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Stimulation of the dorsal half of the rat periaqueductal gray (DPAG) with 60-Hz pulses of increasing intensity, 30-μA pulses of increasing frequency, or increasing doses of an excitatory amino acid elicits sequential defensive responses of exophthalmia, immobility, trotting, galloping, and jumping. These responses may be controlled by voltage-gated calcium channel-specific firing patterns. Indeed, a previous study showed that microinjection of the DPAG with 15 nmol of verapamil, a putative blocker of L-type calcium channels, attenuated all defensive responses to electrical stimulation at the same site as the injection. Accordingly, here we investigated the effects of microinjection of lower doses (0.7 and 7 nmol) of both verapamil and mibefradil, a preferential blocker of T-type calcium channels, on DPAG-evoked defensive behaviors of the male rat. Behaviors were recorded either 24 h before or 10 min, 24 h, and 48 h after microinjection. Effects were analyzed by both threshold logistic analysis and repeated measures analysis of variance for treatment by session interactions. Data showed that the electrodes were all located within the dorsolateral PAG. Compared to the effects of saline, verapamil significantly attenuated exophthalmia, immobility, and trotting. Mibefradil significantly attenuated exophthalmia and marginally attenuated immobility while facilitating trotting. While galloping was not attenuated by either antagonist, jumping was unexpectedly attenuated by 0.7 nmol verapamil only. These results suggest that T-type calcium channels are involved in the low-threshold freezing responses of exophthalmia and immobility, whereas L-type calcium channels are involved in the trotting response that precedes the full-fledged escape responses of galloping and jumping., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Reactive sulfur species including sulfides, polysulfides and cysteine hydropersulfide play extensive roles in health and disease, which involve modification of protein functions through the interaction with metals bound to the proteins, cleavage of cysteine disulfide (S-S) bonds and S-persulfidation of cysteine residues. Sulfides over a wide micromolar concentration range enhance the activity of Ca v 3.2 T-type Ca 2+ channels by eliminating Zn 2+ bound to the channels, thereby promoting somatic and visceral pain. Ca v 3.2 is under inhibition by Zn 2+ in physiological conditions, so that sulfides function to reboot Ca v 3.2 from Zn 2+ inhibition and increase the excitability of nociceptors. On the other hand, polysulfides generated from sulfides activate TRPA1 channels via cysteine S-persulfidation, thereby facilitating somatic, but not visceral, pain. Thus, Ca v 3.2 function enhancement by sulfides and TRPA1 activation by polysulfides, synergistically accelerate somatic pain signals. The increased activity of the sulfide/Ca v 3.2 system, in particular, appears to have a great impact on pathological pain, and may thus serve as a therapeutic target for treatment of neuropathic and inflammatory pain including visceral pain., (Copyright © 2024 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Aim: Bradyarrhythmias result from inhibition of automaticity, prolonged repolarization, or slow conduction in the heart. The ERG channels mediate the repolarizing current I Kr in the cardiac action potential, whereas T-type calcium channels (TTCC) are involved in the sinoatrial pacemaker and atrioventricular conduction in mammals. Zebrafish have become a valuable research model for human cardiac electrophysiology and disease. Here, we investigate the contribution of ERG channels and TTCCs to the pacemaker and atrioventricular conduction in zebrafish larvae and determine the mechanisms causing atrioventricular block., Methods: Zebrafish larvae expressing ratiometric fluorescent Ca 2+ biosensors in the heart were used to measure Ca 2+ levels and rhythm in beating hearts in vivo, concurrently with contraction and hemodynamics. The atrioventricular delay (the time between the start of atrial and ventricular Ca 2+ transients) was used to measure impulse conduction velocity and distinguished between slow conduction and prolonged refractoriness as the cause of the conduction block., Results: ERG blockers caused bradycardia and atrioventricular block by prolonging the refractory period in the atrioventricular canal and in working ventricular myocytes. In contrast, inhibition of TTCCs caused bradycardia and second-degree block (Mobitz type I) by slowing atrioventricular conduction. TTCC block did not affect ventricular contractility, despite being highly expressed in cardiomyocytes. Concomitant measurement of Ca 2+ levels and ventricular size showed mechano-mechanical coupling: increased preload resulted in a stronger heart contraction in vivo., Conclusion: ERG channels and TTCCs influence the heart rate and atrioventricular conduction in zebrafish larvae. The zebrafish lines expressing Ca 2+ biosensors in the heart allow us to investigate physiological feedback mechanisms and complex arrhythmias., (© 2023 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Alzheimer's disease (AD) is the most common dementia in the world characterized by the neuropathological hallmarks consisting of an accumulation of extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles (NFT). There is no fundamental therapeutic treatment. We have developed a novel AD therapeutic candidate SAK3 which improves neuronal plasticity in the brain. SAK3 enhanced the acetylcholine release via T-type calcium channels. T-type calcium channels is highly expressed in neuro-progenitor cells in the hippocampal dentate gyrus. SAK3 enhanced the proliferation and differentiation of the neuro-progenitor cells, thereby improving depressive behaviors. The Cav3.1 null mice impaired the proliferation and differentiation of the neuro-progenitor cells. In addition, SAK3 activated CaMKII involving neuronal plasticity, thereby improving spine regeneration and proteasome activities impaired in AD related App NL-F/NL-F knock-in mice. The improvement of the decreased proteasome activity through enhancement CaMKII/Rpt6 signaling by SAK3 treatment contributed to the amelioration of synaptic abnormalities and cognitive decline. The increased proteasome activity also accounted for inhibition of Aβ deposition. Taken together, the proteasome activation via enhancement of CaMKII/Rpt6 signaling is a new strategy for AD treatment, which rescues the AD pathology including cognitive impairments and Aβ deposition. SAK3 may be a new hopeful drug candidate rescuing dementia patients.

Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132), an organogermanium, is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP) in aqueous solutions, and reduces inflammation, pain and cancer, whereas the underlying mechanisms remain unknown. Sulfides including H 2 S, a gasotransmitter, generated from l-cysteine by some enzymes including cystathionine-γ-lyase (CSE), are pro-nociceptive, since they enhance Ca v 3.2 T-type Ca 2+ channel activity expressed in the primary afferents, most probably by canceling the channel inhibition by Zn 2+ linked via coordinate bonding to His 191 of Ca v 3.2. Given that germanium is reactive to sulfur, we tested whether THGP would directly trap sulfide, and inhibit sulfide-induced enhancement of Ca v 3.2 activity and sulfide-dependent pain in mice. Using mass spectrometry and 1 H NMR techniques, we demonstrated that THGP directly reacted with sulfides including Na 2 S and NaSH, and formed a sulfur-containing reaction product, which decreased in the presence of ZnCl 2 . In Ca v 3.2-transfected HEK293 cells, THGP inhibited the sulfide-induced enhancement of T-type Ca 2+ channel-dependent membrane currents. In mice, THGP, administered systemically or locally, inhibited the mechanical allodynia caused by intraplantar Na 2 S. In the mice with cyclophosphamide-induced cystitis and cerulein-induced pancreatitis, which exhibited upregulation of CSE in the bladder and pancreas, respectively, systemic administration of THGP as well as a selective T-type Ca 2+ channel inhibitor suppressed the cystitis-related and pancreatitis-related visceral pain. These data suggest that THGP traps sulfide and inhibits sulfide-induced enhancement of Ca v 3.2 activity, leading to suppression of Ca v 3.2-dependent pain caused by sulfide applied exogenously and generated endogenously., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Purpose: T-type Ca channels (I Ca ) regulate neuronal excitability and contribute to neurotransmitter release. The phytocannabinoids Δ 9 -tetrahydrocannabinol and cannabidiol effectively modulate T-type I Ca , but effects of other biologically active phytocannabinoids on these channels are unknown. We thus investigated the modulation of T-type I Ca by low abundance phytocannabinoids., Experimental Approach: A fluorometric (fluorescence imaging plate reader [FLIPR]) assay was used to investigate modulation of human T-type I Ca (Ca V 3.1, 3.2 and 3.3) stably expressed in FlpIn-TREx HEK293 cells. The biophysical effects of some compounds were examined using whole-cell patch clamp recordings., Key Results: In the FLIPR assay, all 11 phytocannabinoids tested modulated T-type I Ca , with most inhibiting Ca V 3.1 and Ca V 3.2 more effectively than Ca V 3.3. Cannabigerolic acid was the most potent inhibitor of Ca V 3.1 (pIC 50 6.1 ± 0.6) and Ca V 3.2 (pIC 50 6.4 ± 0.4); in all cases, phytocannabinoid acids were more potent than their corresponding neutral forms. In patch clamp recordings, cannabigerolic acid inhibited Ca V 3.1 and 3.2 with similar potency to the FLIPR assay; the inhibition was associated with significant hyperpolarizing shift in activation and steady-state inactivation of these channels. In contrast, cannabidiol, cannabidivarin, and cannabigerol only affected channel inactivation., Conclusion and Implications: Modulation of T-type calcium channels is a common property of phytocannabinoids, which all increase steady-state inactivation at physiological membrane potentials, with some also affecting channel activation. Thus, T-type I Ca may be a common site of action for phytocannabinoids, and the diverse actions of phytocannabinoids on channel gating may provide insight into structural requirement for selective T-type I Ca modulators., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Conotoxins are tools used by marine Conus snails to hunt and are a significant repository for marine drug research. Conotoxins highly selectively coordinate different subtypes of various ion channels, and a few have been used in pain management. Although more than 8000 conotoxin genes have been found, the biological activity and function of most have not yet been examined. In this report, we selected the toxin gene QcMNCL-XIII0.1 from our previous investigation and studied it in vitro. First, we successfully prepared active recombinant QcMNCL-XIII0.1 using a TrxA (Thioredoxin A)-assisted folding expression vector based on genetic engineering technology. Animal experiments showed that the recombinant QcMNCL-XIII0.1 exhibited nerve conduction inhibition similar to that of pethidine hydrochloride. With flow cytometry combined fluorescent probe Fluo-4 AM, we found that 10 ng/μL recombinant QcMNCL-XIII0.1 inhibited the fluorescence intensity by 31.07% in the 293T cell model transfected with Cav3.1, implying an interaction between α1G T-type calcium channel protein and recombinant QcMNCL-XIII0.1. This toxin could be an important drug in biomedical research and medicine for pain control.

