Low Voltage-activated Calcium Channels Research Articles

Dietary Zinc Deficiency Induces Cav3.2-Dependent Nociceptive Hypersensitivity in Mice.

Cav3.2 channels belong to the T-type calcium channel (T-channel) family, i.e., low voltage-activated calcium channels, and are abundantly expressed in the nociceptors, playing a principal role in the development of pathological pain. The channel activity of Cav3.2 is suppressed by zinc under physiological conditions. We thus tested whether dietary zinc deficiency would cause Cav3.2-dependent nociceptive hypersensitivity in mice. In the mice fed with zinc deficient diet for 2 weeks, plasma zinc levels declined by more than half, and mechanical allodynia developed. The dietary zinc deficiency-induced allodynia was restored by T-channel inhibitors or by Cav3.2 gene silencing. These data demonstrate that zinc deficiency induces Cav3.2-dependent nociceptive hypersensitivity in mice, thereby suggesting that pain experienced by patients with diseases accompanied by zinc deficiency (e.g., chronic kidney disease) might involve the increased Cav3.2 activity.

Regulation of Hippocampal Gamma Oscillations by Modulation of Intrinsic Neuronal Excitability.

Ion channels activated around the subthreshold membrane potential determine the likelihood of neuronal firing in response to synaptic inputs, a process described as intrinsic neuronal excitability. Long-term plasticity of chemical synaptic transmission is traditionally considered the main cellular mechanism of information storage in the brain; however, voltage- and calcium-activated channels modulating the inputs or outputs of neurons are also subjects of plastic changes and play a major role in learning and memory formation. Gamma oscillations are associated with numerous higher cognitive functions such as learning and memory, but our knowledge of their dependence on intrinsic plasticity is by far limited. Here we investigated the roles of potassium and calcium channels activated at near subthreshold membrane potentials in cholinergically induced persistent gamma oscillations measured in the CA3 area of rat hippocampal slices. Among potassium channels, which are responsible for the afterhyperpolarization in CA3 pyramidal cells, we found that blockers of SK (KCa2) and KV7.2/7.3 (KCNQ2/3), but not the BK (KCa1.1) and IK (KCa3.1) channels, increased the power of gamma oscillations. On the contrary, activators of these channels had an attenuating effect without affecting the frequency. Pharmacological blockade of the low voltage-activated T-type calcium channels (CaV3.1–3.3) reduced gamma power and increased the oscillation peak frequency. Enhancement of these channels also inhibited the peak power without altering the frequency of the oscillations. The presented data suggest that voltage- and calcium-activated ion channels involved in intrinsic excitability strongly regulate the power of hippocampal gamma oscillations. Targeting these channels could represent a valuable pharmacological strategy against cognitive impairment.

Targeting the T-type calcium channel Cav3.2 in GABAergic arcuate nucleus neurons to treat obesity

ObjectiveCav3.2, a T-type low voltage-activated calcium channel widely expressed throughout the central nervous system, plays a vital role in neuronal excitability and various physiological functions. However, the effects of Cav3.2 on energy homeostasis remain unclear. Here, we examined the role of Cav3.2 expressed by hypothalamic GABAergic neurons in the regulation of food intake and body weight in mice and explored the underlying mechanisms. MethodsMale congenital Cana1h (the gene coding for Cav3.2) global knockout (Cav3.2KO) mice and their wild type (WT) littermates were first used for metabolic phenotyping studies. By using the CRISPR-Cas9 technique, Cav3.2 was selectively deleted from GABAergic neurons in the arcuate nucleus of the hypothalamus (ARH) by specifically overexpressing Cas9 protein and Cav3.2-targeting sgRNAs in ARH Vgat (VgatARH) neurons. These male mutants (Cav3.2KO-VgatARH) were used to determine whether Cav3.2 expressed by VgatARH neurons is required for the proper regulation of energy balance. Subsequently, we used an electrophysiological patch-clamp recording in ex vivo brain slices to explore the impact of Cav3.2KO on the cellular excitability of VgatARH neurons. ResultsMale Cav3.2KO mice had significantly lower food intake than their WT littermate controls when fed with either a normal chow diet (NCD) or a high-fat diet (HFD). This hypophagia phenotype was associated with increased energy expenditure and decreased fat mass, lean mass, and total body weight. Selective deletion of Cav3.2 in VgatARH neurons resulted in similar feeding inhibition and lean phenotype without changing energy expenditure. These data provides an intrinsic mechanism to support the previous finding on ARH non-AgRP GABA neurons in regulating diet-induced obesity. Lastly, we found that naringenin extract, a predominant flavanone found in various fruits and herbs and known to act on Cav3.2, decreased the firing activity of VgatARH neurons and reduced food intake and body weight. These naringenin-induced inhibitions were fully blocked in Cav3.2KO-VgatARH mice. ConclusionOur results identified Cav3.2 expressed by VgatARH neurons as an essential intrinsic modulator for food intake and energy homeostasis, which is a potential therapeutic target in the treatment of obesity.

