T-type Voltage-gated Calcium Channels Research Articles

Involvement of Voltage-Gated Calcium Channels in Inflammation and Inflammatory Pain.

Voltage-gated calcium channels (VGCCs) are classified into high-voltage-activated (HVA) channels and low-voltage-activated channels consisting of Cav3.1-3.3, known as T ("transient")-type VGCC. There is evidence that certain types of HVA channels are involved in neurogenic inflammation and inflammatory pain, in agreement with reports indicating the therapeutic effectiveness of gabapentinoids, ligands for the α2δ subunit of HVA, in treating not only neuropathic, but also inflammatory, pain. Among the Cav3 family members, Cav3.2 is abundantly expressed in the primary afferents, regulating both neuronal excitability at the peripheral terminals and spontaneous neurotransmitter release at the spinal terminals. The function and expression of Cav3.2 are modulated by a variety of inflammatory mediators including prostanoids and hydrogen sulfide (H2S), a gasotransmitter. The increased activity of Cav3.2 by H2S participates in colonic, bladder and pancreatic pain, and regulates visceral inflammation. Together, VGCCs are involved in inflammation and inflammatory pain, and Cav3.2 T-type VGCC is especially a promising therapeutic target for the treatment of visceral inflammatory pain in patients with irritable bowel syndrome, interstitial cystitis/bladder pain syndrome, pancreatitis, etc., in addition to neuropathic pain.

Oxaliplatin Modulates the Characteristics of Voltage-Gated Calcium Channels and Action Potentials in Small Dorsal Root Ganglion Neurons of Rats.

Oxaliplatin is important for treating colorectal cancer. Although oxaliplatin is highly effective, it has severe side effects, of which neurotoxicity in dorsal root ganglion (DRG) neurons is one of the most common. The key mechanisms of this neurotoxicity are still controversial. However, disturbances of calcium homeostasis in DRG neurons have been suggested to mediate oxaliplatin neurotoxicity. By using whole-cell patch-clamp and current-clamp techniques, as well as immunocytochemical staining, we examined the influence of short- and long-term exposure to oxaliplatin on voltage-gated calcium channels (VGCC) and different VGCC subtypes in small DRG neurons of rats in vitro. Exposure to oxaliplatin reduced VGCC currents (ICa(V)) in a concentration-dependent manner (1-500μM; 13.8-63.3%). Subtype-specific measurements of VGCCs showed differential effects on ICa(V). While acute treatment with oxaliplatin led to a reduction in ICa(V) for P/Q-, T-, and L-type VGCCs, ICa(V) of N-type VGCCs was not affected. Exposure of DRG neurons to oxaliplatin (10 or 100μM) for 24h in vitro significantly increased the ICa(V) current density, with a significant influence on L- and T-type VGCCs. Immunostaining revealed an increase of L- and T-type VGCC protein levels in DRG neurons 24h after oxaliplatin exposure. This effect was mediated by calcium-calmodulin-protein kinase II (CaMKII). Significant alterations in action potentials (AP) and their characteristics were also observed. While the amplitude increased after oxaliplatin treatment, the rise time and time-to-peak decreased, and these effects were reversed by treatment with pimozide and nimodipine, which suggests that VGCCs are critically involved in oxaliplatin-mediated neurotoxicity.

Amplitude-modulated radiofrequency electromagnetic fields target hepatocellular carcinoma stem cells through activation of Cav 3.2 T-type calcium channels.