The CACNA1H gene encodes the α1 subunit of the low voltage-activated Ca v 3.2 T-type calcium channel, an important regulator of neuronal excitability. Alternative mRNA splicing can generate multiple channel variants with distinct biophysical properties and expression patterns. Two major splice variants, containing or lacking exon 26 (± 26) have been found in different human tissues. In this study, we report splice variant specific effects of a Ca v 3.2 mutation found in patients with autosomal dominant writer's cramp, a specific type of focal dystonia. We had previously reported that the R481C missense mutation caused a gain of function effect when expressed in Ca v 3.2 (+ 26) by accelerating its recovery from inactivation. Here, we show that when the mutation is expressed in the short variant of the channel (- 26), we observe a significant increase in current density when compared to wild-type Ca v 3.2 (- 26) but the effect on the recovery from inactivation is lost. Our data add to growing evidence that the functional expression of calcium channel mutations depends on which splice variant is being examined., (© 2021. The Author(s).)

Background: Neuromedin B (Nmb) is implicated in the regulation of nociception of sensory neurons. However, the underlying cellular and molecular mechanisms remain unknown. Methods: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of Nmb on the sensory neuronal excitability and peripheral pain sensitivity mediated by Cav3.2 T-type channels. Results: Nmb reversibly and concentration-dependently increased T-type channel currents ( I T ) in small-sized trigeminal ganglion (TG) neurons through the activation of neuromedin B receptor (NmbR). This NmbR-mediated I T response was G q protein-coupled, but independent of protein kinase C activity. Either intracellular application of the QEHA peptide or shRNA-mediated knockdown of G β abolished the NmbR-induced I T response. Inhibition of protein kinase A (PKA) or AMP-activated protein kinase (AMPK) completely abolished the Nmb-induced I T response. Analysis of phospho-AMPK ( p -AMPK) revealed that Nmb significantly activated AMPK, while AMPK inhibition prevented the Nmb-induced increase in PKA activity. In a heterologous expression system, activation of NmbR significantly enhanced the Cav3.2 channel currents, while the Cav3.1 and Cav3.3 channel currents remained unaffected. Nmb induced TG neuronal hyperexcitability and concomitantly induced mechanical and thermal hypersensitivity, both of which were attenuated by T-type channel blockade. Moreover, blockade of NmbR signalling prevented mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain, and this effect was attenuated by siRNA knockdown of Cav3.2. Conclusions: Our study reveals a novel mechanism by which NmbR stimulates Cav3.2 channels through a G βγ -dependent AMPK/PKA pathway. In mouse models, this mechanism appears to drive the hyperexcitability of TG neurons and induce pain hypersensitivity., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Developmental and epileptic encephalopathies (DEEs) are a group of severe epilepsies that are characterized by seizures and developmental delay. DEEs are primarily attributed to genetic causes and an increasing number of cases have been correlated with variants in ion channel genes. In this study, we report a child with an early severe DEE. Whole exome sequencing showed a de novo heterozygous variant (c.4873-4881 duplication) in the SCN8A gene and an inherited heterozygous variant (c.952G > A) in the CACNA1H gene encoding for Na v 1.6 voltage-gated sodium and Ca v 3.2 voltage-gated calcium channels, respectively. In vitro functional analysis of human Na v 1.6 and Ca v 3.2 channel variants revealed mild but significant alterations of their gating properties that were in general consistent with a gain- and loss-of-channel function, respectively. Although additional studies will be required to confirm the actual pathogenic involvement of SCN8A and CACNA1H, these findings add to the notion that rare ion channel variants may contribute to the etiology of DEEs., (© 2021. The Author(s).)

T-type calcium channels regulate neuronal excitability and are important contributors of pain processing. CaV3.2 channels are the major isoform expressed in nonpeptidergic and peptidergic nociceptive neurons and are emerging as promising targets for pain treatment. Numerous studies have shown that CaV3.2 expression and/or activity are significantly increased in spinal dorsal horn and in dorsal root ganglia neurons in different inflammatory and neuropathic pain models. Pharmacological campaigns to inhibit the functional expression of CaV3.2 for treatment of pain have focused on the development of direct channel blockers, but none have produced lead candidates. Targeting the proteins that regulate the trafficking or transcription, and the ones that modify the channels via post-translational modifications are alternative means to regulate expression and function of CaV3.2 channels and hence to develop new drugs to control pain. Here we synthesize data supporting a role for CaV3.2 in numerous pain modalities and then discuss emerging opportunities for the indirect targeting of CaV3.2 channels., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A family of current-time curves of T-type Ca v 3.1 Ca 2+ channels available in the literature is simulated by a kinetic model differing from that used for the interpretation of all salient features of Na + and Shaker K + channels by the insertion of a multiplying factor expressing the difference between the working potential ϕ and the reversal potential ϕ r . This deterministic model is also used to simulate experimental curves taken from the literature for steady-state 'fast inactivation' and for a gradual passage from fast to 'slow inactivation'. A depolarizing pulse induces fast or slow inactivation depending on whether it lasts 100-500 ms or about 1 min, and is believed to cause a collapse of the central pore near the selectivity filter (SF). A number of features of fast and slow inactivation of Ca v 3.1 Ca 2+ channels are qualitatively interpreted on the basis of a sequence of conformational states. Briefly, the conformation responsible for 'fast inactivation' is assumed to have the activation gate open and the inactivation gate (i.e., the SF) inactive. Immediately after a depolarizing pulse, this conformation is inactive and requires a sufficiently long rest time at a far negative holding potential to recover from inactivation. 'Slow inactivation' is ascribed to a different conformation with the activation gate closed and the SF inactive., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aims: Despite the advanced cancer treatments, there is increased resistance to chemotherapy and subsequent mortality. In lack of reliable data in monolayer cultures and animal models, researchers are shifting to 3D cancer spheroids, which represents the in vivo robust tumour morphology. Calcium is essential in cell signalling and proliferation. It is found that T-type calcium channels (TTCCs) are overexpressed in various cancer cells, supporting their increased proliferation. Many of the TTCCs blockers available could target other channels besides TTCCs, which can cause adverse effects. Therefore, we hypothesise that TTA-A2, a highly selective blocker towards TTCCs, can inhibit the growth of cancer spheroids, and provide an anti-cancer and an adjuvant role in cancer therapy., Methods: We studied TTA-A2 and paclitaxel (PTX-control drug) in lung adenocarcinoma cell line- A549, cancer cells and human embryonic kidney cell line- HEK 293, control cell, in their monolayer and spheroids forms for viability, proliferation, morphology change, migration, and invasion-after 48-96 h of treatment., Key Findings: Though the results varied between the monolayer and spheroids studies, we found both anti-cancer as well as adjuvant effect of TTA-A2 in both the studies. TTA-A2 was able to inhibit the growth, viability, and metastasis of the cancer cells and spheroids. Differences in the results of two modes might explain that why drugs tested successfully in monolayer culture fail in clinical trials., Significance: This study establishes the role of TTA-A2, a potent TTCC blocker as an anti-cancer and adjuvant drug in reducing the viability and metastasis of the cancer cells., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The mating of 77 heterozygous pairs (Ca v 3.2[+|-] x Ca v 3.2[+|-]) revealed a significant deviation of genotype distribution from Mendelian inheritance in weaned pups. The mating of 14 pairs (Ca v 3.2[-|-] female x Ca v 3.2[+|-] male) and 8 pairs (Ca v 3.2[+|-] female x Ca v 3.2[-|-] male) confirmed the significant reduction of deficient homozygous Ca v 3.2[-|-] pups, leading to the conclusion that prenatal lethality may occur, when one or both alleles, encoding the Ca v 3.2T-type Ca 2+ channel, are missing. Also, the mating of 63 heterozygous pairs (Ca v 2.3[+|-] x Ca v 2.3[+|-]) revealed a significant deviation of genotype distribution from Mendelian inheritance in weaned pups, but only for heterozygous male mice, leading to the conclusion that compensation may only occur for Ca v 2.3[-|-] male mice lacking both alleles of the R-type Ca 2+ channel. During the mating of heterozygous parents, the number of female mice within the weaned population does not deviate from the expected Mendelian inheritance. During prenatal development, both, T- and R-type Ca 2+ currents are higher expressed in some tissues than postnatally. It will be discussed that the function of voltage-gated Ca 2+ channels during prenatal development must be investigated in more detail, not least to understand devastative diseases like developmental epileptic encephalopathies (DEE).