Modulation of Recombinant Human T-Type Calcium Channels by Δ9-Tetrahydrocannabinolic Acid In Vitro.

Introduction: Low 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-tetrahydrocannabinolic acid (THCA), the natural nonpsychoactive precursor of THC. The aim of this work was to characterize THCA effects on T-type calcium channels. Materials and Methods: We 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. Results: THCA and THC inhibited the peak current amplitude CaV3.1 with pEC50s of 6.0±0.7 and 5.6±0.4, respectively. THC (1 μM) 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% (10 μM) but did not notably affect CaV3.3 channel activation or inactivation; however, THC caused significant hyperpolarizing shift in CaV3.3 steady-state inactivation. Discussion: THCA 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 through T-type calcium channel modulation without the unwanted psychoactive effects associated with THC.

T-type Calcium Channels in Health and Disease.

Low Voltage-Activated (LVA) T-type calcium channels are characterized by transient current and Low Threshold Spikes (LTS) that trigger neuronal firing and oscillatory behavior. Combined with their preferential localization in dendrites and their specific "window current", T-type calcium channels are considered to be key players in signal amplification and synaptic integration. Assisted by the emerging pharmacological tools, the structural determinants of channel gating and kinetics, as well as novel physiological and pathological functions of T-type calcium channels, are being uncovered. In this review, we provide an overview of structural determinants in T-type calcium channels, their involvement in disorders and diseases, the development of novel channel modulators, as well as Structure-Activity Relationship (SAR) studies that lead to rational drug design.

33-LB: Role of Cav3.2 Channels in Sensory Neuron Abnormalities during Early Hyperglycemia

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes. It is characterized by a constellation of sensory abnormalities, including numbness, tingling, burning, and painful sharp, shooting sensations in the extremities. Patients with DPN are also at high risk for infection and amputation. Traditionally, neuropathy has been considered to result from peripheral nerve damage after long term diabetes, however more recent indicate that functional changes take place in early stages of diabetes. One of those changes is the increased expression and signalling of the receptor for advance glycation end-product (RAGE), which is known to have a pivotal role in the development of diabetic complications. In the current study we concentrate on T-type (Cav 3.2) channels, which are low voltage-activated calcium channels that play a key role in cellular excitability, and have been reported to contribute to pain abnormalities in rat models of DPN. The goal of this project is to elucidate the role of Cav 3.2 during early stages of diabetes in sensory neurons from the dorsal root ganglion (DRG) of mice, and its possible regulation through the RAGE pathway. We used cultured DRG neurons from wilt-type (WT) and RAGE knock-out (RAGE KO) mice maintained in control (5mM) or high glucose (25 mM) for up to 14 days, as well as intact DRG ganglia from streptozotocin (STZ)-induced diabetic mice (WT and RAGE KO) for 4 weeks. Using whole cell patch-clamp electrophysiology and protein biochemistry we revealed changes in the activation kinetics and expression levels of Cav 3.2 channels. Our data suggest that RAGE-dependent changes in Cav 3.2 expression and function may contribute to functional abnormalities in sensory neurons during early hyperglycemia. Disclosure J.T. Neapetung: None. V.A. Campanucci: None.

