Histone methylation-mediated microRNA-32-5p down-regulation in sensory neurons regulates pain behaviors via targeting Cav3.2 channels.

microRNA (miRNA)­mediated gene regulation has been studied as a therapeutic approach, but its functional regulatory mechanism in neuropathic pain is not well understood. Here, we identify that miRNA-32-5p (miR-32-5p) is a functional RNA in regulating trigeminal-mediated neuropathic pain. High-throughput sequencing and qPCR analysis showed that miR-32-5p was the most down-regulated miRNA in the injured trigeminal ganglion (TG) of rats. Intra-TG injection of miR-32-5p agomir or overexpression of miR-32-5p by lentiviral delivery in neurons of the injured TG attenuated established trigeminal neuropathic pain. miR-32-5p overexpression did not affect acute physiological pain, while miR-32-5p down-regulation in intact rats was sufficient to cause pain-related behaviors. Nerve injury increased the methylated histone occupancy of binding sites for the transcription factor glucocorticoid receptor in the miR-32-5p promoter region. Inhibition of the enzymes that catalyze H3K9me2 and H3K27me3 restored the expression of miR-32-5p and markedly attenuated pain behaviors. Further, miR-32-5p­targeted Cav3.2 T-type Ca2+ channels and decreased miR-32-5p associated with neuropathic pain caused an increase in Cav3.2 protein expression and T-type channel currents. Conversely, miR-32-5p overexpression in injured TG suppressed the increased expression of Cav3.2 and reversed mechanical allodynia. Together, we conclude that histone methylation-mediated miR-32-5p down-regulation in TG neurons regulates trigeminal neuropathic pain by targeting Cav3.2 channels.

Enhanced Ca2+ signaling, mild primary aldosteronism, and hypertension in a familial hyperaldosteronism mouse model (Cacna1hM1560V/+ ).

Gain-of-function mutations in the CACNA1H gene (encoding the T-type calcium channel CaV3.2) cause autosomal-dominant familial hyperaldosteronism type IV (FH-IV) and early-onset hypertension in humans. We used CRISPR/Cas9 to generate Cacna1hM1560V/+ knockin mice as a model of the most common FH-IV mutation, along with corresponding knockout mice (Cacna1h-/- ). Adrenal morphology of both Cacna1hM1560V/+ and Cacna1h-/- mice was normal. Cacna1hM1560V/+ mice had elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism). Their adrenal Cyp11b2 (aldosterone synthase) expression was increased and remained elevated on a high-salt diet (relative autonomy, characteristic of primary aldosteronism), but plasma aldosterone was only elevated in male animals. The systolic blood pressure of Cacna1hM1560V/+ mice was 8 mmHg higher than in wild-type littermates and remained elevated on a high-salt diet. Cacna1h-/- mice had elevated renal Ren1 (renin-1) expression but normal adrenal Cyp11b2 levels, suggesting that in the absence of CaV3.2, stimulation of the renin-angiotensin system activates alternative calcium entry pathways to maintain normal aldosterone production. On a cellular level, Cacna1hM1560V/+ adrenal slices showed increased baseline and peak intracellular calcium concentrations in the zona glomerulosa compared to controls, but the frequency of calcium spikes did not rise. We conclude that FH-IV, on a molecular level, is caused by elevated intracellular Ca2+ concentrations as a signal for aldosterone production in adrenal glomerulosa cells. We demonstrate that a germline Cacna1h gain-of-function mutation is sufficient to cause mild primary aldosteronism, whereas loss of CaV3.2 channel function can be compensated for in a chronic setting.

Analgesic Buxus alkaloids with Enhanced Selectivity for the Low-Voltage-Gated Calcium Channel Cav 3.2 over Cav 3.1 through a New Binding Mode.