Loss of T-type calcium channel (TCC) function has been reported to result in decreased cell viability and impaired muscle regeneration, but the underlying mechanisms remain largely unknown. We previously found that expression of TCC is reduced in aged pelvic floor muscle of multiple vaginal delivery mice, and this is related to endoplasmic reticulum stress (ERS) activation and autophagy flux blockade. In the present work, we further investigated the effects of TCC function loss on C2C12 myotubes and skeletal muscle, which is mediated by promotion of ERS and ultimately contributes to mitochondrial-related apoptotic cell death. We found that application of a TCC inhibitor induced mitochondria-related apoptosis in a dose-dependent manner and also reduced mitochondrial transmembrane potential (MMP), induced mito-ROS generation, and enhanced expression of mitochondrial apoptosis proteins. Functional inhibition of TCC induced ERS, resulting in disorder of Ca 2+ homeostasis in endoplasmic reticulum, and ultimately leading to cell apoptosis in C2C12 myotubes. Tibialis anterior muscles of T-type α1H channel knockout mice displayed a smaller skeletal muscle fiber size and elevated ERS-mediated apoptosis signaling. Our data point to a novel mechanism whereby TCC blockade leads to ERS activation and terminal mitochondrial-related apoptotic events in C2C12 myotubes and skeletal muscles., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

In the current study, we sought to investigate whether T-type Ca 2+ channels (TCCs) in the brain are involved in generating post-anesthetic hyperexcitatory behaviors (PAHBs). We found that younger rat pups (postnatal days 9-11) had a higher incidence of PAHBs and higher PAHB scores than older pups (postnatal days 16-18) during emergence from sevoflurane anesthesia. The power spectrum of the theta oscillations (4 Hz-8 Hz) in the prefrontal cortex was significantly enhanced in younger pups when PAHBs occurred, while there were no significant changes in older pups. Both the power of theta oscillations and the level of PAHBs were significantly reduced by the administration of TCC inhibitors. Moreover, the sensitivity of TCCs in the medial dorsal thalamic nucleus to sevoflurane was found to increase with age by investigating the kinetic properties of TCCs in vitro. TCCs were activated by potentiated GABAergic depolarization with a sub-anesthetic dose of sevoflurane (1%). These data suggest that (1) TCCs in the brain contribute to the generation of PAHBs and the concomitant electroencephalographic changes; (2) the stronger inhibitory effect of sevoflurane contributes to the lack of PAHBs in older rats; and (3) the contribution of TCCs to PAHBs is not mediated by a direct effect of sevoflurane on TCCs.

Neocortical GABAergic interneurons expressing vasoactive intestinal polypeptide (VIP) contribute to sensory processing, sensorimotor integration, and behavioral control. In contrast to other major subpopulations of GABAergic interneurons, VIP neurons show a remarkable diversity. Studying morphological and electrophysiological properties of VIP cells, we found a peculiar group of neurons in layer II/III of mouse primary somatosensory (barrel) cortex, which showed a highly dynamic burst firing behavior at resting membrane potential that switched to tonic mode at depolarized membrane potentials. Furthermore, we demonstrate that burst firing depends on T-type calcium channels. The burst-tonic switch could be induced by acetylcholine (ACh) and serotonin. ACh mediated a depolarization via nicotinic receptors whereas serotonin evoked a biphasic depolarization via ionotropic and metabotropic receptors in 48% of the population and a purely monophasic depolarization via metabotropic receptors in the remaining cells. These data disclose an electrophysiologically defined subpopulation of VIP neurons that via neuromodulator-induced changes in firing behavior is likely to regulate the state of cortical circuits in a profound manner., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In mouse cerebellar Purkinje neurons (PNs), the climbing fiber (CF) input provides a signal to parallel fiber (PF) synapses, triggering PF synaptic plasticity. This signal is given by supralinear Ca 2+ transients, associated with the CF synaptic potential and colocalized with the PF Ca 2+ influx, occurring only when PF activity precedes the CF input. Here, we unravel the biophysical determinants of supralinear Ca 2+ signals associated with paired PF-CF synaptic activity. We used membrane potential ( V m ) and Ca 2+ imaging to investigate the local CF-associated Ca 2+ influx following a train of PF synaptic potentials in two cases: (1) when the dendritic V m is hyperpolarized below the resting V m , and (2) when the dendritic V m is at rest. We found that supralinear Ca 2+ signals are mediated by type-1 metabotropic glutamate receptors (mGluR1s) when the CF input is delayed by 100-150 ms from the first PF input in both cases. When the dendrite is hyperpolarized only, however, mGluR1s boost neighboring T-type channels, providing a mechanism for local coincident detection of PF-CF activity. The resulting Ca 2+ elevation is locally amplified by saturation of endogenous Ca 2+ buffers produced by the PF-associated Ca 2+ influx via the mGluR1-mediated nonselective cation conductance. In contrast, when the dendritic V m is at rest, mGluR1s increase dendritic excitability by inactivating A-type K + channels, but this phenomenon is not restricted to the activated PF synapses. Thus, V m is likely a crucial parameter in determining PF synaptic plasticity, and the occurrence of hyperpolarization episodes is expected to play an important role in motor learning. SIGNIFICANCE STATEMENT In Purkinje neurons, parallel fiber synaptic plasticity, determined by coincident activation of the climbing fiber input, underlies cerebellar learning. We unravel the biophysical mechanisms allowing the CF input to produce a local Ca 2+ signal exclusively at the sites of activated parallel fibers. We show that when the membrane potential is hyperpolarized with respect to the resting membrane potential, type-1 metabotropic glutamate receptors locally enhance Ca 2+ influx mediated by T-type Ca 2+ channels, and that this signal is amplified by saturation of endogenous buffer also mediated by the same receptors. The combination of these two mechanisms is therefore capable of producing a Ca 2+ signal at the activated parallel fiber sites, suggesting a role of Purkinje neuron membrane potential in cerebellar learning., (Copyright © 2020 the authors.)

T-type calcium channels are low-threshold voltage-gated calcium channel and characterized by unique electrophysiological properties such as fast inactivation and slow deactivation kinetics. All subtypes of T-type calcium channel (Cav3.1, 3.2 and 3.3) are widely expressed in the central nerve system, and they have an important role in homeostasis of sleep, pain response, and development of epilepsy. Recently, several reports suggest that T-type calcium channels may mediate neuronal plasticity in the mouse brain. We succeeded to develop T-type calcium channel enhancer ethyl 8'-methyl-2',4-dioxo-2-(piperidin-1-yl)-2'H-spiro[cyclopentane-1,3'-imidazo[1,2-a]pyridine]-2-ene-3-carboxylate (SAK3) which enhances Cav3.1 and 3.3 currents in each-channel expressed neuro2A cells. SAK3 can promote acetylcholine (ACh) release in the mouse hippocampus via enhancing T-type calcium channel. In this review, we have introduced the role of T-type calcium channel, especially Cav3.1 channel in the mouse hippocampus based on our previous data using SAK3 and Cav3.1 knockout mice.

