Expression Of Voltage-gated Calcium Channels Research Articles

The PTB and PRR domains of numb regulate neurite outgrowth by influencing voltage-gated calcium channel expression and kinetics

Numb is an evolutionarily conserved protein that regulates the differentiation of neuronal progenitor cells through unknown mechanisms. Numb has four alternative splice variants with different lengths of phosphotyrosine-binding (PTB) and proline-rich regions (PRR) domains. In this study, we demonstrated that Numb expression was increased in the primary cultures of rat cortical and hippocampal neurons over time in vitro, and Numb antisense inhibited neurite outgrowth. We verified that cells overexpressing short PTB (SPTB) or long PTB (LPTB) domains exhibited differentiation or proliferation, respectively. SPTB-mediated differentiation was related to the PRR domains, as cells expressing SPTB/LPRR had longer dendrites and more branched dendrites than cells expressing SPTB/SPRR. The differentiation of both cell types was completely blocked by the Ca2+ chelator. Western blot analysis revealed the increased total protein expression of voltage-gated calcium channel (VGCC) subunit α1C and α1D in cells expressing SPTB and LPTB Numb. The increased expression of the VGCC β3 subunit was only observed in cells expressing SPTB Numb. Immunocytochemistry further showed that SPTB-mediated cell differentiation was associated with increased membrane expression of VGCC subunits α1C, α1D and β3, which corresponded to the higher Ca2+ current (ICa) densities. Furthermore, we found that VGCC of cells transfected with SPTB/SPRR or SPTB/LPRR Numb isoforms exhibit steady-state inactivation (SSI) in both differentiated and undifferentiated phenotypes. A similar SSI of VGCC was observed in the differentiated cells transfected with SPTB/SPRR or SPTB/LPRR Numb isoforms, whereas a left shift SSI of VGCC in cells expressing SPTB/LPRR was detected in the undifferentiated cells. Collectively, these data indicate that SPTB domain is essential for neurite outgrowth involving in membrane expression of VGCC subunits, and LPRR plays a role in neuronal branching and the regulation of VGCC inactivation kinetics.

EXTH-41. COMBINATION TREATMENT OF CALCIUM CHANNEL ANTAGONIST MIBEFRADIL ENHANCES THE EFFECT OF HYDROXYUREA AT THERAPEUTIC RANGES IN HIGH-GRADE MENINGIOMA IN VITRO

Abstract High-grade meningiomas (MNs), WHO Grade II- III, are highly aggressive brain tumors that are difficult to treat. Standard chemotherapy agents are, at best, moderately effective in treating high-grade MNs. There is great interest in developing combinatorial drug treatment regimens to enhance the effects of standard chemotherapy. Many cancer types, including brain tumors, have an increase in the expression of voltage-gated calcium channels (VGCCs). These channels are involved in the regulation of cell cycle, proliferation, and apoptosis, among other functions. FDA-approved VGCC antagonists nimodipine (NIMO) and mibefradil (MIB) target two different VGCCs, L- and T-type channels, and have been of great interest for their use in combination therapy in other cancers. We sought to determine the efficacy of chemotherapy agents tested at clinically relevant concentrations for the treatment of high-grade MNs alone or in combination with NIMO or MIB. We cultured CH-157MN and IOMM-Lee MN cells and assessed their cell survival, apoptosis, and proliferation in the presence of temozolomide, octreotide, sunitinib, or hydroxyurea (HU). We confirmed that HU (150 µM) was the most efficacious chemotherapy agent, decreasing cell viability at therapeutic ranges. Monotherapy treatment with 2 or 4 µM MIB significantly decreased cell viability in both cell lines, whereas 1 µM NIMO only affected IOMM-Lee cells. The combination of HU and MIB further decreased cell viability but only in the most aggressive cell line, IOMM-Lee, whereas NIMO did not enhance HU’s ability to decrease cell survival. Remarkably, we detect an enhancement of HU’s effect by MIB when both agents are used within therapeutic ranges, as reported by pharmacokinetic studies. Here we demonstrate for the first time that T-type calcium channels play an important role in MN survival. The combination of HU and MIB suggests their use could be a promising treatment for high-grade MN.

Using CRISPR knock-in of fluorescent tags to examine isoform-specific expression of EGL-19 in C. elegans.

