T-type Ca Channels Research Articles

Immunohistochemical analysis of T-type calcium channels in acquired melanocytic naevi and melanoma.

Cutaneous malignant melanoma arises from transformed melanocytes de novo or from congenital or acquired melanocytic naevi. We have recently reported that T-type Ca2+ channels (TT-Cs) are upregulated in human melanoma and play an important role in cell proliferation. To describe for the first time in formalin-fixed paraffin-embedded tissue the immunoexpression of TT-Cs in biopsies of normal skin, acquired melanocytic naevi and melanoma, in order to evaluate their role in melanomagenesis and/or tumour progression, their utility as prognostic markers and their possible use in targeted therapies. Tissue samples from normal skin, melanocytic naevi and melanoma were subjected to immunohistochemistry for two TT-Cs (Cav3.1, Cav3.2); markers of proliferation (Ki67), the cell cycle (cyclin D1), hypoxia (Glut1), vascularization (CD31) and autophagy (LC3); BRAF V600E mutation (VE1) and phosphatase and tensin homologue (PTEN). Immunostaining was evaluated by histoscore. In silico analysis was used to assess the prognostic value of TT-C overexpression. TT-C immunoexpression increased gradually from normal skin to common naevi, dysplastic naevi and melanoma samples, but with differences in the distribution of both isoforms. Particularly, Cav3.2 expression was significantly higher in metastatic melanoma than in primary melanoma. Statistical correlation showed a linear interaction between PTEN loss/BRAF V600E/Cav3.1/LC3/ Ki67/cyclin D1/Cav3.2/Glut1. Disease-free survival (DFS) and overall survival correlated inversely with overexpression of Cav3.2. DFS also correlated inversely with overexpression of Cav3.1. TT-C immunoexpression on melanocytic neoplasms is consistent with our previous in vitro studies and appears to be related to tumour progression. TT-C upregulation can be considered as a prognostic marker using The Cancer Genome Atlas database. The high expression of Cav3.2 in metastatic melanoma encourages the investigation of the use of TT-C blockers in targeted therapies.

Ontogenic Changes and Differential Localization of T-type Ca(2+) Channel Subunits Cav3.1 and Cav3.2 in Mouse Hippocampus and Cerebellum.

T-type calcium (Ca2+) channels play a central role in regulating membrane excitability in the brain. Although the contributions of T-type current to neuron output is often proposed to reflect a differential distribution of T-type channel subtypes to somato-dendritic compartments, their precise subcellular distributions in central neurons are not fully determined. Using histoblot and high-resolution immunoelectron microscopic techniques, we have investigated the expression, regional distribution and subcellular localization of T-type Cav3.1 and Cav3.2 channel subunits in the adult brain, as well as the ontogeny of expression during postnatal development. Histoblot analysis showed that Cav3.1 and Cav3.2 proteins were widely expressed in the brain, with mostly non-overlapping patterns. Cav3.1 showed the highest expression level in the molecular layer (ml) of the cerebellum (Cb), and Cav3.2 in the hippocampus (Hp) and the ml of Cb. During development, levels of Cav3.1 and Cav3.2 increased with age, although there were marked region- and developmental stage-specific differences in their expression. At the cellular and subcellular level, immunoelectron microscopy showed that labeling for Cav3.1 was present in somato-dendritic domains of hippocampal interneurons and Purkinje cells (PCs), while Cav3.2 was present in somato-dendritic domains of CA1 pyramidal cells, hippocampal interneurons and PCs. Most of the immunoparticles for Cav3.1 and Cav3.2 were either associated with the plasma membrane or the intracellular membranes, with notable differences depending on the compartment. Thus, Cav3.1 was mainly located in the plasma membrane of interneurons, whereas Cav3.2 was mainly located in the plasma membrane of dendritic spines and had a major intracellular distribution in dendritic shafts. In PCs, Cav3.1 and Cav3.2 showed similar distribution patterns. In addition to its main postsynaptic distribution, Cav3.2 but not Cav3.1 was also detected in axon terminals establishing excitatory synapses. These results shed new light on the subcellular localization of T-type channel subunits and provide evidence for the non-uniform distribution of Cav3.1 and Cav3.2 subunits over the plasma membrane of central neurons, which may account for the functional heterogeneity of T-type mediated current.

Redox-Dependent Modulation of T-Type Ca(2+) Channels in Sensory Neurons Contributes to Acute Anti-Nociceptive Effect of Substance P.

