Voltage-gated Calcium Channel Cav1 Research Articles

Generating a human induced pluripotent stem cell line (XACHi018-A) from a Timothy syndrome infant carrying heterozygous CACNA1C c.1216G>A (p.G406R) mutation

Timothy syndrome, an extremely rare disease, is closely associated with a mutation in CACNA1C gene, which encodes the cardiac L-type voltage-gated calcium channel (Cav1.2). In this study, we generated a human induced pluripotent stem cell (iPSC) line from a Timothy syndrome infant carrying heterozygous CACNA1C mutation (transcript variant NM_000719.7c.1216G>A: p.G406R). The generated iPSC line showed typical stem cell morphology, positively expressed pluripotency and proliferation markers, normal karyotype, and trilineage differentiation potential. Therefore, this patient-specific iPSC can be of great significance in investigating the mechanisms underlying Timothy syndrome, and hence establishing effective intervention strategies

Structural insights into the allosteric effects of the antiepileptic drug topiramate on the CaV2.3 channel

The R-type voltage-gated calcium channel CaV2.3 is predominantly located in the presynapse and is implicated in distinct types of epileptic seizures. It has consequently emerged as a molecular target in seizure treatment. Here, we determined the cryo-EM structure of the CaV2.3-α2δ1-β1 complex in the topiramate-bound state at a 3.0 Å resolution. We provide a snapshot of the binding site of topiramate, a widely prescribed antiepileptic drug, on a voltage-gated ion channel. The binding site is located at an intracellular juxtamembrane hydrophilic cavity. Further structural analysis revealed that topiramate may allosterically facilitate channel inactivation. These findings provide fundamental insights into the mechanism underlying the inhibitory effect of topiramate on CaV and NaV channels, elucidating a previously unseen modulator binding site and thus pointing toward a route for the development of new drugs.

The Alzheimer's disease risk gene BIN1 regulates activity-dependent gene expression in human-induced glutamatergic neurons.

Bridging Integrator 1 (BIN1) is the second most important Alzheimer's disease (AD) risk gene, but its physiological roles in neurons and its contribution to brain pathology remain largely elusive. In this work, we show that BIN1 plays a critical role in the regulation of calcium homeostasis, electrical activity, and gene expression of glutamatergic neurons. Using single-cell RNA-sequencing on cerebral organoids generated from isogenic BIN1 wild type (WT), heterozygous (HET) and homozygous knockout (KO) human-induced pluripotent stem cells (hiPSCs), we show that BIN1 is mainly expressed by oligodendrocytes and glutamatergic neurons, like in the human brain. Both BIN1 HET and KO cerebral organoids show specific transcriptional alterations, mainly associated with ion transport and synapses in glutamatergic neurons. We then demonstrate that BIN1 cell-autonomously regulates gene expression in glutamatergic neurons by using a novel protocol to generate pure culture of hiPSC-derived induced neurons (hiNs). Using this system, we also show that BIN1 plays a key role in the regulation of neuronal calcium transients and electrical activity via its interaction with the L-type voltage-gated calcium channel Cav1.2. BIN1 KO hiNs show reduced activity-dependent internalization and higher Cav1.2 expression compared to WT hiNs. Pharmacological blocking of this channel with clinically relevant doses of nifedipine, a calcium channel blocker, partly rescues electrical and gene expression alterations in BIN1 KO glutamatergic neurons. Further, we show that transcriptional alterations in BIN1 KO hiNs that affect biological processes related to calcium homeostasis are also present in glutamatergic neurons of the human brain at late stages of AD pathology. Together, these findings suggest that BIN1-dependent alterations in neuronal properties could contribute to AD pathophysiology and that treatment with low doses of clinically approved calcium blockers should be considered as an option to slow disease-onset and progression.

The SMA Modifier Plastin 3 Targets Cell Membrane-Associated Proteins in Motoneurons.