332 Background: Amplitude-modulated 27.12 MHz radiofrequency electromagnetic fields (AM RF EMF) delivered via a spoon-shaped antenna placed on the patient’s tongue result in shrinkage of the primary and metastatic tumors in patients with advanced hepatocellular carcinoma (HCC). (Costa FP, et al. Br J Cancer. 2011;105:640-648.) The mechanism by which AM RF EMF have direct antiproliferative effect and disruption of the mitotic spindle on cancer cells is largely unknown. (Zimmerman JW, et al. Br J Cancer. 2012;106:307-313.) Methods: We assessed the Specific Absorption Rate (SAR) level and distribution inside the human body. In vitro experiments with HCC cells were performed as previously described (Zimmerman). Immunodeficient mice were subcutaneously implanted with Huh-7 HCC cells or patient-derived xenografts (PDX). Mice were exposed to HCC-specific AM RF EMF using systems replicating human exposure levels and treatment duration. Tumor samples were examined for EMT and cell cycle markers. Results: Intrabuccal delivery results in whole-body absorption of AM RF EMF. Proliferation of hepatitis B positive and negative cell lines as well as HCC cancer stem cells (CSCs) are blocked by AM RF EMF through Cav 3.2 T-type voltage gated calcium channels (VGCC). Proliferation of HCC is inhibited in vivo and tumor shrinkage occurs by dedifferentiation of HCC cells into quiescent myofibroblasts while the growth of intestinal and blood marrow cells is unaltered. Conclusions: Whole-body SAR ranges from 0.2 to 1 mW/kg and is significantly below the international safety limits for human exposure. Intrabucally-administered AM RF EMF is a novel targeted therapy for systemic treatment of advanced HCC with minimal off target effects. Inhibition of HCC CSCs may explain the extremely long term survival (>5 years) of several patients with advanced HCC.

Cav3.1 overexpression is associated with negative characteristics and prognosis in non-small cell lung cancer

IntroductionVoltage-gated calcium channels (VGCC) have been found to be differentially expressed in several different tumor types, but their role in tumor growth, malignant invasion, metastases and impact on clinical outcomes has not been clarified.Materials and MethodsFrom a cohort database of 193 patients with early-stage NSCLC, 163 formalin-fixed paraffin-embedded specimens were available for analysis to construct tissue microarrays. Cav3.1 protein expression was detected using fluorescence immunohistochemistry, and quantified using automated image acquisition and analysis.ResultsAmong the cohort of 193 NSCLC patients, adenocarcinoma (53.9%) and squamous cell carcinoma (SCC) (30.1%) were the most common histologies. There was no difference between SCC and non-SCC subtypes in overall survival (OS) or relapse-free survival (RFS); 74.2 vs 90.1 months (p = 0.543) and 48.8 vs 52.6 months (p = 0.766), respectively. T-type VGCC 3.1 (Cav3.1) overexpression was assessed by tissue microarray immunohistochemistry analysis from 163 available patient samples. Eighteen (11.0%) NSCLC primaries were found to have Cav3.1 overexpression levels, and were significantly associated with SCC histology (p < 0.001), larger tumor size (p < 0.001) and later stage disease at diagnosis (p = 0.019). Median OS was 48.6 vs 106.7 months for Cav3.1 overexpressing and non-overexpressing patients, respectively (p = 0.032). Regression analysis revealed a significantly negative effect for Cav3.1 overexpression on RFS (Hazard ratio [HR] = 2.02, p = 0.048).ConclusionsCav3.1 overexpression is a potential biomarker for poorer patient outcomes. These results bring supportive evidence for calcium channels inducing an aggressive phenotype in NSCLC and potentially may serve as a therapeutic target in overexpressing tumors.

L- and T-type calcium channel blockers protect against the inhibitory effects of mipafox on neurite outgrowth and plasticity-related proteins in SH-SY5Y cells

ABSTRACTSome organophosphorus compounds (OP), including the pesticide mipafox, produce late onset distal axonal degeneration, known as organophosphorus-induced delayed neuropathy (OPIDN). The underlying mechanism involves irreversible inhibition of neuropathy target esterase (NTE) activity, elevated intracellular calcium levels, increased activity of calcium-activated proteases and impaired neuritogenesis. Voltage-gated calcium channels (VGCC) appear to play a role in several neurologic disorders, including OPIDN. Therefore, this study aimed to examine and compare the neuroprotective effects of T-type (amiloride) and L-type (nimodipine) VGCC blockers induced by the inhibitory actions of mipafox on neurite outgrowth and axonal proteins of retinoic-acid-stimulated SH-SY5Y human neuroblastoma cells, a neuronal model widely employed to determine the neurotoxicity attributed to OP. Both nimodipine and amiloride significantly blocked augmentation of intracellular calcium levels and activity of calpains, as well as decreased neurite length, number of differentiated cells, and lowered concentrations of growth-associated protein 43 (GAP-43) and synapsin induced by mipafox. Only nimodipine inhibited reduction of synaptophysin levels produced by mipafox. These findings demonstrate a role for calcium and VGCC in the impairment of neuronal plasticity mediated by mipafox. Data also demonstrated the neuroprotective potential of T-type and L-type VGCC blockers to inhibit OP-mediated actions, which may be beneficial to counteract cases of pesticide poisoning.