Although the central medial nucleus (CeM) of the thalamus is an essential part of the arousal system for sleep and anesthesia initiation, the precise mechanisms that regulate its activity are not well studied. We examined the role of CaV3.1 isoform of T-type calcium channels (T-channels) in the excitability and rhythmic activity of CeM neurons during isoflurane (ISO)-induced anesthesia by using mouse genetics and selective pharmacology. Patch-clamp recordings taken from acute brain slices revealed that CaV3.1 channels in CeM are inhibited by prototypical volatile anesthetic ISO (250 and 500 μM) and selective T-channels blocker 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2). Both TTA-P2 and ISO attenuated tonic and burst firing modes, and hyperpolarized CeM neurons from wild type (WT) mice. These effects were greatly diminished or abolished in CaV3.1 null mice. Our ensuing in vivo local field potential (LFP) recordings from CeM indicated that the ability of TTA-P2 and anesthetic concentrations of ISO to promote δ oscillation was substantially weakened in CaV3.1 null mice. Furthermore, escalating ISO concentrations induced stronger burst-suppression LFP pattern in mutant than in WT mice. Our results demonstrate for the first time the importance of CaV3.1 channels in thalamocortical oscillations from the non-specific thalamic nuclei that underlie clinically important effects of ISO., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Neuromuscular disorders encompass a wide range of conditions often associated with a genetic component. In the present study, we report a patient with severe infantile-onset amyotrophy in whom two compound heterozygous variants in the gene CACNA1H encoding for Ca v 3.2 T-type calcium channels were identified. Functional analysis of Ca v 3.2 variants revealed several alterations of the gating properties of the channel that were in general consistent with a loss-of-channel function. Taken together, these findings suggest that severe congenital amyoplasia may be related to CACNA1H and would represent a new phenotype associated with mutations in this gene.

Although brain-derived neurotrophic factor (BDNF) is implicated in the nociceptive signaling of peripheral sensory neurons, the underlying mechanisms remain largely unknown. Here, we elucidated the effects of BDNF on the neuronal excitability of trigeminal ganglion (TG) neurons and the pain sensitivity of rats mediated by T-type Ca 2+ channels. BDNF reversibly and dose-dependently enhanced T-type channel currents through the activation of tropomyosin receptor kinase B (TrkB). Antagonism of phosphatidylinositol 3-kinase (PI3K) but not of its downstream target, the kinase AKT, abolished the BDNF-induced T-type channel response. BDNF application activated p38 mitogen-activated protein kinase (MAPK), and this effect was prevented by inhibition of PI3K but not of protein kinase A (PKA). Antagonism of either PI3K or p38 MAPK prevented the BDNF-induced stimulation of PKA activity, whereas PKA inhibition blocked the BDNF-mediated increase in T-type currents. BDNF increased the rate of action potential firing in TG neurons and enhanced the pain sensitivity of rats to mechanical stimuli. Moreover, inhibition of TrkB signaling abolished the increased mechanical sensitivity in a rat model of chronic inflammatory pain, and this effect was attenuated by either T-type channel blockade or knockdown of the channel Ca v 3.2. Together, our findings indicate that BDNF enhances T-type currents through the stimulation of TrkB coupled to PI3K-p38-PKA signaling, thereby inducing neuronal hyperexcitability of TG neurons and pain hypersensitivity in rats., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Aim: The present study was designed to investigate the possible role of T-type Ca 2+ channels and HCN channels in the development of stress adaptation in cold-water immersion stress-subjected mice., Material and Methods: The mice were subjected to cold-water immersion stress by placing them individually in a water tank (depth = 15.5 cm; temperature = 15 ± 2 °C) for 5 min. The mice were subjected to single episode of cold-water immersion stress for inducing acute stress; while for inducing stress adaptation, mice were subjected to repeated episodes of homotypic stressor (5 min) for 5 consecutive days. Animals were administered with ethosuximide (100 and 200 mg/kg, i.p.) and ivabradine (5 and 10 mg/kg, i.p.) before subjecting them to stress for five days. The stress-related behavioral alterations were assessed using the actophotometer, the hole board, the open field and the social interaction tests. The plasma corticosterone levels were quantified as a biochemical parameter of hypothalamic-pituitary-adrenal (HPA) axis activation., Results: Acute stress altered the behavioral and biochemical parameters of the animals. However, repeated stress significantly restored the behavioral and biochemical alterations signifying the development of adaptation. Administration of ethosuximide and ivabradine abolished the restoration of behavioral and biochemical changes in the animals subjected to repeated stress., Conclusion: The ethosuximide and ivabradine mediated attenuation of stress adaptation demonstrates that the opening of T-type Ca 2+ channels and activation of HCN channels are involved in inducing stress adaptation in repeated stress-subjected animals., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Temporal lobe epilepsy (TLE) is a form of adult epilepsy involving the entorhinal cortex (EC). Layer II neurons of the medial EC (mEC) are spared and become hyperexcitable in TLE. Studies have suggested a role for T-type calcium channels (T-type Ca 2+ channels) in facilitating increases in neuronal activity associated with TLE within the hippocampus. We sought to determine if T-type Ca 2+ channels play a role in facilitating neuronal hyperexcitability of layer II mEC stellate neurons in TLE. TLE was induced in rats by electrical stimulation of the hippocampus to induce status epilepticus (SE). Brain slices were prepared from rats exhibiting spontaneous seizures and compared with age-matched control rats. Action potentials (APs) were evoked either by current injection steps or via presynaptic stimulation of mEC deep layers. The selective T-type Ca 2+ channel antagonist, TTA-P2 (1 μM), was applied to determine the role of T-type Ca 2+ channels in maintaining neuronal excitability. Quantitative PCR techniques were used to assess T-type Ca 2+ channel isoform mRNA levels within the mEC layer II. TLE mEC layer II stellate neurons were hyperexcitable compared to control neurons, evoking a higher frequency of APs and generating bursts of APs when synaptically stimulated. TTA-P2 (1 μM) reduced firing frequencies in TLE and control neurons and reduced AP burst firing in TLE stellate neurons. TTA-P2 had little effect on synaptically evoked AP's in control neurons. TTA-P2 also inhibited rebound APs evoked in TLE neurons to a greater degree than in control neurons. TLE tissue had almost a 3-fold increase in Ca v 3.1 mRNA compared to controls. Ca v 3.2 or Ca v 3.3 levels were unchanged. These findings support a role for T-type Ca 2+ channel in establishing neuronal hyperexcitability of mEC layer II stellate neurons in TLE. Increased expression of Ca v 3.1 may be important for establishing neuronal hyperexcitability of mEC layer II neurons in TLE., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Heat can trigger testicular damage and impair fertility. Leydig cells produce testosterone in response to stimulation by luteinizing hormone (LH), which induces Ca 2+ entry and K + efflux through ion channels in their plasma membrane. Considering that mechanisms coordinating the Leydig cell responses to hyperthermic stress remain unclear; the present study analyzed the effects of heat stress (HS, 43°C, 15 min) and inhibition of Hsp90 on T-type calcium currents and voltage-dependent potassium currents (VKC) in mice Leydig cells. Results show that HS reduced the VKC steady state currents at +80 mV (45.3%) and maximum conductance (71.5%), as well as increased the activation time constant (31.7%) and the voltage for which half the channels are open (30%). Hsp90 inhibition did not change the VKC currents. T-type calcium currents were not affected by HS or Hsp90 inhibition. In conclusion, HS can slow the activation, reduce the currents and voltage dependence of the VKC, suggesting a possible role of these currents in the response to hyperthermic stress in Leydig cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background and Purpose: T-type calcium (Ca v 3) and transient receptor potential vanilloid-1 (TRPV1) channels play central roles in the control of excitability in the peripheral nervous system and are regarded as potential therapeutic pain targets. Modulators that either activate or inhibit TRPV1-mediated currents display analgesic properties in various pain models despite opposing effects on their connate target, TRPV1. We explored the effects of TRPV1-active compounds on Ca v 3-mediated currents., Experimental Approach: Whole-cell patch clamp recordings were used to examine the effects of TRPV1-active compounds on rat dorsal root ganglion low voltage-activated calcium currents and recombinant Ca v 3 isoforms in expression systems., Key Results: The classical TRPV1 agonist capsaicin as well as TRPV1 antagonists A-889425, BCTC, and capsazepine directly inhibited Ca v 3 channels. These compounds altered the voltage-dependence of activation and inactivation of Ca v 3 channels and delayed their recovery from inactivation, leading to a concomitant decrease in T-type current availability. The TRPV1 antagonist capsazepine potently inhibited Ca v 3.1 and 3.2 channels (K D  < 120 nM), as demonstrated by its slow off rate. In contrast, neither the TRPV1 agonists, Palvanil and resiniferatoxin, nor the TRPV1 antagonist AMG9810 modulated Ca v 3-mediated currents., Conclusions and Implications: Analgesic TRPV1-active compounds inhibit Ca v 3 currents in native and heterologous systems. Hence, their analgesic effects may not be exclusively attributed to their actions on TRPV1, which has important implications in the current understanding of nociceptive pathways. Importantly, our results highlight the need for attention in the experimental design used to address the analgesic properties of Ca v 3 channel inhibitors., (© 2019 The British Pharmacological Society.)