Loss of the Survival Motor Neuron (SMN) gene inevitably leads to spinal muscular atrophy (SMA), one of the most common fatal neuromuscular diseases in children with FDA and EMA approved therapies. However, the cellular mechanisms leading to neuromuscular junction (NMJ) dysfunction due to impaired Ca2+ homeostasis in the presynaptic compartment remain largely unexplained. In the last decade, the so-called SMA modifiers have gained attention. The F-actin bundler Plastin 3 (PLS3) is one of them and counteracts neurotransmission defects, including altered vesicle endocytosis, in Smn-deficient NMJs. Properly bundled F-actin is the basis for the translocation and arrangement of transmembrane proteins at the cell surface. Our recently published data by Hennlein et al., J Cell Biol. (2023) clearly showed that Smn deficiency impairs the F-actin dependent translocation of the high-affinity BDNF receptor TrkB to the cell surface resulting in reduced BDNF-mediated TrkB activation in motor axon terminals. Strikingly, the overexpression of PLS3 restores TrkB availability, and significantly improves the clustering of the active zone-associated voltage-gated calcium channel Cav2.2 in growth cones of Smn-deficient motoneurons. These observations raise the question of how PLS3 mediates the proper cell surface localization and cluster-like formation of Cav2.2 in motor axon terminals.

Epilepsy-linked kinase CDKL5 phosphorylates voltage-gated calcium channel Cav2.3, altering inactivation kinetics and neuronal excitability

Developmental and epileptic encephalopathies (DEEs) are a group of rare childhood disorders characterized by severe epilepsy and cognitive deficits. Numerous DEE genes have been discovered thanks to advances in genomic diagnosis, yet putative molecular links between these disorders are unknown. CDKL5 deficiency disorder (CDD, DEE2), one of the most common genetic epilepsies, is caused by loss-of-function mutations in the brain-enriched kinase CDKL5. To elucidate CDKL5 function, we looked for CDKL5 substrates using a SILAC-based phosphoproteomic screen. We identified the voltage-gated Ca2+ channel Cav2.3 (encoded by CACNA1E) as a physiological target of CDKL5 in mice and humans. Recombinant channel electrophysiology and interdisciplinary characterization of Cav2.3 phosphomutant mice revealed that loss of Cav2.3 phosphorylation leads to channel gain-of-function via slower inactivation and enhanced cholinergic stimulation, resulting in increased neuronal excitability. Our results thus show that CDD is partly a channelopathy. The properties of unphosphorylated Cav2.3 closely resemble those described for CACNA1E gain-of-function mutations causing DEE69, a disorder sharing clinical features with CDD. We show that these two single-gene diseases are mechanistically related and could be ameliorated with Cav2.3 inhibitors.

Reduced subjective sleep quality in people rating themselves as electro-hypersensitive: An observational study

BackgroundDisturbed sleep is among the most frequent health complaints of people exposed to radio frequency electromagnetic fields (RF-EMF) used in mobile telecommunication, particularly in individuals who consider themselves as EMF hypersensitive (EHS). We aimed at investigating whether the EHS status per se is associated with sleep complaints. Because allelic variants of the gene encoding the L-type, voltage-gated calcium channel Cav1.2 (CACNA1C) were previously associated with sleep complaints reminiscent of those reported by EHS individuals, we also explored whether self-rated EHS status and sleep quality associate with these gene variants. MethodsA total of 2′040 participants (1′381 females) aged 18–30 years completed online, validated questionnaires on EMF sensitivity, subjective sleep quality, daytime sleepiness, mentation during sleep, and diurnal preference. They also provided a saliva sample for genotyping three functional variants of CACNA1C (rs7304986, rs16929277 and rs2302729). Eligible participants endorsing the question “Are you electro-hypersensitive?” were considered as “EHS” (n = 105), those denying this question yet believing to develop detrimental health symptoms due to prevailing electromagnetic pollution as “attributers” (n = 254), and the remaining participants as “non-EHS” (n = 1′406). We combined the EHS and attributers into one group for binary analyses. In exploratory analyses, we then tested possible associations between EMF sensitivity, subjective sleep variables and CACNA1C variants using linear and logistic regression. We used age, sex, level of education, presence of sleep disorders and habitual mobile phone use as covariates and corrected with Benjamini-Hochberg False Discovery Rate for multiple comparisons. ResultsThe EHS/attributers consistently reported prolonged sleep latency, reduced sleep quality, higher sleepiness and more nocturnal mentation when compared to non-EHS. Habitual mobile phone use was not associated with self-rated sleep latency and sleep quality scores. While the T-allele of variant rs2302729 of CACNA1C was associated with both, self-reported EMF sensitivity and reduced subjective sleep quality, we found no evidence for the hypothesis that EHS mediates impaired sleep quality via this allelic variant. ConclusionsIrrespective of reported RF-EMF exposure, self-rated EHS/attributers rated subjective sleep quality worse than non-EHS individuals. Trial registrationSwiss National Clinical Trials Portal (SNCTP000002285) and ClinicalTrials.gov (NCT03074617).

