Novel Scorpion Toxin ω-Buthitoxin-Hf1a Selectively Inhibits Calcium Influx via CaV3.3 and CaV3.2 and Alleviates Allodynia in a Mouse Model of Acute Postsurgical Pain.

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 µM) and CaV3.3 (IC50 = 0.49 µM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.

Elevated microRNA-214-3p level ameliorates neuroinflammation after spinal cord ischemia-reperfusion injury by inhibiting Nmb/Cav3.2 pathway.

BACKGROUND: Neuromedin B (Nmb) plays a pivotal role in the transmission of neuroinflammation, particularly during spinal cord ischemia-reperfusion injury (SCII). However, the detailed molecular mechanisms underlying this process remain elusive. METHODS: The SCII model was established by clamping the abdominal aorta of male Sprague-Dawley (SD) rats for 60 min. The protein expression levels of Nmb, Cav3.2, and IL-1ß were detected by Western blotting, while miR-214-3p expression was quantified by qRT-PCR. The targeted regulation between miR-214-3p and Nmb was investigated using a dual-luciferase reporter gene assay. The cellular localization of Nmb and Cav3.2 with cell-specific markers was visualized by immunofluorescence staining. The specific roles of miR-214-3p on the Nmb/Cav3.2 interactions in SCII-injured rats were explored by intrathecal injection of Cav3.2-siRNA, PD168368 (a specific NmbR inhibitor) and synthetic miR-214-3p agomir and antagomir in separate experiments. Additionally, hind-limb motor function was evaluated using the modified Tarlov scores. RESULTS: Compared to the Sham group, the protein expression levels of Nmb, Cav3.2, and the proinflammatory factor Interleukin(IL)-1ß were significantly elevated at 24 h post-SCII. Intrathecal injection of PD168368 and Cav3.2-siRNA significantly suppressed the expression of Cav3.2 and IL-1ß compared to the SCII group. The miRDB database and dual-luciferase reporter gene assay identified Nmb as a direct target of miR-214-3p. As expected, in vivo overexpression of miR-214-3p by agomir-214-3p pretreatment significantly inhibited the increases in Nmb, Cav3.2 and IL-1ß expression and improved lower limb motor function in SCII-injured rats, while antagomiR-214-3p pretreatment reversed these effects. CONCLUSIONS: Nmb protein levels positively correlated with Cav3.2 expression in SCII rats. Upregulating miR-214-3p ameliorated hind-limb motor function and protected against neuroinflammation via inhibiting the aberrant Nmb/Cav3.2 interactions and downstream IL-1ß release. These findings provide novel therapeutic targets for clinical prevention and treatment of SCII.

The role of T-type calcium channels in elderly human vascular function: A pilot randomized controlled trial.

Endothelial dysfunction develops with age and may precede cardiovascular disease. Animal data suggest that T-type calcium channels play an important role in endothelial function, but data from humans are lacking. This study included 15 healthy, sedentary, elderly males for a double blinded, randomized controlled trial. For 8 weeks, they were given 40 mg/day of either efonidipine (L- and T-type calcium channel blocker (CCB)) or nifedipine (L-type CCB). Vascular function was evaluated by graded femoral arterial infusions of acetylcholine (ACh; endothelium-dependent vasodilator) and sodium nitroprusside (endothelium-independent vasodilator) both with and without co-infusion of N-acetylcysteine (NAC; antioxidant). We measured leg blood flow and mean arterial pressure and calculated leg vascular conductance to evaluate the leg vascular responses. Despite no significant change in blood pressure in either group, we observed higher leg blood flow responses (Δ 0.43 ± 0.45 l/min, P = 0.006) and leg vascular conductance (Δ 5.38 ± 5.67 ml/min/mmHg, P = 0.005) to intra-arterial ACh after efonidipine, whereas there was no change in the nifedipine group, and no differences between groups. We found no upregulation of endothelial nitric oxide synthase in vastus lateralis muscle biopsies within or between groups. Smooth muscle cell responsiveness was unaltered by efonidipine or nifedipine. Intravenous co-infusion of NAC did not affect endothelium-dependent vasodilatation in either of the CCB groups. These results suggest that 8 weeks' inhibition of T- and L-type calcium channels augments endothelium-dependent vasodilatory function in healthy elderly males. Further studies are required to elucidate if T-type calcium channel inhibition can counteract endothelial dysfunction.