Ciguatoxins Evoke Potent CGRP Release by Activation of Voltage-Gated Sodium Channel Subtypes NaV1.9, NaV1.7 and NaV1.1.

Ciguatoxins (CTXs) are marine toxins that cause ciguatera fish poisoning, a debilitating disease dominated by sensory and neurological disturbances that include cold allodynia and various painful symptoms as well as long-lasting pruritus. Although CTXs are known as the most potent mammalian sodium channel activator toxins, the etiology of many of its neurosensory symptoms remains unresolved. We recently described that local application of 1 nM Pacific Ciguatoxin-1 (P-CTX-1) into the skin of human subjects induces a long-lasting, painful axon reflex flare and that CTXs are particularly effective in releasing calcitonin-gene related peptide (CGRP) from nerve terminals. In this study, we used mouse and rat skin preparations and enzyme-linked immunosorbent assays (ELISA) to study the molecular mechanism by which P-CTX-1 induces CGRP release. We show that P-CTX-1 induces CGRP release more effectively in mouse as compared to rat skin, exhibiting EC50 concentrations in the low nanomolar range. P-CTX-1-induced CGRP release from skin is dependent on extracellular calcium and sodium, but independent from the activation of various thermosensory transient receptor potential (TRP) ion channels. In contrast, lidocaine and tetrodotoxin (TTX) reduce CGRP release by 53–75%, with the remaining fraction involving L-type and T-type voltage-gated calcium channels (VGCC). Using transgenic mice, we revealed that the TTX-resistant voltage-gated sodium channel (VGSC) NaV1.9, but not NaV1.8 or NaV1.7 alone and the combined activation of the TTX-sensitive VGSC subtypes NaV1.7 and NaV1.1 carry the largest part of the P-CTX-1-caused CGRP release of 42% and 34%, respectively. Given the contribution of CGRP to nociceptive and itch sensing pathways, our findings contribute to a better understanding of sensory symptoms of acute and chronic ciguatera that may help in the identification of potential therapeutics.

Inhibition of human N- and T-type calcium channels by an ortho-phenoxyanilide derivative, MONIRO-1.

Voltage-gated calcium channels are involved in nociception in the CNS and in the periphery. N-type (Cav 2.2) and T-type (Cav 3.1, Cav 3.2 and Cav 3.3) voltage-gated calcium channels are particularly important in studying and treating pain and epilepsy. In this study, whole-cell patch clamp electrophysiology was used to assess the potency and mechanism of action of a novel ortho-phenoxylanilide derivative, MONIRO-1, against a panel of voltage-gated calcium channels including Cav 1.2, Cav 1.3, Cav 2.1, Cav 2.2, Cav 2.3, Cav 3.1, Cav 3.2 and Cav 3.3. MONIRO-1 was 5- to 20-fold more potent at inhibiting human T-type calcium channels, hCav 3.1, hCav 3.2 and hCav 3.3 (IC50 : 3.3±0.3, 1.7±0.1 and 7.2±0.3μM, respectively) than N-type calcium channel, hCav 2.2 (IC50 : 34.0±3.6μM). It interacted with L-type calcium channels Cav 1.2 and Cav 1.3 with significantly lower potency (IC50 >100μM) and did not inhibit hCav 2.1 or hCav 2.3 channels at concentrations as high as 100μM. State- and use-dependent inhibition of hCav 2.2 channels was observed, whereas stronger inhibition occurred at high stimulation frequencies for hCav 3.1 channels suggesting a different mode of action between these two channels. Selectivity, potency, reversibility and multi-modal effects distinguish MONIRO-1 from other low MW inhibitors acting on Cav channels involved in pain and/or epilepsy pathways. High-frequency firing increased the affinity for MONIRO-1 for both hCav 2.2 and hCav 3.1 channels. Such Cav channel modulators have potential clinical use in the treatment of epilepsies, neuropathic pain and other nociceptive pathophysiologies. This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

