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Abstract: Epilepsy affects more than 40 million people worldwide, constituting one of the most debilitating disorders of the Central Nervous System (CNS). It results from an imbalance in the electrical activity of neurons, which is primarily mediated by calcium ions. In many cases, treatment with Antiepileptic Drugs (AEDs) that regulate calcium channel activity results in successful seizure control. However, AEDs frequently cause adverse effects that range in severity from minimal impairment of the CNS to death from aplastic anemia or hepatic failure. Moreover, 30% of epileptic patients show drug-resistant epilepsy and do not respond to any form of medical treatment. In this context, nanotechnology has emerged as an excellent tool to overcome AEDs limitations. Numerous nano-strategies have been proposed as therapeutics and diagnostics for epilepsy through inhibition of different calcium channel types in the brain. In addition, limited brain access of classical AEDs in patients showing refractory epilepsy could be improved through the design of targeted drug delivery nanosystems. This report presents a review of the nanocarriers developed so far that could facilitate the interaction with calcium channels in the brain and the transport of AEDs through the blood-brain-barrier, mapping out a potential future direction in the research of epilepsy treatment.

Initiated by mathematical modelling of extracellular interactions between G-protein coupled receptors (GPCRs) and ligands in normal versus diseased (inflamed) environments, we previously reported the successful design, synthesis and testing of the prototype opioid painkiller NFEPP that does not elicit adverse side effects. Uniquely, this design recognised that GPCRs function differently under pathological versus healthy conditions. We now present a novel stochastic model of GPCR function that includes intracellular dissociation of G-protein subunits and modulation of plasma membrane calcium channels associated with parameters of inflamed tissue (pH, radicals). By means of molecular dynamics simulations, we also assessed qualitative changes of the reaction rates due to additional disulfide bridges inside the GPCR binding pocket and used these rates for stochastic simulations of the corresponding reaction jump process. The modelling results were validated with in vitro experiments measuring calcium currents and G-protein activation. We found markedly reduced G-protein dissociation and calcium channel inhibition induced by NFEPP at normal pH, and enhanced constitutive G-protein activation but lower probability of ligand binding with increasing radical concentrations. These results suggest that, compared to radicals, low pH is a more important determinant of overall GPCR function in an inflamed environment. Future drug design efforts should take this into account.

Calcium channel antagonists inhibit voltage-dependent L-type calcium channels found on cardiac myocytes and peripheral vascular smooth muscle cells (primarily arteries and arterioles). By blocking calcium from entering the cell, calcium antagonists produce negative chronotropic effects (decreased heart rate), negative inotropic effects (decreased force of myocardial contraction), and negative dromotropic effects (decreased conduction velocity through the atrioventricular node). They cause direct vasodilation of peripheral vasculature and interfere with pancreatic release of insulin by blocking L-type calcium ion channels of pancreatic beta-cells. Following oral administration, absorption of calcium channel blockers is rapid and nearly complete (80–100%), although their bioavailability is limited and variable (15–94%) due to a significant first-pass metabolism in the liver. Calcium channel blockers are used in the treatment of hypertension, angina, abnormal heart rhythms (e.g., atrial fibrillation and paroxysmal supraventricular tachycardia), pulmonary hypertension, Raynaud's syndrome, cardiomyopathy and subarachnoid hemorrhage. They are also used in the prevention of migraine headaches and as a tocolytic agent (interrupt uterine contractions when premature labor occurs).

Background Chronic kidney disease (CKD) is involved in a progressive deterioration in renal function over the years and is now a global public health problem. Currently, reducing the number of patients progressing to end-stage renal failure is urgently necessary. Hypertension and CKD interact with each other, and good control of blood pressure (BP) can improve CKD patients’ prognosis. With the current global trend for more strict BP control, the importance of BP management and the need for medication to achieve this strict goal are increasing. Calcium channel blockers (CCBs), which target voltage-dependent calcium channels, are frequently used in combination with renin–angiotensin–aldosterone system inhibitors for CKD patients because of their strong BP-lowering properties and relatively few adverse side effects. Calcium channels have several subtypes, including L, N, T, P/Q, and R, and three types of CCBs, L-type CCBs, L-/T-type CCBs, and L-/N-type CCBs, that are available. Nowadays, the new functions and effects of the CCBs are being elucidated. Conclusion We should use different types of CCBs properly depending on their pharmacological effects, such as the strength of antihypertensive effects and the organ protection effects, taking into account the pathophysiology of the patients. In this article, the role and the use of CCBs in CKD patients are reviewed.

Each heartbeat is initiated by the action potential, an electrical signal that depolarizes the plasma membrane and activates a cycle of calcium influx via voltage-gated calcium channels, calcium release via ryanodine receptors, and calcium reuptake and efflux via calcium-ATPase pumps and sodium-calcium exchangers. Agonists of the sympathetic nervous system bind to adrenergic receptors in cardiomyocytes, which, via cascading signal transduction pathways and protein kinase A (PKA), increase the heart rate (chronotropy), the strength of myocardial contraction (inotropy), and the rate of myocardial relaxation (lusitropy). These effects correlate with increased intracellular concentration of calcium, which is required for the augmentation of cardiomyocyte contraction. Despite extensive investigations, the molecular mechanisms underlying sympathetic nervous system regulation of calcium influx in cardiomyocytes have remained elusive over the last 40 years. Recent studies have uncovered the mechanisms underlying this fundamental biologic process, namely that PKA phosphorylates a calcium channel inhibitor, Rad, thereby releasing inhibition and increasing calcium influx. Here, we describe an updated model for how signals from adrenergic agonists are transduced to stimulate calcium influx and contractility in the heart. Expected final online publication date for the Annual Review of Physiology, Volume 84 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