Analysis of tetrodotoxin-sensitive sodium and low voltage-activated calcium channels in developing mouse retinal horizontal cells

Expression patterns of voltage-gated ion channels determine the spatio-temporal dynamics of ion currents that supply excitable neurons in developing tissue with proper electrophysiological properties. The purpose of the study was to identify fast cationic inward currents in mouse retinal horizontal cells (HCs) and describe their biophysical properties at different developmental stages. We also aimed to reveal their physiological role in shaping light responses (LRs) in adult HCs. HCs were recorded in horizontal slices of wild-type mouse retina at postnatal stages ranging from p8 through p60. Voltage-dependent inward currents were isolated with appropriate voltage protocols and blockers specific for sodium and T-type calcium channels. LRs were evoked with full-field flashes (130 μW/cm2). Transient and steady inward currents were identified at all developmental stages. Transient currents were mediated by T-type calcium and TTX-sensitive sodium channels, whereas steady currents were blocked by cadmium, indicating the presence of high voltage-activated calcium channels. Activation and steady-state inactivation kinetics of T-type calcium channels revealed a contribution to the resting membrane potential during postnatal development. Additionally, both sodium and T-type calcium channels had an impact on HC LRs at light offset in adult animals. Our results showed that the voltage-dependent inward currents of postnatally developing mouse HCs consist of T-type calcium, TTX-sensitive sodium, and high voltage-activated calcium channels, and that transient ionic currents contributed to light-evoked responses of adult HCs, suggesting a role in HC information processing.

Dual role of T-type calcium channels in anxiety-related behavior.

T-type calcium channels are low voltage activated calcium channels that are widely expressed in various brain regions including stress-responsive regions. These channels regulate the diverse functions of the central nervous system, and modulation of these channels is shown to modulate the anxiety. Studies have described that modulation of T-type calcium channels may either aggravate or ameliorate anxiety-related behavior, suggesting the dual role of these channels. The studies employing animals with overexpression of T-type calcium channels reported their anxiety-inducing role. Therefore, the blockade of these channels using various pharmacological agents such as ethosuximide, plant extracts of linalool or rosemary, and corticotropin-releasing factor (CRF) is reported to ameliorate anxiety. On the contrary, knockout of the gene encoding these channels predisposes the rodents to anxiety-related disorders, suggesting the anxiety-attenuating role of these channels. It may be possible that these channels in normal or basal state attenuate anxiety, whereas activation of these channels in stressful condition may produce anxiety. The present review describes the dual role of T-type calcium channels in anxiety-related behavior in both preclinical and clinical studies.

Upregulation of interleukin-6 on Cav3.2 T-type calcium channels in dorsal root ganglion neurons contributes to neuropathic pain in rats with spinal nerve ligation

The T-type calcium channels Cav3.2, one of the low voltage-activated (LVA) calcium channels, have been found to play important roles in the neuronal excitability. Recently, we and others have demonstrated that accumulation of Cav3.2 channels in the dorsal root ganglion (DRG) neurons and sensory nerves contributes to neuropathic pain after peripheral nerve injury. In the present study, we aimed to further investigate the regulation of Cav3.2 channels by interleukin-6 (IL-6) in DRG neurons in neuropathic pain rats after spinal nerve ligation (SNL). The results showed that Cav3.2 channel protein expression in L5 DRG neurons was upregulated and blockade of this channel decreased the hyperexcitability of DRG neurons and mechanical allodynia in SNL neuropathic pain rats. Furthermore, inhibition of IL-6 trans-signaling reduced the upregulation of Cav3.2 T-type channel induced by FIL-6 (a fusion protein of IL-6 and sIL-6R) in primary cultured DRG neurons in vitro. In vivo, inhibition of IL-6 trans-signaling reversed the upregulation of Cav3.2, reduced the hyperexcitability of L5 DRG neurons and alleviated mechanical allodynia in SNL rats. Our results suggest that IL-6 upregulates Cav3.2 T-type channels expression and function through the IL-6/sIL-6R trans-signaling pathway in DRG neurons, thus contributes to the development of neuropathic pain in SNL rats.