Effect of ABT-639 on Cav3.2 channel activity and its analgesic actions in mouse models of inflammatory and neuropathic pain.

Cav3.2 T-type calcium channels are important targets for pain relief in rodent models of inflammatory and neuropathic pain. Even though many T-type channel blockers have been tested in mice, only one molecule, ABT-639, has been tested in phase II clinical studies and did not produce analgesic effects over placebo. Here we examined the effects of ABT-639 on Cav3.2 channel activity in tsA-201 cells and dorsal root ganglion (DRG) neurons, in comparison with another established Cav3.2 inhibitor Z944. These experiments revealed that Z944 mediated ∼100-fold more potent inhibition of Cav3.2 currents than ABT-639, with the latter blocking channel activity by less than 15 percent when applied at a concentration of 30 µM. A slight increase in ABT-639 potency was observed at more depolarized holding potentials, suggesting that this compound may act preferentially on inactivated channels. We tested the effects of both compounds in the Complete Freund's Adjuvant (CFA) model of chronic inflammatory pain, and in partial sciatic nerve injury model of neuropathic pain in mice. In the neuropathic pain model, both Z944 and ABT-639 reversed mechanical hypersensitivity to similar degrees when delivered systemically, but remarkably, when delivered intrathecally, only Z944 was effective. In the CFA model, both compounds reversed thermal hyperalgesia upon systemic delivery, but only Z944 mediated pain relief upon intrathecal delivery, indicating that ABT-639 acts primarily at peripheral sites. ABT-639 lost its analgesic effects in CFA treated Cav3.2 null mice, indicating that these channels are essential for ABT-639-mediated pain relief despite its poor inhibition of Cav3.2 currents.

ERG potassium channels and T-type calcium channels contribute to the pacemaker and atrioventricular conduction in zebrafish larvae.

AIM: Bradyarrhythmias result from inhibition of automaticity, prolonged repolarization, or slow conduction in the heart. The ERG channels mediate the repolarizing current IKr in the cardiac action potential, whereas T-type calcium channels (TTCC) are involved in the sinoatrial pacemaker and atrioventricular conduction in mammals. Zebrafish have become a valuable research model for human cardiac electrophysiology and disease. Here, we investigate the contribution of ERG channels and TTCCs to the pacemaker and atrioventricular conduction in zebrafish larvae and determine the mechanisms causing atrioventricular block. METHODS: Zebrafish larvae expressing ratiometric fluorescent Ca2+ biosensors in the heart were used to measure Ca2+ levels and rhythm in beating hearts in vivo, concurrently with contraction and hemodynamics. The atrioventricular delay (the time between the start of atrial and ventricular Ca2+ transients) was used to measure impulse conduction velocity and distinguished between slow conduction and prolonged refractoriness as the cause of the conduction block. RESULTS: ERG blockers caused bradycardia and atrioventricular block by prolonging the refractory period in the atrioventricular canal and in working ventricular myocytes. In contrast, inhibition of TTCCs caused bradycardia and second-degree block (Mobitz type I) by slowing atrioventricular conduction. TTCC block did not affect ventricular contractility, despite being highly expressed in cardiomyocytes. Concomitant measurement of Ca2+ levels and ventricular size showed mechano-mechanical coupling: increased preload resulted in a stronger heart contraction in vivo. CONCLUSION: ERG channels and TTCCs influence the heart rate and atrioventricular conduction in zebrafish larvae. The zebrafish lines expressing Ca2+ biosensors in the heart allow us to investigate physiological feedback mechanisms and complex arrhythmias.