We tested whether NNC 55-0396 (NNC), a T-type calcium channel (T-channel) blocker, reduces the brain injury caused by middle cerebral artery occlusion and reperfusion (MCAO/R) in mice. NNC, administered i.c.v. before the occlusion, greatly reduced the MCAO/R-induced brain infarct and neurological dysfunctions, although it, given toward the end of occlusion, was less effective. Systemic administration of NNC before the occlusion also attenuated the infarct and neurological dysfunctions. Our data imply that blood-brain-barrier-permeable T-channel blockers such as NNC are capable of reducing MCAO/R-induced brain damage, and that T-channels are involved in neuronal damage induced by ischemia rather than reperfusion., (Copyright © 2019 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Background and Purpose: Abdominal pain associated with low-grade inflammation is frequently encountered in irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) during remission. Current treatments are not very effective and new therapeutic approaches are needed. The role of Ca V 3.2 channels, which are important in other chronic pain contexts, was investigated in a murine model of colonic hypersensitivity (CHS) associated with low-grade inflammation., Experimental Approach: Low doses of dextran sulfate sodium (DSS; 0.5%) were chronically administered to C57BL/6j mice in drinking water. Their inflammatory state was assessed by systemic and local measures of IL-6, myeloperoxidase, and lipocalin-2 using elisa. Colonic sensitivity was evaluated by the visceromotor responses to colorectal distension. Functional involvement of Ca V 3.2 channels was assessed with different pharmacological (TTA-A2, ABT-639, and ethosuximide) and genetic tools., Key Results: DSS induced low-grade inflammation associated with CHS in mice. Genetic or pharmacological inhibition of Ca V 3.2 channels reduced CHS. Cav3.2 channel deletion in primary nociceptive neurons in dorsal root ganglia (Ca V 3.2 Nav1.8 KO mice) suppressed CHS. Spinal, but not systemic, administration of ABT-639, a peripherally acting T-type channel blocker, reduced CHS. ABT-639 given intrathecally to Ca V 3.2 Nav1.8 KO mice had no effect, demonstrating involvement of Ca V 3.2 channels located presynaptically in afferent fibre terminals. Finally, ethosuximide, which is a T-type channel blocker used clinically, reduced CHS., Conclusions and Implications: These results suggest that ethosuximide represents a promising drug reposition strategy and that inhibition of Ca V 3.2 channels is an attractive therapeutic approach for relieving CHS in IBS or IBD., (© 2019 The British Pharmacological Society.)

Objectives: Voltage-gated Ca 2+ channels (VGCCs) are of central relevance in regulating Ca 2+ influx into living cells. The low-voltage activated (LVA) Ca v 3 T-type Ca 2+ channels are widely distributed throughout the brain including the peripheral auditory system and ascending auditory tract. Their exact role in auditory information processing is still not fully understood. Within the LVA subgroup, Ca v 3.2 T-type Ca 2+ channels seem to be of special importance as qPCR revealed a steady increase in Ca v 3.2 transcript levels over age, e.g. in the cochlea and spiral ganglion neurons (SGN). Furthermore, pharmacological studies suggested an association between Ca v 3.2 expression and both age-related and noise-induced hearing loss. Given the potential functional relevance of Ca v 3.2 VGGCs in sensorineural hearing loss, we recorded gender specific auditory evoked brainstem responses (ABRs) upon both click and tone burst presentation. Here we present auditory brainstem response (ABR) data from Ca v 3.2 +/+ , Ca v 3.2 +/- and Ca v 3.2 -/- mice from both genders which are of value for researchers who want to evaluate how Ca v 3.2 loss affects basic auditory parameters, e.g. click and tone burst based hearing thresholds, amplitude growth function and peak latencies., Data Description: Information presented here includes ABR data from age-matched female and male Ca v 3.2 +/+ , Ca v 3.2 +/- and Ca v 3.2 -/- mice and technical aspects of the auditory recording protocol. Data were recorded using a commercially available ABR setup from Tucker Davis Technologies Inc. (TDT). Raw data files (arf.-file format) were exported as txt.-files with free access for analysis.

In cerebellar Purkinje neuron dendrites, the transient depolarization associated with a climbing fiber (CF) EPSP activates voltage-gated Ca 2+ channels (VGCCs), voltage-gated K + channels (VGKCs), and Ca 2+ -activated SK and BK K + channels. The resulting membrane potential ( V m ) and Ca 2+ transients play a fundamental role in dendritic integration and synaptic plasticity of parallel fiber inputs. Here we report a detailed investigation of the kinetics of dendritic Ca 2+ and K + channels activated by CF-EPSPs, based on optical measurements of V m and Ca 2+ transients and on a single-compartment NEURON model reproducing experimental data. We first measured V m and Ca 2+ transients associated with CF-EPSPs at different initial V m , and we analyzed the changes in the Ca 2+ transients produced by the block of each individual VGCCs, of A-type VGKCs and of SK and BK channels. Then, we constructed a model that includes six active ion channels to accurately match experimental signals and extract the physiological kinetics of each channel. We found that two different sets of channels are selectively activated. When the dendrite is hyperpolarized, CF-EPSPs mainly activate T-type VGCCs, SK channels, and A-type VGKCs that limit the transient V m ∼ <0 mV. In contrast, when the dendrite is depolarized, T-type VGCCs and A-type VGKCs are inactivated and CF-EPSPs activate P/Q-type VGCCs, high-voltage activated VGKCs, and BK channels, leading to Ca 2+ spikes. Thus, the potentially activity-dependent regulation of A-type VGKCs, controlling the activation of this second set of channels, is likely to play a crucial role in signal integration and plasticity in Purkinje neuron dendrites. SIGNIFICANCE STATEMENT The climbing fiber synaptic input transiently depolarizes the dendrite of cerebellar Purkinje neurons generating a signal that plays a fundamental role in dendritic integration. This signal is mediated by two types of Ca 2+ channels and four types of K + channels. Thus, understanding the kinetics of all of these channels is crucial for understanding PN function. To obtain this information, we used an innovative strategy that merges ultrafast optical membrane potential and Ca 2+ measurements, pharmacological analysis, and computational modeling. We found that, according to the initial membrane potential, the climbing fiber depolarizing transient activates two distinct sets of channels. Moreover, A-type K + channels limit the activation of P/Q-type Ca 2+ channels and associated K + channels, thus preventing the generation of Ca 2+ spikes., (Copyright © 2019 the authors 0270-6474/19/391969-13$15.00/0.)