IntroductionLow voltage-activated T-type calcium channels (T-type ICa), CaV3.1, CaV3.2, and CaV3.3 are opened by small depolarizations from the resting membrane potential in many cells and have been associated with neurological disorders including absence epilepsy and pain. Δ9-tetrahydrocannabinol (THC) is the principal psychoactive compound in Cannabis and also directly modulates T-type ICa, however, there is no information about functional activity of most phytocannabinoids on T-type calcium channels, including Δ9-tetrahydrocannabinol acid (THCA), the natural non-psychoactive precursor of THC. The aim of this work was to characterize THCA effects on T-type calcium channels.Materials and MethodsWe used HEK293 Flp-In-TREx cells stably expressing CaV3.1, 3.2 or 3.3. Whole-cell patch clamp recordings were made to investigate cannabinoid modulation of ICa.ResultsTHCA and THC inhibited the peak current amplitude CaV3.1 with a pEC50s of 6.0 ± 0.7 and 5.6 ± 0.4, respectively. 1μM THCA or THC produced a significant negative shift in half activation and inactivation of CaV3.1 and both drugs prolonged CaV3.1 deactivation kinetics. THCA (10 μM) inhibited CaV3.2 by 53% ± 4 and both THCA and THC produced a substantial negative shift in the voltage for half inactivation and modest negative shift in half activation of CaV3.2. THC prolonged the deactivation time of CaV3.2 while THCA did not. THCA inhibited the peak current of CaV3.3 by 43% ± 2 (10μM) but did not notably affect CaV3.3 channel activation or inactivation, however, THC caused significant hyperpolarizing shift in CaV3.3 steady state inactivation.DiscussionTHCA modulated T-type ICa currents in vitro, with significant modulation of kinetics and voltage dependence at low μM concentrations. This study suggests that THCA may have potential for therapeutic use in pain and epilepsy via T-type channel modulation without the unwanted psychoactive effects associated with THC.

Copy Number Variation (CNV) at the 1q21.1 locus is associated with a range of neurodevelopmental and psychiatric disorders in humans, including abnormalities in head size and motor deficits. Yet, the functional consequences of these CNVs (both deletion and duplication) on neuronal development remain unknown. To determine the impact of CNV at the 1q21.1 locus on neuronal development, we generated induced pluripotent stem cells from individuals harbouring 1q21.1 deletion or duplication and differentiated them into functional cortical neurons. We show that neurons with 1q21.1 deletion or duplication display reciprocal phenotype with respect to proliferation, differentiation potential, neuronal maturation, synaptic density and functional activity. Deletion of the 1q21.1 locus was also associated with an increased expression of lower cortical layer markers. This difference was conserved in the mouse model of 1q21.1 deletion, which displayed altered corticogenesis. Importantly, we show that neurons with 1q21.1 deletion and duplication are associated with differential expression of calcium channels and demonstrate that physiological deficits in neurons with 1q21.1 deletion or duplication can be pharmacologically modulated by targeting Ca2+ channel activity. These findings provide biological insight into the neuropathological mechanism underlying 1q21.1 associated brain disorder and indicate a potential target for therapeutic interventions.

Efficient and reliable neurotransmission requires precise coupling between action potentials (APs), Ca2+entry and neurotransmitter release. However, Ca2+requirements for release, including the number of channels required, their subtypes, and their location with respect to primed vesicles, remains to be precisely defined for central synapses. Indeed, Ca2+entry may occur through small numbers or even single open Ca2+channels, but these questions remain largely unexplored in simple active zone (AZ) synapses common in the nervous system, and key to addressing Ca2+channel and synaptic dysfunction underlying numerous neurologic and neuropsychiatric disorders. Here, we present single channel analysis of evoked AZ Ca2+entry, using cell-attached patch clamp and lattice light-sheet microscopy (LLSM), resolving small channel numbers evoking Ca2+entry following depolarization, at single AZs in individual central lamprey reticulospinal presynaptic terminals from male and females. We show a small pool (mean of 23) of Ca2+channels at each terminal, comprising N-(CaV2.2), P/Q-(CaV2.1), and R-(CaV2.3) subtypes, available to gate neurotransmitter release. Significantly, of this pool only one to seven channels (mean of 4) open on depolarization. High temporal fidelity lattice light-sheet imaging reveals AP-evoked Ca2+transients exhibiting quantal amplitude variations of 0–6 event sizes between individual APs and stochastic variation of precise locations of Ca2+entry within the AZ. Further, total Ca2+channel numbers at each AZ correlate to the number of presynaptic primed synaptic vesicles. Dispersion of channel openings across the AZ and the similar number of primed vesicles and channels indicate that Ca2+entry via as few as one channel may trigger neurotransmitter release.SIGNIFICANCE STATEMENTPresynaptic Ca2+entry through voltage-gated calcium channels (VGCCs) causes neurotransmitter release. To understand neurotransmission, its modulation, and plasticity, we must quantify Ca2+entry and its relationship to vesicle fusion. This requires direct recordings from active zones (AZs), previously possible only at calyceal terminals containing many AZs, where few channels open following action potentials (APs; Sheng et al., 2012), and even single channel openings may trigger release (Stanley, 1991, 1993). However, recording from more conventional terminals with single AZs commonly found centrally has thus far been impossible. We addressed this by cell-attached recordings from acutely dissociated single lamprey giant axon AZs, and by lattice light sheet microscopy of presynaptic Ca2+entry. We demonstrate nanodomains of presynaptic VGCCs coupling with primed vesicles with 1:1 stoichiometry.

The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel β subunit (CaVβ), and the binding between CaVα1/CaVβ appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVβ. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVβ independent promotion of dynamin-dependent endocytosis.