Aims: Neuropeptide substance P (SP) is produced and released by a subset of peripheral sensory neurons that respond to tissue damage (nociceptors). SP exerts excitatory effects in the central nervous system, but peripheral SP actions are still poorly understood; therefore, here, we aimed at investigating these peripheral mechanisms. Results: SP acutely inhibited T-type voltage-gated Ca2+ channels in nociceptors. The effect was mediated by neurokinin 1 (NK1) receptor-induced stimulation of intracellular release of reactive oxygen species (ROS), as it can be prevented or reversed by the reducing agent dithiothreitol and mimicked by exogenous or endogenous ROS. This redox-mediated T-type Ca2+ channel inhibition operated through the modulation of CaV3.2 channel sensitivity to ambient zinc, as it can be prevented or reversed by zinc chelation and mimicked by exogenous zinc. Elimination of the zinc-binding site in CaV3.2 rendered the channel insensitive to SP-mediated inhibition. Importantly, peripherally applied SP significantly reduced bradykinin-induced nociception in rats in vivo; knock-down of CaV3.2 significantly reduced this anti-nociceptive effect. This atypical signaling cascade shared the initial steps with the SP-mediated augmentation of M-type K+ channels described earlier. Innovation: Our study established a mechanism underlying the peripheral anti-nociceptive effect of SP whereby this neuropeptide produces ROS-dependent inhibition of pro-algesic T-type Ca2+ current and concurrent enhancement of anti-algesic M-type K+ current. These findings will lead to a better understanding of mechanisms of endogenous analgesia. Conclusion: SP modulates T-type channel activity in nociceptors by a redox-dependent tuning of channel sensitivity to zinc; this novel modulatory pathway contributes to the peripheral anti-nociceptive effect of SP. Antioxid. Redox Signal. 25, 233–251.

Ionic Basis for Membrane Potential Resonance in Neurons of the Inferior Olive

Some neurons have the ability to enhance output voltage to input current with a preferred frequency, which is called resonance. Resonance is thought tobe a basis for membrane potential oscillation. Although ion channels responsible for resonance have been reported, the precise mechanisms by which these channels work remain poorly understood. We have found that resonance is reduced but clearly present in the inferior olivary neurons ofCav3.1 T-type voltage-dependent Ca(2+) channel knockout (KO) mice. The activation of Cav3.1 channels is strongly membrane potential dependent, but less frequency dependent. Residual resonance in Cav3.1 KO mice is abolished by a hyper-polarization-activated cyclic nucleotide-gated (HCN) channelblocker, ZD7288, and is partially suppressed byvoltage-dependent K(+) channel blockers. Resonance is inhibited by ZD7288 in wild-type mice andimpaired in HCN1 KO mice, suggesting that theHCN1 channel is essentialfor resonance. The ZD7288-sensitive current is nearly sinusoidal and strongly frequency dependent. These results suggest that Cav3.1 and HCN1 channels act as amplifying and resonating conductances, respectively.

Expression and function of T-type α1H Ca(2)(+) channels in the rat model of Hirschsprung disease

To investigate the role of T-type α1H Ca(2+) channels(Cav 3.2) in the pathogenesis of Hirschsprung disease(HD). Eighty neonatal SD rats 6 to 8 days of age were randomly divided into 2 groups, 40 in each. Microinjector catheters were carefully placed into the bowl of one group, and 0.2% benzalkonium chloride (BAC) solution was injected to establish HD rat model. Control group was treated with saline instead of benzalkonium chloride. At postoperative 4, 6 and 8 weeks, ten rats were sacrificed randomly and examined through general observation, histopathological observation and immunofluorescent staining of PGP 9.5 to identify the animal models. Meanwhile, the distribution of Cav 3.2 in abnormal colon of HD rat model was studied by immunohistochemical staining, Western blot, and the co-localization of Cav 3.2 and c-kit was studied by double immunofluorescent staining. Besides, function of Cav 3.2 was surveyed with the model rats tensile force and frequency of colonic muscle in vitro. After 8 weeks of BAC treatment, the rat model was successfully established according to the results of histopathological and immunofluorescent staining, which showed decrease or lack of ganglion cells within the area of BAC treatment. The distribution of Cav 3.2 was detected by immunohistochemical staining. In the normal colon, Cav 3.2 were mainly distributed between circular muscle and longitudinal muscle, and showed continuous distribution. However, in the narrow segment of HD rat model, the distribution of Cav 3.2 was decreased significantly, and its continuity was destroyed . The results of Western blot were quite consistent with immunohistochemistry staining, in the narrow segment of HD rat model, the expression of Cav 3.2 was decreased gradually after the BAC treatment, especially at postoperative 8 weeks. The relative expression of Cav 3.2 of the control group was 0.63±0.06, 0.62±0.09, 0.63±0.06 at postoperative 4, 6 and 8 weeks respectively. While that of HD group was 0.63±0.06, 0.38±0.06, 0.26±0.07 respectively, which was significantly lower as compared with that of control group at postoperative 6 and 8 weeks respectively (t-value 5.27 and 8.63 respectively, both P<0.05). The co-localization of Cav 3.2 and c-kit was studied by double immunofluorescent staining. Similarly, compared with control groups, the co-localization of Cav 3.2 and c-kit were reduced obviously or vanished in the narrow segment of HD rat model. In motility studies, the control rats show cyclic depolarization regularly. However, the cyclic depolarization largely disappeared in model rats. Besides, on the premise of cyclic depolarization in control muscle strips, when we added ZnCl2, known to inhibit Cav 3.2 selectively among three T-channel isoforms, into tissue chambers, the pattern of muscle contractions appear similar to HD model rats. The abnormal alteration of Cav 3.2 probably mediate the functional change of interstitial cells of cajal in the HD, and finally induce the intestinal dysfunction of HD.