Song learning in zebra finches ( Taeniopygia guttata ) requires exposure to the song of a tutor, resulting in an auditory memory. This memory is the foundation for later sensorimotor learning, resulting in the production of a copy of the tutor's song. The cortical premotor nucleus HVC (proper name) is necessary for auditory and sensorimotor learning as well as the eventual production of adult song. We recently discovered that the intrinsic physiology of HVC neurons changes across stages of song learning, but are those changes the result of learning or are they experience-independent developmental changes? To test the role of auditory experience in driving intrinsic changes, patch-clamp experiments were performed comparing HVC neurons in juvenile birds with varying amounts of tutor exposure. The intrinsic physiology of HVC neurons changed as a function of tutor exposure. Counterintuitively, tutor deprivation resulted in juvenile HVC neurons showing an adult-like phenotype not present in tutor-exposed juveniles. Biophysical models were developed to predict which ion channels were modulated by experience. The models indicate that tutor exposure transiently suppressed the I h and T-type Ca 2+ currents in HVC neurons that target the basal ganglia, whereas tutor exposure increased the resting membrane potential and decreased the spike amplitude in HVC neurons that drive singing. Our findings suggest that intrinsic plasticity may be part of the mechanism for auditory learning in the HVC. More broadly, models of learning and memory should consider intrinsic plasticity as a possible mechanism by which the nervous system encodes the lasting effects of experience. SIGNIFICANCE STATEMENT It is well established that learning involves plasticity of the synapses between neurons. However, the activity of a neural circuit can also be dramatically altered by changes in the intrinsic properties (ion channels) of the component neurons. The present experiments show experience-dependent changes in the intrinsic physiology of neurons in the cortical premotor nucleus HVC (proper name) in juvenile zebra finches ( Taeniopygia guttata ) during auditory learning of a tutor's song. Tutor deprivation does not "arrest" development of intrinsic properties, but rather results in neurons with a premature adult-like physiological phenotype. It is possible that auditory learning involves a form of nonsynaptic plasticity and that experience-dependent suppression of specific ion channels may work in concert with synaptic plasticity to promote vocal learning., (Copyright © 2019 the authors 0270-6474/19/391206-16$15.00/0.)

Low-threshold Ca 2+ spikes are mediated by T-type Ca 2+ channels, which have electrophysiological properties of fast inactivation and slow deactivation kinetics. A low membrane potential of approximately -60 mV is sufficient to trigger channel opening. We recently introduced a novel T-type Ca 2+ channel enhancer that improves cognition and inhibits amyloid beta aggregation in an Alzheimer's disease (AD) mouse model. The enhancer stimulates ACh release, Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) activity, and neurogenesis in the hippocampus. Then, we discuss how T-type Ca 2+ channel enhancer improves cognition and impaired neurogenesis and how CaMKII signaling in neurodegenerative diseases reduces amyloid beta aggregation. We provide a perspective of the potential AD therapies to target CaMKII signaling. In this context, we overview our attempts leading to the development of a T-type Ca 2+ channel enhancer as cognitive enhancer, the action of which has been associated with CaMKII and presumably proteasome activity., (Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Among voltage-gated Ca 2+ channels, T-type Ca 2+ channels, which are activated by low voltages, regulate neuronal excitability, spontaneous neurotransmitter release, hormone secretion, etc. and also participate in proliferation of distinct cancer cells. Among three isoforms of T-type Ca 2+ channels, Ca v 3.2 is detectable in 100% of biopsy samples from prostate cancer patients. In general, prostate cancer cells are highly sensitive to androgen deprivation therapy, but often acquire hormone-therapy resistance. The androgen deprivation may trigger neuroendocrine (NE)-like differentiation of some prostate cancer cells. We have analyzed the expression and function of Ca v 3.2 in human prostate cancer LNCaP cells during NE-like differentiation. NE-like LNCaP cells overexpress Ca v 3.2 through the CREB/Egr-1 pathway and also cystathionine-γ-lyase (CSE), which generates H 2 S that enhances the channel activity of Ca v 3.2. H 2 S generated by upregulated CSE appears to enhance the activity of upregulated Ca v 3.2 after the differentiation. The enhanced Ca v 3.2 activity in NE-like cells may contribute to increased secretion of mitogenic factors essential for androgen-independent proliferation of surrounding prostate cancer cells. It is known that increased extracellular glucose levels enhance Ca v 3.2 activity through asparagine (N)-linked glycosylation of Ca v 3.2, which might contribute to diabetic neuropathy. We then found that high glucose accelerates the enhanced channel function and overexpression of Ca v 3.2 in NE-like LNCaP cells, which might be associated with clinical evidence for diabetes-related poor prognosis of prostate cancer and development of hormone therapy resistance. Thus, Ca v 3.2 is considered to play a role in the pathophysiology of prostate cancer, and may serve as a therapeutic target.

Rationale: Currently available antipsychotics are unsatisfactory given their side effects and limited efficacy for the cognitive symptoms of schizophrenia. Many currently available drugs, such as haloperidol, are T-type calcium channel antagonists in addition to their well-established antagonism of dopamine D2 receptors. Thus, preclinical research into the effects of T-type calcium channel antagonists/blockers in behavioral assays related to schizophrenia may inform novel therapeutic strategies., Objectives: We explored the effects of a recently developed highly selective T-type calcium channel antagonist, Z944 (2.5, 5.0, 10.0 mg/kg), on the MK-801 (0.15 mg/kg) model of acute psychosis., Methods: To examine the effects of Z944 on behaviors relevant to schizophrenia, we tested touchscreen-based paired associates learning given its relevance to the cognitive symptoms of the disorder and locomotor activity given its relevance to the positive symptoms., Results: Acute treatment with Z944 failed to reverse the visuospatial associative memory impairments caused by MK-801 in paired associates learning. The highest dose of drug (10.0 mg/kg) given alone produced subtle impairments on paired associates learning. In contrast, Z944 (5.0 mg/kg) blocked the expected increase in locomotion following MK-801 treatment in a locomotor assay., Conclusions: These experiments provide support that Z944 may reduce behaviors relevant to positive symptoms of schizophrenia, although additional study of its effects on cognition is required. These findings and other research suggest T-type calcium channel antagonists may be an alternative to currently available antipsychotics with less serious side effects.

Many disease-causing missense mutations affect intrinsically disordered regions (IDRs) of proteins, but the molecular mechanism of their pathogenicity is enigmatic. Here, we employ a peptide-based proteomic screen to investigate the impact of mutations in IDRs on protein-protein interactions. We find that mutations in disordered cytosolic regions of three transmembrane proteins (GLUT1, ITPR1, and CACNA1H) lead to an increased clathrin binding. All three mutations create dileucine motifs known to mediate clathrin-dependent trafficking. Follow-up experiments on GLUT1 (SLC2A1), the glucose transporter causative of GLUT1 deficiency syndrome, revealed that the mutated protein mislocalizes to intracellular compartments. Mutant GLUT1 interacts with adaptor proteins (APs) in vitro, and knocking down AP-2 reverts the cellular mislocalization and restores glucose transport. A systematic analysis of other known disease-causing variants revealed a significant and specific overrepresentation of gained dileucine motifs in structurally disordered cytosolic domains of transmembrane proteins. Thus, several mutations in disordered regions appear to cause "dileucineopathies.", (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aims: We have previously demonstrated that blockade of T-type calcium channels by the non-selective antagonist, ethosuximide (ETX), is effective at reducing electrographical and behavioral correlates of alcohol-withdrawal (WD) seizure. Here, we investigated whether blockade of these calcium channels with the selective antagonist TTA-P2 also reduces alcohol-WD seizure., Short Summary: The non-specific T-type calcium channel antagonist, ETX, is protective against alcohol-WD seizure. However, the mechanism of this effect is unclear. Here, we provide evidence that further suggests selective blockade of T-type calcium channels are protective against alcohol-WD seizure and WD-related mortality., Methods: We used an intermittent ethanol exposure model to produce WD-induced hyperexcitability in DBA/2 J mice. Seizure severity was intensified with the chemoconvulsant pentylenetetrazole (PTZ)., Results: TTA-P2 (10 mg/kg) reduced seizure severity in mice undergoing alcohol WD with concurrent PTZ treatment (20 mg/kg). Moreover, TTA-P2 (20 and 40 mg/kg) was also protective against PTZ-induced (40 mg/kg) seizure and mortality., Conclusions: These results are consistent with prior results using ETX, and suggest that the protective effects of ETX and TTA-P2 against EtOH WD seizures are mediated by T-type calcium channels.

The roles of low-voltage-activated (T-type) calcium channels in brain diseases have been studied extensively. Less is known regarding the involvement of T-type channels in cognition and behavior. Sensory integration (SI) is a cognitive process whereby the brain uses unimodal or multimodal sensory features to create a comprehensive representation of the environment. The multisensory object oddity (MSO) task assesses SI using combinations of sensory features of objects, either in the same or different sensory modalities. The regulation of SI involves the orbitofrontal cortex (OFC), an area which shows high levels of T-type calcium channel expression. We tested the effects of blocking T-type calcium channels on the MSO task with the selective T-type antagonist, Z944 (5 mg/kg; i.p. systemic; 100 or 500 µM OFC infusion), in male Long Evans rats. With systemic treatment, Z944 impaired the visual and visual-olfactory versions of the task. Infusion of 100 and 500 µM Z944 produced deficits in the olfactory version of the task. In addition, only vehicle-infused, but not Z944-infused, rats showed significant performance above chance for all task variants. Thus, the present results suggest that T-type calcium channels in OFC are involved in SI of features in an oddity task. Given that unimodal SI was disrupted by OFC infusions of Z944, the deficits in the multimodal task must be interpreted with caution. As SI is disrupted in psychiatric disorders, further investigations elucidating the brain regions implicated in SI regulation by T-type calcium channels may help inform therapeutic development for those suffering from SI impairments., (© 2018 Marks et al.; Published by Cold Spring Harbor Laboratory Press.)