KEY POINTS Chronic stress (CS) is predicted to modify the electrical excitability of anterior pituitary corticotrophs. Electrophysiological recordings from isolated corticotrophs from CS male mice display spontaneous electrical bursting behaviour compared to the tonic spiking behaviour of unstressed corticotrophs. The increased spontaneous bursting from CS corticotrophs is BK-dependent and mathematical modelling reveals that the time constant of activation, properties and proportion of BK channels functionally coupled to L-type calcium channels determines the promotion of bursting activity. CS (but not unstressed) corticotrophs display CRH-induced bursting even when the majority of BK channels are pharmacologically inhibited, which can be explained by the stochastic behaviour of a small number of BK channels with distinct properties. Corticotroph excitability can be finely tuned by the stochastic behaviour of a small number of BK channels dependent on their properties and functional co-localisation with L-type calcium channels to control corticotroph excitability over diverse time domains and physiological challenges. ABSTRACT Coordination of an appropriate stress response is dependent upon anterior pituitary corticotroph excitability in response to hypothalamic secretagogues and glucocorticoid negative feedback. A key determinant of corticotroph excitability is large conductance calcium- and voltage- activated (BK) potassium channels that are critical for promoting CRH-induced bursting that enhances ACTH secretion. Previous studies revealed HPA axis hyperexcitability following chronic stress (CS) is partly a function of increased corticotroph output. Thus, we hypothesise that chronic stress promotes corticotroph excitability through a BK-dependent mechanism. Corticotrophs from CS mice displayed significant increases in spontaneous bursting, that was suppressed by the BK blocker paxilline. Mathematical modelling reveals that the time constant of BK channel activation, plus properties and proportion of BK channels functionally coupled to L-type calcium (Ca2+ ) channels determines bursting activity. Surprisingly, CS corticotrophs (but not unstressed) display CRH-induced bursting even when the majority of BK channels are inhibited by paxilline; that modelling suggests is a consequence of the stochastic behaviour of a small number of BK channels coupled to L-type Ca2+ channels. Our data reveal that changes in the stochastic behaviour of a small number of BK channels can finely tune corticotroph excitability through stress-induced changes in BK channel properties. Importantly, regulation of BK channel function is highly context dependent allowing dynamic control of corticotroph excitability over a large range of time domains and physiological challenges in health and disease. This is likely to occur in other BK-expressing endocrine cells, with important implications for the physiological processes they regulate and the potential for therapy. This article is protected by copyright. All rights reserved.

Kisspeptin (Kiss1) neurons are essential for reproduction, but their role in the control of energy balance and other homeostatic functions remains unclear. High-frequency firing of hypothalamic arcuate Kiss1 (Kiss1ARH) neurons releases kisspeptin into the median eminence, and neurokinin B (NKB) and dynorphin onto neighboring Kiss1ARHneurons to generate a slow EPSP mediated by TRPC5 channels that entrains intermittent, synchronous firing of Kiss1ARHneurons. High-frequency optogenetic stimulation of Kiss1ARHneurons also releases glutamate to excite the anorexigenic proopiomelanocortin (POMC) neurons and inhibit the orexigenic neuropeptide Y/agouti-related peptide (AgRP) neurons via metabotropic glutamate receptors. At the molecular level, the endoplasmic reticulum (ER) calcium-sensing protein stromal interaction molecule 1 (STIM1) is critically involved in the regulation of neuronal Ca2+signaling and neuronal excitability through its interaction with plasma membrane (PM) calcium (e.g., TRPC) channels. Therefore, we hypothesized that deletion ofStim1in Kiss1ARHneurons would increase neuronal excitability and their synchronous firing, which ultimately would affect energy homeostasis. Using optogenetics in combination with whole-cell recording and GCaMP6 imaging in slices, we discovered that deletion ofStim1in Kiss1 neurons significantly increased the amplitude and duration of the slow EPSP and augmented synchronous [Ca2+]i oscillations in Kiss1ARHneurons. Deletion ofStim1in Kiss1ARHneurons amplified the actions of NKB and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting (HFD). Therefore, STIM1 appears to play a critical role in regulating synchronous firing of Kiss1ARHneurons, which ultimately affects the coordination between energy homeostasis and reproduction.SIGNIFICANCE STATEMENTHypothalamic arcuate kisspeptin (Kiss1ARH) neurons are essential for stimulating the pulsatile release of gonadotropin-releasing hormone (GnRH) and maintaining fertility. However, Kiss1ARHneurons appear to be a key player in coordinating energy balance with reproduction. The regulation of calcium channels and hence calcium signaling is critically dependent on the endoplasmic reticulum (ER) calcium-sensing protein stromal interaction molecule 1 (STIM1), which interacts with the plasma membrane (PM) calcium channels. We have conditionally deletedStim1in Kiss1ARHneurons and found that it significantly increased the excitability of Kiss1ARHneurons and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting (HFD).

This study describes the interaction between CaV3.2 calcium channels and the receptor for activated C kinase 1 (Rack-1), a scaffold protein which has recently been implicated in neuropathic pain. The coexpression of CaV3.2 and Rack-1 in tsA-201 cells led to a reduction in the magnitude of whole-cell CaV3.2 currents and CaV3.2 channel expression at the plasma membrane. Co-immunoprecipitations from transfected cells show the formation of a molecular protein complex between Cav3.2 channels and Rack-1. We determined that the interaction of Rack-1 occurs at the intracellular II–III loop and the C-terminus of the channel. Finally, the coexpression of PKCβII abolished the effect of Rack-1 on current densities. Altogether, our findings show that Rack-1 regulates CaV3.2-mediated calcium entry in a PKC-dependent manner.

[Image: see text] The synthesis and pharmacological activities of a new series of piperazinyl quinazolin-4-(3H)-one derivatives acting toward the α2δ-1 subunit of voltage-gated calcium channels (Ca(v)α2δ-1) are reported. Different positions of a micromolar HTS hit were explored, and best activities were obtained for compounds containing a small alkyl group in position 3 of the quinazolin-4-(3H)-one scaffold and a 3-methyl-piperazin-1-yl- or 3,5-dimethyl-piperazin-1-yl-butyl group in position 2. The activity was shown to reside in the R enantiomer of the chain in position 2, and several eutomers reached single digit nanomolar affinities. Final modification of the central scaffold to reduce lipophilicity provided the pyrido[4,3-d]pyrimidin-4(3H)-one 16RR, which showed high selectivity for Ca(v)α2δ-1 versus Ca(v)α2δ-2, probably linked to its improved analgesic efficacy-safety ratio in mice over pregabalin.