T-type voltage gated calcium channels are involved in endothelium-dependent relaxation of mice pulmonary artery

In pulmonary arterial endothelial cells, Ca2+ channels and intracellular Ca2+ concentration ([Ca2+]i) control the release of vasorelaxant factors such as nitric oxide and are involved in the regulation of pulmonary arterial blood pressure. The present study was undertaken to investigate the implication of T-type voltage-gated Ca2+ channels (T-VGCCs, Cav3.1 channel) in the endothelium-dependent relaxation of intrapulmonary arteries. Relaxation was quantified by means of a myograph in wild type and Cav3.1−/− mice. Endothelial [Ca2+]i and NO production were measured, on whole vessels, with the fluo-4 and DAF-fm probes. Acetylcholine (ACh) induced a nitric oxide- and endothelium-dependent relaxation that was significantly reduced in pulmonary arteries from Cav3.1−/− compared to wild type mice as well as in the presence of T-VGCC inhibitors (NNC 55-0396 or mibefradil). ACh also increased endothelial [Ca2+]i and NO production that were both reduced in Cav3.1−/− compared to wild type mice or in the presence of T-VGCC inhibitors. Immunofluorescence labeling revealed the presence of Cav3.1 channels in endothelial cells that co-localized with endothelial nitric oxide synthase in arteries from wild type mice. TRPV4-, beta2 adrenergic- and nitric oxide donors (SNP)-mediated relaxation were not altered in Cav3.1−/− compared to wild type mice. Finally, in chronically hypoxic mice, a model of pulmonary hypertension, ACh relaxation was reduced but still depended on Cav3.1 channels activity. The present study thus demonstrates that T-VGCCs, mainly Cav3.1 channel, contribute to intrapulmonary vascular reactivity in mice by controlling endothelial [Ca2+]i and ACh-mediated relaxation.

Deltamethrin affects the expression of voltage-gated calcium channel α1 subunits and the locomotion, egg-laying, foraging behavior of Caenorhabditis elegans

Deltamethrin belongs to the class of synthetic pyrethroids, which are being widely used as insecticides in agricultural practices. Voltage-gated sodium channels (VGSCs) are the primary targets of these chemicals for toxicity to insects. Caenorhabditis elegans (C. elegans) does not have VGSCs but is susceptible to deltamethrin. Recent findings have suggested that pyrethroids can affect voltage-gated calcium channels (VGCCs). However, it remains elusive whether deltamethrin induces toxicity to C. elegans via modulating the activity of VGCCs. To identify the potential target of deltamethrin, we exposed C. elegans to different concentrations of deltamethrin and Ca2+ channel blockers for different times, characterized the behavioral toxicity of deltamethrin on C. elegans, and determined the expression of egl-19, unc-2, and cca-1, which encode the α1-subunit of the L-, R/N/P/Q-, and T-type VGCC, respectively. We found that deltamethrin inhibited the locomotion, egg-laying and foraging ability of C. elegans in a concentration dependent manner. We also showed that body length of worms on agar plates containing 200mgL−1 deltamethrin for 12h was not significantly different from controls, whereas the cholinesterase inhibitor carbofuran caused hypercontraction which is a characteristic of organophosphates and carbamates, suggesting that deltamethrin's mode of action is distinct from those nematicides. In addition, unc-2 was significantly up-regulated following 0.05mgL−1 deltamethrin exposure for 24h; while egl-19 and cca-1 were significantly up-regulated following 5 and 50mgL−1 deltamethrin exposure for 24h. Further tests of worms' sensitivity and expression of three α1-subunits of VGCC to Ca2+ channel blockers indicate that deltamethrin may induce toxic behavior C. elegans via modulation of the expression of the α1-subunits of VGCC. This study provides insights into the linkage between deltamethrin-induced toxic behavior and the regulation of α1-subunits of VGCC in C. elegans.