Low-voltage-gated calcium channels (LVGCCs; Cav 3.1-3.3) represent promising drug targets for epilepsy, pain, and essential tremor. At present, modulators with heightened selectivity for a subtype of LVGCCs are still highly desired. In this study we explored three classes of Buxus alkaloids and identified 9(10/19)abeo-artanes Buxusemine H and Buxusemine L (BXSL) as an unprecedented type of Cav 3.2 inhibitors. Particularly, BXSL exhibited Cav 3.2 inhibition comparable to Z944, a non-subtype-selective LVGCCs inhibitor under clinical trial. While lacking specificity for Cav 3.3, BXSL showed a 30-fold selectivity of Cav 3.2 over Cav 3.1. As compared to several well-known inhibitors, the experimental and computational studies suggested BXSL exhibits a distinct binding mode to Cav 3.2, notably through the essential interaction with serine-1543 in domain III. Furthermore, BXSL showed minimal impact on various recombinant and native nociceptive ion channels, while significantly reducing the excitability of isolated mouse dorsal root ganglion neurons. Animal studies in wild-type and Cav 3.2 knock-out mice revealed that BXSL (5 mg/kg), by inhibiting Cav 3.2, exhibits an analgesic effect equivalent to Z944 (10 mg/kg) or mibefradil (10 mg/kg). Moreover, we proposed a structural rationale for the high selectivity of 9(10/19)abeo-artane-type alkaloids towards Cav 3.2 over Cav 3.1. This study introduces a novel analgesic agent and valuable molecular insight for structure-based innovative Cav 3.2 drug development.

T-Type Ca2+ Channels Boost Neurotransmission in Mammalian Cone Photoreceptors.

It is a commonly accepted view that light stimulation of mammalian photoreceptors causes a graded change in membrane potential instead of developing a spike. The presynaptic Ca2+ channels serve as a crucial link for the coding of membrane potential variations into neurotransmitter release. Cav1.4 L-type Ca2+ channels are expressed in photoreceptor terminals, but the complete pool of Ca2+ channels in cone photoreceptors appears to be more diverse. Here, we discovered, employing whole-cell patch-clamp recording from cone photoreceptor terminals in both sexes of mice, that their Ca2+ currents are composed of low- (T-type Ca2+ channels) and high- (L-type Ca2+ channels) voltage-activated components. Furthermore, Ca2+ channels exerted self-generated spike behavior in dark membrane potentials, and spikes were generated in response to light/dark transition. The application of fast and slow Ca2+ chelators revealed that T-type Ca2+ channels are located close to the release machinery. Furthermore, capacitance measurements indicated that they are involved in evoked vesicle release. Additionally, RT-PCR experiments showed the presence of Cav3.2 T-type Ca2+ channels in cone photoreceptors but not in rod photoreceptors. Altogether, we found several crucial functions of T-type Ca2+ channels, which increase the functional repertoire of cone photoreceptors. Namely, they extend cone photoreceptor light-responsive membrane potential range, amplify dark responses, generate spikes, increase intracellular Ca2+ levels, and boost synaptic transmission.SIGNIFICANCE STATEMENT Photoreceptors provide the first synapse for coding light information. The key elements in synaptic transmission are the voltage-sensitive Ca2+ channels. Here, we provide evidence that mouse cone photoreceptors express low-voltage-activated Cav3.2 T-type Ca2+ channels in addition to high-voltage-activated L-type Ca2+ channels. The presence of T-type Ca2+ channels in cone photoreceptors appears to extend their light-responsive membrane potential range, amplify dark response, generate spikes, increase intracellular Ca2+ levels, and boost synaptic transmission. By these functions, Cav3.2 T-type Ca2+ channels increase the functional repertoire of cone photoreceptors.

CaMKII and CaV3.2 T-type calcium channel mediate Connexin-43-dependent inflammation by activating astrocytes in vincristine-induced neuropathic pain.

Vincristine (VCR), an alkaloid isolated from vinca, is a commonly used chemotherapeutic drug. However, VCR therapy can lead to dose-dependent peripheral neurotoxicity, mainly manifesting as neuropathic pain, which is one of the dominant reasons for limiting its utility. Experimentally, we discovered that VCR-induced neuropathic pain (VINP) was accompanied by astrocyte activation; the upregulation of phospho-CaMKII (p-CaMKII), CaV3.2, and Connexin-43 (Cx43) expression; and the production and release of inflammatory cytokines and chemokines in the spinal cord. Similar situations were also observed in astrocyte cultures. Interestingly, these alterations were all reversed by intrathecal injection of KN-93 (a CaMKII inhibitor) or L-Ascorbic acid (a CaV3.2 inhibitor). In addition, KN-93 and L-Ascorbic acid inhibited the increase in [Ca2+]i associated with astrocyte activation. We also verified that knocking down or inhibiting Cx43 level via intrathecal injection of Cx43 siRNA or Gap27 (a Cx43 mimetic peptide) relieved pain hypersensitivity and reduced the release of inflammatory factors; however, they did not affect astrocyte activation or p-CaMKII and CaV3.2 expression. Besides, the overexpression of Cx43 through the transfection of the Cx43 plasmid did not affect p-CaMKII and CaV3.2 expressions in vitro. Therefore, CaMKII and CaV3.2 may activate astrocytes by increasing [Ca2+]i, thereby mediating Cx43-dependent inflammation in VINP. Moreover, we demonstrated that the CaMKII signalling pathway was involved in VCR-induced inflammation, apoptosis, and mitochondrial damage. Collectively, our findings show a novel mechanism by which CaMKII and CaV3.2 mediate Cx43-dependent inflammation by activating astrocytes in neuropathic pain induced by VCR.