Background Familial hemiplegic migraine (FHM) is an inherited autosomal dominant disorder characterized by migraine with reversible hemiplegia. FHM1 is caused by variants in CACNA1A, encoding a P/Q type neuronal voltage-gated calcium channel α subunit, which is also associated with episodic ataxia type 2 (EA2). FHM2 is associated with ATP1A2, which codes for an Na+/K+-ATPase isoform 2 subunit. Case presentation We identified an FHM2 family, the mother and her daughter, with a novel variant in ATP1A2, p.Gly377Asp, located in a well-conserved P-type ATPase motif. Additionally, the mother harbored deletion in the CACNA1A, associated with EA2, but her daughter did not. The mother presented migraine with typical aura without motor deficit, whereas her daughter had migraine accompanied by recurrent motor deficit and altered consciousness. The additional CACNA1A deletion in the mother might serve as a modifier. Conclusion Our report emphasizes the importance of genetic analysis to diagnose neurological ion channel/transporter diseases.

This review describes interactions between compounds, primarily dihydropyridines, that block L-type calcium channels and drugs that cause dependence, and the potential importance of these interactions. The main dependence-inducing drugs covered are alcohol, psychostimulants, opioids, and nicotine. In preclinical studies, L-type calcium channel blockers prevent or reduce important components of dependence on these drugs, particularly their reinforcing actions and the withdrawal syndromes. The channel blockers also reduce the development of tolerance and/or sensitization, and they have no intrinsic dependence liability. In some instances, their effects include reversal of brain changes established during drug dependence. Prolonged treatment with alcohol, opioids, psychostimulant drugs, or nicotine causes upregulation of dihydropyridine binding sites. Few clinical studies have been carried out so far, and reports are conflicting, although there is some evidence of effectiveness of L-channel blockers in opioid withdrawal. However, the doses of L-type channel blockers used clinically so far have necessarily been limited by potential cardiovascular problems and may not have provided sufficient central levels of the drugs to affect neuronal dihydropyridine binding sites. New L-type calcium channel blocking compounds are being developed with more selective actions on subtypes of L-channel. The preclinical evidence suggests that L-type calcium channels may play a crucial role in the development of dependence to different types of drugs. Mechanisms for this are proposed, including changes in the activity of mesolimbic dopamine neurons, genomic effects, and alterations in synaptic plasticity. Newly developed, more selective L-type calcium channel blockers could be of considerable value in the treatment of drug dependence. SIGNIFICANCE STATEMENT: Dependence on drugs is a very serious health problem with little effective treatment. Preclinical evidence shows drugs that block particular calcium channels, the L-type, reduce dependence-related effects of alcohol, opioids, psychostimulants, and nicotine. Clinical studies have been restricted by potential cardiovascular side effects, but new, more selective L-channel blockers are becoming available. L-channel blockers have no intrinsic dependence liability, and laboratory evidence suggests they reverse previously developed effects of dependence-inducing drugs. They could provide a novel approach to addiction treatment.

The voltage-gated calcium channel CaV1.1 belongs to the family of pseudo-heterotetrameric cation channels, which are built of four structurally and functionally distinct voltage-sensing domains (VSDs) arranged around a common channel pore. Upon depolarization, positive gating charges in the S4 helices of each VSD are moved across the membrane electric field, thus generating the conformational change that prompts channel opening. This sliding helix mechanism is aided by the transient formation of ion-pair interactions with countercharges located in the S2 and S3 helices within the VSDs. Recently, we identified a domain-specific ion-pair partner of R1 and R2 in VSD IV of CaV1.1 that stabilizes the activated state of this VSD and regulates the voltage dependence of current activation in a splicing-dependent manner. Structure modeling of the entire CaV1.1 in a membrane environment now revealed the participation in this process of an additional putative ion-pair partner (E216) located outside VSD IV, in the pore domain of the first repeat (IS5). This interdomain interaction is specific for CaV1.1 and CaV1.2 L-type calcium channels. Moreover, in CaV1.1 it is sensitive to insertion of the 19 amino acid peptide encoded by exon 29. Whole-cell patch-clamp recordings in dysgenic myotubes reconstituted with wild-type or E216 mutants of GFP-CaV1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V1/2) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-CaV1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V1/2, whereas no further shift of V1/2 was observed on substitution of E216 with A or Q. Together with our previous findings, these results demonstrate that inter- and intradomain ion-pair interactions cooperate in the molecular mechanism regulating VSD function and channel gating in CaV1.1.

Thyroid hormones influence brain development through the regulation of gene expression. Ca2+-dependent gene expression is a major pathway controlled by the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), which in turn is induced by the thyroid hormone T3, as also demonstrated in a mouse embryonic stem cell line. In addition, T3 controls the expression of neurexin, synaptotagmin2 (SYT2), synaptotagmin-related gene1 (SRG1), and a number of other genes involved in neurotransmitter release in a Ca2+-dependent manner. It has been noticed that the development of dopaminergic neurons by evoking significant calcium entry occurs through TRPC calcium channels. It was also demonstrated that the T3-mediated development of an early neuronal network is characteristic for depolarizing GABAergic neurons concomitant with intracellular calcium transients. An important aspect of T3-dependent regulation of gene expression in the developing brain is its modulation by the transcription activator COUP-TF1. Regulation of alternative splicing by CaMKIV is another important aspect for embryonal neural development since it can lead to the expression of PMCA1a, the neuronal-specific isoform of the plasma membrane calcium pump. Maternal hypothyroidism or CaMKIV deficiency can have a severe influence on fetal brain development.

