Germline de novo variant F747S extends the phenotypic spectrum of CACNA1D Ca2+ channelopathies.

Germline gain-of-function missense variants in the pore-forming Cav1.3 α1-subunit (CACNA1D gene) confer high risk for a severe neurodevelopmental disorder with or without endocrine symptoms. Here, we report a 4-week-old new-born with the novel de novo missense variant F747S with a so far not described prominent jittering phenotype in addition to symptoms previously reported for CACNA1D mutations including developmental delay, elevated aldosterone level and transient hypoglycemia. We confirmed the pathogenicity of this variant in whole-cell patch-clamp experiments with wild-type and F747S mutant channels heterologously expressed together with α2δ1 and cytosolic ß3 or membrane-bound ß2a subunits. Mutation F747S caused the quantitatively largest shift in the voltage dependence of activation (-28 mV) reported so far for CACNA1D germline mutations. It also shifted inactivation to more negative voltages, slowed the time course of current inactivation and slowed current deactivation upon repolarization with both co-expressed ß-subunits. In silico modelling and molecular docking, simulations revealed that this gain-of-function phenotype can be explained by formation of a novel inter-domain hydrogen bond between mutant residues S747 (IIS6) with N1145 (IIIS6) stabilizing selectively the activated open channel state. F747S displayed 2-6-fold increased sensitivity for the L-type Ca2+ channel blocker isradipine compared to wild type. Our data confirm the pathogenicity of the F747S variant with very strong gain-of-function gating changes, which may contribute to the novel jittering phenotype. Increased sensitivity for isradipine suggests this drug for potential symptomatic off-label treatment for carriers of this mutation.

The Potential of L-Type Calcium Channels as a Drug Target for Neuroprotective Therapy in Parkinson's Disease.

The motor symptoms of Parkinson's disease (PD) mainly arise from degeneration of dopamine neurons within the substantia nigra. As no disease-modifying PD therapies are available, and side effects limit long-term benefits of current symptomatic therapies, novel treatment approaches are needed. The ongoing phase III clinical study STEADY-PD is investigating the potential of the dihydropyridine isradipine, an L-type Ca2+ channel (LTCC) blocker, for neuroprotective PD therapy. Here we review the clinical and preclinical rationale for this trial and discuss potential reasons for the ambiguous outcomes of in vivo animal model studies that address PD-protective dihydropyridine effects. We summarize current views about the roles of Cav1.2 and Cav1.3 LTCC isoforms for substantia nigra neuron function, and their high vulnerability to degenerative stressors, and for PD pathophysiology. We discuss different dihydropyridine sensitivities of LTCC isoforms in view of their potential as drug targets for PD neuroprotection, and we conclude by considering how these aspects could guide further drug development.

Novel CACNA1A Variant p.Cys256Phe Disrupts Disulfide Bonds and Causes Spinocerebellar Ataxia.

BACKGROUND: Spinocerebellar ataxia (SCA) is a progressive, autosomal dominant neurodegenerative disorder typically associated with CAG repeat expansions. OBJECTIVE: We assessed the pathogenicity of the novel, heterozygous missense variant p.Cys256Phe (C256F) in the pore-forming α1-subunit of the Cav2.1 Ca2+ channel found in a 63-year-old woman with SCA with no CAG repeat expansion. METHODS: We examined the effect of the C256F variant on channel function using whole-cell patch-clamp recordings in transfected tsA-201 cells. RESULTS: The maximum Ca2+ current density was significantly reduced in the mutant compared to wild-type, which could not be explained by lower expression levels of mutant Cav2.1 α1- protein. Together with a significant increase in current inactivation, this is consistent with a loss of channel function. Molecular modeling predicted disruption of a conserved disulfide bond through the C256F variant. CONCLUSIONS: Our results support the pathogenicity of the C256F variant for the SCA phenotype and provide further insight into Cav2.1 structure and function.