Suppression of Sleep Spindle Rhythmogenesis in Mice with Deletion of CaV3.2 and CaV3.3 T-type Ca2+ Channels

Low-threshold voltage-gated T-type Ca(2+) channels (T-channels or CaV3 channels) sustain oscillatory discharges of thalamocortical (TC) and nucleus Reticularis thalami (nRt) cells. The CaV3.3 subtype dominates nRt rhythmic bursting and mediates a substantial fraction of spindle power in the NREM sleep EEG. CaV3.2 channels are also found in nRt, but whether these contribute to nRt-dependent spindle generation is unexplored. We investigated thalamic rhythmogenesis in mice lacking this subtype in isolation (CaV3.2KO mice) or in concomitance with CaV3.3 deletion (CaV3.double-knockout (DKO) mice). We examined discharge characteristics of thalamic cells and intrathalamic evoked synaptic transmission in brain slices from wild-type, CaV3.2KO and CaV3.DKO mice through patch-clamp recordings. The sleep profile of freely behaving CaV3.2KO and CaV3.DKO mice was assessed by polysomnographic recordings. CaV3.2 channel deficiency left nRt discharge properties largely unaltered, but additional deletion of CaV3.3 channels fully abolished low-threshold whole-cell Ca(2+) currents and bursting, and suppressed burst-mediated inhibitory responses in TC cells. CaV3.DKO mice had more fragmented sleep, with shorter NREM sleep episodes and more frequent microarousals. The NREM sleep EEG power spectrum displayed a relative suppression of the σ frequency band (10-15 Hz), which was accompanied by an increase in the δ band (1-4 Hz). Consistent with previous findings, CaV3.3 channels dominate nRt rhythmogenesis, but the lack of CaV3.2 channels further aggravates neuronal, synaptic, and EEG deficits. Therefore, CaV3.2 channels can boost intrathalamic synaptic transmission, and might play a modulatory role adjusting the relative presence of NREM sleep EEG rhythms.

Endostatin is protective against monocrotaline-induced right heart disease through the inhibition of T-type Ca(2+) channel.

Endostatin (ES), a C-terminal fragment of collagen XVIIIα1, has a potent anti-angiogenic effect. ES prevents tumor proliferation through inhibiting T-type Ca(2+) channel. T-type Ca(2+) channel is re-expressed during heart diseases including monocrotaline (MCT)-induced right heart failure. The present study aimed to clarify the effects of ES on T-type Ca(2+) channel and pathogenesis of MCT-induced right ventricular disease. MCT or saline was injected intraperitoneally to rats. After cardiomyocytes were isolated from right ventricles (RVs), T-type Ca(2+) channel current (I CaT) was measured by a patch-clamp method. After ES small interfering RNA (siRNA) or control siRNA (20μg) was administrated for 1week via the right jugular vein 1week after MCT injection, echocardiography and histological analysis were done. I CaT was significantly increased in RV from MCT-injected rats, and ES significantly inhibited it. The survival rate of ES siRNA-administrated MCT rats (MCT ES si group) was decreased. In echocardiography, although ES siRNA did not affect pulmonary arterial pressure, RV systolic function was impaired in MCT ES si group compared with control siRNA-administrated MCT rats (MCT cont si group). In the histological analysis of RV, ES expression was increased in MCT cont si group, and ES siRNA inhibited it. Furthermore, although MCT cont si group showed only cardiomyocyte hypertrophy, MCT ES si group showed notable enlargement of intercellular spaces. The present study for the first time revealed that ES inhibits T-type Ca(2+) channel activity in RV from MCT-injected rats. ES gene knockdown deteriorates MCT-induced right heart disease. ES is thus cardioprotective possibly through inhibiting T-type Ca(2+) channel activity.