Mutations in P/Q type voltage gated calcium channel (VGCC) lead severe human neurological diseases such as episodic ataxia 2, familial hemiplegic migraine 1, absence epilepsy, progressive ataxia and spinocerebellar ataxia 6. The pathogenesis of these diseases remains unclear. Mice with spontaneous mutation in the Cacna1a gene encoding the pore-forming subunit of P/Q type VGCC also exhibit ataxia, epilepsy and neurodegeneration. Based on the previous work showing that the P/Q type VGCC in neurons regulates lysosomal fusion through its calcium channel activity on lysosomes, we utilized CACNA1A mutant mice to further investigate the mechanism by which P/Q-type VGCCs regulate lysosomal function and neuronal homeostasis. We found CACNA1A mutant neurons have reduced lysosomal calcium storage without changing the resting calcium concentration in cytoplasm and the acidification of lysosomes. Immunohistochemistry and transmission electron microscopy reveal axonal degeneration due to lysosome dysfunction in the CACNA1A mutant cerebella. The calcium modulating drug thapsigargin, by depleting the ER calcium store, which locally increases the calcium concentration can alleviate the defective lysosomal fusion in mutant neurons. We propose a model that in cerebellar neurons, P/Q-type VGCC maintains the integrity of the nervous system by regulating lysosomal calcium homeostasis to affect lysosomal fusion, which in turn regulates multiple important cellular processes such as autophagy and endocytosis. This study helps us to better understand the pathogenesis of P/Q-type VGCC related neurodegenerative diseases and provides a feasible direction for future pharmacological treatment.

Multiple myeloma (MM) is a common malignant tumor of plasma cells. Despite several treatment approaches in the past two decades, MM remains an aggressive and incurable disease in dire need of new treatment strategies. Approximately 70–80% of patients with MM have myeloma bone disease (MBD), often accompanied by pathological fractures and hypercalcemia, which seriously affect the prognosis of the patients. Calcium channels and transporters can mediate Ca2+ balance inside and outside of the membrane, indicating that they may be closely related to the prognosis of MM. Therefore, this review focuses on the roles of some critical calcium channels and transporters in MM prognosis, which located in the plasma membrane, endoplasmic reticulum and mitochondria. The goal of this review is to facilitate the identification of new targets for the treatment and prognosis of MM. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12964-021-00781-4.

ω-Conotoxins inhibit N-type voltage-gated calcium (CaV2.2) channels and exhibit efficacy in attenuating neuropathic pain but have a low therapeutic index. Here, we synthesized and characterized a novel ω-conotoxin, Bu8 from Conus bullatus, which consists of 25 amino acid residues and three disulfide bridges. Bu8 selectively and potently inhibits depolarization-activated Ba2+ currents mediated by rat CaV2.2 expressed in HEK293T cells (IC50 = 89 nmol/L). Bu8 is two-fold more potent than ω-conotoxin MVIIA, a ω-conotoxin currently used for the treatment of severe chronic pain. It also displays potent analgesic activity in animal pain models of hot plate and acetic acid writhing but has fewer side effects on mouse motor function and lower toxicity in goldfish. Its lower side effects may be attributed to its faster binding rate and higher recovery ratios. The NMR structure demonstrates that Bu8 contains a small irregular triple β-strand. The structure–activity relationships of Bu8 Ala mutants and Bu8/MVIIA hybrid mutants demonstrate that the binding mode of CaV2.2 with the amino acid residues in loop 1 and loop 2 of Bu8 is different from that of MVIIA. This study characterizes a novel, more potent ω-conotoxin and provides new insights for designing CaV2.2 antagonists.

Voltage gated calcium currents (VGCCs) in sensory neurons underlie processes ranging from neurotransmitter release to gene expression and remain a therapeutic target for the treatment of pain. Yet virtually all we know about VGCCs has been obtained through the study of rodent sensory neurons and heterologously expressed channels. To address this, high voltage activated (HVA) Ca2+ currents in dissociated human and rat dorsal root ganglion (DRG) neurons were characterized with whole cell patch clamp techniques. The HVA currents from both species shared basic biophysical and pharmacological properties. However, HVA currents in human neurons differed from those in the rat in at least three potentially important ways: (1) Ca2+ current density was significantly smaller, (2) the proportion of nifedipine-sensitive currents was far greater, and (3) a subpopulation of human neurons displayed relatively large constitutive current inhibition. These results highlight the need to for the study of native proteins in their native environment prior to initiating costly clinical trials.

Unilateral traumatic brain injury (TBI) causes cortical dysfunctions spreading to the primarily undamaged hemisphere. This phenomenon, called transhemispheric diaschisis, is mediated by an imbalance of glutamatergic versus GABAergic neurotransmission. This study investigated the role of GABAergic, somatostatin-positive (SST) interneurons in the contralateral hemisphere 72 h after unilateral TBI. The brain injury was induced to the primary motor/somatosensory cortex of glutamate decarboxylase 67–green fluorescent protein (GAD67-GFP) knock-in mice at postnatal days 19–21 under anesthesia in vivo. Single GFP+ interneurons of the undamaged, contralateral cortex were isolated by fluorescence-activated cell sorting and analyzed by mass spectrometry. TBI caused a switch of 2 α subunits of pore-forming L-type voltage-gated calcium channels (VGCC) in GABAergic interneurons, an increased expression of CaV1.3, and simultaneous ablation of CaV1.2. This switch was associated with 1) increased excitability of single SST interneurons in patch-clamp recordings and (2) a recovery from early network hyperactivity in the contralateral hemisphere in microelectrode array recordings of acute slices. The electrophysiological changes were sensitive to pharmacological blockade of CaV1.3 (isradipine, 100 nM). These data identify a switch of 2 α subunits of VGCCs in SST interneurons early after TBI as a mechanism to counterbalance post-traumatic hyperexcitability.