Whole Exome Sequencing Identifies a Heterozygous Variant in the Cav1.3 Gene CACNA1D Associated with Familial Sinus Node Dysfunction and Focal Idiopathic Epilepsy.

Cav1.3 voltage-gated L-type calcium channels (LTCCs) are involved in cardiac pacemaking, hearing and hormone secretion, but are also expressed postsynaptically in neurons. So far, homozygous loss of function mutations in CACNA1D encoding the Cav1.3 α1-subunit are described in congenital sinus node dysfunction and deafness. In addition, germline mutations in CACNA1D have been linked to neurodevelopmental syndromes including epileptic seizures, autism, intellectual disability and primary hyperaldosteronism. Here, a three-generation family with a syndromal phenotype of sinus node dysfunction, idiopathic epilepsy and attention deficit hyperactivity disorder (ADHD) is investigated. Whole genome sequencing and functional heterologous expression studies were used to identify the disease-causing mechanisms in this novel syndromal disorder. We identified a heterozygous non-synonymous variant (p.Arg930His) in the CACNA1D gene that cosegregated with the combined clinical phenotype in an autosomal dominant manner. Functional heterologous expression studies showed that the CACNA1D variant induces isoform-specific alterations of Cav1.3 channel gating: a gain of ion channel function was observed in the brain-specific short CACNA1D isoform (Cav1.3S), whereas a loss of ion channel function was seen in the long (Cav1.3L) isoform. The combined gain-of-function (GOF) and loss-of-function (LOF) induced by the R930H variant are likely to be associated with the rare combined clinical and syndromal phenotypes in the family. The GOF in the Cav1.3S variant with high neuronal expression is likely to result in epilepsy, whereas the LOF in the long Cav1.3L variant results in sinus node dysfunction.

Correction to: De novo CACNA1D Ca2+ channelopathies: clinical phenotypes and molecular mechanism.

The above article was published online with an error in Article title. Author mispronounced the name of a gene (CACNA1D instead of CACAN1D). The correct gene is presented above.

De novo CACNA1D Ca2+ channelopathies: clinical phenotypes and molecular mechanism.

The identification of rare disease-causing variants in humans by large-scale next-generation sequencing (NGS) studies has also provided us with new insights into the pathophysiological role of de novo missense variants in the CACNA1D gene that encodes the pore-forming α1-subunit of voltage-gated Cav1.3 L-type Ca2+ channels. These CACNA1D variants have been identified somatically in aldosterone-producing adenomas as well as germline in patients with neurodevelopmental and in some cases endocrine symptoms. In vitro studies in heterologous expression systems have revealed typical gating changes that indicate enhanced Ca2+ influx through Cav1.3 channels as the underlying disease-causing mechanism. Here we summarize the clinical findings of 12 well-characterized individuals with a total of 9 high-risk pathogenic CACNA1D variants. Moreover, we propose how information from somatic mutations in aldosterone-producing adenomas could be used to predict the potential pathogenicity of novel germline variants. Since these pathogenic de novo variants can cause a channel-gain-of function, we also discuss the use of L-type Ca2+ channel blockers as a potential therapeutic option.

RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells.

Cav1.3 L-type Ca2+ channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca2+ signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca2+-binding proteins inhibits Ca2+-dependent inactivation, while the mechanisms underlying slow voltage-dependent inactivation (VDI) are not completely understood. Here we studied if the complex formation of Cav1.3 LTCCs with the presynaptic active zone proteins RIM2α and RIM-binding protein 2 (RBP2) can stabilize slow VDI. We detected both RIM2α and RBP isoforms in adult mouse IHCs, where they co-localized with Cav1.3 and synaptic ribbons. Using whole-cell patch-clamp recordings (tsA-201 cells), we assessed their effect on the VDI of the C-terminal full-length Cav1.3 (Cav1.3L) and a short splice variant (Cav1.342A) that lacks the C-terminal RBP2 interaction site. When co-expressed with the auxiliary ß3 subunit, RIM2α alone (Cav1.342A) or RIM2α/RBP2 (Cav1.3L) reduced Cav1.3 VDI to a similar extent as observed in IHCs. Membrane-anchored ß2 variants (ß2a, ß2e) that inhibit inactivation on their own allowed no further modulation of inactivation kinetics by RIM2α/RBP2. Moreover, association with RIM2α and/or RBP2 consolidated the negative Cav1.3 voltage operating range by shifting the channel's activation threshold toward more hyperpolarized potentials. Taken together, the association with "slow" ß subunits (ß2a, ß2e) or presynaptic scaffolding proteins such as RIM2α and RBP2 stabilizes physiological gating properties of IHC Cav1.3 LTCCs in a splice variant-dependent manner ensuring proper IHC function.