Therapeutic potential of RQ-00311651, a novel T-type Ca2+ channel blocker, in distinct rodent models for neuropathic and visceral pain.

T-type Ca channels (T channels), particularly Cav3.2 among the 3 isoforms, play a role in neuropathic and visceral pain. We thus characterized the effects of RQ-00311651 (RQ), a novel T-channel blocker, in HEK293 cells transfected with human Cav3.1 or Cav3.2 by electrophysiological and fluorescent Ca signaling assays, and also evaluated the antiallodynic/antihyperalgesic activity of RQ in somatic, visceral, and neuropathic pain models in rodents. RQ-00311651 strongly suppressed T currents when tested at holding potentials of -65 ∼ -60 mV, but not -80 mV, in the Cav3.1- or Cav3.2-expressing cells. RQ-00311651 also inhibited high K-induced Ca signaling in those cells. In mice, RQ, administered intraperitoneally (i.p.) at 5 to 20 mg/kg or orally at 20 to 40 mg/kg, significantly suppressed the somatic hyperalgesia and visceral pain-like nociceptive behavior/referred hyperalgesia caused by intraplantar and intracolonic administration of NaHS or Na2S, H2S donors, respectively, which involve the enhanced activity of Cav3.2 channels. RQ-00311651, given i.p. at 5 to 20 mg/kg, exhibited antiallodynic or antihyperalgesic activity in rats with spinal nerve injury-induced neuropathy or in rats and mice with paclitaxel-induced neuropathy. Oral and i.p. RQ at 10 to 20 mg/kg also suppressed the visceral nociceptive behavior and/or referred hyperalgesia accompanying cerulein-induced acute pancreatitis and cyclophosphamide-induced cystitis in mice. The analgesic and antihyperalgesic/antiallodynic doses of oral and i.p. RQ did not significantly affect the locomotor activity and motor coordination. Together, RQ is considered a state-dependent blocker of Cav3.1/Cav3.2 T channels and may serve as an orally available analgesic for treatment of neuropathic and inflammatory pain including distinct visceral pain with minimum central side effects.

Regulation of the T‐type Ca2+ channel Cav3.2 by hydrogen sulfide: emerging controversies concerning the role of H2S in nociception

Ion channels represent a large and growing family of target proteins regulated by gasotransmitters such as nitric oxide, carbon monoxide and, as described more recently, hydrogen sulfide. Indeed, many of the biological actions of these gases can be accounted for by their ability to modulate ion channel activity. Here, we report recent evidence that H2 S is a modulator of low voltage-activated T-type Ca(2+) channels, and discriminates between the different subtypes of T-type Ca(2+) channel in that it selectively modulates Cav3.2, whilst Cav3.1 and Cav3.3 are unaffected. At high concentrations, H2 S augments Cav3.2 currents, an observation which has led to the suggestion that H2 S exerts its pro-nociceptive effects via this channel, since Cav3.2 plays a central role in sensory nerve excitability. However, at more physiological concentrations, H2 S is seen to inhibit Cav3.2. This inhibitory action requires the presence of the redox-sensitive, extracellular region of the channel which is responsible for tonic metal ion binding and which particularly distinguishes this channel isoform from Cav3.1 and 3.3. Further studies indicate that H2 S may act in a novel manner to alter channel activity by potentiating the zinc sensitivity/affinity of this binding site. This review discusses the different reports of H2 S modulation of T-type Ca(2+) channels, and how such varying effects may impact on nociception given the role of this channel in sensory activity. This subject remains controversial, and future studies are required before the impact of T-type Ca(2+) channel modulation by H2 S might be exploited as a novel approach to pain management.

Pathophysiological implication of CaV3.1 T-type Ca2+ channels in trigeminal neuropathic pain

A crucial pathophysiological issue concerning central neuropathic pain is the modification of sensory processing by abnormally increased low-frequency brain rhythms. Here we explore the molecular mechanisms responsible for such abnormal rhythmicity and its relation to neuropathic pain syndrome. Toward this aim, we investigated the behavioral and electrophysiological consequences of trigeminal neuropathic pain following infraorbital nerve ligations in CaV3.1 T-type Ca(2+) channel knockout and wild-type mice. CaV3.1 knockout mice had decreased mechanical hypersensitivity and reduced low-frequency rhythms in the primary somatosensory cortex and related thalamic nuclei than wild-type mice. Lateral inhibition of gamma rhythm in primary somatosensory cortex layer 4, reflecting intact sensory contrast, was present in knockout mice but severely impaired in wild-type mice. Moreover, cross-frequency coupling between low-frequency and gamma rhythms, which may serve in sensory processing, was pronounced in wild-type mice but not in CaV3.1 knockout mice. Our results suggest that the presence of CaV3.1 channels is a key element in the pathophysiology of trigeminal neuropathic pain.