Bioelectric Reprogramming of the Bone Marrow Niche in Acute Myeloid Leukemia

Background. A large body of evidence indicates that cancer is not comprised solely of malignant cells, but that it represents part of a complex network of transformed and non-transformed cells, including mesenchymal stromal cells (MSCs), immune, nervous, endothelial, stem cells and fibroblasts that, together with soluble mediators, make up the tumor microenvironment. More recently, molecular mechanisms underlying a novel system of developmental control have been described to define the role of bioelectric gradients. Differences in endogenous resting membrane potential (V mem) of non-neural cells have importance in cell-cell interactions and regulation or dysregulation in embryogenesis, regeneration patterns, and cell behavior, especially proliferation and differentiation. Recently, the same issues have been suggested to be related to cancer where abnormal depolarization of resting V mem is associated to neoplastic cells and the role of ion channels conductivity in cancer initiation and progression deserves great attention in tumor microenvironment remodeling and therapy response. Methods and results. We characterized MSCs derived from bone marrow (BM) of pediatric acute myeloid leukemia patients (AML-MSCs) and identified significant changes in ion channel expression profile, including the Voltage-gated L-type calcium channel CaV1.2. We investigated the role of CaV1.2 and documented a reduced expression of CaV1.2 channel in 14 primary AML-MSCs (<53% by flow cytometry, p<0.001) mediating more frequent spontaneous calcium oscillations with higher amplitude than those found in MSCs derived from healthy donors (h-MSCs) (21% and 7%, respectively, p<0.05). Measurement of V mem by patch clamp and a fluorescent potential-sensitive probe DiBAC revealed that AML-MSCs showed a more depolarized V mem (-14.7 mV) with respect to h-MSCs (-30.5 mV, p<0.01). We hypothesized that depolarization may play a role in the AML-MSCs gene expression and functional reprogramming. Hence, we exposed h-MSCs to AML blasts demonstrating that MSCs V mem depolarized (-11.8 mV, n=10), resembling the AML-MSCs V mem, with a concomitant decrease of CaV1.2 channel expression level (<44% by flow cytometry, p<0.01). We treated h-MSCs with depolarizing pharmacological agents (ouabain and potassium gluconate) finding that the pharmacological-induced V mem depolarization promotes a pro-leukemia cell phenotype like the AML-MSCs. Briefly, MSCs exhibited higher proliferation level (ouabain, 2.5-fold; Kgluc, 1.9-fold, p<0.05), higher release of IL-6 (1.7-fold average, p<0.05), lower immunomodulatory potential and reduced the CaV1.2 levels (<35% by flow cytometry, p<0.05) with respect to untreated h-MSCs. On the contrary, we treated AML-MSCs with hyperpolarizing agents (lubiprostone and ivermectin), which reverted their phenotype to that of h-MSCs, including lower proliferation level (ivermectin -1.9-fold, p<0.05), higher immunomodulatory potential, and higher CaV1.2 levels (1.8-fold by flow cytometry, p<0.05) with respect to untreated AML-MSCs, suggesting that depolarization is reversible and that by modeling MSCs V mem we can restore an healthy stroma within the BM niche. We transduced AML-MSCs with a CaV1.2 expressing lentiviral vector (namely re-AML-MSCs, n=7) inducing a stable hyperpolarized V mem and documented that their gene expression profile reverted toward the h-MSCs profile. Finally, we observed that re-AML-MSCs propagated their acquired bioelectrical state to the contiguous AML-MSCs establishing a healthy bio-electric field. Conclusions. We demonstrated an unprecedented AML niche bioelectrical code that governs the state of MSCs, demonstrating that the contact with AML blasts is the driving force inducing the h-MSCs V mem depolarization shift and the acquisition of pro-leukemia functions. In addition, we unveiled CaV1.2 channels as a key player in this process and postulated that rewriting the bioelectric code of the patient' AML-MSCs, through the restoration of voltage-dependent calcium channel CaV1.2 activity, could be a strategy to restore their healthy phenotype. These findings provide the biological rationale to evaluate the potential of novel MSCs gene or pharmacological therapies that could both disadvantage AML progression and restore a permissive BM niche.