Increased Calcium Influx through L-type Calcium Channels in Human and Mouse Neural Progenitors Lacking Fragile X Mental Retardation Protein.

SummaryThe absence of FMR1 protein (FMRP) causes fragile X syndrome (FXS) and disturbed FMRP function is implicated in several forms of human psychopathology. We show that intracellular calcium responses to depolarization are augmented in neural progenitors derived from human induced pluripotent stem cells and mouse brain with FXS. Increased calcium influx via nifedipine-sensitive voltage-gated calcium (Cav) channels contributes to the exaggerated responses to depolarization and type 1 metabotropic glutamate receptor activation. The ratio of L-type/T-type Cav channel expression is increased in FXS progenitors and correlates with enhanced progenitor differentiation to glutamate-responsive cells. Genetic reduction of brain-derived neurotrophic factor in FXS mouse progenitors diminishes the expression of Cav channels and activity-dependent responses, which are associated with increased phosphorylation of the phospholipase C-γ1 site within TrkB receptors and changes of differentiating progenitor subpopulations. Our results show developmental effects of increased calcium influx via L-type Cav channels in FXS neural progenitors.

CACHD1 is an α2δ-Like Protein That Modulates CaV3 Voltage-Gated Calcium Channel Activity.

The putative cache (Ca2+ channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the α2δ voltage-gated Ca2+ channel auxiliary subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus, and cerebellum, with a broadly similar tissue distribution to CaV3 subunits, in particular CaV3.1. In expression studies, CACHD1 increased cell-surface localization of CaV3.1, and these proteins were in close proximity at the cell surface, consistent with the formation of CACHD1-CaV3.1 complexes. In functional electrophysiological studies, coexpression of human CACHD1 with CaV3.1, CaV3.2, and CaV3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, α2δ-1 had no effect on peak current density or maximal conductance in CaV3.1, CaV3.2, or CaV3.3. A comparison of CACHD1-mediated increases in CaV3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female embryonic day 19 rats, CACHD1 overexpression increased CaV3-mediated action potential firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an α2δ-like protein that functionally modulates CaV3 voltage-gated calcium channel activity.SIGNIFICANCE STATEMENT This is the first study to characterize the Ca2+ channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is widely expressed in the CNS, in particular in the thalamus, hippocampus, and cerebellum. CACHD1 distribution is similar to that of low voltage-activated (CaV3, T-type) calcium channels, in particular to CaV3.1, a protein that regulates neuronal excitability and is a potential therapeutic target in conditions such as epilepsy and pain. CACHD1 is structurally an α2δ-like protein that functionally increases CaV3 calcium current. CACHD1 increases the presence of CaV3.1 at the cell surface, forms complexes with CaV3.1 at the cell surface, and causes an increase in channel open probability. In hippocampal neurons, CACHD1 causes increases in neuronal firing. Thus, CACHD1 represents a novel protein that modulates CaV3 activity.

T-Type Calcium Channels Are Required to Maintain Viability of Neural Progenitor Cells.

T-type calcium channels are low voltage-activated calcium channels that evoke small and transient calcium currents. Recently, T-type calcium channels have been implicated in neurodevelopmental disorders such as autism spectrum disorder and neural tube defects. However, their function during embryonic development is largely unknown. Here, we investigated the function and expression of T-type calcium channels in embryonic neural progenitor cells (NPCs). First, we compared the expression of T-type calcium channel subtypes (CaV3.1, 3.2, and 3.3) in NPCs and differentiated neural cells (neurons and astrocytes). We detected all subtypes in neurons but not in astrocytes. In NPCs, CaV3.1 was the dominant subtype, whereas CaV3.2 was weakly expressed, and CaV3.3 was not detected. Next, we determined CaV3.1 expression levels in the cortex during early brain development. Expression levels of CaV3.1 in the embryonic period were transiently decreased during the perinatal period and increased at postnatal day 11. We then pharmacologically blocked T-type calcium channels to determine the effects in neuronal cells. The blockade of T-type calcium channels reduced cell viability, and induced apoptotic cell death in NPCs but not in differentiated astrocytes. Furthermore, blocking T-type calcium channels rapidly reduced AKT-phosphorylation (Ser473) and GSK3β-phosphorylation (Ser9). Our results suggest that T-type calcium channels play essential roles in maintaining NPC viability, and T-type calcium channel blockers are toxic to embryonic neural cells, and may potentially be responsible for neurodevelopmental disorders.