The T-type calcium channelosome.

T-type calcium channels perform crucial physiological roles across a wide spectrum of tissues, spanning both neuronal and non-neuronal system. For instance, they serve as pivotal regulators of neuronal excitability, contribute to cardiac pacemaking, and mediate the secretion of hormones. These functions significantly hinge upon the intricate interplay of T-type channels with interacting proteins that modulate their expression and function at the plasma membrane. In this review, we offer a panoramic exploration of the current knowledge surrounding these T-type channel interactors, and spotlight certain aspects of their potential for drug-based therapeutic intervention.

Cav3.2 T-Type Calcium Channel Mediates Acute Itch and Contributes to Chronic Itch and Inflammation in Experimental Atopic Dermatitis.

Voltage-gated calcium channels regulate neuronal excitability. The Cav3.2 isoform of the T-type voltage-activated calcium channel is expressed in sensory neurons and is implicated in pain transmission. However, its role in itch remains unclear. In this study, we demonstrated that Cav3.2 is expressed by mechanosensory and peptidergic subsets of mouse dorsal root ganglion neurons and colocalized with TRPV1 and receptors for type 2 cytokines. Cav3.2-positive neurons innervate human skin. A deficiency of Cav3.2 reduces histamine, IL-4/IL-13, and TSLP-induced itch in mice. Cav3.2 channels were upregulated in the dorsal root ganglia of an atopic dermatitis (AD)-like mouse model and mediated neuronal excitability. Genetic knockout of Cav3.2 or T-type calcium channel blocker mibefradil treatment reduced spontaneous and mechanically induced scratching behaviors and skin inflammation in an AD-like mouse model. Substance P and vasoactive intestinal polypeptide levels were increased in the trigeminal ganglia from AD-like mouse model, and genetic ablation or pharmacological inhibition of Cav3.2 reduced their gene expression. Cav3.2 knockout also attenuated the pathologic changes in ex vivo skin explants cocultured with trigeminal ganglia neurons from AD-induced mice. Our study identifies the role of Cav3.2 in both histaminergic and nonhistaminergic acute itch. Cav3.2 channel also contributes to AD-related chronic itch and neuroinflammation.

The role of HCN channels on the effects of T-type calcium channels and GABAA receptors in the absence epilepsy model of WAG/Rij rats.

In this study we used ivabradine (IVA), a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, to identify its effect on spike-wave discharges (SWDs); and aimed to determine the role of IVA on the effects of T-type calcium channel blocker NNC 55-0396, GABAA receptor agonist muscimol and antagonist bicuculline in male WAG/Rij rats. After tripolar electrodes for electrocorticogram (ECoG) recordings were placed on the WAG/Rij rats' skulls, 5, 10, and 20 mg/kg IVA were intraperitoneally administered for 7 consecutive days and ECoG recordings were obtained on days 0th, 3rd, 6th, and 7th for three hours before and after injections. While acute injection of 5, 10, and 20 mg/kg IVA did not affect the total number and the mean duration of SWDs, subacute administration (7 days) of IVA decreased the SWDs parameters 24 hours after the 7th injection. Interestingly, when IVA was administered again 24 hours after the 6th IVA injection, it increased the SWDs parameters. Western-blot analyses showed that HCN1 and HCN2 expressions decreased and HCN4 increased in the 5-month-old WAG/Rij rats compared to the 1-month-old WAG/Rij and 5-month-old native Wistar rats, while subacute IVA administration increased the levels of HCN1 and HCN2 channels, except HCN4. Subacute administration of IVA reduced the antiepileptic activity of NNC, while the proepileptic activity of muscimol and the antiepileptic activity of bicuculline were abolished. It might be suggested that subacute IVA administration reduces absence seizures by changing the HCN channel expressions in WAG/Rij rats, and this affects the T-type calcium channels and GABAA receptors.

Direct Current Stimulation Modulates Synaptic Facilitation via Distinct Presynaptic Calcium Channels.