Benchmarking of Small Molecule Feature Representations for hERG, Nav1.5, and Cav1.2 Cardiotoxicity Prediction.

In the field of drug discovery, there is a substantial challenge in seeking out chemical structures that possess desirable pharmacological, toxicological, and pharmacokinetic properties. Complications arise when drugs interfere with the functioning of cardiac ion channels, leading to serious cardiovascular consequences. The discontinuation and removal of numerous approved drugs from the market or at late development stages in the pipeline due to such inhibitory effects further highlight the urgency of addressing this issue. Consequently, the early prediction of potential blockers targeting cardiac ion channels during the drug discovery process is of paramount importance. This study introduces a deep learning framework that computationally determines the cardiotoxicity associated with the voltage-gated potassium channel (hERG), the voltage-gated calcium channel (Cav1.2), and the voltage-gated sodium channel (Nav1.5) for drug candidates. The predictive capabilities of three feature representations─molecular fingerprints, descriptors, and graph-based numerical representations─are rigorously benchmarked. Additionally, a novel training and evaluation data set framework is presented, enabling predictive model training of drug off-target cardiotoxicity using a comprehensive and large curated data set covering these three cardiac ion channels. To facilitate these predictions, a robust and comprehensive small molecule cardiotoxicity prediction tool named CToxPred has been developed. It is made available as open source under the permissive MIT license at https://github.com/issararab/CToxPred.

Abstract P3145: Enhancing Cardiac Cav1.2 Ion Channel Function By Targeted Deubiquitination

Ventricular excitation-contraction coupling relies on precise spatial arrangement of voltage-gated calcium channels CaV1.2 within T-tubular dyadic junctions proximal to ryanodine receptors (RyR2) that gate Ca2+ release from the sarcoplasmic reticulum. Impaired CaV1.2 trafficking is linked to devastating cardiovascular diseases such as arrythmias and cardiac hypertrophy. Ubiquitination is an important regulatory mechanism that controls ion channel surface trafficking and turnover. Emerging evidence hint at a prominent role for CaV1.2 ubiquitination to underlie heart failure disease progression, that suppresses CaV1.2 currents (ICa,L), resulting in harmful compensatory mechanisms. Targeted protein stabilization (TPS) is a burgeoning therapeutic approach for undruggable targets. We hypothesized that harnessing deubiquitinases using TPS can counteract pathological ubiquitination of CaV1.2, overcoming the need for hormonal remodeling and lead to novel treatment. To this end, we sought to develop engineered deubiquitinases (enDUBs) that can increase functional expression of CaV1.2 channels in the heart. The catalytic domain of ubiquitin specific protease 21 (USP21) fused to nb.F3 (F3-USP21) robustly increased CaV1.2 currents reconstituted in HEK293 cells as well as endogenous ICa,L in mammalian ventricular cardiomyocytes. This increase was within the physiological range of the impact of β-adrenergic stimulation of CaV1.2 and did not alter ventricular action potential duration. The mechanism of nbF3-USP21-induced increase in ICa,L is augmented CaV1.2 surface density. Reassuringly, this approach did not adversely interfere with the correct placement of CaV1.2 channels at dyadic junctions as cardiomyocytes expressing nb.F3-USP21 displayed normal colocalization of CaV1.2 with RyR2 at T-tubules. In conclusion, our study provides evidence that targeting deubiquitinases using TPS could be a viable therapeutic approach for rescuing CaV1.2 functional expression and these findings may pave the way for future development of TPS-based therapies for cardiovascular diseases such as arrhythmias and cardiac hypertrophy.

Active Zone Trafficking of CaV2/UNC-2 Channels Is Independent of β/CCB-1 and α2δ/UNC-36 Subunits.