Abstract P3-07-03: Targeting low voltage-activated calcium channels in HER2-positive breast cancer

Abstract Under physiological conditions low voltage-activated calcium channels (LVA, also called T-type channels) occur mostly in the brain, peripheral nervous system, smooth muscles, and pacemaker of the heart. However, LVA are often aberrantly expressed in various cancers and their overexpression is inversely correlated with outcomes. Active LVA contribute to proliferation, progression and resistance to therapy in several human cancers, including breast cancer (BC). The molecular mechanism of this activity is still not fully understood, and there is a lack of pre-clinical studies that target LVA in animal models of BC.Data from the Cancer Genome Atlas (TCGA) show a positive correlation between HER2 gene amplification and LVA channel overexpression, particularly of Cav3.1 (CACNA1G) subunit. The goal of this study is to identify functional links between HER2 receptor signaling and LVA channels, and validate LVA Ca2+ channels as a potential target for BC therapy. Inhibition of LVA, by small molecule antagonist mibefradil (MIB) or shRNA, significantly decreased the activity of PI3K/mTOR/AKT pathway in HER2-positive BC cell lines, blocked cell cycle progression, and at higher concentrations induced apoptotic cell death. Furthermore, LVA antagonist, mibefradil, sensitized cells to trastuzumab, lapatinib and paclitaxel, commonly used standard treatments for HER2-positive BC. To test the effects of LVA inhibition in vivo, the MMTV-PyMT mouse model of BC was used. In this model several features of HER2-positive BC are recapitulated, such as activation of PI3K/AKT/mTOR pathway, overexpression of HER2, BIRC5 and cyclin D1, and low expression of ERα, PGR and FOXA1. In PyMT animals palpable tumors can be detected at the age of 10-12 weeks, and 100% of animals will develop high tumor burden, including lung metastases, at the age of 15-20 weeks. Expression of CACNA1G subunit was detected in breast epithelial cells of 7 week old mice, and throughout the adult life, in both wild-type and PyMT animals, but CACNA1G was not expressed in lung or skeletal muscles. In in vitro experiments, cell lines derived from PyMT tumors were sensitive to MIB. In in vivo experiment, 5 to 7 weeks-old PyMT mice were fed either control or MIB-containing diet ad lib. Analysis of mouse serum revealed stable MIB concentration of ˜1500 ng/mL achieved within one week of treatment. In MIB-fed animals tumor appearance was delayed by 2 weeks, and the tumors were smaller as compared to control, thus suggesting a supporting role for LVA channels in breast tumor development and progression. Together, our observations provide new insights into the role of Ca2+ channels in HER2 driven BC and posit a future use of LVA channel antagonists for the treatment HER2-positive breast tumors. Citation Format: Dziegielewska B, Dunlap-Brown ME, Warnock WT, Dillon PM, Bouton AH, Dziegielewski J. Targeting low voltage-activated calcium channels in HER2-positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-03.

Regulation of the CaV3.2 calcium channels in health and disease Regulácia CaV3.2 vápnikových kanálov v zdraví a chorobe

Abstract Family of T-type or low-voltage activated calcium channels consists of three members: CaV3.1, CaV3.2, and CaV3.3. CaV3.2 channel has almost identical biophysical properties as the CaV3.1 channel, but is distinguished by a specific tissue expression profile and a prominent role in several pathologies, including neuropathic pain, epilepsy, and dysregulation of cardiac rhythm. Further, it may be involved in phenotype of autism spectrum disorders, and amyotrophic lateral sclerosis. It represents a promising target for future pharmacotherapies.