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

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

CaV3.2 (CACNA1H) in Primary Aldosteronism.

Aldosterone is a steroid hormone produced in the zona glomerulosa (ZG) of the adrenal cortex. The most prominent function of aldosterone is the control of electrolyte homeostasis and blood pressure via the kidneys. The primary factors regulating aldosterone synthesis are the serum concentrations of angiotensin II and potassium. The T-type voltage-gated calcium channel CaV3.2 (encoded by CACNA1H) is an important component of electrical as well as intracellular calcium oscillations, which govern aldosterone production in the ZG. Excessive aldosterone production that is (partially) uncoupled from physiological stimuli leads to primary aldosteronism, the most common cause of secondary hypertension. Germline gain-of-function mutations in CACNA1H were identified in familial hyperaldosteronism, whereas somatic mutations are a rare cause of aldosterone-producing adenomas. In this review, we summarize these findings, put them in perspective, and highlight missing knowledge.

CaV3.2 calcium channels control NMDA receptor-mediated transmission: a new mechanism for absence epilepsy.

CaV3.2 T-type calcium channels, encoded by CACNA1H, are expressed throughout the brain, yet their general function remains unclear. We discovered that CaV3.2 channels control NMDA-sensitive glutamatergic receptor (NMDA-R)-mediated transmission and subsequent NMDA-R-dependent plasticity of AMPA-R-mediated transmission at rat central synapses. Interestingly, functional CaV3.2 channels primarily incorporate into synapses, replace existing CaV3.2 channels, and can induce local calcium influx to control NMDA transmission strength in an activity-dependent manner. Moreover, human childhood absence epilepsy (CAE)-linked hCaV3.2(C456S) mutant channels have a higher channel open probability, induce more calcium influx, and enhance glutamatergic transmission. Remarkably, cortical expression of hCaV3.2(C456S) channels in rats induces 2- to 4-Hz spike and wave discharges and absence-like epilepsy characteristic of CAE patients, which can be suppressed by AMPA-R and NMDA-R antagonists but not T-type calcium channel antagonists. These results reveal an unexpected role of CaV3.2 channels in regulating NMDA-R-mediated transmission and a novel epileptogenic mechanism for human CAE.

Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

BACKGROUND: Adipokines, including adiponectin, are implicated in nociceptive pain; however, the underlying cellular and molecular mechanisms remain unknown. METHODS: Using electrophysiological recording, immunostaining, molecular biological approaches and animal behaviour tests, we elucidated a pivotal role of adiponectin in regulating membrane excitability and pain sensitivity by manipulating Cav3.2 channels in trigeminal ganglion (TG) neurons. RESULTS: Adiponectin enhanced T-type Ca2+ channel currents (IT) in TG neurons through the activation of adiponectin receptor 1 (adipoR1) but independently of heterotrimeric G protein-mediated signaling. Coimmunoprecipitation revealed a physical association between AdipoR1 and casein kinase II alpha-subunits (CK2α) in the TG, and inhibiting CK2 activity by chemical inhibitor or siRNA targeting CK2α prevented the adiponectin-induced IT response. Adiponectin significantly activated protein kinase C (PKC), and this effect was abrogated by CK2α knockdown. Adiponectin increased the membrane abundance of PKC beta1 (PKCß1). Blocking PKCß1 pharmacologically or genetically abrogated the adiponectin-induced IT increase. In heterologous expression systems, activation of adipoR1 induced a selective enhancement of Cav3.2 channel currents, dependent on PKCß1 signaling. Functionally, adiponectin increased TG neuronal excitability and induced mechanical pain hypersensitivity, both attenuated by T-type channel blockade. In a trigeminal neuralgia model induced by chronic constriction injury of infraorbital nerve, blockade of adipoR1 signaling suppressed mechanical allodynia, which was prevented by silencing Cav3.2. CONCLUSION: Our study elucidates a novel signaling cascade wherein adiponectin stimulates TG Cav3.2 channels via adipoR1 coupled to a novel CK2α-dependent PKCß1. This process induces neuronal hyperexcitability and pain hypersensitivity. Insight into adipoR-Cav3.2 signaling in sensory neurons provides attractive targets for pain treatment.