The CaV2 voltage-gated calcium channel is the major conduit of calcium ions necessary for neurotransmitter release at presynaptic active zones (AZs). The CaV2 channel is a multimeric complex that consists of a pore-forming α1 subunit and two auxiliary β and α2δ subunits. Although auxiliary subunits are critical for channel function, whether they are required for α1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans, we show that UNC-2/α1 localizes to AZs even in the absence of CCB-1/β or UNC-36/α2δ, albeit at low levels. When UNC-2 is manipulated to be trapped in the endoplasmic reticulum (ER), CCB-1 and UNC-36 fail to colocalize with UNC-2 in the ER, indicating that they do not coassemble with UNC-2 in the ER. Moreover, blocking ER-associated degradation does not further increase presynaptic UNC-2 channels in ccb-1 or unc-36 mutants, indicating that UNC-2 levels are not regulated in the ER. An unc-2 mutant lacking C-terminal AZ protein interaction sites with intact auxiliary subunit binding sites displays persistent presynaptic UNC-2 localization and a prominent increase of UNC-2 channels in nonsynaptic axonal regions, underscoring a protective role of auxiliary subunits against UNC-2 degradation. In the absence of UNC-2, presynaptic CCB-1 and UNC-36 are profoundly diminished to barely detectable levels, indicating that UNC-2 is required for the presynaptic localization of CCB-1 and UNC-36. Together, our findings demonstrate that although the pore-forming subunit does not require auxiliary subunits for its trafficking and transport to AZs, it recruits auxiliary subunits to stabilize and expand calcium channel signalosomes.SIGNIFICANCE STATEMENT Synaptic transmission in the neuron hinges on the coupling of synaptic vesicle exocytosis with calcium influx. This calcium influx is mediated by CaV2 voltage-gated calcium channels. These channels consist of one pore-forming α1 subunit and two auxiliary β and α2δ subunits. The auxiliary subunits enhance channel function and regulate the overall level of channels at presynaptic terminals. However, it is not settled how these auxiliary subunits regulate the overall channel level. Our study in C. elegans finds that although the auxiliary subunits do not coassemble with α1 and aid trafficking, they are recruited to α1 and stabilize the channel complex at presynaptic terminals. Our study suggests that drugs that target the auxiliary subunits can directly destabilize and have an impact on CaV2 channels.

Angelica decursiva exerts antihypertensive activity by inhibiting L-type calcium channel

Ethnopharmacological relevanceAngelica decursiva is a perennial herb that belongs to the Umbelliferae family. It is traditionally used to treat fever, upper respiratory tract infections, bleeding and hypertension. However, despite its extensive pharmacological potential, literature reports on its antihypertensive pharmacological properties are scarce. Aim of the studyIn the study, crude extract from A. decursiva roots was examined for its antihypertensive activity and its molecular basis was explored. Materials and methodsA. decursiva roots were extracted with ethanol, and isolated with silica gel normal-phase chromatography and reverse-phase high performance liquid chromatography. L-NAME-induced hypertensive mouse model was used to detect in vivo hypertensive activity. Thoracic aorta ring contraction activity and electrophysiology recordings were employed to evaluate in vitro antihypertensive activity and revealed an antihypertensive target, which was transiently expressed in HEK293T cells. ResultsAngelica decursiva ethanol decoction (ADED) exhibited significant antihypertensive effects in L-NAME-induced hypertension models and phenylephrine-induced vasoconstriction. Further screening revealed that demethylsuberosin is an essential component accounting for the antihypertension effects of A. decursiva. Voltage-gated calcium channel CaV1.2 is the likely target of A. decursiva for its antihypertension effects. ConclusionThe study suggests that A. decursiva and demethylsuberosin may be effective antihypertensive agents in preclinical studies. It appears that A. decursiva and demethylsuberosin exert antihypertensive effects by inhibiting the CaV1.2 channel, which contributes to the vasodilatory effect. The present study provides experimental evidence that A. decursiva is an effective remedy for hypertension in folklore. Demethylsuberosin could be a lead molecule for antihypertension drug development.

