A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.

L-type voltage-gated Ca(2+) channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The pore-forming α1-subunit comprises four repeated domains (I-IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca(2+) levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane. Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function

Lower Affinity of Isradipine for L-Type Ca2+ Channels during Substantia Nigra Dopamine Neuron-Like Activity: Implications for Neuroprotection in Parkinson's Disease

Ca2+ -influx through L-type Ca2+ -channels (LTCCs) is associated with activity-related stressful oscillations of Ca2+ 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 in vivo pretreatment with therapeutically relevant isradipine plasma levels, or Ca(v)1.3 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 (Ca(v)1.2 alpha 1-subunit, long and short Cav1.3 1-subunit splice variants; beta 3/alpha 2 delta 1). During SN DA-like pacemaking, only Ca(v)1.3 variants conducted Ca2+ current (I-Ca) at subthreshold potentials between action potentials. SN DA-like burst activity increased integrated I-Ca during (Ca(v)1.2 plus Ca(v)1.3) and after (Ca(v)1.3) the burst. Isradipine inhibition was splice variant and isoform dependent, with a 5to11-fold lower sensitivity to Ca(v)1.3 variants during SN DA-like pacemaking compared with Ca(v)1.2 during aSM-like activity. Supratherapeutic isradipineconcentrationsreducedthepacemakerprecisionofadultmouseSNDAneuronsbutdidnotaffecttheirsomaticCa(2+) oscillations. Our data predict that Ca(v)1.2 and Ca(v)1.3 splice variants contribute differentially to Ca2+ load inSNDAneurons, with prominent Ca(v)1.3-mediated ICa between action potentials and after bursts. The failure of therapeutically relevant isradipine levels to protectSNDAneurons can be explained by weaker state-dependent inhibition of SN DA LTCCs compared with aSM Ca(v)1.2