Spontaneous activity in the microvasculature of visceral organs: role of pericytes and voltage-dependent Ca(2+) channels.

The microvasculature plays a primary role in the interchange of substances between tissues and the circulation. In visceral organs that undergo considerable distension upon filling, the microvasculature appears to display intrinsic contractile properties to maintain their flow. Submucosal venules in the bladder or gastrointestinal tract generate rhythmic spontaneous phasic constrictions and associated Ca(2+) transients. These events are initiated within either venular pericytes or smooth muscle cells (SMCs) arising from spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) and the opening of Ca(2+) -activated chloride channels (CaCCs) that trigger Ca(2+) influx through L-type voltage-dependent Ca(2+) channels (VDCCs). L-type VDCCs also play a critical role in maintaining synchrony within the contractile mural cells. In the stomach myenteric layer, spontaneous Ca(2+) transients originating in capillary pericytes appear to spread to their neighbouring arteriolar SMCs. Capillary Ca(2+) transients primarily rely on SR Ca(2+) release, but also require Ca(2+) influx through T-type VDCCs for their synchrony. The opening of T-type VDCCs also contribute to the propagation of Ca(2+) transients into SMCs. In visceral microvasculature, pericytes act as either spontaneously active contractile machinery of the venules or as pacemaker cells generating synchronous Ca(2+) transients that drive spontaneous contractions in upstream arterioles. Thus pericytes play different roles in different vascular beds in a manner that may well depend on the selective expression of T-type and L-type Ca(2+) channels.

Mitochondria and carbon monoxide: cytoprotection and control of cell metabolism - a role for Ca(2+) ?

Carbon monoxide (CO) is an endogenously produced gasotransmitter with important biological functions: anti-inflammation, anti-apoptosis, vasomodulation and cell metabolism modulation. The most recognized cellular target for CO is the mitochondria. Physiological concentrations of CO generate mitochondrial reactive oxygen species (ROS), which are signalling molecules for CO-induced pathways. Indeed, small amounts of ROS promote cytoprotection by a preconditioning effect. Furthermore, CO prevents cell death by limiting mitochondrial membrane permeabilization, which inhibits the release of pro-apoptotic factors into the cytosol; both events are ROS dependent. CO also increases the ability of mitochondria to take up Ca(2+) . Mitochondrial metabolism is modulated by CO, namely by increasing TCA cycle rate, oxidative phosphorylation and mitochondrial biogenesis, which, in turn, increases ATP production. CO's modulation of metabolism might be important for cellular response to diseases, namely cancer and ischaemic diseases. Finally, another cytoprotective role of CO involves the control of Ca(2+) channels. By limiting the activity of T-type and L-type Ca(2+) channels, CO prevents excitotoxicity-induced cell death and modulates cell proliferation. Several questions concerning Ca(2+) signalling, mitochondria and CO can be asked, for instance whether CO modulation of cell metabolism would be dependent on the mitochondrial Ca(2+) uptake capacity, since small amounts of Ca(2+) can increase mitochondrial metabolism. Whether CO controls Ca(2+) communication between mitochondria and endoplasmic reticulum is another open field of research. In summary, CO emerges as a key gasotransmitter in the control of several cellular functions of mitochondria: metabolism, cell death and Ca(2+) signalling.

Critical Roles of STAT3 in β-Adrenergic Functions in the Heart.

β-Adrenergic receptors (βARs) play paradoxical roles in the heart. On one hand, βARs augment cardiac performance to fulfill the physiological demands, but on the other hand, prolonged activations of βARs exert deleterious effects that result in heart failure. The signal transducer and activator of transcription 3 (STAT3) plays a dynamic role in integrating multiple cytokine signaling pathways in a number of tissues. Altered activation of STAT3 has been observed in failing hearts in both human patients and animal models. Our objective is to determine the potential regulatory roles of STAT3 in cardiac βAR-mediated signaling and function. We observed that STAT3 can be directly activated in cardiomyocytes by β-adrenergic agonists. To follow up this finding, we analyzed βAR function in cardiomyocyte-restricted STAT3 knockouts and discovered that the conditional loss of STAT3 in cardiomyocytes markedly reduced the cardiac contractile response to acute βAR stimulation, and caused disengagement of calcium coupling and muscle contraction. Under chronic β-adrenergic stimulation, Stat3cKO hearts exhibited pronounced cardiomyocyte hypertrophy, cell death, and subsequent cardiac fibrosis. Biochemical and genetic data supported that Gαs and Src kinases are required for βAR-mediated activation of STAT3. Finally, we demonstrated that STAT3 transcriptionally regulates several key components of βAR pathway, including β1AR, protein kinase A, and T-type Ca(2+) channels. Our data demonstrate for the first time that STAT3 has a fundamental role in βAR signaling and functions in the heart. STAT3 serves as a critical transcriptional regulator for βAR-mediated cardiac stress adaption, pathological remodeling, and heart failure.