An Intrinsic Transcriptional Program Underlying Synaptic Scaling during Activity Suppression

Homeostatic scaling allows neurons to maintain stable activity patterns by globally altering their synaptic strength in response to changing activity levels. Suppression of activity by the blocking of action potentials increases synaptic strength through anupregulation of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Although this synaptic upscaling was shown to require transcription, the molecular nature of the intrinsic transcription program underlying this process and its functional significance have been unclear. Using RNA-seq, we identified 73 genes that were specifically upregulated in response to activity suppression. In particular, Neuronal pentraxin-1 (Nptx1) increased within 6hr of activity blockade, and knockdown of this gene blocked the increase in synaptic strength. Nptx1 induction is mediated by calcium influx through the T-type voltage-gated calcium channel, as well as two transcription factors, SRF and ELK1. Altogether, these results uncover a transcriptional program that specifically operates when neuronal activity is suppressed to globally coordinate the increase in synaptic strength.

Indispensable role of the voltage-gated calcium channels in the procognitive effects of angiotensin IV.

Voltage-gated calcium channels (VGCCs) play a major role in brain functioning, including that of cognition-related structures such as cerebral cortex and hippocampus. Cellular mechanisms underlying learning and memory enhancing effect of the neuropeptide angiotensin IV (Ang IV) have been linked to VGCCs but only in respect of its long-term potentiation (LTP)-inducing effect. To assess behaviorally effects of L- and T-type VGCCs blocking drugs in low, behaviorally inactive, doses on Ang IV facilitation of recall of aversively (foot-shock) and appetitively (curiosity for novelty) motivated behaviors. About 240 male Wistar rats were used. All animals received oral (p.o.) dose of nimodipine (12mg/kg) or mibefradil (1mg/kg) dissolved in saline or saline alone followed by an intracerebroventricular (i.c.v.) injection of 1nmol of Ang IV dissolved in 2μl of normal saline or saline alone 15min later. Groups of about 10 rats, separate for each experiment, were tested for recall of aversively (inhibitory avoidance, IA) and appetitively (object recognition, OR) reinforced behaviors. To verify lack of unspecific motor and emotional effects of our treatments separate groups of rats were tested in open field (OF) and elevated 'plus' maze (EPM), respectively. Both, nimodipine and mibefradil prevented recall facilitating effects of subsequently injected Ang IV. The peptide as well as both VGCCs blocking drugs had no (OF), or only negligible (EPM) effects on motor performance and emotionality of rats. The data support a notion about key role of the functional VGCCs in neuronal procognitive effects of Ang IV.

(±)-Ganoapplanin, a Pair of Polycyclic Meroterpenoid Enantiomers from Ganoderma applanatum.

(±)-Ganoapplanin (1), a pair of novel meroterpenoid enantiomers featuring an unprecedented dioxaspirocyclic skeleton constructed from a 6/6/6/6 tetracyclic system and an unusual tricyclo[4.3.3.03',7']dodecane motif, were isolated from Ganoderma applanatum. Its structure and absolute configurations were determined by spectroscopic analyses, X-ray crystallography, and ECD (electronic circular dichroism calculations). A plausible biogenetic pathway, involving a key Gomberg-Bachmann reaction, was also proposed for (±)-1. Biological studies showed that (±)-1 and its enantiomers exhibited different inhibitory activities on T-type voltage-gated calcium channels.