A T-type channel-calmodulin complex triggers \u03b1CaMKII activation

Calmodulin (CaM) is an important signaling molecule that regulates a vast array of cellular functions by activating second messengers involved in cell function and plasticity. Low voltage-activated calcium channels of the Cav3 family have the important role of mediating low threshold calcium influx, but were not believed to interact with CaM. We find a constitutive association between CaM and the Cav3.1 channel at rest that is lost through an activity-dependent and Cav3.1 calcium-dependent CaM dissociation. Moreover, Cav3 calcium influx is sufficient to activate αCaMKII in the cytoplasm in a manner that depends on an intact Cav3.1 C-terminus needed to support the CaM interaction. Our findings thus establish that T-type channel calcium influx invokes a novel dynamic interaction between CaM and Cav3.1 channels to trigger a signaling cascade that leads to αCaMKII activation.

Role of low-voltage-activated calcium current and extracellular calcium in controlling the firing pattern of developing CA1 pyramidal neurons

The firing pattern of individual neurons is an important element for information processing and storing. During the first weeks of development, there is a transitional period during which CA1 pyramidal neurons display burst-spiking behavior in contrast to the adult regular-firing pattern. Spike after-depolarizations (ADPs) constitute a major factor underlying burst-spiking behavior. Using current-clamp recordings, we studied ADP waveforms and firing patterns in CA1 pyramidal neurons of Wistar rats from 9 to 19 postnatal days (P9–19). The percentage of burst-spiking neurons increased up to P16, in correlation with the emergence of an active component in the ADP. The application of low-voltage-activated (LVA) calcium channel blockers such as nickel or mibefradil suppressed the generation of the active ADP component and burst-spiking behavior. In agreement with the development of the ADP waveform and burst-spiking behavior, voltage-clamp experiments in dissociated pyramidal neurons showed an increase in the LVA calcium current in P16–19 vs P9–12. Finally, we found that a reduction of extracellular calcium levels decreases the percentage of burst-spiking cells due to a reduction in the active component of the ADP. We conclude that a major contribution of LVA calcium channels to ADP determines the bursting capability of CA1 pyramidal neurons during a transitional postnatal period in contrast to adulthood.

Compensatory reduction of Cav3.1 expression in thalamocortical neurons of juvenile rats of WAG/Rij model of absence epilepsy

Absence seizures are the non-convulsive form of generalized epilepsy critically dependent on T-type calcium channels (Cav3) in thalamic neurons. In humans, absences accompany only childhood or adolescent epileptic syndromes—though in its polygenic rat models WAG/Rij and GAERS the opposite developmental pattern is observed. Hereby we address this issue by transcriptional and functional study of thalamic Cav3 in juvenile (i.e., free of seizures) rats of the absence-prone WAG/Rij strain and their coevals of the maternal Wistar strain. First, we measured the low voltage-activated (LVA) Ca2+ current in freshly isolated thalamocortical neurons from laterodorsal nucleus of thalamus. The difference between current densities in control (12.9±1.8pA/pF) and absence epilepsy (7.9±1.8pA/pF) groups reached ∼39%. Second, we assessed the contribution of different T-channel isoforms into the reduction of Cav3-mediated current in WAG/Rij juveniles by means of RT PCR. The expression of all three LVA calcium channels was revealed with the prevalence of G and I isoforms. The expression level of G isoform (Cav3.1) was 35% smaller in WAG/Rij strain if compared to the control animals while that of H and I isoforms (Cav3.2 and Cav3.3, respectively) remained stable. The weakened expression of Cav3.1 in juveniles of WAG/Rij rats could represent a compensatory mechanism determining the pattern of the age dependency in the disease manifestation by this model of absence epilepsy.