Role of TPEN in Amyloid-β25-35-Induced Neuronal Damage Correlating with Recovery of Intracellular Zn2+ and Intracellular Ca2+ Overloading.

The overproduction of neurotoxic amyloid-β (Aβ) peptides in the brain is a hallmark of Alzheimer's disease (AD). To determine the role of intracellular zinc ion (iZn2+) dysregulation in mediating Aβ-related neurotoxicity, this study aimed to investigate whether N, N, N', N'‑tetrakis (2‑pyridylmethyl) ethylenediamine (TPEN), a Zn2+‑specific chelator, could attenuate Aβ25-35‑induced neurotoxicity and the underlying mechanism. We used the 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay to measure the viability of primary hippocampal neurons. We also determined intracellular Zn2+ and Ca2+ concentrations, mitochondrial and lysosomal functions, and intracellular reactive oxygen species (ROS) content in hippocampal neurons using live-cell confocal imaging. We detected L-type voltage-gated calcium channel currents (L-ICa) in hippocampal neurons using the whole‑cell patch‑clamp technique. Furthermore, we measured the mRNA expression levels of proteins related to the iZn2+ buffer system (ZnT-3, MT-3) and voltage-gated calcium channels (Cav1.2, Cav1.3) in hippocampal neurons using RT-PCR. The results showed that TPEN attenuated Aβ25-35‑induced neuronal death, relieved the Aβ25-35‑induced increase in intracellular Zn2+ and Ca2+ concentrations; reversed the Aβ25-35‑induced increase in ROS content, the Aβ25-35‑induced increase in the L-ICa peak amplitude at different membrane potentials, the Aβ25-35‑induced the dysfunction of the mitochondria and lysosomes, and the Aβ25-35‑induced decrease in ZnT-3 and MT-3 mRNA expressions; and increased the Cav1.2 mRNA expression in the hippocampal neurons. These results suggest that TPEN, the Zn2+-specific chelator, attenuated Aβ25-35‑induced neuronal damage, correlating with the recovery of intracellular Zn2+ and modulation of abnormal Ca2+-related signaling pathways.

Molecular insights into the gating mechanisms of voltage-gated calcium channel CaV2.3

High-voltage-activated R-type CaV2.3 channel plays pivotal roles in many physiological activities and is implicated in epilepsy, convulsions, and other neurodevelopmental impairments. Here, we determine the high-resolution cryo-electron microscopy (cryo-EM) structure of human CaV2.3 in complex with the α2δ1 and β1 subunits. The VSDII is stabilized in the resting state. Electrophysiological experiments elucidate that the VSDII is not required for channel activation, whereas the other VSDs are essential for channel opening. The intracellular gate is blocked by the W-helix. A pre-W-helix adjacent to the W-helix can significantly regulate closed-state inactivation (CSI) by modulating the association and dissociation of the W-helix with the gate. Electrostatic interactions formed between the negatively charged domain on S6II, which is exclusively conserved in the CaV2 family, and nearby regions at the alpha-interacting domain (AID) and S4-S5II helix are identified. Further functional analyses indicate that these interactions are critical for the open-state inactivation (OSI) of CaV2 channels.

A Cross-Sectional Study of the Neuropsychiatric Phenotype of CACNA1C-Related Disorder

BackgroundCACNA1C encodes the voltage-gated L-type calcium channel CaV1.2. A specific gain-of-function pathogenic variant in CACNA1C causes Timothy syndrome type 1 (TS1) with cardiac long QT syndrome, syndactyly, and neuropsychiatric symptoms. Our previous work found that the TS1 mutation alters neuronal activity-dependent signaling and interneuron migration. Recent case series highlighted a broader spectrum of CACNA1C-related disorder (CRD) that includes isolated cardiac disease, isolated neurologic deficits, and TS, but it is unknown how the clinical presentation of other CRD variants relates to neural defects. We surveyed individuals with CRD to define the neuropsychiatric and developmental phenotype in an effort to guide future research into the role of calcium channels in neural development. MethodsCaregivers of and individuals with CRD completed an online survey of pre- and perinatal events, cardiac events, developmental milestones, neuropsychiatric symptoms, and neuropsychiatric diagnoses. Multiple Mann-Whitney tests were used for comparison of categorical values and Fisher exact test for comparison of categorical variables between participants with and without cardiac arrhythmia. ResultsTwenty-four participants with germline CACNA1C variants including TS1 completed the survey. The most common neuropsychiatric symptoms and/or diagnoses were developmental delay in 92%, incoordination in 71%, hypotonia in 67%, autism spectrum disorder in 50% (autistic features in 92%), seizures in 37.5%, and attention-deficit/hyperactivity disorder in 21% of participants. There were no significant differences in symptoms between participants with and without arrhythmia. ConclusionsIn our CRD cohort, there was an increased prevalence of multiple neuropsychiatric symptoms compared with the general population. These findings indicate the key role of CaV1.2 in brain development and the clinical importance of screening and therapeutically addressing neuropsychiatric symptoms in all individuals with CRD. Future directions include deep phenotyping of neuropsychiatric symptoms and efforts to relate these symptoms to cellular defects.