New gain-of-function mutation shows CACNA1D as recurrently mutated gene in autism spectrum disorders and epilepsy.

CACNA1D encodes the pore-forming α1-subunit of Cav1.3, an L-type voltage-gated Ca2+-channel. Despite the recent discovery of two de novo missense gain-of-function mutations in Cav1.3 in two individuals with autism spectrum disorder (ASD) and intellectual disability CACNA1D has not been considered a prominent ASD-risk gene in large scale genetic analyses, since such studies primarily focus on likely-disruptive genetic variants. Here we report the discovery and characterization of a third de novo missense mutation in CACNA1D (V401L) in a patient with ASD and epilepsy. For the functional characterization we introduced mutation V401L into two major C-terminal long and short Cav1.3 splice variants, expressed wild-type or mutant channel complexes in tsA-201 cells and performed whole-cell patch-clamp recordings. Mutation V401L, localized within the channel's activation gate, significantly enhanced current densities, shifted voltage dependence of activation and inactivation to more negative voltages and reduced channel inactivation in both Cav1.3 splice variants. Altogether, these gating changes are expected to result in enhanced Ca2+-influx through the channel, thus representing a strong gain-of-function phenotype. Additionally, we also found that mutant channels retained full sensitivity towards the clinically available Ca2+ -channel blocker isradipine. Our findings strengthen the evidence for CACNA1D as a novel candidate autism risk gene and encourage experimental therapy with available channel-blockers for this mutation. The additional presence of seizures and neurological abnormalities in our patient define a novel phenotype partially overlapping with symptoms in two individuals with PASNA (congenital primary aldosteronism, seizures and neurological abnormalities) caused by similar Cav1.3 gain-of-function mutations.

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 Cav1.3 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 (Cav1.2 α1-subunit, long and short Cav1.3 α1-subunit splice variants; ß3/α2δ1). During SN DA-like pacemaking, only Cav1.3 variants conducted Ca2+ current (ICa) at subthreshold potentials between action potentials. SN DA-like burst activity increased integrated ICa during (Cav1.2 plus Cav1.3) and after (Cav1.3) the burst. Isradipine inhibition was splice variant and isoform dependent, with a 5- to 11-fold lower sensitivity to Cav1.3 variants during SN DA-like pacemaking compared with Cav1.2 during aSM-like activity. Supratherapeutic isradipine concentrations reduced the pacemaker precision of adult mouse SN DA neurons but did not affect their somatic Ca2+ oscillations. Our data predict that Cav1.2 and Cav1.3 splice variants contribute differentially to Ca2+ load in SN DA neurons, with prominent Cav1.3-mediated ICa 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 Cav1.2.SIGNIFICANCE STATEMENT The high vulnerability of dopamine (DA) neurons in the substantia nigra (SN) to neurodegenerative stressors causes Parkinson's disease (PD). Ca2+ influx through voltage-gated L-type Ca2+ channels (LTCCs), in particular Cav1.3, 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 Cav1.2 LTCCs in resistance blood vessels (mediating dose-limiting vasodilating effects) and even at supratherapeutic concentrations isradipine fails to reduce somatic Ca2+ oscillations of SN DA neurons.

Mechanisms Responsible for ω-Pore Currents in Cav Calcium Channel Voltage-Sensing Domains.