L-type voltage-gated calcium channels (VGCCs) regulate calcium influx and excitation-contraction coupling in many types of muscle cells. Thus, VGCC mutations can cause skeletal and cardiac muscle diseases in humans, such as Duchenne muscular dystrophy and Timothy syndrome. To better understand the genetics and native expression of VGCCs, we have chosen to use the microscopic roundworm, C. elegans . The egl-19 locus is the sole L-type VGCC gene and it encodes three distinct isoforms (a, b, and c). Isoform c is curious because the protein is truncated, lacking the transmembrane domains that form the physical calcium channel. In this study, we have characterized egl-19 expression using CRISPR/Cas9 genome editing to 'knock-in' fluorescent tags of differing colors, allowing us to distinguish the expression pattern of each isoform. Not surprisingly, we found that EGL-19 is expressed in all types of muscle. In addition, we provide evidence that the truncated c isoform is expressed. Finally, although we find evidence that specific isoforms can have unique subcellular distributions, we also observed some expression patterns that appear to be artifacts. Overall, our results show interesting patterns of egl-19 expression, but also highlight the need for caution when interpreting expression of reporter genes even when they represent endogenous tags.

Role of GABAA receptor depolarization-mediated VGCC activation in sevoflurane-induced cognitive impairment in neonatal mice.

BackgroundIn neonatal mice, anesthesia with sevoflurane depolarizes the GABA Type A receptor (GABAAR), which leads to cognitive impairment. Calcium accumulation in neurons can lead to neurotoxicity. Voltage-gated calcium channels (VGCCs) can increase intracellular calcium concentration under isoflurane and hypoxic conditions. The underlying mechanisms remain largely unknown.MethodsSix-day-old mice were anesthetized with 3% sevoflurane for 2 h/day for 3 days. The Y-Maze, new object recognition (NOR) test, the Barnes maze test, immunoassay, immunoblotting, the TUNEL test, and Golgi–Cox staining were used to assess cognition, calcium concentration, inflammatory response, GABAAR activation, VGCC expression, apoptosis, and proliferation of hippocampal nerve cells in mice and HT22 cells.ResultsCompared with the control group, mice in the sevoflurane group had impaired cognitive function. In the sevoflurane group, the expression of Gabrb3 and Cav1.2 in the hippocampal neurons increased (p < 0.01), the concentration of calcium ions increased (p < 0.01), inflammatory reaction and apoptosis of neurons increased (p < 0.01), the proliferation of neurons in the DG area decreased (p < 0.01), and dendritic spine density decreased (p < 0.05). However, the inhibition of Gabrb3 and Cav1.2 alleviated cognitive impairment and reduced neurotoxicity.ConclusionsSevoflurane activates VGCCs by inducing GABAAR depolarization, resulting in cognitive impairment. Activated VGCCs cause an increase in intracellular calcium concentration and an inflammatory response, resulting in neurotoxicity and cognitive impairment.

Inhibitory effects on L- and N-type calcium channels by a novel CaV\u03b21 variant identified in a patient with autism spectrum disorder

Voltage-gated calcium channel (VGCC) subunits have been genetically associated with autism spectrum disorders (ASD). The properties of the pore-forming VGCC subunit are modulated by auxiliary β-subunits, which exist in four isoforms (CaVβ1-4). Our previous findings suggested that activation of L-type VGCCs is a common feature of CaVβ2 subunit mutations found in ASD patients. In the current study, we functionally characterized a novel CaVβ1b variant (p.R296C) identified in an ASD patient. We used whole-cell and single-channel patch clamp to study the effect of CaVβ1b_R296C on the function of L- and N-type VGCCs. Furthermore, we used co-immunoprecipitation followed by Western blot to evaluate the interaction of the CaVβ1b-subunits with the RGK-protein Gem. Our data obtained at both, whole-cell and single-channel levels, show that compared to a wild-type CaVβ1b, the CaVβ1b_R296C variant inhibits L- and N-type VGCCs. Interaction with and modulation by the RGK-protein Gem seems to be intact. Our findings indicate functional effects of the CaVβ1b_R296C variant differing from that attributed to CaVβ2 variants found in ASD patients. Further studies have to detail the effects on different VGCC subtypes and on VGCC expression.