Voltage-dependent CaV3.2 and CaV2.2 channels in nociceptive pathways.

Noxious stimuli like cold, heat, pH change, tissue damage, and inflammation depolarize a membrane of peripheral endings of specialized nociceptive neurons which eventually results in the generation of an action potential. The electrical signal is carried along a long axon of nociceptive neurons from peripheral organs to soma located in dorsal root ganglions and further to the dorsal horn of the spinal cord where it is transmitted through a chemical synapse and is carried through the spinal thalamic tract into the brain. Two subtypes of voltage-activated calcium play a major role in signal transmission: a low voltage-activated CaV3.2 channel and a high voltage-activated CaV2.2 channel. The CaV3.2 channel contributes mainly to the signal conductance along nociceptive neurons while the principal role of the CaV2.2 channel is in the synaptic transmission at the dorsal horn. Both channels contribute to the signal initiation at peripheral nerve endings. This review summarizes current knowledge about the expression and distribution of these channels in a nociceptive pathway, the regulation of their expression and gating during pain pathology, and their suitability as targets for pharmacological therapy.

T-type Ca2+ channels play a dual role in modulating the excitability of dorsal root ganglia neurons.

A subgroup of low-threshold dorsal root ganglia (DRG) neurons discharge action potentials (APs) with an afterdepolarizing potential (ADP). The ADP is formed by T-type Ca2+ currents. It is known that T-type Ca2+ currents contribute to neuropathic pain. However, the change in ADP-firing of injured DRG neurons has not been widely studied yet. Here we applied patch clamp to record ADP-firing and T-type Ca2+ currents in intact and chronically compressed DRG (CCD) neurons and examined T-type Ca2+ channel proteins expression with western blotting. After CCD injury, the incidences of both ADP firing and non-ADP burst firing increased, and T-type Ca2+ channels contributed to both of these firing patterns. The neurons discharging large-amplitude-ADP firing were TTX-insensitive, implying that high-density T-type Ca2+ channels might cooperate with TTX-insensitive Na+ channels to reduce the AP threshold. By contrast, the neurons displaying non-ADP burst firing were TTX-sensitive, implying that low density T-type Ca2+ channels may cooperate with TTX-sensitive Na+ channels to increase AP number. In DRG neurons, T-type Ca2+ currents density varied widely, ranging between 100 pA/pF and 5 pA/pF. After injury, the proportion of DRG neurons with large T-type Ca2+ currents increased in parallel with the increase in the incidence of large-amplitude-ADP firing. And in addition to Cav3.2, Cav3.3 channels are also likely to contribute to low-threshold firing. The data revealed that T-type Ca2+ channels may play a dual role in modulating the injured neurons' high excitability through a cooperative process with Na+ channels, thereby contributing to neuropathic pain.

Effects of Bepridil and Pimozide, Existing Medicines Capable of Blocking T-Type Ca2+ Channels, on Visceral Pain in Mice.

T-Type Ca2+ channels (T-channels), particularly Cav3.2, are now considered as therapeutic targets for treatment of intractable pain including visceral pain. Among existing medicines, bepridil, a multi-channel blocker, used for treatment of arrhythmia and angina, and pimozide, a dopamine D2 receptor antagonist, known as a typical antipsychotic, have potent T-channel blocking activity. We thus tested whether bepridil and pimozide could suppress visceral pain in mice. Colonic and bladder pain were induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) and systemic administration of cyclophosphamide (CPA), respectively. Referred hyperalgesia was assessed by von Frey test, and colonic hypersensitivity to distension by a volume load with intracolonic water injection and spontaneous bladder pain were evaluated by observing nociceptive behaviors in conscious mice. The mice exhibited referred hyperalgesia and colonic hypersensitivity to distension on day 6 after TNBS treatment. Systemic administration of bepridil at 10-20 mg/kg or pimozide at 0.1-0.5 mg/kg strongly reduced the referred hyperalgesia on the TNBS-induced referred hyperalgesia and colonic hypersensitivity to distension. CPA treatment caused bladder pain-like nociceptive behavior and referred hyperalgesia, which were reversed by bepridil at 10-20 mg/kg or pimozide at 0.5-1 mg/kg. Our data thus suggest that bepridil and pimozide, existing medicines capable of blocking T-channels, are useful for treatment of colonic and bladder pain, and serve as seeds for the development of new medicines for visceral pain treatment.