Mutations of positively charged amino acids in the S4 transmembrane segment of a voltage-gated ion channel form ion-conducting pathways through the voltage-sensing domain, named ω-current. Here, we used structure modeling and MD simulations to predict pathogenic ω-currents in CaV1.1 and CaV1.3 Ca2+ channels bearing several S4 charge mutations. Our modeling predicts that mutations of CaV1.1-R1 (R528H/G, R897S) or CaV1.1-R2 (R900S, R1239H) linked to hypokalemic periodic paralysis type 1 and of CaV1.3-R3 (R990H) identified in aldosterone-producing adenomas conducts ω-currents in resting state, but not during voltage-sensing domain activation. The mechanism responsible for the ω-current and its amplitude depend on the number of charges in S4, the position of the mutated S4 charge and countercharges, and the nature of the replacing amino acid. Functional characterization validates the modeling prediction showing that CaV1.3-R990H channels conduct ω-currents at hyperpolarizing potentials, but not upon membrane depolarization compared with wild-type channels.

Cav 1.3 (CACNA1D) L-type Ca2+ channel dysfunction in CNS disorders.

Cav 1.3 belongs to the family of voltage-gated L-type Ca2+ channels and is encoded by the CACNA1D gene. Cav 1.3 channels are not only essential for cardiac pacemaking, hearing and hormone secretion but are also expressed postsynaptically in neurons, where they shape neuronal firing and plasticity. Recent findings provide evidence that human mutations in the CACNA1D gene can confer risk for the development of neuropsychiatric disease and perhaps also epilepsy. Loss of Cav 1.3 function, as shown in knock-out mouse models and by human mutations, does not result in neuropsychiatric or neurological disease symptoms, whereas their acute selective pharmacological activation results in a depressive-like behaviour in mice. Therefore it is likely that CACNA1D mutations enhancing activity may be disease relevant also in humans. Indeed, whole exome sequencing studies, originally prompted to identify mutations in primary aldosteronism, revealed de novo CACNA1D missense mutations permitting enhanced Ca2+ signalling through Cav 1.3. Remarkably, apart from primary aldosteronism, heterozygous carriers of these mutations also showed seizures and neurological abnormalities. Different missense mutations with very similar gain-of-function properties were recently reported in patients with autism spectrum disorders (ASD). These data strongly suggest that CACNA1D mutations enhancing Cav 1.3 activity confer a strong risk for - or even cause - CNS disorders, such as ASD.

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.

CaV1.2 and CaV1.3 channel hyperactivation in mouse islet ß cells exposed to type 1 diabetic serum.

The voltage-gated Ca(2+) (CaV) channel acts as a key player in ß cell physiology and pathophysiology. ß cell CaV channels undergo hyperactivation subsequent to exposure to type 1 diabetic (T1D) serum resulting in increased cytosolic free Ca(2+) concentration and thereby Ca(2+)-triggered ß cell apoptosis. The present study was aimed at revealing the subtypes of CaV1 channels hyperactivated by T1D serum as well as the biophysical mechanisms responsible for T1D serum-induced hyperactivation of ß cell CaV1 channels. Patch-clamp recordings and single-cell RT-PCR analysis were performed in pancreatic ß cells from CaV1 channel knockout and corresponding control mice. We now show that functional CaV1.3 channels are expressed in a subgroup of islet ß cells from CaV1.2 knockout mice (CaV1.2(-/-)). T1D serum enhanced whole-cell CaV currents in islet ß cells from CaV1.3 knockout mice (CaV1.3(-/-)). T1D serum increased the open probability and number of functional unitary CaV1 channels in CaV1.2(-/-) and CaV1.3(-/-) ß cells. These data demonstrate that T1D serum hyperactivates both CaV1.2 and CaV1.3 channels by increasing their conductivity and number. These findings suggest CaV1.2 and CaV1.3 channels as potential targets for anti-diabetes therapy.