Transcranial direct current stimulation (tDCS) is a subthreshold neurostimulation technique known for ameliorating neuropsychiatric conditions. The principal mechanism of tDCS is the differential polarization of subcellular neuronal compartments, particularly the axon terminals that are sensitive to external electrical fields. Yet, the underlying mechanism of tDCS is not fully clear. Here, we hypothesized that direct current stimulation (DCS)-induced modulation of presynaptic calcium channel conductance alters axon terminal dynamics with regard to synaptic vesicle release. To examine the involvement of calcium-channel subtypes in tDCS, we recorded spontaneous excitatory postsynaptic currents (sEPSCs) from cortical layer-V pyramidal neurons under DCS while selectively inhibiting distinct subtypes of voltage-dependent calcium channels. Blocking P/Q or N-type calcium channels occluded the effects of DCS on sEPSCs, demonstrating their critical role in the process of DCS-induced modulation of spontaneous vesicle release. However, inhibiting T-type calcium channels did not occlude DCS-induced modulation of sEPSCs, suggesting that despite being active in the subthreshold range, T-type calcium channels are not involved in the axonal effects of DCS. DCS modulates synaptic facilitation by regulating calcium channels in axon terminals, primarily via controlling P/Q and N-type calcium channels, while T-type calcium channels are not involved in this mechanism.

KTt-45, a T-type calcium channel blocker, acts as an anticancer agent by inducing apoptosis on HeLa cervical cancer cell line.

The abnormal expression in the T-type calcium channels is involved in various cancer types, thus inhibiting T-type calcium channels is one of approaches in cancer treatment. The fact that KTt-45 acted as a T-type calcium channel inhibitor as well as a pain-relief agent prompts us to address if KTt-45 plays any role against cancer cells. The results showed that KTt-45 caused cytotoxic effects towards HeLa cervical, Raji lymphoma, MCF-7 breast cancer, and A549 lung cancer cell lines with IC50 values less than 100 µM, in which highly selective toxicity was against HeLa cells (IC50 = 37.4 µM, SI > 3.2). Strikingly, the KTt-45 induced an accumulation of cytoplasmic vacuoles after 48 h treatment and mitochondrial-dependent apoptosis activation as evidenced by morphological features, chromatin condensation, nuclear fragmentation, and significant activation of caspase-9 as well as caspase-3. In conclusion, KTt-45 could inhibit cell growth and trigger mitochondrial-dependent apoptosis in HeLa cervical cancer cells. The results, taken together, strongly demonstrated that KTt-45 is a potential agent for further study on anticancer drug development which not only targets cancer cells but also helps to relieve neuropathic pain in cancer patients.

CaV3.1 channels facilitate calcium wave generation and myogenic tone development in mouse mesenteric arteries.

The arterial myogenic response to intraluminal pressure elicits constriction to maintain tissue perfusion. Smooth muscle [Ca2+] is a key determinant of constriction, tied to L-type (CaV1.2) Ca2+ channels. While important, other Ca2+ channels, particularly T-type could contribute to pressure regulation within defined voltage ranges. This study examined the role of one T-type Ca2+ channel (CaV3.1) using C57BL/6 wild type and CaV3.1-/- mice. Patch-clamp electrophysiology, pressure myography, blood pressure and Ca2+ imaging defined the CaV3.1-/- phenotype relative to C57BL/6. CaV3.1-/- mice had absent CaV3.1 expression and whole-cell current, coinciding with lower blood pressure and reduced mesenteric artery myogenic tone, particularly at lower pressures (20-60 mmHg) where membrane potential is hyperpolarized. This reduction coincided with diminished Ca2+ wave generation, asynchronous events of Ca2+ release from the sarcoplasmic reticulum, insensitive to L-type Ca2+ channel blockade (Nifedipine, 0.3 µM). Proximity ligation assay (PLA) confirmed IP3R1/CaV3.1 close physical association. IP3R blockade (2-APB, 50 µM or xestospongin C, 3 µM) in nifedipine-treated C57BL/6 arteries rendered a CaV3.1-/- contractile phenotype. Findings indicate that Ca2+ influx through CaV3.1 contributes to myogenic tone at hyperpolarized voltages through Ca2+-induced Ca2+ release tied to the sarcoplasmic reticulum. This study helps establish CaV3.1 as a potential therapeutic target to control blood pressure.