Objective: Lercanidipine is a calcium antagonist with no cardiodepressant activity, long lasting antihypertensive action and reno-protective effect. Our previous data demonstrated that lercanidipine blocks L-type calcium channels (CaL). However, no data are available concerning its effects on T-type calcium channels (CaT). The aim of this study was to evaluate the effect on both CaL and CaT and the selectivity ratio of R-lercanidipine, S-lercanidipine and RS-lercanidipine. A comparison with other dihydropyridines (amlodipine and lacidipine) and the CaT blocker mibefradil was also performed., Materials and Methods: In patch-clamped guinea-pig ventricular myocytes, a voltage protocol was applied mimicking a normal action potential: HP of -90 mV, 200 ms depolarizing steps to -50/+50 mV. Lercanidipine was tested at concentrations (1-10 µM) able to block ≈ 50% CaL evoked from a HP in the range of -50 to -30 mV. Cells were superfused with a Na+ and K+ free solution pre-warmed to 35°C to abolish overlapping currents., Results: Using the described voltage protocol, all dihydropyridines at 1 µM blocked less than 20% CaL, with the exception of lacidipine, that reduced CaL >60% of control. All calcium channel blockers (CCBs) blocked a significant amount of CaT, varying from 28% (mibefradil) to 4.3% (amlodipine). Based on the ratio between CaT and CaL blockade in each cell (T/L), mibefradil, as expected, showed the highest T affinity (T/L=1.3). Lercanidipine, either racemate or enantiomers, showed a noticeable T selectivity, T/L varying from 1.05 (S-lercanidipine) to 1.15 (R-lercanidipine)., Conclusions: All CCBs examined in this study showed both T- and L-channel blocking activities and can be differentiated based on their relative affinity. Among tested dihydropyridines, lercanidipine showed the highest T/L selectivity.

Objective: Genetic alterations have been identified in the CACNA1H gene, encoding the Ca V 3.2 T-type calcium channel in patients with absence epilepsy, yet the precise mechanisms relating to seizure propagation and spike-wave-discharge (SWD) pacemaking remain unknown. Neurons of the thalamic reticular nucleus (TRN) express high levels of Ca V 3.2 calcium channels, and we investigated whether a gain-of-function mutation in the Cacna1h gene in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) contributes to seizure propagation and pacemaking in the TRN., Methods: Pathophysiological contributions of Ca V 3.2 calcium channels to burst firing and absence seizures were assessed in vitro using acute brain slice electrophysiology and quantitative real-time polymerase chain reaction (PCR) and in vivo using free-moving electrocorticography recordings., Results: TRN neurons from GAERS display sustained oscillatory burst-firing that is both age- and frequency-dependent, occurring only in the frequencies overlapping with GAERS SWDs and correlating with the expression of a Ca V 3.2 mutation-sensitive splice variant. In vivo knock-down of Ca V 3.2 using direct thalamic injection of lipid nanoparticles containing Ca V 3.2 dicer small interfering (Dsi) RNA normalized TRN burst-firing, and in free-moving GAERS significantly shortened seizures., Significance: This supports a role for TRN Ca V 3.2 T-type channels in propagating thalamocortical network seizures and setting the pacemaking frequency of SWDs., (© 2018 The Authors. Epilepsia published by Wiley Periodicals, Inc. on behalf of International League Against Epilepsy.)

Key Points: Cav3.1 T-type Ca 2+ channel current (I Ca-T ) contributes to heart rate genesis but is not known to contribute to heart rate regulation by the sympathetic/β-adrenergic system (SAS). We show that the loss of Cav3.1 makes the beating rates of the heart in vivo and perfused hearts ex vivo, as well as sinoatrial node cells, less sensitive to β-adrenergic stimulation; it also renders less conduction acceleration through the atrioventricular node by β-adrenergic stimulation. Increasing Cav3.1 in cardiomyocytes has the opposite effects. I Ca-T in sinoatrial nodal cells can be upregulated by β-adrenergic stimulation. The results of the present study add a new contribution to heart rate regulation by the SAS system and provide potential new mechanisms for the dysregulation of heart rate and conduction by the SAS in the heart. T-type Ca 2+ channel can be a target for heart disease treatments that aim to slow down the heart rate ABSTRACT: Cav3.1 (α 1G ) T-type Ca 2+ channel (TTCC) is expressed in mouse sinoatrial node cells (SANCs) and atrioventricular (AV) nodal cells and contributes to heart rate (HR) genesis and AV conduction. However, its role in HR regulation and AV conduction acceleration by the β-adrenergic system (SAS) is unclear. In the present study, L- (I Ca-L ) and T-type (I Ca-T ) Ca 2+ currents were recorded in SANCs from Cav3.1 transgenic (TG) and knockout (KO), and control mice. I Ca-T was absent in KO SANCs but enhanced in TG SANCs. In anaesthetized animals, different doses of isoproterenol (ISO) were infused via the jugular vein and the HR was recorded. The EC 50 of the HR response to ISO was lower in TG mice but higher in KO mice, and the maximal percentage of HR increase by ISO was greater in TG mice but less in KO mice. In Langendorff-perfused hearts, ISO increased HR and shortened PR intervals to a greater extent in TG but to a less extent in KO hearts. KO SANCs had significantly slower spontaneous beating rates than control SANCs before and after ISO; TG SANCs had similar basal beating rates as control SANCs probably as a result of decreased I Ca-L but a greater response to ISO than control SANCs. I Ca-T in SANCs was significantly increased by ISO. I Ca-T upregulation by β-adrenergic stimulation contributes to HR and conduction regulation by the SAS. TTCC can be a target for slowing the HR., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

The central medial nucleus (CeM) is a part of the intralaminar thalamus, which is involved in the control of arousal and sensory processing. However, ionic conductances and mechanisms that regulate the activity of the CeM are not well studied. Here, we used in vitro electrophysiology in acute brain slices from adolescent rats to demonstrate that T-type calcium currents (T-currents) are prominent in the majority of the studied CeM neurons and are critical determinants of low-threshold calcium spikes (LTSs), which in turn regulate excitability of these neurons. Using an ATP-free internal solution decreased T-current density and induced a profound hyperpolarizing shift in steady-state inactivation curves while voltage-dependent activation kinetics were spared. Furthermore, selective pharmacological blockade of T-channels or use of an ATP-free solution reduced both tonic action potential (AP) frequency and rebound burst firing in CeM neurons. Our results indicate that T-channels are critical regulators of a thalamocortical circuit output and suggest that cytosolic ATP could be an endogenous regulatory mechanism in which T-channels may functionally gate sensory transmission and arousal in vivo .

Introduction: There are few medications that are available for the treatment of essential tremor (ET) and they are only moderately effective. Areas covered: Data were obtained from a PubMed search. Original articles, review articles, and clinical guidelines were included. Two disease models for ET have been proposed: 1) the olivary model, which attributes ET to a pathological pacemaker in the inferior olivary nucleus, and 2) the cerebellar degeneration model, which postulates that ET originates in the cerebellum and could be related to deficient or abnormal Purkinje cell (PC) output. Underlying biochemical dysfunction in T-type calcium channels (T-tCaC) may loosely be linked to the first model and deficiency/abnormality in γ-aminobutyric acid (GABA) neurotransmission, to the second. Expert commentary: Human data points robustly to the role of GABA in ET. Numerous medications that target the GABA system have been tried, with variable success. Given the many different types of GABA-ergic neurons, and the multitude of GABA A receptor subtypes, a given medication could have competing/cancelling effects. It would seem that influencing GABA receptors broadly is not as effective as targeting certain GABA A receptor subtypes. Future research should seek to identify molecular candidates that have a more targeted effect within the GABA system.