Electrical therapies act on the Ca2+/CaM signaling pathway to enhance bone regeneration with bioactive glass [S53P4] and allogeneic grafts

This study aimed to investigate the application of low-intensity electrostimulation (ES) and electromagnetic stimulation (EM) associated with bioactive glass (BG) or allogeneic grafts (BB) in bone regeneration. A cell viability test on osteoblasts (UMR-106) was performed in the presence of BB and BG grafts associated with ES (10μA/5min) and EM (500 Hz/2min). Critical defects (25 mm2 ) in calvaria were generated in male Wistar rats, and bone regeneration was evaluated on the 30th, 60th, and 120th days after surgery. Cell proliferation increased with the application of ES in both grafts and after EM with BG. Bone remodeling was more effective using the allogeneic graft in both therapies, with increased angiogenesis, osteoblast proliferation, and OPN expression in the BB + EM group. A higher number of osteoblasts and osteoclasts, and an increase in bone sialoprotein, Runx-2, and Opn gene expression were found in the BB + ES group. The BG graft associated with EM therapy had an increased proliferation of osteoblasts and increased expression of Runx-2 and Opn. Groups that had BG and ES therapy had increased numbers of osteoblasts, osteoclasts, and increased OPN expression. The expression of voltage-gated calcium channels increased in groups with ES, while calmodulin expression increased in therapies without grafting. ES and EM therapies favored the repair of bone defects upon grafting by improving angiogenesis, osteogenic gene expression, and tissue reorganization. Despite activating different pathways, both therapies increased the intracellular concentrations of calmodulin, leading to cell proliferation and bone regeneration.

Gabapentinoid Benefit and Risk Stratification: Mechanisms Over Myth.

IntroductionRecent years have seen a dramatic escalation of off-label prescribing for gabapentin and pregabalin (gabapentinoids) owing in part to generic versions of each being released over the past two decades, but also in part as a response to increasing calls for multimodal and non-opioid pain management strategies. In this context, several recent articles have been published alleging widespread misuse, with speculations on the unappreciated addictive potential of the gabapentinoid class of drugs. Reports of a 1% population-level abuse prevalence stem from a single internet survey in the UK, and the vanishingly small adverse event outcomes data do not support such frequency. In this targeted narrative review, we aim to disabuse pain physicians and other clinicians, pharmacists, and policymakers of both the positive and negative myths concerning gabapentinoid medications.ResultsGabapentinoids inhibit the joint action of voltage-gated calcium channel (VGCC) α2δ subunits in conjunction with the n-methyl-d-aspartate (NMDA) receptor, with subsequent downregulation of VGCC expression and excitatory neurotransmitter release, and possibly synaptogenesis as well, through actions on thrombospondins. These activities reduce the likelihood of central sensitization, which explains in part the efficacy of the gabapentinoids in the management of neuropathic pain. Gabapentinoids also facilitate slow-wave sleep, a relatively rare phenomenon among central nerve system-acting agents, which is also thought to explain some of the therapeutic benefit of the class in conditions such as fibromyalgia. The number needed to treat to see benefit overlaps that of the nonsteroidal anti-inflammatory drugs, but with a considerably improved safety profile. Along these lines, in the context of over 50 million prescriptions per year in the USA alone, the gabapentinoids display remarkably low risk, including risks of misuse, abuse, and dependence. Furthermore, the neurobiology of these agents does not lend plausibility to the allegations, as they have never been shown to elicit dopaminergic activity within the nucleus accumbens, and in addition likely confer a "negative-feedback loop" for habituation and dependence by serving as functional NMDA antagonists, possibly through their actions on thrombospondins. Clinical and epidemiological addictionology studies corroborate the lack of any significant addictive potential of the gabapentinoids, and these drugs are increasingly being used in the treatment of addiction to other substances, with excellent results and no evidence of cross-addiction. However, among individuals with other substance use disorders and, in particular opioid use disorder, there are consistent data showing misuse of gabapentinoids in up to 20% of this population. Although there are allegations of using gabapentinoids to amplify the hedonic effects of opioids, the vast majority of misuse events appear to occur in an attempt to ameliorate opioid withdrawal symptoms. Furthermore, rare but potentially serious respiratory depression may occur, again amplified in the context of opioid or other sedative use. Careful risk:benefit assessment and stratification are warranted when prescription of a gabapentinoid is under consideration, in particular among individuals using opioids.ConclusionsGabapentinoids remain a vital tool in the pain physician’s multimodal armamentarium, but these drugs may not be effective in every clinical situation. Individuals with central sensitization and pain associated with slow-wave sleep deficits and potentially persons with comorbid addictions may benefit the most. The gabapentinoids appear to possess no addictive potential on their own, based on laboratory and clinical data, but they may be abused by persons with opioid use disorders; consequently, cautious risk stratification must take place.