Cisplatin-induced neuropathic pain is mediated by upregulation of N-type voltage-gated calcium channels in dorsal root ganglion neurons

Cisplatin is important in the treatment of various types of cancer. Although it is highly effective, it also has severe side effects, with neurotoxicity in dorsal root ganglion (DRG) neurons being one of the most common. The key mechanisms of neurotoxicity are still controversially discussed; however, disturbances of the calcium homeostasis in DRG neurons have been suggested to mediate cisplatin neurotoxicity. By using the whole-cell patch-clamp technique, immunostaining and behavioral experiments with Sprague-Dawley rats, we examined the influence of short- and long-term exposure to cisplatin on voltage-gated calcium channel (VGCC) currents (ICa(V)) in small DRG neurons. In vitro exposure to cisplatin reduced ICa(V) in a concentration-dependent manner (0.01–50μM; 13.8–77.3%; IC50 5.07μM). Subtype-specific measurements of VGCCs showed differential effects on ICa(V). While the ICa(V) of P/Q-, L- and T-type VGCCs were reduced, ICa(V) of N-type VGCCs were increased by 30.3% during depolarization to 0mV. Exposure of DRG neurons to cisplatin (0.5 or 5μM) for 24–48h in vitro significantly increased a CaMK II-mediated ICa(V) current density. Immunostaining and western blot analysis revealed an increase of N-type VGCC protein level in DRG neurons 24h after cisplatin exposure. Cisplatin-mediated activation of caspase-3 was prevented by inhibition of N-type VGCCs using Ɯ-conotoxin MVIIA. Behavioral experiments showed that Ɯ-conotoxin MVIIA treatment prevented neuropathic syndromes in vivo by inhibiting upregulation of the N-type protein level. Here we show evidence for the first time for a crucial role of N-type VGCC in the genesis of cisplatin-induced polyneuropathy.

Ion channels, ion channel receptors, and visceral hypersensitivity in irritable bowel syndrome.

Ion channels are expressed throughout the gastrointestinal system and regulate nearly every aspect of digestion, including fluid secretion and absorption, motility, and visceral sensitivity. It is therefore not surprising that in the setting of functional bowel disorders, such as irritable bowel syndrome (IBS), ion channels are often altered in terms of expression level and function and are a target of pharmacological intervention. This is particularly true of their role in driving abdominal pain through visceral hypersensitivity (VH), which is the main reason IBS patients seek medical care. In the study by Scanzi etal., in the current issue of this journal, they provide evidence that the T-type voltage-gated calcium channel (Cav ) Cav 3.2 is upregulated in human IBS patients, and is necessary for the induction of an IBS-like disease state in mice. In this mini-review, we will discuss the contribution of specific ion channels to VH in IBS, both in human patients and rodent models. We will also discuss how Cav 3.2 may play a role as an integrator of multiple environmental stimuli contributing toward VH.

Ca2+ transients in astrocyte fine processes occur via Ca2+ influx in the adult mouse hippocampus.

Astrocytes display complex morphologies with an array of fine extensions extending from the soma and the primary thick processes. Until the use of genetically encoded calcium indicators (GECIs) selectively expressed in astrocytes, Ca2+ signaling was only examined in soma and thick primary processes of astrocytes where Ca2+ -sensitive fluorescent dyes could be imaged. GECI imaging in astrocytes revealed a previously unsuspected pattern of spontaneous Ca2+ transients in fine processes that has not been observed without chronic expression of GECIs, raising potential concerns about the effects of GECI expression. Here, we perform two-photon imaging of Ca2+ transients in adult CA1 hippocampal astrocytes using a new single-cell patch-loading strategy to image Ca2+ -sensitive fluorescent dyes in the cytoplasm of fine processes. We observed that astrocyte fine processes exhibited a high frequency of spontaneous Ca2+ transients whereas astrocyte soma rarely showed spontaneous Ca2+ oscillations similar to previous reports using GECIs. We exploited this new approach to show these signals were independent of neuronal spiking, metabotropic glutamate receptor (mGluR) activity, TRPA1 channels, and L- or T-type voltage-gated calcium channels. Removal of extracellular Ca2+ almost completely and reversibly abolished the spontaneous signals while IP3 R2 KO mice also exhibited spontaneous and compartmentalized signals, suggesting they rely on influx of extracellular Ca2+ . The Ca2+ influx dependency of the spontaneous signals in patch-loaded astrocytes was also observed in astrocytes expressing GCaMP3, further highlighting the presence of Ca2+ influx pathways in astrocytes. The mechanisms underlying these localized Ca2+ signals are critical for understanding how astrocytes regulate important functions in the adult brain. GLIA 2016;64:2093-2103.

