine is currently undergoing testing in a phase III clinical trial in early PD. We report no evidence for neuroprotection by pretreatment with therapeutically relevant isradipine plasma levels, or Ca1.3 LTCC deficiency in 6-OHDA-treated male mice. To explain this finding, we investigated the pharmacological properties of human LTCCs during SN DA-like and arterial smooth muscle (aSM)-like activity patterns using whole-cell patch-clamp recordings in HEK293 cells (Ca1.2 α1-subunit, long and short Ca1.3 α1-subunit splice variants; β3/α2δ1). During SN DA-like pacemaking, only Ca1.3 variants conducted Ca current (I) at subthreshold potentials between action potentials. SN DA-like burst activity increased integrated I during (Ca1.2 plus Ca1.3) and after (Ca1.3) the burst. Isradipine inhibition was splice variant and isoform dependent, with a 5- to 11-fold lower sensitivity to Ca1.3 variants during SN DA-like pacemaking compared with Ca1.2 during aSM-like activity. Supratherapeutic isradipine concentrations reduced the pacemaker precision of adult mouse SN DA neurons but did not affect their somatic Ca oscillations. Our data predict that Ca1.2 and Ca1.3 splice variants contribute differentially to Ca load in SN DA neurons, with prominent Ca1.3-mediated I between action potentials and after bursts. The failure of therapeutically relevant isradipine levels to protect SN DA neurons can be explained by weaker state-dependent inhibition of SN DA LTCCs compared with aSM Ca1.2. The high vulnerability of dopamine (DA) neurons in the substantia nigra (SN) to neurodegenerative stressors causes Parkinson's disease (PD). Ca influx through voltage-gated L-type Ca channels (LTCCs), in particular Ca1.3, appears to contribute to this vulnerability, and the LTCC inhibitor isradipine is currently being tested as a neuroprotective agent for PD in a phase III clinical trial. However, in our study isradipine plasma concentrations approved for therapy were not neuroprotective in a PD mouse model. We provide an explanation for this observation by demonstrating that during SN DA-like neuronal activity LTCCs are less sensitive to isradipine than Ca1.2 LTCCs in resistance blood vessels (mediating dose-limiting vasodilating effects) and even at supratherapeutic concentrations isradipine fails to reduce somatic Ca oscillations of SN DA neurons.