Effect of Low Intensity Magnetic Field Stimulation on Calcium-Mediated Cytotoxicity After Mild Spinal Cord Contusion Injury in Rats.

Background:Magnetic field (MF) stimulation has the potential to reduce secondary damage and promote functional recovery after neural tissue injury. The study aimed to observe the effect of very low intensity (17.96µT) MF on general body condition, secondary damage, pain status, and locomotion.Methods:We exposed rats to MF (2 h/day × 3 weeks) after 6.25 mm contusion spinal injury. Locomotor behavior was evaluated by BBB score, pain assessment was done by recording threshold for tail flick, expression of voltage-gated calcium channels and extent of secondary damage in the spinal cord was assessed by immunofluorescence and Cresyl violet staining, respectively.Results:A significant (p ≤ .001) improvement in bladder function as well as BBB score was observed after MF exposure in comparison with sham and SCI over the observation period of 3 weeks. SCI group showed an increase in the threshold for vocalization after discharge, which decreased following MF exposure. Cresyl violet staining showed significantly higher tissue sparing (73%) at the epicenter after MF exposure when compared to SCI group. This was accompanied with a significant decrease in calcium channel expression in MF group as compared to SCI.Conclusion:The results suggest facilitation of sensory-motor recovery after MF exposure, which could be due to attenuation of secondary damage and calcium-mediated excitotoxicity in a mild contusion rat model of SCI.

Calcium signaling and expression of voltage-gated calcium channels in the mouse ovary throughout the estrous cycle†.

The estrous cycle is an iterative change in the anatomy, endocrinology, physiology, and behavior to provide maximum fecundity. Ovarian steroid production involves gonadotropin-induced [Ca2+]i raises due in part to voltage-gated Ca2+ channels (VGCCs) whose identity and tissue distribution in situ is largely unknown. Using fluorescence Ca2+ imaging and confocal microscopy, we recorded both spontaneous and depolarization-induced Ca2+ signals in living mouse ovarian slices. They were most prominent in theca cells (TCs) and oocytes. The presence of Ca2+ channel subunits CaV 1.2, CaV 1.3, CaV 2.1, CaV 2.2, and CaV 3 was examined with immunofluorescence of ovarian sections. CaV 1.2 and CaV 1.3 (L-type Ca2+ channels) are present in the stroma, granulosa cells (GCs), and corpora lutea (CL). Intriguingly subunits that are characteristic of nerve cells are also expressed: P/Q-type (CaV 2.1; α1A) in the stroma and CL cells and N-type (CaV 2.2; α1B) in perifollicular smooth muscle cells. The expression of α1 subunits fluctuates along the estrous cycle: in metestrus-diestrus (the quiescent stage of the cycle), CL and GCs are similarly stained, while in proestrus (stage of maximal ovarian stimulation) CL staining increases relatively to GCs. Also in proestrus, CaV 3 Ca2+ channel subunits are expressed more in CL compared to GC suggesting a more significant role of Ca2+ channels. In estrus, CaV 3 subunits from mesenchymal and interfollicular stromal cells become intensely stained. Our study represents an important step in understanding the role of VGCCs in ovarian physiology and possibly in ovarian cancer and other reproductive pathologies.

Expression of voltage-gated calcium channels augments cell susceptibility to membrane disruption by nanosecond pulsed electric field

We compared membrane permeabilization by nanosecond pulsed electric field (nsPEF) in HEK293 cells with and without assembled CaV1.3 L-type voltage-gated calcium channel (VGCC). Individual cells were subjected to one 300-ns pulse at 0 (sham exposure); 1.4; 1.8; or 2.3 kV/cm, and membrane permeabilization was evaluated by measuring whole-cell currents and by optical monitoring of cytosolic Ca2+. nsPEF had either no effect (0 and 1.4 kV/cm), or caused a lasting (>80 s) increase in the membrane conductance in about 50% of cells (1.8 kV/cm), or in all cells (2.3 kV/cm). The conductance pathway opened by nsPEF showed strong inward rectification, with maximum conductance increase for the inward current at the most negative membrane potentials. Although these potentials were below the depolarization threshold for VGCC activation, the increase in conductance in cells which expressed VGCC (VGCC+ cells) was about twofold greater than in cells which did not (VGCC− cells). Among VGCC+ cells, the nsPEF-induced increase in membrane conductance showed a positive correlation with the amplitude of VGCC current measured in the same cells prior to nsPEF exposure. These findings demonstrate that the expression of VGCC makes cells more susceptible to membrane permeabilization by nsPEF. Time-lapse imaging of nsPEF-induced Ca2+ transients confirmed permeabilization by a single 300-ns pulse at 1.8 or 2.3 kV/cm, but not at 1.4 kV/cm, and the transients were expectedly larger in VGCC+ cells. However, it remains to be established whether larger transients reflected additional Ca2+ entry through VGCC, or were a result of more severe electropermeabilization of VGCC+ cells.

Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium.

Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs) contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1), estrogen receptors (ESR1, ESR2, GPR30), and nuclear receptor coactivator 3 (NCOA3). Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM) downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM) antagonized the effect of BAY K8644 (2.5 or 5 µM) in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant human uterine myometrium and their gene expression regulation by beta-estradiol in pregnant conditions. Further exploration of the calcium signaling in TCs and its modulation by estrogen hormones will contribute to the understanding of labor and pregnancy mechanisms and to the development of effective strategies to reduce the risk of premature birth.

Calcium Channels in Postnatal Development of Rat Pancreatic Beta Cells and Their Role in Insulin Secretion.

Pancreatic beta cells during the first month of development acquire functional maturity, allowing them to respond to variations in extracellular glucose concentration by secreting insulin. Changes in ionic channel activity are important for this maturation. Within the voltage-gated calcium channels (VGCC), the most studied channels are high-voltage-activated (HVA), principally L-type; while low-voltage-activated (LVA) channels have been poorly studied in native beta cells. We analyzed the changes in the expression and activity of VGCC during the postnatal development in rat beta cells. We observed that the percentage of detection of T-type current increased with the stage of development. T-type calcium current density in adult cells was higher than in neonatal and P20 beta cells. Mean HVA current density also increased with age. Calcium current behavior in P20 beta cells was heterogeneous; almost half of the cells had HVA current densities higher than the adult cells, and this was independent of the presence of T-type current. We detected the presence of α1G, α1H, and α1I subunits of LVA channels at all ages. The Cav 3.1 subunit (α1G) was the most expressed. T-type channel blockers mibefradil and TTA-A2 significantly inhibited insulin secretion at 5.6 mM glucose, which suggests a physiological role for T-type channels at basal glucose conditions. Both, nifedipine and TTA-A2, drastically decreased the beta-cell subpopulation that secretes more insulin, in both basal and stimulating glucose conditions. We conclude that changes in expression and activity of VGCC during the development play an important role in physiological maturation of beta cells.

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on micropillar substrates

In this study, poly-L-lactic acid micropillar substrates were fabricated to evaluate the influence of topographic substrates on cell morphological and functional characteristics, such as spreading area, voltage-gated calcium channels (VGCCs) and membrane potential. The proliferation, spreading area, perimeter and circularity of SH-SY5Y cells interfaced with different substrates were first investigated. In addition, the cytoskeleton and focal adhesion of a cell as important manifestations of cell morphology were analyzed by immunofluorescence. VGCC responsiveness was evaluated by measuring the dynamic changes in intracellular Ca2+ evoked by 50 mM extracellular K+. To determine study whether the differences in VGCC responsiveness were caused by the differences in VGCC gene expression, the expression of N/L- type VGCCs was determined by qPCR and fluorescence staining. Notably, improved measurement of the membrane potential with potentiometric fluorescent dye TMRM was applied to determine the membrane potential of SH-SY5Y cells. Results indicated that the SH-SY5Y cells were deformed significantly to adapt to the substrates; however, no distinct effect on the proliferative ability of SH-SY5Y cells was observed. The micropillar substrates markedly influenced VGCC responsiveness, which correlated strongly with cell spreading but not with VGCC expression. The resting membrane potential of SH-SY5Y cells cultured on different substrates also changed, but no effect on responsiveness of VGCC was observed. These results suggest that the effect of the micropillar substrates on cell VGCC responsiveness may be attributed to changes in the functionality of the ion channel itself. Thus, topographic substrates can be used to engineer cell functionality in cell-based drug screening.