Exploring poisonous mechanism of honeybee, Apis mellifera ligustica Spinola, caused by pyrethroids

As the important intracellular secondary messengers, calcium channel is the target of many neurotoxic pesticides as calcium homeostasis in the neuroplasm play important role in neuronal functions and behavior in insects. This study investigated the effect of deltamethrin (DM) on calcium channel in the brain nerve cells of adult workers of Apis mellifera ligustica Spinola that were cultured in vitro. The results showed that the intracellular calcium concentration was significantly elevated even with a very low concentration of the DM (3.125×10−2mg/L). Further testing revealed that T-type voltage-gated calcium channels (VGCCs), except for sodium channels, was one of the target of DM on toxicity of Apis mellifera, while DM has no significant effect on the L-type VGCCs, N-methyl-d-aspartate receptor-gated calcium channels and calcium store. These results suggesting that the DM may act on T-type VGCCs in brain cells of honeybees and result in behavioral abnormalities including swarming, feeding, learning, and acquisition.

P/Q-type and T-type voltage-gated calcium channels are involved in the contraction of mammary and brain blood vessels from hypertensive patients.

Calcium channel blockers are widely used in cardiovascular diseases. Besides L-type channels, T- and P/Q-type calcium channels are involved in the contraction of human renal blood vessels. It was hypothesized that T- and P/Q-type channels are involved in the contraction of human brain and mammary blood vessels. Internal mammary arteries from bypass surgery patients and cerebral arterioles from patients with brain tumours with and without hypertension were tested in a myograph and perfusion set-up. PCR and immunohistochemistry were performed on isolated blood vessels. The P/Q-type antagonist ω-agatoxin IVA (10-8 molL-1 ) and the T-type calcium blocker mibefradil (10-7 molL-1 ) inhibited KCl depolarization-induced contraction in mammary arteries from hypertensive patients with no effect on blood vessels from normotensive patients. ω-Agatoxin IVA decreased contraction in cerebral arterioles from hypertensive patients. L-type blocker nifedipine abolished the contraction in mammary arteries. PCR analysis showed expression of P/Q-type (Cav 2.1), T-type (Cav 3.1 and Cav 3.2) and L-type (Cav 1.2) calcium channels in mammary and cerebral arteries. Immunohistochemical labelling of mammary and cerebral arteries revealed the presence of Cav 2.1 in endothelial and smooth muscle cells. Cav 3.1 was also detected in mammary arteries. P/Q- and T-type Cav are present in human internal mammary arteries and in cerebral penetrating arterioles. P/Q- and T-type calcium channels are involved in the contraction of mammary arteries from hypertensive patients but not from normotensive patients. Furthermore, in cerebral arterioles P/Q-type channels importance was restricted to hypertensive patients might lead to that T- and P/Q-type channels could be a new target in hypertensive patients.

Genetic alteration of the metal/redox modulation of Cav3.2 T‐type calcium channel reveals its role in neuronal excitability