Stimulation-induced Ca(2+) influx at nodes of Ranvier in mouse peripheral motor axons.

In peripheral myelinated axons of mammalian spinal motor neurons, Ca(2+) influx was thought to occur only in pathological conditions such as ischaemia. Using Ca(2+) imaging in mouse large motor axons, we find that physiological stimulation with trains of action potentials transiently elevates axoplasmic [C(2+)] around nodes of Ranvier. These stimulation-induced [Ca(2+)] elevations require Ca(2+) influx, and are partially reduced by blocking T-type Ca(2+) channels (e.g. mibefradil) and by blocking the Na(+)/Ca(2+) exchanger (NCX), suggesting an important contribution of Ca(2+) influx via reverse-mode NCX activity. Acute disruption of paranodal myelin dramatically increases stimulation-induced [Ca(2+)] elevations around nodes by allowing activation of sub-myelin L-type (nimodipine-sensitive) Ca(2+) channels. The Ca(2+) that enters myelinated motor axons during normal activity is likely to contribute to several signalling pathways; the larger Ca(2+) influx that occurs following demyelination may contribute to the axonal degeneration that occurs in peripheral demyelinating diseases. Activity-dependent Ca(2+) signalling is well established for somata and terminals of mammalian spinal motor neurons, but not for their axons. Imaging of an intra-axonally injected fluorescent [Ca(2+)] indicator revealed that during repetitive action potential stimulation, [Ca(2+)] elevations localized to nodal regions occurred in mouse motor axons from ventral roots, phrenic nerve and intramuscular branches. These [Ca(2+)] elevations (∼ 0.1 μm with stimulation at 50 Hz, 10 s) were blocked by removal of Ca(2+) from the extracellular solution. Effects of pharmacological blockers indicated contributions from both T-type Ca(2+) channels and reverse mode Na(+)/Ca(2+) exchange (NCX). Acute disruption of paranodal myelin (by stretch or lysophosphatidylcholine) increased the stimulation-induced [Ca(2+)] elevations, which now included a prominent contribution from L-type Ca(2+) channels. These results suggest that the peri-nodal axolemma of motor axons includes multiple pathways for stimulation-induced Ca(2+) influx, some active in normally-myelinated axons (T-type channels, NCX), others active only when exposed by myelin disruption (L-type channels). The modest axoplasmic peri-nodal [Ca(2+)] elevations measured in intact motor axons might mediate local responses to axonal activation. The larger [Ca(2+) ] elevations measured after myelin disruption might, over time, contribute to the axonal degeneration observed in peripheral demyelinating neuropathies.

Hypoxic induction of T-type Ca(2+) channels in rat cardiac myocytes: role of HIF-1α and RhoA/ROCK signalling.

T-type Ca(2+) channels are expressed in the ventricular myocytes of the fetal and perinatal heart, but are normally downregulated as development progresses. Interestingly, however, these channels are re-expressed in adult cardiomyocytes under pathological conditions. We investigated low voltage-activated T-type Ca(2+) channel regulation in hypoxia in rat cardiomyocytes. Molecular studies revealed that hypoxia induces the upregulation of Cav 3.2 mRNA, whereas Cav 3.1 mRNA is not significantly altered. The effect of hypoxia on Cav 3.2 mRNA was time- and dose-dependent, and required hypoxia inducible factor-1α (HIF-1α) stabilization. Patch-clamp recordings confirmed that T-type Ca(2+) channel currents were upregulated in hypoxic conditions, and the addition of 50 μm NiCl2 (a T-type channel blocker) demonstrated that the Cav 3.2 channel is responsible for this upregulation. This increase in current density was not accompanied by significant changes in the Cav 3.2 channel electrophysiological properties. The small monomeric G-protein RhoA and its effector Rho-associated kinase I (ROCKI), which are known to play important roles in cardiovascular physiology, were also upregulated in neonatal rat ventricular myocytes subjected to hypoxia. Pharmacological experiments indicated that both proteins were involved in the observed upregulation of the Cav 3.2 channel and the stabilization of HIF-1α that occurred in response to hypoxia. These results suggest a possible role for Cav 3.2 channels in the increased probability of developing arrhythmias observed in ischaemic situations, and in the pathogenesis of diseases associated with hypoxic Ca(2+) overload.