C-terminal modulatory domain controls coupling of voltage-sensing to pore opening in Cav1.3 L-type Ca(2+) channels.

Activity of voltage-gated Cav1.3 L-type Ca(2+) channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Cav1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (QON-V) and of current activation (ICa-V) of the long (Cav1.3L) and a short Cav1.3 splice variant (Cav1.342A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (QON) of Cav1.3L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Cav1.2 and Cav3.1. However, a significantly higher fraction of the total charge had to move for activation of Cav1.3 half-maximal conductance (Cav1.3: 68%; Cav1.2: 52%; Cav3.1: 22%). This indicated a weaker coupling of Cav1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Cav1.342A, thereby shifting ICa-V by 7.2 mV to potentials that were more negative without changing QON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of QON-V and a more negative activation of ICa-V of all three channels. These findings illustrate that the voltage sensors of Cav1.3 channels respond more sensitively to depolarization than those of Cav1.2 or Cav3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Cav1.342A splice variants to activate at lower voltages without affecting QON-V.

Cav1.2 and Cav1.3 L-type calcium channels regulate dopaminergic firing activity in the mouse ventral tegmental area.

Dopaminergic projections from the ventral tegmental area (VTA) constitute the mesolimbocortical system that underlies addiction and psychosis primarily as a result of increased dopaminergic transmission. Dopamine release is spike dependent. L-type calcium channels (LTCCs) play an important role in regulating firing activities, but the contribution of specific subtypes remains unclear. This article describes different functions of Cav1.2 and Cav1.3 subtypes in regulating firing properties with two transgenic mouse strains. For basal firing, Cav1.3-deficient (Cav1.3(-/-)) mice had a lower basal firing frequency. The dihydropyridine (DHP) channel blocker nifedipine reduced single-spike firing in mice expressing DHP-insensitive Cav1.2 channels (Cav1.2DHP(-/-) mice), confirming the significant contribution from the Cav1.3 subtype in basal firing. Moreover, the DHP channel activator (S)-(-)-Bay K8644 and the non-DHP channel activator FPL 64176 converted firing patterns from single spiking to bursting in Cav1.2DHP(-/-) mice. Nifedipine inhibited burst firing induced by both activators, suggesting that Cav1.3 also serves an essential role in burst firing. However, FPL 64176 also induced bursting in Cav1.3(-/-) mice. These results indicate that the Cav1.3 subtype is crucial to regulation of basal single-spike firing, while activation of both Cav1.2 and Cav1.3 can support burst firing of VTA neurons.

Novel protocol for multiple-dose oral administration of the L-type Ca2+ channel blocker isradipine in mice: A dose-finding pharmacokinetic study.

Studies in genetically modified animals and human genetics have recently provided new insight into the role of voltage-gated L-type Ca2+ channels in human disease. Therefore, the inhibition of L-type Ca2+ channels in vivo in wildtype and mutant mice by potent dihydropyridine (DHP) Ca2+ channel blockers serves as an important pharmacological tool. These drugs have a short plasma half-life in humans and especially in rodents and show high first-pass metabolism upon oral application. In the vast majority of in vivo studies, they have therefore been delivered through parenteral routes, mostly subcutaneously or intraperitoneally. High peak plasma concentrations of DHPs cause side effects, evident as DHP-induced aversive behaviors confounding the interpretation of behavioral readouts. Nevertheless, pharmacokinetic data measuring the exposure achieved with these applications are sparse. Moreover, parenteral injections require animal handling and can be associated with pain, discomfort and stress which could influence a variety of physiological processes, behavioral and other functional readouts. Here, we describe a noninvasive oral application of the DHP isradipine by training mice to quickly consume small volumes of flavored yogurt that can serve as drug vehicle. This procedure does not require animal handling, allows repeated drug application over several days and reproducibly achieves peak plasma concentrations over a wide range previously shown to be well-tolerated in humans. This protocol should facilitate ongoing nonclinical studies in mice exploring new indications for DHP Ca2+ channel blockers.