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.

Electric field stimulation unmasks a subtle role for T-type calcium channels in regulating lymphatic contraction.

We previously identified two isoforms of T-type, voltage-gated calcium (Cav3) channels (Cav3.1, Cav3.2) that are functionally expressed in murine lymphatic muscle cells; however, contractile tests of lymphatic vessels from single and double Cav3 knock-out (DKO) mice, exhibited nearly identical parameters of spontaneous twitch contractions as wild-type (WT) vessels, suggesting that Cav3 channels play no significant role. Here, we considered the possibility that the contribution of Cav3 channels might be too subtle to detect in standard contraction analyses. We compared the sensitivity of lymphatic vessels from WT and Cav3 DKO mice to the L-type calcium channel (Cav1.2) inhibitor nifedipine and found that the latter vessels were significantly more sensitive to inhibition, suggesting that the contribution of Cav3 channels might normally be masked by Cav1.2 channel activity. We hypothesized that shifting the resting membrane potential (Vm) of lymphatic muscle to a more negative voltage might enhance the contribution of Cav3 channels. Because even slight hyperpolarization is known to completely silence spontaneous contractions, we devised a method to evoke nerve-independent, twitch contractions from mouse lymphatic vessels using single, short pulses of electric field stimulation (EFS). TTX was present throughout to block the potential contributions of voltage-gated Na+ channels in perivascular nerves and lymphatic muscle. In WT vessels, EFS evoked single contractions that were comparable in amplitude and degree of entrainment to those occurring spontaneously. When Cav1.2 channels were blocked or deleted, only small residual EFS-evoked contractions (~ 5% of normal amplitude) were present. These residual, EFS-evoked contractions were enhanced (to 10-15%) by the KATP channel activator pinacidil (PIN) but were absent in Cav3 DKO vessels. Our results point to a subtle contribution of Cav3 channels to lymphatic contractions that can be unmasked in the absence of Cav1.2 channel activity and when the resting Vm is more hyperpolarized than normal.

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.

Englerin A Inhibits T-Type Voltage-Gated Calcium Channels at Low Micromolar Concentrations.

Englerin A (EA) is a potent agonist of tetrameric transient receptor potential canonical (TRPC) ion channels containing TRPC4 and TRPC5 subunits. TRPC proteins form cation channels that are activated by plasma membrane receptors. They convert extracellular signals such as angiotensin II into cellular responses, whereupon Na+ and Ca2+ influx and depolarization of the plasma membrane occur. Via depolarization, voltage-gated Ca2+ (CaV) channels can be activated, further increasing Ca2+ influx. We investigated the extent to which EA also affects the functions of CaV channels using the high-voltage-activated L-type Ca2+ channel CaV1.2 and the low-voltage-activated T-type Ca2+ channels CaV3.1, CaV3.2, and CaV3.3. After expression of cDNAs in human embryonic kidney (HEK293) cells, EA inhibited currents through all T-type channels at half-maximal inhibitory concentrations (IC50) of 7.5 to 10.3 µM. In zona glomerulosa cells of the adrenal gland, angiotensin II-induced elevation of cytoplasmic Ca2+ concentration leads to aldosterone release. We identified transcripts of low- and high-voltage-activated CaV channels and of TRPC1 and TRPC5 in the human adrenocortical (HAC15) zona glomerulosa cell line. Although no EA-induced TRPC activity was measurable, Ca2+ channel blockers distinguished T- and L-type Ca2+ currents. EA blocked 60% of the CaV current in HAC15 cells and T- and L-type channels analyzed at -30 mV and 10 mV were inhibited with IC50 values of 2.3 and 2.6 µM, respectively. Although the T-type blocker Z944 reduced basal and angiotensin II-induced 24-hour aldosterone release, EA was not effective. In summary, we show here that EA blocks CaV1.2 and T-type CaV channels at low-micromolar concentrations. SIGNIFICANCE STATEMENT: In this study we showed that englerin A (EA), a potent agonist of tetrameric transient receptor potential canonical (TRPC)4- or TRPC5-containing channels and currently under investigation to treat certain types of cancer, also inhibits the L-type voltage-gated Ca2+ (CaV) channel CaV1.2 and the T-type CaV channels CaV3.1, CaV3.2, and CaV3.3 channels at low micromolar concentrations.