A concerted action of L- and T-type Ca(2+) channels regulates locus coeruleus pacemaking.

Dysfunction of noradrenergic locus coeruleus (LC) neurons is involved in psychiatric and neurodegenerative diseases and is an early hallmark of Parkinson's disease (PD). The analysis of ion channels underlying the autonomous electrical activity of LC neurons, which is ultimately coupled to cell survival signaling pathways, can lead to a better understanding of the vulnerability of these neurons. In LC neurons somatodendritic Ca(2+) oscillations, mediated by L-type Ca(2+) channels, accompany spontaneous spiking and are linked to mitochondrial oxidant stress. However, the expression and functional implication of low-threshold activated T-type Ca(2+) channels in LC neurons were not yet studied. To this end we performed RT-PCR expression analysis in LC neurons. In addition, we utilized slice patch clamp recordings of in vitro brainstem slices in combination with L-type and T-type Ca(2+) channel blockers. We found the expression of a distinct set of L-type and T-type Ca(2+) channel subtypes mediating a pronounced low-threshold activated Ca(2+) current component. Analyzing spike trains, we revealed that neither L-type Ca(2+) channel nor T-type Ca(2+) channel blockade alone leads to a change in firing properties. In contrast, a combined application of antagonists significantly decreased the afterhyperpolarization amplitude, resulting in an increased firing frequency. Hence, we report the functional expression of T-type Ca(2+) channels in LC neurons and demonstrate their role in increasing the robustness of LC pacemaking by working in concert with Cav1 channels.

Enhanced BDNF signalling following chronic hypoxia potentiates catecholamine release from cultured rat adrenal chromaffin cells

We investigated the role of the neurotrophin BDNF signalling via the TrkB receptor in rat adrenomedullary chromaffin cells (AMCs) exposed to normoxia (Nox; 21% O2) and chronic hypoxia (CHox; 2% O2) in vitro for ∼ 48 h. TrkB receptor expression was upregulated in primary AMCs and in immortalized chromaffin (MAH) cells exposed to CHox; this effect was absent in MAH cells deficient in the transcription factor, hypoxia inducible factor (HIF)-2α. Relative to normoxic controls, activation of the TrkB receptor in chronically hypoxic AMCs led to a marked increase in membrane excitability, intracellular [Ca(2+)], and catecholamine secretion. The BDNF-induced rise of intracellular [Ca(2+)] in CHox cells was sensitive to the selective T-type Ca(2+) channel blocker TTA-P2 and tetrodotoxin (TTX), suggesting key roles of low threshold T-type Ca(2+) and voltage-gated Na(+) channels in the signalling pathway. Environmental stressors, including chronic hypoxia, enhance the ability of adrenomedullary chromaffin cells (AMCs) to secrete catecholamines; however, the underlying molecular mechanisms remain unclear. Here, we investigated the role of brain-derived neurotrophic factor (BDNF) signalling in rat AMCs exposed to chronic hypoxia. In rat adrenal glands, BDNF and its tropomyosin-related kinase B (TrkB) receptor are highly expressed in the cortex and medulla, respectively. Exposure of AMCs to chronic hypoxia (2% O2; 48 h) in vitro caused a significant increase to TrkB mRNA expression. A similar increase was observed in an immortalized chromaffin cell line (MAH cells); however, it was absent in MAH cells deficient in the transcription factor HIF-2α. A specific TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF), stimulated quantal catecholamine secretion from chronically hypoxic (CHox; 2% O2) AMCs to a greater extent than normoxic (Nox; 21% O2) controls. Activation of TrkB by BDNF or 7,8-DHF increased intracellular Ca(2+) ([Ca(2+)]i), an effect that was significantly larger in CHox cells. The 7,8-DHF-induced [Ca(2+)]i rise was sensitive to the tyrosine kinase inhibitor K252a and nickel (2 mm), but not the Ca(2+) store-depleting agent cyclopiazonic acid. Blockade of T-type calcium channels with TTA-P2 (1 μm) or voltage-gated Na(+) channels with TTX inhibited BDNF-induced [Ca(2+)]i increases. BDNF also induced a dose-dependent enhancement of action potential firing in CHox cells. These data demonstrate that during chronic hypoxia, enhancement of BDNF-TrkB signalling increases voltage-dependent Ca(2+) influx and catecholamine secretion in chromaffin cells, and that T-type Ca(2+) channels play a key role in the signalling pathway.