Pathogenicity of de novo CACNA1D Ca2+ channel variants predicted from sequence co-variation.

Voltage-gated L-type Cav1.3 Ca2+ channels support numerous physiological functions including neuronal excitability, sinoatrial node pacemaking, hearing, and hormone secretion. De novo missense mutations in the gene of their pore-forming α1-subunit (CACNA1D) induce severe gating defects which lead to autism spectrum disorder and a more severe neurological disorder with and without endocrine symptoms. The number of CACNA1D variants reported is constantly rising, but their pathogenic potential often remains unclear, which complicates clinical decision-making. Since functional tests are time-consuming and not always available, bioinformatic tools further improving pathogenicity potential prediction of novel variants are needed. Here we employed evolutionary analysis considering sequences of the Cav1.3 α1-subunit throughout the animal kingdom to predict the pathogenicity of human disease-associated CACNA1D missense variants. Co-variation analyses of evolutionary information revealed residue-residue couplings and allowed to generate a score, which correctly predicted previously identified pathogenic variants, supported pathogenicity in variants previously classified as likely pathogenic and even led to the re-classification or re-examination of 18 out of 80 variants previously assessed with clinical and electrophysiological data. Based on the prediction score, we electrophysiologically tested one variant (V584I) and found significant gating changes associated with pathogenic risks. Thus, our co-variation model represents a valuable addition to complement the assessment of the pathogenicity of CACNA1D variants completely independent of clinical diagnoses, electrophysiology, structural or biophysical considerations, and solely based on evolutionary analyses.

Quantitative proteomics of the Cav2 channel nano-environments in the mammalian brain.

Local Ca(2+) signaling occurring within nanometers of voltage-gated Ca(2+) (Cav) channels is crucial for CNS function, yet the molecular composition of Cav channel nano-environments is largely unresolved. Here, we used a proteomic strategy combining knockout-controlled multiepitope affinity purifications with high-resolution quantitative MS for comprehensive analysis of the molecular nano-environments of the Cav2 channel family in the whole rodent brain. The analysis shows that Cav2 channels, composed of pore-forming alpha1 and auxiliary beta subunits, are embedded into protein networks that may be assembled from a pool of approximately 200 proteins with distinct abundance, stability of assembly, and preference for the three Cav2 subtypes. The majority of these proteins have not previously been linked to Cav channels; about two-thirds are dedicated to the control of intracellular Ca(2+) concentration, including G protein-coupled receptor-mediated signaling, to activity-dependent cytoskeleton remodeling or Ca(2+)-dependent effector systems that comprise a high portion of the priming and release machinery of synaptic vesicles. The identified protein networks reflect the cellular processes that can be initiated by Cav2 channel activity and define the molecular framework for organization and operation of local Ca(2+) signaling by Cav2 channels in the brain.

Cav 1.3-selective inhibitors of voltage-gated L-type Ca2+ channels: Fact or (still) fiction?

Voltage-gated L-type Ca2+ -channels (LTCCs) are the target of Ca2+ -channel blockers (CCBs), which are in clinical use for the evidence-based treatment of hypertension and angina. Their cardiovascular effects are largely mediated by the Cav 1.2-subtype. However, based on our current understanding of their physiological and pathophysiological roles, Cav 1.3 LTCCs also appear as attractive drug targets for the therapy of various diseases, including treatment-resistant hypertension, spasticity after spinal cord injury and neuroprotection in Parkinson's disease. Since CCBs inhibit both Cav 1.2 and Cav 1.3, Cav 1.3-selective inhibitors would be valuable tools to validate the therapeutic potential of Cav 1.3 channel inhibition in preclinical models. Despite a number of publications reporting the discovery of Cav 1.3-selective blockers, their selectivity remains controversial. We conclude that at present no pharmacological tools exist that are suitable to confirm or refute a role of Cav 1.3 channels in cellular responses. We also suggest essential criteria for a small molecule to be considered Cav 1.3-selective.