We studied the pronociceptive role of proteinase-activated receptor-2 (PAR2) in mouse bladder. In female mice, intravesical infusion of the PAR2-activating peptide, SLIGRL-amide (SL), caused delayed mechanical hypersensitivity in the lower abdomen, namely 'referred hyperalgesia', 6-24 h after the administration. The PAR2-triggered referred hyperalgesia was prevented by indomethacin or a selective TRPV1 blocker, and restored by a T-type Ca 2+ channel blocker. In human urothelial T24 cells, SL caused delayed prostaglandin E 2 production and COX-2 upregulation. Our data suggest that luminal PAR2 stimulation in the bladder causes prostanoid-dependent referred hyperalgesia in mice, which involves the activation of TRPV1 and T-type Ca 2+ channels., (Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

In patients with thalassemia, iron overload cardiomyopathy is a major cause of cardiac dysfunction and mortality. Despite many advances in the development of new iron chelating agents, heart failure still occurs in some patients and can lead to an increase in mortality rate. Recently, potential novel therapeutic strategies in the treatment of these patients have focused on L-type and T-type calcium channel blockers. These 2 channels have been reported as being the main routes for cardiac iron uptake under conditions of iron overload. In this review, the effects of these calcium channel blockers on both cardiac iron uptake and cardiac function under conditions of iron overload are discussed, and both consistent and inconsistent findings published in various studies are summarized and reviewed. Through this we hope to provide a greater insight into how future studies can most effectively use these drugs at the clinical trial stage.

Medicinal plants represent a significant reservoir of unexplored substances for early-stage drug discovery. Of interest, two flowering Mediterranean plants have been used for thousands of years for their beneficial effects on nervous disorders, including anxiety and mood. However, the therapeutic potential of these plants regarding their ability to target ion channels and neuronal excitability remains largely unknown. Towards this goal, we have investigated the ability of Lavender and Rosemary to modulate T-type calcium channels (TTCCs). TTCCs play important roles in neuronal excitability, neuroprotection, sensory processes and sleep. These channels are also involved in epilepsy and pain. Using the whole-cell patch-clamp technique, we have characterized how Lavender and Rosemary extracts, as well as their major active compounds Linalool and Rosmarinic acid, modulate the electrophysiological properties of recombinant TTCCs (CaV3.2) expressed in HEK-293T cells. Both the methanolic and essential oil extracts as well as the active compounds of these plants inhibit Cav3.2 current in a concentration-dependent manner. In addition, these products also induce a negative shift of the steady-state inactivation of CaV3.2 current with no change in the activation properties. Taken together, our findings reveal that TTCCs are a molecular target of the Lavender and Rosemary compounds, suggesting that inhibition of TTCCs could contribute to the anxiolytic and the neuroprotective effects of these plants.

We describe the discovery and optimization of new, brain-penetrant T-type calcium channel blockers. We present optimized compounds with excellent efficacy in a rodent model of generalized absence-like epilepsy. Along the fine optimization of a chemical series with a pharmacological target located in the CNS (target potency, brain penetration, and solubility), we successfully identified an Ames negative aminopyrazole as putative metabolite of this compound series. Our efforts culminated in the selection of compound 20, which was elected as a preclinical candidate.

Compelling evidence links amyloid beta (Aβ) peptide accumulation in the brains of Alzheimer's disease (AD) patients with the emergence of learning and memory deficits, yet a clear understanding of the events that drive this synaptic pathology are lacking. We present evidence that neurons exposed to Aβ are unable to form new synapses, resulting in learning deficits in vivo. We demonstrate the Nogo receptor family (NgR1-3) acts as Aβ receptors mediating an inhibition of synapse assembly, plasticity, and learning. Live imaging studies reveal Aβ activates NgRs on the dendritic shaft of neurons, triggering an inhibition of calcium signaling. We define T-type calcium channels as a target of Aβ-NgR signaling, mediating Aβ's inhibitory effects on calcium, synapse assembly, plasticity, and learning. These studies highlight deficits in new synapse assembly as a potential initiator of cognitive pathology in AD, and pinpoint calcium dysregulation mediated by NgRs and T-type channels as key components. VIDEO ABSTRACT., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Endocannabinoid signaling system is involved in regulating multiple neuronal functions in the central nervous system by activating G-protein coupled cannabinoid CB1 and CB2 receptors (CB1Rs and CB2Rs). Growing evidence has shown that CB1Rs and CB2Rs are extensively expressed in retinal ganglion cells (RGCs). Here, modulation of L- and T-types Ca 2+ channels by activating CB1Rs and CB2Rs in RGCs was investigated. Triple immunofluorescent staining showed that L-type subunit Ca V 1.2 was co-localized with T-type subunits (Ca V 3.1, Ca V 3.2 and Ca V 3.3) in rat RGCs. In acutely isolated rat RGCs, the CB1R agonist WIN55212-2 suppressed both peak and steady-state Ca 2+ currents in a dose-dependent manner, with IC 50 being 9.6 μM and 8.4 μM, respectively. It was further shown that activation of CB1Rs by WIN55212-2 or ACEA, another CB1R agonist, significantly suppressed both L- and T-type Ca 2+ currents, and shifted inactivation curve of T-type one toward hyperpolarization direction. While the effect on L-type Ca 2+ channels was mediated by intracellular cAMP/protein kinase A (PKA), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and calcium/calmodulin-dependent protein kinase II (CaMKII) signaling pathways, only CaMKII signaling pathway was involved in the effect on T-type Ca 2+ channels. Furthermore, CB65 and HU308, two specific CB2R agonists, significantly suppressed T-type Ca 2+ channels, which was mediated by intracellular cAMP/PKA and CaMKII signaling pathways, but had no effect on L-type channels. These results imply that endogenous cannabinoids may modulate the excitability and the output of RGCs by differentially suppressing the activity of L- and T-type Ca 2+ channels through activation of CB1Rs and CB2Rs. This article is part of the Special Issue entitled "A New Dawn in Cannabinoid Neurobiology"., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Calcium-dependent inactivation of high voltage-activated Ca 2+ channels plays a crucial role in limiting rises in intracellular calcium (Ca 2+ i ). A key mediator of these effects is calmodulin, which has been found to bind the C-terminus of the pore-forming α subunit. In contrast, little is known about how Ca 2+ i can regulate low voltage-activated T-type Ca 2+ channels. Using whole cell patch clamp, we examined the biophysical properties of Ca 2+ current through the three T-type Ca 2+ channel isoforms, Ca v 3.1, Ca v 3.2, or Ca v 3.3, comparing internal solutions containing 27 nM and l μM free Ca 2+ Both activation and inactivation kinetics of Ca v 3.3 current in l μM Ca 2+ i solution were more rapid than those in 27 nM Ca 2+ i solution. In addition, both activation and steady-state inactivation curves of Ca v 3.3 were negatively shifted in the higher Ca 2+ i solution. In contrast, the biophysical properties of Ca v 3.1 and Ca v 3.2 isoforms were not significantly different between the two internal solutions. Overexpression of CaM 1234 (a calmodulin mutant that doesn't bind Ca 2+ ) occluded the effects of l μM Ca 2+ i on Ca v 3.3, implying that CaM is involved in the Ca 2+ i regulation effects on Ca v 3.3. Yeast two-hybrid screening and co-immunoprecipitation experiments revealed a direct interaction of CaM with the carboxyl terminus of Ca v 3.3. Taken together, our results suggest that Ca v 3.3 T-type channel is potently regulated by Ca 2+ i via interaction of Ca 2+ /CaM with the carboxyl terminus of Ca v 3.3., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Basal ganglia (BG) circuits orchestrate complex motor behaviors predominantly via inhibitory synaptic outputs. Although these inhibitory BG outputs are known to reduce the excitability of postsynaptic target neurons, precisely how this change impairs motor performance remains poorly understood. Here, we show that optogenetic photostimulation of inhibitory BG inputs from the globus pallidus induces a surge of action potentials in the ventrolateral thalamic (VL) neurons and muscle contractions during the post-inhibitory period. Reduction of the neuronal population with this post-inhibitory rebound firing by knockout of T-type Ca 2+ channels or photoinhibition abolishes multiple motor responses induced by the inhibitory BG input. In a low dopamine state, the number of VL neurons showing post-inhibitory firing increases, while reducing the number of active VL neurons via photoinhibition of BG input, effectively prevents Parkinson disease (PD)-like motor symptoms. Thus, BG inhibitory input generates excitatory motor signals in the thalamus and, in excess, promotes PD-like motor abnormalities. VIDEO ABSTRACT., (Copyright © 2017 Elsevier Inc. All rights reserved.)