TRPM7 and CaV3.2 channels mediate Ca2+ influx required for egg activation at fertilization.

The success of mammalian development following fertilization depends on a series of transient increases in egg cytoplasmic Ca2+, referred to as Ca2+ oscillations. Maintenance of these oscillations requires Ca2+ influx across the plasma membrane, which is mediated in part by T-type, CaV3.2 channels. Here we show using genetic mouse models that TRPM7 channels are required to support this Ca2+ influx. Eggs lacking both TRPM7 and CaV3.2 stop oscillating prematurely, indicating that together they are responsible for the majority of Ca2+ influx immediately following fertilization. Fertilized eggs lacking both channels also frequently display delayed resumption of Ca2+ oscillations, which appears to require sperm-egg fusion. TRPM7 and CaV3.2 channels almost completely account for Ca2+ influx observed following store depletion, a process previously attributed to canonical store-operated Ca2+ entry mediated by STIM/ORAI interactions. TRPM7 serves as a membrane sensor of extracellular Mg2+ and Ca2+ concentrations and mediates the effects of these ions on Ca2+ oscillation frequency. When bred to wild-type males, female mice carrying eggs lacking TRPM7 and CaV3.2 are subfertile, and their offspring have increased variance in postnatal weight. These in vivo findings confirm previous observations linking in vitro experimental alterations in Ca2+ oscillatory patterns with developmental potential and offspring growth. The identification of TRPM7 and CaV3.2 as key mediators of Ca2+ influx following fertilization provides a mechanistic basis for the rational design of culture media that optimize developmental potential in research animals, domestic animals, and humans.

Calcium Channel Subunit α2δ4 Is Regulated by Early Growth Response 1 and Facilitates Epileptogenesis.

Transient brain insults, including status epilepticus (SE), can trigger a period of epileptogenesis during which functional and structural reorganization of neuronal networks occurs resulting in the onset of focal epileptic seizures. In recent years, mechanisms that regulate the dynamic transcription of individual genes during epileptogenesis and thereby contribute to the development of a hyperexcitable neuronal network have been elucidated. Our own results have shown early growth response 1 (Egr1) to transiently increase expression of the T-type voltage-dependent Ca2+ channel (VDCC) subunit CaV3.2, a key proepileptogenic protein. However, epileptogenesis involves complex and dynamic transcriptomic alterations; and so far, our understanding of the transcriptional control mechanism of gene regulatory networks that act in the same processes is limited. Here, we have analyzed whether Egr1 acts as a key transcriptional regulator for genes contributing to the development of hyperexcitability during epileptogenesis. We found Egr1 to drive the expression of the VDCC subunit α2δ4, which was augmented early and persistently after pilocarpine-induced SE. Furthermore, we show that increasing levels of α2δ4 in the CA1 region of the hippocampus elevate seizure susceptibility of mice by slightly decreasing local network activity. Interestingly, we also detected increased expression levels of Egr1 and α2δ4 in human hippocampal biopsies obtained from epilepsy surgery. In conclusion, Egr1 controls the abundance of the VDCC subunits CaV3.2 and α2δ4, which act synergistically in epileptogenesis, and thereby contributes to a seizure-induced "transcriptional Ca2+ channelopathy."SIGNIFICANCE STATEMENT The onset of focal recurrent seizures often occurs after an epileptogenic process induced by transient insults to the brain. Recently, transcriptional control mechanisms for individual genes involved in converting neurons hyperexcitable have been identified, including early growth response 1 (Egr1), which activates transcription of the T-type Ca2+ channel subunit CaV3.2. Here, we find Egr1 to regulate also the expression of the voltage-dependent Ca2+ channel subunit α2δ4, which was augmented after pilocarpine- and kainic acid-induced status epilepticus. In addition, we observed that α2δ4 affected spontaneous network activity and the susceptibility for seizure induction. Furthermore, we detected corresponding dynamics in human biopsies from epilepsy patients. In conclusion, Egr1 orchestrates a seizure-induced "transcriptional Ca2+ channelopathy" consisting of CaV3.2 and α2δ4, which act synergistically in epileptogenesis.