Disease-associated calmodulin mutations disrupt L-type Ca2+ channel (Cav1.2) activity and CaMKIIdelta phosphorylation in long QT syndrome

Abstract Introduction Long QT Syndrome (LQTS) is a major inherited arrhythmia syndrome that can cause sudden cardiac death. Using genome sequencing in human patients, mutations in the ubiquitous calcium (Ca2+) sensor protein calmodulin (CaM) have been associated to LQTS. CaM is an ion channel regulator and can modulate the activity of the voltage-gated calcium channel (Cav1.2) and Ca2+/CaM-dependent protein kinase II (CaMKIIδ), involved in cardiac muscle contraction. However the molecular mechanism by which CaM mutations contribute to irregular heartbeats remains unclear. Methods Interaction of CaM proteins with Cav1.2 and CaMKIId synthetic peptides (Cav1.2-NSCaTE51–68, Cav1.2-IQ1665–1685, Cav1.2-C1627–1652, CaMKIIδ294–315,) was investigated using Isothermal Titration Calorimetry (ITC) and X-ray crystallography. Whole-cell patch clamp electrophysiology was used to determine the effect of CaM mutations on L-type Ca2+ currents and Ca2+-dependent inactivation (CDI). CaMKIIδ phosphorylation activity was determined by western blot and fluorescence kinase assay. Results Binding affinity of CaMKIId and Cav1.2 peptides to the LQTS-associated CaM variants was significantly reduced, up to 7-fold. Interestingly, the Cav1.2-IQ1665–1685 peptide showed a stronger binding, up to 2-fold, towards LQTS-CaM mutants. Crystal structures of Ca2+-CaM:CaMKIId294–315 showed structural alterations induced by LQTS associated mutations. In addition, we demonstrated that CaMKIIδ autophosphorylation and kinase activity can be significantly reduced by LQTS-associated CaM mutants. Electrophysiological examination of Cav1.2 function revealed that CaM mutations significantly impaired channel CDI, without affecting the voltage dependence of activation and inactivation. Conclusions These data demonstrate a strong correlation between LQTS-associated CaM mutations and Cav1.2 activity. We provide molecular insights into the diverse factors contributing to CaM-mediated arrhythmias. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Intermediate Basic Science Research Fellowship

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels.

Voltage-gated calcium channel Cav2.1 undergoes Ca2+-dependent facilitation and inactivation, which are important in short-term synaptic plasticity. In presynaptic terminals, Cav2.1 forms large protein complexes that include synaptotagmins. Synaptotagmin-7 (Syt-7) is essential to mediate short-term synaptic plasticity in many synapses. Here, based on evidence that Cav2.1 and Syt-7 are both required for short-term synaptic facilitation, we investigated the direct interaction of Syt-7 with Cav2.1 and probed its regulation of Cav2.1 function. We found that Syt-7 binds specifically to the α1A subunit of Cav2.1 through interaction with the synaptic-protein interaction (synprint) site. Surprisingly, this interaction enhances facilitation in paired-pulse protocols and accelerates the onset of facilitation. Syt-7α induces a depolarizing shift in the voltage dependence of activation of Cav2.1 and slows Ca2+-dependent inactivation, whereas Syt-7β and Syt-7γ have smaller effects. Our results identify an unexpected, isoform-specific interaction between Cav2.1 and Syt-7 through the synprint site, which enhances Cav2.1 facilitation and modulates its inactivation.