The shear wave elastic modulus and the increased nuclear factor kappa B (NF-kB/p65) and cyclooxygenase-2 (COX-2) expression in the area of myofascial trigger points activated in a rat model by blunt trauma to the vastus medialis

We aimed to elucidate the increased inflammatory cytokines expression such as nuclear factor kappa B (NF-kB/p65), cyclooxygenase-2 (COX-2), and voltage-gated calcium channels (VGCC) in the area of activated myofascial trigger points (MTrPs) in a rat model by blunt trauma to the vastus medialis and to evaluate the feasibility of a quantitative analysis of muscle elastic modulus using shear wave elastography (SWE). Twelve 7-week-old male SD rats were divided into normal (NM, n = 6) and model groups (MO, n = 6). In the MO group, MTrPs were activated with a blunt strike to the left vastus medialis and subsequent eccentric exercise for 8 weeks. After 4 weeks of rest, the elastic modulus in the focal site was evaluated using SWE. Electromyography (EMG) data were collected at MTrPs and muscle tissues were evaluated for expression of nuclear factor kappa B (NF-kB/p65), cyclooxygenase-2 (COX-2) protein, and voltage-gated calcium channels (VGCC). The number of the palpable taut bands; EMG frequency and amplitude; elastic modulus values; and NFkB/p65, COX-2, and VGCC expression levels were significantly higher for the left focal area in the MO group compared to those for the NM group (p’s < 0.05). These findings suggest that elastic modulus measurement using ultrasound SWE may be effective in evaluating MPS. In addition, increased COX-2, NFkB/p65, and VGCC expression may expand the integrated hypothesis of trigger points.

Trafficking of neuronal calcium channels.

Neuronal voltage-gated calcium channels (VGCCs) serve complex yet essential physiological functions via their pivotal role in translating electrical signals into intracellular calcium elevations and associated downstream signalling pathways. There are a number of regulatory mechanisms to ensure a dynamic control of the number of channels embedded in the plasma membrane, whereas alteration of the surface expression of VGCCs has been linked to various disease conditions. Here, we provide an overview of the mechanisms that control the trafficking of VGCCs to and from the plasma membrane, and discuss their implication in pathophysiological conditions and their potential as therapeutic targets.

EFFECTS OF METHAMPHETAMINE ON LOCOMOTOR ACTIVITY AND THALAMIC GENE EXPRESSION IN LEPTIN-DEFICIENT OBESE MICE.

Leptin is an adipose-derived hormone that regulates energy balance. Leptin receptors are expressed in extrahypothalamic sites and several reports showed that leptin can influence feeding and locomotor behavior via direct actions on dopaminergic neurons. The leptin deficient mouse (ob/ob) has been used as an animal model of blunted leptin action, and presents with obesity and mild type 2 diabetes. We used ob/ob mice to study the effect of repeated 7-day methamphetamine (METH) administration analyzing locomotion, behavioral sensitization, and somatosensory thalamic mRNA expression of voltage-gated calcium channels and glutamatergic receptors using RT-PCR. We observed reduced METH-mediated responses in ob/ob mice associated with enhanced in mRNA expression of key voltage-gated and glutamate receptors in the somatosensory thalamus. Results described here are important for understanding the control of locomotion and thalamocortical excitability by leptin.

The Role of Calcium Channels in Epilepsy

A central theme in the quest to unravel the genetic basis of epilepsy has been the effort to elucidate the roles played by inherited defects in ion channels. The ubiquitous expression of voltage-gated calcium channels (VGCCs) throughout the central nervous system (CNS), along with their involvement in fundamental processes, such as neuronal excitability and synaptic transmission, has made them attractive candidates. Recent insights provided by the identification of mutations in the P/Q-type calcium channel in humans and rodents with epilepsy and the finding of thalamic T-type calcium channel dysfunction in the absence of seizures have raised expectations of a causal role of calcium channels in the polygenic inheritance of idiopathic epilepsy. In this review, we consider how genetic variation in neuronal VGCCs may influence the development of epilepsy.