Retinoblastoma is the most common malignant intraocular tumor in children. Y79 human retinoblastoma cells are in vitro models of retinal tumors used for drug screening. Undifferentiated Y79 cells originate from a primitive multi-potential neuroectodermal cell and express neuronal and glial properties. However, the nature of cellular heterogeneity in Y79 cells is unclear because functional methods to characterize neurons or glial cells have not been employed to Y79 cells. Here, we perform patch-clamp recordings to characterize electrophysiological properties in retinoblastoma cells. We identified a population of large-sized Y79 cells (i.e., giant cells, ~ 40-µm diameter), hyperpolarized resting membrane potential (–54 mV), and low input resistance (~ 600 MΩ), indicating electrically mature cells. We also found that giant Y79 cells contain increased density of T-type calcium channels. Finally, we found that T-type calcium channels are active only in giant cells suggesting that cancer treatments aimed to prevent calcium influx in retinoblastomas should be tested in giant cells.

Asymptomatic sudden death is the principal cause of mortality in Chagas disease. There is little information about molecular mechanisms involved in the pathophysiology of malignant arrhythmias in Chagasic patients. Previous studies have involved Trypanosoma cruzi secretion proteins in the genesis of arrhythmias ex vivo, but the molecular mechanisms involved are still unresolved. Thus, the aim was to determine the effect of these secreted proteins on the cellular excitability throughout to test its effects on catecholamine secretion, sodium-, calcium-, and potassium-conductance and action potential (AP) firing. Conditioned medium was obtained from the co-culture of T. cruzi and Vero cells (African green monkey kidney cells) and ultra-filtered for concentrating immunogenic high molecular weight parasite proteins. Chromaffin cells were assessed with the parasite and Vero cells control medium. Parasite-secreted proteins induce catecholamine secretion in a dose-dependent manner. Additionally, T. cruzi conditioned medium induced depression of both calcium conductance and calcium and voltage-dependent potassium current. Interestingly, this fact was related to the abolishment of the hyperpolarization phase of the AP produced by the parasite medium. Taken together, these results suggest that T. cruzi proteins may be involved in the genesis of pro-arrhythmic conditions that could influence the appearance of malignant arrhythmias in Chagasic patients.

The sigma-1 receptor (Sig-1R) is encoded by the SIGMAR1 gene and is a nonopioid transmembrane receptor located in the mitochondrial-associated endoplasmic reticulum membrane (MAM). It helps to locate endoplasmic reticulum calcium channels, regulates calcium homeostasis, and acts as a molecular chaperone to control cell fate and participate in signal transduction. It plays an important role in protecting neurons through a variety of signaling pathways and participates in the regulation of cognition and motor behavior closely related to neurodegenerative diseases. Based on its neuroprotective effects, Sig-1R has now become a breakthrough target for alleviating Alzheimer's disease and other neurodegenerative diseases. This article reviews the most cutting-edge research on the function of Sig-1R under normal or pathologic conditions and target drugs of the sigma-1 receptor in neurodegenerative diseases.

Voltage-gated CaV2.2 calcium channels are expressed in nociceptors at presynaptic terminals, soma, and axons. CaV2.2 channel inhibitors applied to the spinal cord relieve pain in humans and rodents, especially during pathological pain, but a biological function of nociceptor CaV2.2 channels in processing of nociception, outside presynaptic terminals in the spinal cord, is underappreciated. Here, we demonstrate that functional CaV2.2 channels in peripheral axons innervating skin are required for capsaicin-induced heat hypersensitivity in male and female mice. We show that CaV2.2 channels in TRPV1-nociceptor endings are activated by capsaicin-induced depolarization and contribute to increased intracellular calcium. Capsaicin induces hypersensitivity of both thermal nociceptors and mechanoreceptors, but only heat hypersensitivity depends on peripheral CaV2.2 channel activity, and especially a cell type-specific CaV2.2 splice isoform. CaV2.2 channels at peripheral nerve endings might be important therapeutic targets to mitigate certain forms of chronic pain. SIGNIFICANCE STATEMENT It is generally assumed that nociceptor termini in spinal cord dorsal horn are the functionally significant sites of CaV2.2 channel in control of transmitter release, and the transmission of sensory information from the periphery to central sites. We show that peripheral CaV2.2 channels are essential for the classic, heat hypersensitivity response to develop in skin following capsaicin exposure. This function of CaV2.2 is highly selective for heat, but not mechanical hypersensitivity induced by capsaicin exposure, and is not a property of closely related CaV2.1 channels. Our findings suggest that interrupting CaV2.2-dependent calcium entry in skin might reduce heat hypersensitivity that develops after noxious heat exposure, and may limit the degree of heat hypersensitivity associated with certain other forms of pain.

Approximately one-third of estrogen receptor (ER) positive breast tumors fail to respond to or become resistant to hormonal therapy. Although the mechanisms responsible for hormone resistance are not completely understood, resistance is associated with alterations in ERα; overexpression of proteins that interact with the receptor; and hormone-independent activation of the receptor by growth factor signal transduction pathways. Our previous studies show that in estrogen dependent breast cancer cells, activation of the epidermal growth factor signaling pathway increases intracellular calcium which binds to and activates ERα through sites in the ligand-binding domain of the receptor and that treatment with extracellular calcium increases the concentration of intracellular calcium which activates ERα and induces hormone-independent cell growth. The present study asked whether overexpression of calcium channels contributes to the hormone-independent and -resistant phenotype of breast cancer cells and whether clinically used calcium channel blockers reverse hormone independence and resistance. The results show that hormone-independent and -resistant cells overexpress calcium channels, have high concentrations of intracellular calcium, overexpress estrogen responsive genes and, as expected, grow in the absence of estradiol and that treatment with calcium channel blockers decreased the concentration of intracellular calcium, the expression of estrogen responsive genes and cell growth. More importantly, in hormone-resistant cells, treatment that combined a calcium channel blocker with an antiestrogen reversed resistance to the antiestrogen.