Appetitive 50 kHz calls in a pavlovian conditioned approach task in Cacna1c haploinsufficient rats

We have previously shown that rats emit high-frequency 50 kHz ultrasonic vocalizations (USV) during sign- and goal-tracking in a common Pavlovian conditioned approach task. Such 50 kHz calls are probably related to positive affect and are associated with meso-limbic dopamine function. In humans, the CACNA1C gene, encoding for the α1C subunit of the L-type voltage-gated calcium channel CaV1.2, is implicated in several mental disorders, including mood disorders associated with altered dopamine signaling. In the present study, we investigated sign- and goal-tracking behavior and the emission of 50 kHz USV in Cacna1c haploinsufficent rats in a task where food pellet delivery is signaled by an appearance of an otherwise inoperable lever. Over the course of this Pavlovian training, these rats not only increased their approach to the reward site, but also their rates of pressing the inoperable lever. During subsequent extinction tests, where reward delivery was omitted, extinction patterns differed between reward site (i.e. magazine entries) and lever, since magazine entries quickly declined whereas behavior towards the lever transiently increased. Based on established criteria to define sign- or goal-tracking individuals, no CACNA1C rat met a sign-tracking criterion, since around 42% of rats tested where goal-trackers and the other 58% fell into an intermediate range. Regarding USV, we found that the CACNA1C rats emitted 50 kHz calls with a clear subject-dependent pattern; also, most of them were of a flat subtype and occurred mainly during initial habituation phases without cues or rewards. Compared, to previously published wildtype controls, Cacna1c haploinsufficent rats displayed reduced numbers of appetitive 50 kHz calls. Moreover, similar to wildtype littermate controls, 50 kHz call emission in Cacna1c haploinsufficent rats was intra-individually stable over training days and was negatively associated with goal-tracking. Together, these findings provide evidence in support of 50 kHz calls as trait marker. The finding that Cacna1c haploinsufficent rats show reductions of 50 kHz calls accompanied with more goal-tracking, is consistent with the assumption of altered dopamine signaling in these rats, a finding which supports their applicability in models of mental disorders.

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria

CaV1 and CaV2 voltage-gated calcium channels evolved from an ancestral CaV1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian CaV1 channels are uniquely subject to pronounced, buffer-resistant Ca2+/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca2+ rises. Early diverging, nonbilaterian invertebrates also possess CaV1 and CaV2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both CaV1 and CaV2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of CaV1 and CaV2 channels. Here, we build upon previous characterization of Trichoplax CaV channels by determining the cellular expression and ion-conducting properties of the CaV1 channel orthologue, TCaV1. We show that TCaV1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage–activated Ca2+ currents with kinetics resembling those of rat CaV1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCaV1, but not TCaV2, exhibits buffer-resistant Ca2+/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca2+ signaling within various cellular contexts.

The Builders of the Junction: Roles of Junctophilin1 and Junctophilin2 in the Assembly of the Sarcoplasmic Reticulum-Plasma Membrane Junctions in Striated Muscle.

Contraction of striated muscle is triggered by a massive release of calcium from the sarcoplasmic reticulum (SR) into the cytoplasm. This intracellular calcium release is initiated by membrane depolarization, which is sensed by voltage-gated calcium channels CaV1.1 (in skeletal muscle) and CaV1.2 (in cardiac muscle) in the plasma membrane (PM), which in turn activate the calcium-releasing channel ryanodine receptor (RyR) embedded in the SR membrane. This cross-communication between channels in the PM and in the SR happens at specialized regions, the SR-PM junctions, where these two compartments come in close proximity. Junctophilin1 and Junctophilin2 are responsible for the formation and stabilization of SR-PM junctions in striated muscle and actively participate in the recruitment of the two essential players in intracellular calcium release, CaV and RyR. This short review focuses on the roles of junctophilins1 and 2 in the formation and organization of SR-PM junctions in skeletal and cardiac muscle and on the functional consequences of the absence or malfunction of these proteins in striated muscle in light of recently published data and recent advancements in protein structure prediction.