IL-6 induced upregulation of T-type Ca2+ currents and sensitization of DRG nociceptors is attenuated by MNK inhibition.

Phosphorylation of the 5' cap-binding protein eIF4E by MAPK-interacting kinases (MNK1/2) is important for nociceptor sensitization and the development of chronic pain. IL-6-induced dorsal root ganglion (DRG) nociceptor excitability is attenuated in mice lacking eIF4E phosphorylation, in MNK1/2-/- mice, and by the nonselective MNK1/2 inhibitor cercosporamide. Here, we sought to better understand the neurophysiological mechanisms underlying how IL-6 causes nociceptor excitability via MNK-eIF4E signaling using the highly selective MNK inhibitor eFT508. DRG neurons were cultured from male and female ICR mice, 4-7 wk old. DRG cultures were treated with vehicle, IL-6, eFT508 (pretreat) followed by IL-6, or eFT508 alone. Whole cell patch-clamp recordings were done on small-diameter neurons (20-30 pF) to measure membrane excitability in response to ramp depolarization. IL-6 treatment (1 h) resulted in increased action potential firing compared with vehicle at all ramp intensities, an effect that was blocked by pretreatment with eFT508. Basic membrane properties, including resting membrane potential, input resistance, and rheobase, were similar across groups. Latency to the first action potential in the ramp protocol was lower in the IL-6 group and rescued by eFT508 pretreatment. We also found that the amplitudes of T-type voltage-gated calcium channels (VGCCs) were increased in the DRG following IL-6 treatment, but not in the eFT508 cotreatment group. Our findings are consistent with a model wherein MNK-eIF4E signaling controls the translation of signaling factors that regulate T-type VGCCs in response to IL-6 treatment. Inhibition of MNK with eFT508 disrupts these events, thereby preventing nociceptor hyperexcitability.NEW & NOTEWORTHY In this study, we show that the MNK inhibitor and anti-tumor agent eFT508 (tomivosertib) is effective in attenuating IL-6 induced sensitization of dorsal root ganglion (DRG) nociceptors. Pretreatment with eFT508 in DRG cultures from mice helps mitigate the development of hyperexcitability in response to IL-6. Furthermore, our data reveal that the upregulation of T-type voltage-gated calcium channels following IL-6 application can be blocked by eFT508, implicating the MNK-eIF4E signaling pathway in membrane trafficking of ion channels.

Characterisation of d-Conotoxin TxVIA as a Mammalian T-Type Calcium Channel Modulator.

The 27-amino acid (aa)-long d-conotoxin TxVIA, originally isolated from the mollusc-hunting cone snail Conus textile, slows voltage-gated sodium (NaV) channel inactivation in molluscan neurons, but its mammalian ion channel targets remain undetermined. In this study, we confirmed that TxVIA was inactive on mammalian NaV1.2 and NaV1.7 even at high concentrations (10 µM). Given the fact that invertebrate NaV channel and T-type calcium channels (CaV3.x) are evolutionarily related, we examined the possibility that TxVIA may act on CaV3.x. Electrophysiological characterisation of the native TxVIA on CaV3.1, 3.2 and 3.3 revealed that TxVIA preferentially inhibits CaV3.2 current (IC50 = 0.24 mM) and enhances CaV3.1 current at higher concentrations. In fish bioassays TxVIA showed little effect on zebrafish behaviours when injected intramuscular at 250 ng/100 mg fish. The binding sites for TxVIA at NaV1.7 and CaV3.1 revealed that their channel binding sites contained a common epitope.

Dietary ascorbic acid restriction in GNL/SMP30-knockout mice unveils the role of ascorbic acid in regulation of somatic and visceral pain sensitivity.