Chronic intrathecal infusion of mibefradil, ethosuximide and nickel attenuates nerve ligation-induced pain in rats

T-type Ca(2+) channels (TCC) are important for pain transmission, especially the Ca(V)3.2 subtype. In this study, we examined the effects of intrathecal TCC blockers in the L5/6 spinal nerve ligation pain rat model. Under isoflurane anaesthesia, rats received right L5/6 spinal nerve ligation and intrathecal catheters (attached to an infusion pump) were sited. After surgery, saline, mibefradil, ethosuximide or NiCl2 were given intrathecally for seven days. The right hindpaw withdrawal thresholds to von Frey hair stimuli and withdrawal latencies to radiant heat were measured before and once daily for seven days after surgery. Double immunofluorescence and western blotting were used to examine the expression of Ca(V)3.2 in dorsal root ganglion (DRG) and spinal cord. On post-ligation day seven, rats receiving mibefradil, ethosuximide or NiCl2 had significant higher median withdrawal thresholds (15.0, 10.2, and 10.9 g) and latencies (8.0, 7.6 and 7.6 s) than saline-treated rats (1.6 g and 4.3 s, respectively). Ca(V)3.2 was expressed in parvalbumin(+), IB4(+), CGRP(+) and VR1(+) neurones in DRG and most neurones in spinal dorsal horn. Ca(V)3.2 was up-regulated in the right L5/6 DRG and spinal cord seven days after nerve ligation. In this study, we demonstrated that intrathecal TCC blockers attenuate the development of nerve injury-induced mechanical allodynia and thermal hyperalgesia. Our data suggest that continuous intrathecal infusion of TCC or Ca(V)3.2 blockers may be a promising alternative for the management of nerve injury-induced pain.

Altered thalamocortical rhythmicity and connectivity in mice lacking CaV3.1 T-type Ca2+ channels in unconsciousness

In unconscious status (e.g., deep sleep and anesthetic unconsciousness) where cognitive functions are not generated there is still a significant level of brain activity present. Indeed, the electrophysiology of the unconscious brain is characterized by well-defined thalamocortical rhythmicity. Here we address the ionic basis for such thalamocortical rhythms during unconsciousness. In particular, we address the role of CaV3.1 T-type Ca(2+) channels, which are richly expressed in thalamic neurons. Toward this aim, we examined the electrophysiological and behavioral phenotypes of mice lacking CaV3.1 channels (CaV3.1 knockout) during unconsciousness induced by ketamine or ethanol administration. Our findings indicate that CaV3.1 KO mice displayed attenuated low-frequency oscillations in thalamocortical loops, especially in the 1- to 4-Hz delta band, compared with control mice (CaV3.1 WT). Intriguingly, we also found that CaV3.1 KO mice exhibited augmented high-frequency oscillations during unconsciousness. In a behavioral measure of unconsciousness dynamics, CaV3.1 KO mice took longer to fall into the unconscious state than controls. In addition, such unconscious events had a shorter duration than those of control mice. The thalamocortical interaction level between mediodorsal thalamus and frontal cortex in CaV3.1 KO mice was significantly lower, especially for delta band oscillations, compared with that of CaV3.1 WT mice, during unconsciousness. These results suggest that the CaV3.1 channel is required for the generation of a given set of thalamocortical rhythms during unconsciousness. Further, that thalamocortical resonant neuronal activity supported by this channel is important for the control of vigilance states.

T-type calcium channel antagonists, mibefradil and NNC-55-0396 inhibit cell proliferation and induce cell apoptosis in leukemia cell lines.

BackgroundT-type Ca2+ channels are often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation and death. Methods: RT-PCR, Q-PCR, western blotting and whole-cell patch-clamp recording were employed to assess the expression of T-type Ca2+ channels in leukemia cell lines. The function of T-type Ca2+ channels in leukemia cell growth and the possible mechanism of the effect of T-type Ca2+ channel antagonists on cell proliferation and apoptosis were examined in T-lymphoma cell lines.ResultsWe show that leukemia cell lines exhibited reduced cell growth when treated with T-type Ca2+ channel inhibitors, mibefradil and NNC-55-0396 in a concentration-dependent manner. Mechanistically, these inhibitors played a dual role on cell viability: (i) blunting proliferation, through a halt in the progression to the G1-S phase; and (ii) promoting cell apoptosis, partially dependent on the endoplasmic reticulum Ca2+ release. In addition, we observed a reduced phosphorylation of ERK1/2 in MOLT-4 cells in response to mibefradil and NNC-55-0396 treatment.ConclusionsThese results indicate that mibefradil and NNC-55-0396 regulate proliferation and apoptosis in T-type Ca2+ channel expressing leukemia cell lines and suggest a potential therapeutic target for leukemia.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0171-4) contains supplementary material, which is available to authorized users.