Injury-specific functional alteration of N-type voltage-gated calcium channels in synaptic transmission of primary afferent C-fibers in the rat spinal superficial dorsal horn

We investigated functional alterations of voltage-gated calcium channels (VGCCs) in excitatory synaptic transmission from primary afferent A- and C-fibers after peripheral nerve injury. Patch-clamp recordings were performed on substantia gelatinosa (SG) neurons of spinal cord slices with an attached dorsal root, prepared from L5 spinal nerve-ligated (SNL) rats. The effects of neuronal VGCC blockers, ω-conotoxin GVIA (ω-CgTX) for N-type channels and ω-agatoxin IVA (ω-AgaIVA) for P/Q-type channels, on evoked excitatory postsynaptic currents (eEPSCs) by stimulation of A- or C-fibers were studied. Besides, electrophysiological assay using dorsal root ganglion (DRG) and immunohistochemistry were done. In naïve rats, ω-CgTX (0.1–1μM) reduced more effectively A-fiber eEPSCs than C-fiber ones. After nerve injury, ω-CgTX produced great inhibition of C-fiber eEPSCs in slices with the injured L5 dorsal root of SNL model rats, as compared to sham-operated rats. By contrast, in slices with the non-injured L4 one, inhibitory effects of ω-CgTX were not changed. This occurred concurrently with increased expression of N-type VGCCs in L5 spinal dorsal horn and with enhanced Ca2+ currents through N-type VGCCs in small-sized (C-type) L5 DRG. In terms of A-fiber eEPSCs, ω-CgTX elicited similar inhibition in nerve-injured and sham-operated rats. ω-AgaIVA (0.1μM) had less effect on A- or C-fiber eEPSCs. These results indicate that N-type, but not P/Q-type, VGCCs mainly contribute to excitatory synaptic transmission from A- and C-fibers in the spinal dorsal horn. More importantly, following nerve injury, the functional contribution of N-type VGCCs to nociceptive transmission is increased in the pre-synaptic terminals of injured C-fibers.

Meta-Analysis of Public Microarray Datasets Reveals Voltage-Gated Calcium Gene Signatures in Clinical Cancer Patients.

Voltage-gated calcium channels (VGCCs) are well documented to play roles in cell proliferation, migration, and apoptosis; however, whether VGCCs regulate the onset and progression of cancer is still under investigation. The VGCC family consists of five members, which are L-type, N-type, T-type, R-type and P/Q type. To date, no holistic approach has been used to screen VGCC family genes in different types of cancer. We analyzed the transcript expression of VGCCs in clinical cancer tissue samples by accessing ONCOMINE (www.oncomine.org), a web-based microarray database, to perform a systematic analysis. Every member of the VGCCs was examined across 21 different types of cancer by comparing mRNA expression in cancer to that in normal tissue. A previous study showed that altered expression of mRNA in cancer tissue may play an oncogenic role and promote tumor development; therefore, in the present findings, we focus only on the overexpression of VGCCs in different types of cancer. This bioinformatics analysis revealed that different subtypes of VGCCs (CACNA1C, CACNA1D, CACNA1B, CACNA1G, and CACNA1I) are implicated in the development and progression of diverse types of cancer and show dramatic up-regulation in breast cancer. CACNA1F only showed high expression in testis cancer, whereas CACNA1A, CACNA1C, and CACNA1D were highly expressed in most types of cancer. The current analysis revealed that specific VGCCs likely play essential roles in specific types of cancer. Collectively, we identified several VGCC targets and classified them according to different cancer subtypes for prospective studies on the underlying carcinogenic mechanisms. The present findings suggest that VGCCs are possible targets for prospective investigation in cancer treatment.

Regulation of L-type Voltage Gated Calcium Channel CACNA1S in Macrophages upon Mycobacterium tuberculosis Infection.

We demonstrated earlier the inhibitory role played by Voltage Gated Calcium Channels (VGCCs) in regulating Mycobacterium tuberculosis (M. tb) survival and pathogenesis. In this report, we investigated mechanisms and key players that regulate the surface expression of VGCC-CACNA1S by Rv2463 and M. tb infection in macrophages. Our earlier work identified Rv2463 to be expressed at early times post infection in macrophages that induced suppressor responses to dendritic cells and macrophages. Our results in this study demonstrate a role of MyD88 independent TLR pathway in mediating CACNA1S expression. Dissecting the role for second messengers, we show that calcium homeostasis plays a key role in CACNA1S expression during M. tb infection. Using siRNAs against molecular sensors of calcium regulation, we show an involvement of ER associated Stromal Interaction Molecules 1 and 2 (STIM1 and STIM2), and transcription factor pCREB, towards CACNA1S expression that also involved the MyD88 independent pathway. Interestingly, reactive oxygen species played a negative role in M. tb mediated CACNA1S expression. Further, a cross-regulation of ROS and pCREB was noted that governed CACNA1S expression. Characterizing the mechanisms governing CACNA1S expression would improve our understanding of the regulation of VGCC expression and its role in M. tb pathogenesis during M. tb infection.