BACKGROUND AND PURPOSE Endothelium-derived hyperpolarizing factor (EDHF) has been suggested as a therapeutic target for vascular protection against ischaemic brain injury. However, the molecular entity of EDHF and its action on neurons remains unclear. This study was undertaken to demonstrate whether the hydrogen sulfide (H2 S) acts as EDHF and exerts neuroprotective effect via large-conductance Ca2+ -activated K+ (BKCa /KCa 1.1) channels. EXPERIMENTAL APPROACH The whole-cell patch-clamp technology was used to record the changes of BKCa currents in rat neurons induced by EDHF. The cerebral ischaemia/reperfusion model of mice and oxygen-glucose deprivation/reoxygenation (OGD/R) model of neurons were used to explore the neuroprotection of EDHF by activating BKCa channels in these neurons. KEY RESULTS Increases of BKCa currents and membrane hyperpolarization in hippocampal neurons induced by EDHF could be markedly inhibited by BKCa channel inhibitor iberiotoxin or endothelial H2 S synthase inhibitor propargylglycine. The H2 S donor, NaHS-induced BKCa current and membrane hyperpolarization in neurons were also inhibited by iberiotoxin, suggesting that H2 S acts as EDHF and activates the neuronal BKCa channels. Besides, we found that the protective effect of endothelium-derived H2 S against mice cerebral ischaemia/reperfusion injury was disrupted by iberiotoxin. Importantly, the inhibitory effect of NaHS or BKCa channel opener on OGD/R-induced neuron injury and the increment of intracellular Ca2+ level could be inhibited by iberiotoxin but enhanced by co-application with L-type but not T-type calcium channel inhibitor. CONCLUSION AND IMPLICATIONS Endothelium-derived H2 S acts as EDHF and exerts neuroprotective effects via activating the BKCa channels and then inhibiting the T-type calcium channels in hippocampal neurons.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing second messenger which uniquely mobilizes Ca2+ from acidic endolysosomal organelles. However, the molecular identity of the NAADP receptor remains unknown. Given the necessity of the endolysosomal two-pore channel (TPC1 or TPC2) in NAADP signaling, we performed affinity purification and quantitative proteomic analysis of the interacting proteins of NAADP and TPCs. We identified a Sm-like protein Lsm12 complexed with NAADP, TPC1, and TPC2. Lsm12 directly binds to NAADP via its Lsm domain, colocalizes with TPC2, and mediates the apparent association of NAADP to isolated TPC2 or TPC2-containing membranes. Lsm12 is essential and immediately participates in NAADP-evoked TPC activation and Ca2+ mobilization from acidic stores. These findings reveal a putative RNA-binding protein to function as an NAADP receptor and a TPC regulatory protein and provides a molecular basis for understanding the mechanisms of NAADP signaling., Nicotinic acid adenine dinucleotide phosphate (NAADP) potent Ca2+ mobilizing second messenger which uniquely triggers Ca2+ release from acidic endolysosomal organelles. Here the authors identify Lsm12 as an NAADP receptor essential for NAADP-evoked Ca2+ release from lysosomes via NAADP binding on its Lsm domain.

Exercise is known to improve cardiac recovery following coronary occlusion. However, whether short-term exercise can improve cardiac function and hypoxia tolerance ex vivo independent of reperfusion injury and the possible role of calcium channels in improved hypoxia tolerance remains unknown. Therefore, in the current study, heart function was measured ex vivo using the Langendorff method at different oxygen levels after a 4-week voluntary wheel-running regimen in trained and untrained male mice (C57Bl/6NCrl). The levels of cardiac Ca2+-channels: L-type Ca2+-channel (CACNA1C), ryanodine receptor (RyR-2), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2), and sodium-calcium exchanger were measured using western blot. Trained mice displayed lower cardiac afterload pressure generation capacity (rate and amplitude), but unaltered hypoxia tolerance when compared to untrained mice with similar heart rates. The level of CACNA1C positively correlated with the pressure generation rate and amplitude. Furthermore, the CACNA1C-RYR-2 ratio also positively correlated with the pressure generation rate. While the 4-week training period was not enough to alter the intrinsic cardiac hypoxia tolerance, interestingly it decreased pressure generation capacity and slowed pressure decreasing capacity in the mouse hearts ex vivo. This reduction in pressure generation rate could be linked to the level of channel proteins in sarcolemmal Ca2+-cycling in trained mice. However, the Ca2+-channel levels did not differ significantly between the groups, and thus, the level of calcium channels cannot fully explain all the functional alterations, despite the detected correlations. Therefore, additional studies are warranted to reveal further mechanisms that contribute to the reduced intrinsic capacity for pressure production in trained mouse hearts.

Peptidylarginine deiminases (PAD) enzymes were initially characterized in uteri, but since then little research has examined their function in this tissue. PADs post-translationally convert arginine residues in target proteins to citrulline and are highly expressed in ovine caruncle epithelia and ovine uterine luminal epithelial (OLE)-derived cell line. Progesterone (P4) not only maintains the uterine epithelia but also regulates the expression of endometrial genes that code for proteins that comprise the histotroph and are critical during early pregnancy. Given this, we tested whether P4 stimulates PAD-catalyzed histone citrullination to epigenetically regulate expression of the histotroph gene insulin-like growth factor binding protein 1 (IGFBP1) in OLE cells. 100 nM P4 significantly increases IGFBP1 mRNA expression; however, this increase is attenuated by pre-treating OLE cells with 100 nM progesterone receptor antagonist RU486 or 2 µM of a pan-PAD inhibitor. P4 treatment of OLE cells also stimulates citrullination of histone H3 arginine residues 2, 8, and 17 leading to enrichment of the ovine IGFBP1 gene promoter. Since PAD2 nuclear translocation and catalytic activity require calcium, we next investigated whether P4 triggers calcium influx in OLE cells. OLE cells were pre-treated with 10 nM nicardipine, an L-type calcium channel blocker, followed by stimulation with P4. Using fura2-AM imaging, we found that P4 initiates a rapid calcium influx through L-type calcium channels in OLE cells. Furthermore, this influx is necessary for PAD2 nuclear translocation and resulting citrullination of histone H3 arginine residues 2, 8, and 17. Our work suggests that P4 stimulates rapid calcium influx through L-type calcium channels initiating PAD-catalyzed histone citrullination and an increase in IGFBP1 expression.