Mammalian neurotoxins, Blarina paralytic peptides, cause hyperpolarization of human T-type Ca channel hCav3.2 activation.

Among the rare venomous mammals, the short-tailed shrew Blarina brevicauda has been suggested to produce potent neurotoxins in its saliva to effectively capture prey. Several kallikrein-like lethal proteases have been identified, but the active substances of B. brevicauda remained unclear. Here, we report Blarina paralytic peptides (BPPs) 1 and 2 isolated from its submaxillary glands. Synthetic BPP2 showed mealworm paralysis and a hyperpolarization shift (-11 mV) of a human T-type Ca2+ channel (hCav3.2) activation. The amino acid sequences of BPPs were similar to those of synenkephalins, which are precursors of brain opioid peptide hormones that are highly conserved among mammals. However, BPPs rather resembled centipede neurotoxic peptides SLPTXs in terms of disulfide bond connectivity and stereostructure. Our results suggested that the neurotoxin BPPs were the result of convergent evolution as homologs of nontoxic endogenous peptides that are widely conserved in mammals. This finding is of great interest from the viewpoint of the chemical evolution of vertebrate venoms.

Antihyperalgesic effect of joint mobilization requires Cav3.2 calcium channels.

The present study was undertaken to explore the relative contributions of Cav3.2 T-type channels to mediating the antihyperalgesic activity of joint manipulation (JM) therapy. We used the chronic constriction injury model (CCI) to induce peripheral neuropathy and chronic pain in male mice, followed by JM. We demonstrate that JM produces long-lasting mechanical anti-hyperalgesia that is abolished in Cav3.2 null mice. Moreover, we found that JM displays a similar analgesic profile as the fatty acid amide hydrolase inhibitor URB597, suggesting a possible converging mechanism of action involving endocannabinoids. Overall, our findings advance our understanding of the mechanisms through which JM produces analgesia.

The relationship of promoter methylation of calcium voltage-gated channel alpha 1 and interleukin-16 to primary osteoarthritis.

OBJECTIVE: Osteoarthritis is the most prevalent joint disease worldwide and the primary cause of musculoskeletal dysfunction. Epigenetic changes in various genes, particularly methylation, have been implicated as possible underlying causes of primary osteoarthritis. The aim of our study was to investigate the promoter methylation status of the calcium voltage-gated channel alpha 1 subunit G (CACNA1G) and interleukin-16 (IL-16) genes, which are strongly associated with calcium channel activity and antigen presentation, respectively, in primary osteoarthritis patients. PATIENTS AND METHODS: Twenty-one patients with primary osteoarthritis and 25 healthy controls were included in our study. The methylation status of CACNA1G and IL-16 genes was analyzed with methylation-specific Polymerase Chain Reaction (PCR), and the serum levels of IL-16 were determined with Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: The age of the patients was 63.95±8.41 years, and they were 15 females and 6 males. The promoter of the CACNA1G gene was found to be hypermethylated in primary osteoarthritis patients (p<0.001). In contrast, the promoter of the IL-16 gene was found to be hypomethylated compared to the control (p<0.001). The serum levels of IL-16 increased in parallel with the hypomethylated promoter status of IL-16 gene in primary osteoarthritis patients compared to the control (p<0.001). CONCLUSIONS: Our study indicates that the methylation status of CACNA1G and IL-16 gene promoters are epigenetically altered in patients with primary osteoarthritis. Moreover, increased serum IL-16 levels in osteoarthritis patients may be associated with hypomethylation of the IL-16 gene promoter.