Phosphorylation of the Cav3.2 T-type calcium channel directly regulates its gating properties

Phosphorylation is a major mechanism regulating the activity of ion channels that remains poorly understood with respect to T-type calcium channels (Cav3). These channels are low voltage-activated calcium channels that play a key role in cellular excitability and various physiological functions. Their dysfunction has been linked to several neurological disorders, including absence epilepsy and neuropathic pain. Recent studies have revealed that T-type channels are modulated by a variety of serine/threonine protein kinase pathways, which indicates the need for a systematic analysis of T-type channel phosphorylation. Here, we immunopurified Cav3.2 channels from rat brain, and we used high-resolution MS to construct the first, to our knowledge, in vivo phosphorylation map of a voltage-gated calcium channel in a mammalian brain. We identified as many as 34 phosphorylation sites, and we show that the vast majority of these sites are also phosphorylated on the human Cav3.2 expressed in HEK293T cells. In patch-clamp studies, treatment of the channel with alkaline phosphatase as well as analysis of dephosphomimetic mutants revealed that phosphorylation regulates important functional properties of Cav3.2 channels, including voltage-dependent activation and inactivation and kinetics. We also identified that the phosphorylation of a locus situated in the loop I-II S442/S445/T446 is crucial for this regulation. Our data show that Cav3.2 channels are highly phosphorylated in the mammalian brain and establish phosphorylation as an important mechanism involved in the dynamic regulation of Cav3.2 channel gating properties.

Low threshold T‐type calcium channels as targets for novel epilepsy treatments

Low voltage-activated T-type calcium channels were originally cloned in the 1990s and much research has since focused on identifying the physiological roles of these channels in health and disease states. T-type calcium channels are expressed widely throughout the brain and peripheral tissues, and thus have been proposed as therapeutic targets for a variety of diseases such as epilepsy, insomnia, pain, cancer and hypertension. This review discusses the literature concerning the role of T-type calcium channels in physiological and pathological processes related to epilepsy. T-type calcium channels have been implicated in pathology of both the genetic and acquired epilepsies and several anti-epileptic drugs (AEDs) in clinical use are known to suppress seizures via inhibition of T-type calcium channels. Despite the fact that more than 15 new AEDs have become clinically available over the past 20 years at least 30% of epilepsy patients still fail to achieve seizure control, and many patients experience unwanted side effects. Furthermore there are no treatments that prevent the development of epilepsy or mitigate the epileptic state once established. Therefore there is an urgent need for the development of new AEDs that are effective in patients with drug resistant epilepsy, are anti-epileptogenic and are better tolerated. We also review the mechanisms of action of the current AEDs with known effects on T-type calcium channels and discuss novel compounds that are being investigated as new treatments for epilepsy.

Endogenous release of 5-HT modulates the plateau phase of NMDA-induced membrane potential oscillations in lamprey spinal neurons.

The lamprey central nervous system has been used extensively as a model system for investigating the networks underlying vertebrate motor behavior. The locomotor networks can be activated by application of glutamate agonists, such as N-methyl-D-aspartic acid (NMDA), to the isolated spinal cord preparation. Many spinal neurons are capable of generating pacemaker-like membrane potential oscillations upon activation of NMDA receptors. These oscillations rely on the voltage-dependent properties of NMDA receptors in interaction with voltage-dependent potassium and calcium-dependent potassium (K(Ca)) channels, as well as low voltage-activated calcium channels. Upon membrane depolarization, influx of calcium will activate K(Ca) channels, which in turn, will contribute to repolarization and termination of the depolarized phase. The appearance of the NMDA-induced oscillations varies markedly between spinal cord preparations; they may either have a pronounced, depolarized plateau phase or be characterized by a short-lasting depolarization lasting approximately 200-300 ms without a plateau. Both types of oscillations increase in frequency with increased concentrations of NMDA. Here, we characterize these two types of membrane potential oscillations and show that they depend on the level of endogenous release of 5-HT in the spinal cord preparations. In the lamprey, 5-HT acts to block voltage-dependent calcium channels and will thereby modulate the activity of K(Ca) channels. When 5-HT antagonists were administered, the plateau-like oscillations were converted to the second type of oscillations lacking a plateau phase. Conversely, plateau-like oscillations can be induced or prolonged by 5-HT agonists. These properties are most likely of significance for the modulatory action of 5-HT on the spinal networks for locomotion.