SummaryIn this work, the presence of calcium-dependent calcium channels and their receptors (RyR) has been investigated in Paracentrotus lividus eggs and early embryos, from unfertilized egg to four-blastomere stages. Electrophysiological recordings of RyR single-channel current fluctuations showed that RyRs are functional during the first developmental events with a maximum at zygote stage, c. 40 min after fertilization, corresponding to the first cleavage. The nature of vertebrate-like RyRs active at this stage was established by specific activation/blockade experiments.

Light activation of the classical light-sensing retinal neurons, the photoreceptors, results in a graded change in membrane potential that ultimately leads to a reduction in neurotransmitter release to the post-synaptic retinal neurons. Photoreceptors show striking powers of adaptation, and for visual processing to function optimally, they must adjust their gain to remain responsive to different levels of ambient light intensity. The presence of a tightly controlled balance of inward and outward currents modulated by several different types of ion channels is what gives photoreceptors their remarkably dynamic operating range. Part of the resetting and modulation of this operating range is controlled by potassium and calcium voltage-gated channels, which are involved in setting the dark resting potential and synapse signal processing, respectively. Their essential contribution to visual processing is further confirmed in patients suffering from cone dystrophy with supernormal rod response (CDSRR) and congenital stationary night blindness type 2 (CSNB2), both conditions that lead to irreversible vision loss. This review will discuss these two types of voltage-gated ion channels present in photoreceptors, focussing on their structure and physiology, and their role in visual processing. It will also discuss the use and benefits of knockout mouse models to further study the function of these channels and what routes to potential treatments could be applied for CDSRR and CSNB2.

The neuronal-type (N-type) voltage-gated calcium (Cav) channels, which are designated Cav2.2, have an important role in the release of neurotransmitters1-3. Ziconotide is a Cav2.2-specific peptide pore blocker that has been clinically used for treating intractable pain4-6. Here we present cryo-electron microscopy structures of human Cav2.2 (comprising the core α1 and the ancillary α2δ-1 and β3 subunits) in the presence or absence of ziconotide. Ziconotide is thoroughly coordinated by helices P1 and P2, which support the selectivity filter, and the extracellular loops (ECLs) in repeats II, III and IV of α1. To accommodate ziconotide, the ECL of repeat III and α2δ-1 have to tilt upward concertedly. Three of the voltage-sensing domains (VSDs) are in a depolarized state, whereas the VSD of repeat II exhibits a down conformation that is stabilized by Cav2-unique intracellular segments and a phosphatidylinositol 4,5-bisphosphate molecule. Our studies reveal the molecular basis for Cav2.2-specific pore blocking by ziconotide and establish the framework for investigating electromechanical coupling in Cav channels.

Retigabine (RTG, Ezogabine, DC23129) is the first neuronal potassium channel opener in the treatment of epilepsy and exerts its effects through the activation of neuronal KCNQ2/3 potassium channels; in higher doses, it acts also on sodium and voltage-gated calcium channels. The aim of this study was to investigate possible age-dependent therapeutic effects of RTG on spike-and-wave discharges (SWD) in an animal model of absence epilepsy using WAG/Rij rats. In this study, 6- and 12-month-old WAG/Rij rats were used. For both age categories, three sub-groups that consisted of one control group (n=7) by the administration of 20% DMSO (control) and two study groups by the administration of 5 mg/kg (n=7) and 15 mg/kg RTG (n=7) were designed. EEG electrodes were placed onto the skull of anaesthetized animals; and baseline EEG was recorded for one hour after a recovery period from surgery. Then, the pre-determined two distinct doses of RTG and 20% DMSO were administered as a solvent via intraperitoneal injections, and EEG was recorded for 3 hours. After injection, both doses of RTG increased the total SWD number and duration of SWD in the first and second hours in 12-month-old rats. These parameters were elevated compared to 6-month-old rats. Age-dependent effects of RTG were observed in SWD activity. Pro-epileptic effects in middle-aged WAG/Rij rats were demonstrated in both RTG doses. Differences in the distribution of KCNQ2/3 channels and switch of GABAergic system from inhibitory to excitatory with age might contribute to increased SWD activity in middle-aged rats.

T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here, we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy, the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analysed possible effects on Cav3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with Cav3.3-I860N showing the strongest and Cav3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in Cav3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the Cav3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the Cav3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Cav3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Cav3.3 to slow oscillations with Cav3.3-I860N and an intermediate firing mode with Cav3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy., T-type calcium channels regulate neuronal firing, are implicated in brain disease and are targets for anti-epileptic drugs. El Ghaleb et al. identify heterozygous missense variants of CACNA1I associated with variable neurodevelopmental disorders and epilepsy, and show how gain-of-function mutations cause hyper-excitability.

T-type calcium channels are important regulators of neuronal excitability. The mammalian brain expresses three T-type channel isoforms (Cav3.1, Cav3.2 and Cav3.3) with distinct biophysical properties that are critically regulated by temperature. Here, we test the effects of how temperature affects spike output in a reduced firing neuron model expressing specific Cav3 channel isoforms. The modeling data revealed only a minimal effect on baseline spontaneous firing near rest, but a dramatic increase in rebound burst discharge frequency for Cav3.1 compared to Cav3.2 or Cav3.3 due to differences in window current or activation/recovery time constants. The reduced response by Cav3.2 could optimize its activity where it is expressed in peripheral tissues more subject to temperature variations than Cav3.1 or Cav3.3 channels expressed prominently in the brain. These tests thus reveal that aspects of neuronal firing behavior are critically dependent on both temperature and T-type calcium channel subtype. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00813-7.