Effects of the T-type calcium channel CaV3.2 R1584P mutation on absence seizure susceptibility in GAERS and NEC congenic rats models.

RATIONALE: Low-voltage-activated or T-type Ca2+ channels play a key role in the generation of seizures in absence epilepsy. We have described a homozygous, gain of function substitution mutation (R1584P) in the CaV3.2 T-type Ca2+ channel gene (Cacna1h) in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). The non-epileptic control (NEC) rats, derived from the same original Wistar strains as GAERS but selectively in-breed not to express seizures, are null for the R1584P mutation. To study the effects of this mutation in rats who otherwise have a GAERS or NEC genetic background, we bred congenic GAERS-Cacna1hNEC (GAERS null for R1584P mutation) and congenic NEC-Cacna1hGAERS (NEC homozygous for R1584P mutation) and evaluated the seizure and behavioral phenotype of these strains in comparison to the original GAERS and NEC strains. METHODS: To evaluate seizure expression in the congenic strains, EEG electrodes were implanted in NEC, GAERS, GAERS-Cacna1hNEC without the R1584P mutation, and NEC-Cacna1hGAERS with the R1584P mutation rats. In the first study, continuous EEG recordings were acquired from week 4 (when seizures begin to develop in GAERS) to week 14 of age (when GAERS display hundreds of seizures per day). In the second study, the seizure and behavioral phenotype of GAERS and NEC-Cacna1hGAERS strains were evaluated during young age (6 weeks of age) and adulthood (16 weeks of age) of GAERS, NEC, GAERS-Cacna1hNEC and NEC-Cacna1hGAERS. The Open field test (OFT) and sucrose preference test (SPT) were performed to evaluate anxiety-like and depressive-like behavior, respectively. This was followed by EEG recordings at 18 weeks of age to quantify the seizures, and spike-wave discharge (SWD) cycle frequency. At the end of the study, the whole thalamus was collected for T-type calcium channel mRNA expression analysis. RESULTS: GAERS had a significantly shorter latency to first seizures and an increased number of seizures per day compared to GAERS-Cacna1hNEC. On the other hand, the presence of the R1584P mutation in the NEC-Cacna1hGAERS was not enough to generate spontaneous seizures in their seizure-resistant background. 6 and 16-week-old GAERS and GAERS-Cacna1hNEC rats showed anxiety-like behavior in the OFT, in contrast to NEC and NEC-Cacna1hGAERS. Results from the SPT showed that the GAERS developed depressive-like in the SPT compared to GAERS-Cacna1hNEC, NEC, and NEC-Cacna1hGAERS. Analysis of the EEG at 18 weeks of age showed that the GAERS had an increased number of seizures per day, increased total seizure duration and a higher cycle frequency of SWD relative to GAERS-Cacna1hNEC. However, the average seizure duration was not significantly different between strains. Quantitative real-time PCR showed that the T-type Ca2+ channel isoform CaV3.2 channel expression was significantly increased in GAERS compared to NEC, GAERS-Cacna1hNEC and NEC-Cacna1hGAERS. The presence of the R1584P mutation increased the total ratio of CaV3.2 + 25/-25 splice variants in GAERS and NEC-Cacna1hGAERS compared to NEC and GAERS-Cacna1hNEC. DISCUSSION: The data from this study demonstrate that the R1584P mutation in isolation on a seizure-resistant NEC genetic background was insufficient to generate absence seizures, and that a GAERS genetic background can cause seizures even without the mutation. However, the study provides evidence that the R1584P mutation acts as a modulator of seizures development and expression, and depressive-like behavior in the SPT, but not the anxiety phenotype of the GAERS model of absence epilepsy.