In this study, we describe a new knock-in (KI) mouse model that allows the study of the H191-dependent regulation of T-type Cav3.2 channels. Sensitivity to zinc, nickel and ascorbate of native Cav3.2 channels is significantly impeded in the dorsal root ganglion (DRG) neurons of this KI mouse. Importantly, we describe that this H191-dependent regulation has discrete but significant effects on the excitability properties of D-hair (down-hair) cells, a sub-population of DRG neurons in which Cav3.2 currents prominently regulate excitability. Overall, this study reveals that the native H191-dependent regulation of Cav3.2 channels plays a role in the excitability of Cav3.2-expressing neurons. This animal model will be valuable in addressing the potential in vivo roles of the trace metal and redox modulation of Cav3.2 T-type channels in a wide range of physiological and pathological conditions. Cav3.2 channels are T-type voltage-gated calcium channels that play important roles in controlling neuronal excitability, particularly in dorsal root ganglion (DRG) neurons where they are involved in touch and pain signalling. Cav3.2 channels are modulated by low concentrations of metal ions (nickel, zinc) and redox agents, which involves the histidine 191 (H191) in the channel's extracellular IS3-IS4 loop. It is hypothesized that this metal/redox modulation would contribute to the tuning of the excitability properties of DRG neurons. However, the precise role of this H191-dependent modulation of Cav3.2 channel remains unresolved. Towards this goal, we have generated a knock-in (KI) mouse carrying the mutation H191Q in the Cav3.2 protein. Electrophysiological studies were performed on a subpopulation of DRG neurons, the D-hair cells, which express large Cav3.2 currents. We describe an impaired sensitivity to zinc, nickel and ascorbate of the T-type current in D-hair neurons from KI mice. Analysis of the action potential and low-threshold calcium spike (LTCS) properties revealed that, contrary to that observed in WT D-hair neurons, a low concentration of zinc and nickel is unable to modulate (1) the rheobase threshold current, (2) the afterdepolarization amplitude, (3) the threshold potential necessary to trigger an LTCS or (4) the LTCS amplitude in D-hair neurons from KI mice. Together, our data demonstrate that this H191-dependent metal/redox regulation of Cav3.2 channels can tune neuronal excitability. This study validates the use of this Cav3.2-H191Q mouse model for further investigations of the physiological roles thought to rely on this Cav3.2 modulation.

Bioelectric Signals and Calcium Waves Coordinate Skin Progenitor Cell Movement Patterns during the Polarization of Feather Buds

A crucial challenge for tissue engineering is how to guide stem cells and their progeny to assemble into the unique organ shapes and structures. Currently known organizers for organogenesis include biochemical molecules, physical forces and bioelectric signals. One manifestation of bioelectric signaling in developing embryos is a relatively steady and long-lasting direct current (DC) and associated electric field. Using a vibrating probe, we detected the endogenous DC currents at different developmental stages of chicken feather buds. Interestingly, reversion of the direction of DC currents was observed at embryonic day 9 in anterior regions of feather buds. Perturbation of the endogenous DC currents by applying pulsed electric fields in the developing feather buds led to reorientation of the feather buds. Molecular studies revealed that T-type voltage-gated calcium channels (VGCCs) were expressed in anterior bud epithelial cells and mesenchymal cells, but rarely in posterior regions. Perfusion of high KCl solution induced cytoplasmic calcium elevations in epithelial and mesenchymal cells. The epithelial cells showed mosaic patterns of calcium fluctuations, while the mesenchymal cells had synchronized calcium fluctuations. Long-term calcium imaging demonstrated brief calcium transients in epithelial cells but systematic calcium waves in mesenchymal cells during feather polarization. Moreover, cell nucleus imaging revealed the movement of anterior bud epithelial cells toward posterior regions and the upward movement of mesenchymal cells during feather polarization. Functional perturbation of T-type VGCCs disrupted the patterns of cell movement and feather polarity, confirming the importance of bioelectric signals and calcium waves in feather polarity establishment.

T-type calcium channels in neuropathic pain.

Pain is a quite frequent complaint accompanying numerous pathologies. Among these pathological cases, numerous neuropathies are retrieved with identified etiologies (chemotherapies, diabetes, surgeries…) and also more diffuse syndromes such as fibromyalgia. More broadly, pain is one of the first consequences of most inherited diseases. Despite its importance for the quality of life, current pain management is limited to drugs that are either old or with a limited efficacy or that possess a bad risk benefit ratio. As no new pharmacological concept has led to new analgesics in the last decades, the discovery of new medications is needed, and to this aim, the identification of new druggable targets in pain transmission is a first step. Therefore, studies of ion channels in pain pathways are extremely active. This is particularly true with ion channels in peripheral sensory neurons in dorsal root ganglia known how to express unique sets of these channels. Moreover, both spinal and supraspinal levels are clearly important in pain modulation. Among these ion channels, we and others revealed the important role of low voltage-gated calcium channels in cellular excitability in different steps of the pain pathways. These channels, by being activated nearby resting membrane potential, have biophysical characteristics suited to facilitate action potential generation and rhythmicity. In this review, we will present the current knowledge on the role of these channels in the perception and modulation of pain.