Cav3.2 T-type Ca2+ channels are expressed in the primary afferents and play a pronociceptive role. The activity of Cav3.2 is enhanced by H2S, a gasotransmitter, and suppressed by ascorbic acid (vitamin C) through metal-catalyzed oxidation of the Zn2+-binding His191 in Cav3.2. Since rodents, but not humans, are capable of synthesizing ascorbic acid, the present study examined the role of ascorbic acid in nociceptive processing, using the mice lacking GNL/SMP30, an enzyme essential for ascorbic acid biosynthesis. Intraplantar and intracolonic administration of NaHS, an H2S donor, caused somatic allodynia and referred hyperalgesia, respectively, and repeated treatment with paclitaxel produced neuropathic allodynia in wild-type mice, all of which were suppressed by ascorbic acid or T-type Ca2+ channel blockers. Dietary ascorbic acid restriction caused dramatic decreases in plasma and tissue ascorbic acid levels in GNL/SMP30-knockout, but not wild-type, mice. The ascorbic acid restriction enhanced the somatic and visceral hypersensitivity following intraplantar and intracolonic NaHS, respectively, and paclitaxel-induced neuropathy in GNL/SMP30-knockout mice, while it had no such effect in wild-type mice. Together, our data unveil the critical role of ascorbic acid in regulating somatic and visceral pain sensitivity and support accumulating clinical evidence for the usefulness of ascorbic acid in pain management.

Cav 3.2 T-type calcium channel mutation influences kindling-induced thalamic neuronal firing patterns in Genetic Absence Epilepsy Rats From Strasbourg.

OBJECTIVE: Recent data indicate that amygdala kindling leads to significant changes in interictal neuronal firing patterns of thalamic reticular nucleus (TRN) neurons by decreasing the spontaneous firing rate and increasing burst firing in nonepileptic control (NEC) rats. Genetic Absence Epilepsy Rats From Strasbourg (GAERS) were resistant to these kindling-induced firing changes in TRN neurons, and are also resistant to the progression of kindling. We investigated whether a homozygous, missense, single nucleotide mutation (R1584P) in the Cav 3.2 T-type Ca2+ channel gene, which has been correlated with the expression of absence seizures in GAERS, influenced kindling progression and TRN firing patterns. METHODS: Double-crossed (GAERS vs NEC; F2) rats that were homozygous for the Cav 3.2 mutation (PP) and those negative for the mutation (RR) were implanted with a stimulating electrode in the amygdala. Rats received a total of 30 kindling stimulations at their afterdischarge threshold current twice daily, and kindling progression was evaluated. Thereafter, the extracellular neuronal activity of TRN neurons was recorded in vivo under neuroleptanesthesia to investigate the influence of Cav 3.2 mutation on TRN firing patterns. RESULTS: We found that the R1584P mutation did not affect kindling progression in F2 crosses (P = 0.78). However, it influenced kindling-induced neuronal firing of TRN neurons. After 30 stimulations, RR rats exhibited a lower firing rate and a higher percentage of burst firing compared to PP rats. The decrease in firing frequency was correlated with the increase in the amount of burst firing in RR rats (R2  = 0.497). SIGNIFICANCE: Our findings suggest that mutation in Cav 3.2 T-type Ca2+ channels may play a role in the resistance to kindling-induced changes in TRN neurons to a low-frequency and high-percentage bursting pattern seen in association with the convulsive stages of amygdala kindling, but is not in itself enough to explain the resistance to kindling progression observed in GAERS.

Increased expression of CaV3.2 T-type calcium channels in damaged DRG neurons contributes to neuropathic pain in rats with spared nerve injury.

Ion channels are very important in the peripheral sensitization in neuropathic pain. Our present study aims to investigate the possible contribution of CaV3.2 T-type calcium channels in damaged dorsal root ganglion neurons in neuropathic pain. We established a neuropathic pain model of rats with spared nerve injury. In these model rats, it was easy to distinguish damaged dorsal root ganglion neurons (of tibial nerve and common peroneal nerve) from intact dorsal root ganglion neurons (of sural nerves). Our results showed that CaV3.2 protein expression increased in medium-sized neurons from the damaged dorsal root ganglions but not in the intact ones. With whole cell patch clamp recording technique, it was found that after-depolarizing amplitudes of the damaged medium-sized dorsal root ganglion neurons increased significantly at membrane potentials of -85 mV and -95 mV. These results indicate a functional up-regulation of CaV3.2 T-type calcium channels in the damaged medium-sized neurons after spared nerve injury. Behaviorally, blockade of CaV3.2 with antisense oligodeoxynucleotides could significantly reverse mechanical allodynia. These results suggest that CaV3.2 T-type calcium channels in damaged medium-sized dorsal root ganglion neurons might contribute to neuropathic pain after peripheral nerve injury.

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.