High Voltage-activated Calcium Research Articles

The α2δ Calcium Channel Subunit Accessorily and Independently Affects the Biological Function of Ditylenchus destructor.

The α2δ subunit is a high-voltage activated (HVA) calcium channel (Cav1 and Cav2) auxiliary subunit that increases the density and function of HVA calcium channels in the plasma membrane of mammals. However, its function in plant parasitic nematodes remains unknown. In this study, we cloned the full-length cDNA sequence of the voltage-gated calcium channel (VGCC) α2δ subunit (named DdCavα2δ) in Ditylenchus destructor. We found that DdCavα2δ tends to be expressed in the egg stage, followed by the J3 stage. RNA-DIG in situ hybridization experiments showed that the DdCavα2δ subunit was expressed in the body wall, esophageal gland, uterus, post uterine, and spicules of D. destructor. The in vitro application of RNA interference (RNAi) affected the motility, reproduction, chemotaxis, stylet thrusting, and protein secretion of D. destructor to different degrees by targeting DdCα1D, DdCα1A, and DdCavα2δ in J3 stages, respectively. Based on the results of RNAi experiments, it was hypothesized that L-type VGCC may affect the motility, chemotaxis, and stylet thrusting of D. destructor. Non-L-type VGCC may affect the protein secretion and reproduction of D. destructor. The DdCavα2δ subunit gene also affected the motility, chemotaxis, and reproduction of D. destructor. These findings reveal the independent function of the VGCC α2δ subunit in D. destructor as well as give a theoretical foundation for future research on plant parasitic nematode VGCC.

Analgesic effects of Phα1β toxin: a review of mechanisms of action involving pain pathways.

Phα1β is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1β to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1β (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1β antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

Inhibition of multiple voltage-gated calcium channels may contribute to spinally mediated analgesia by epipregnanolone in a rat model of surgical paw incision

ABSTRACTVoltage-activated calcium channels play an important role in excitability of sensory nociceptive neurons in acute and chronic pain models. We have previously shown that low-voltage-activated calcium channels, or T-type channels (T-channels), increase excitability of sensory neurons after surgical incision in rats. We have also found that endogenous 5β-reduced neuroactive steroid epipregnanolone [(3β,5β)-3-hydroxypregnan-20-one] blocked isolated T-currents in dorsal root ganglion (DRG) cells in vitro, and reduced nociceptive behavior in vivo, after local intraplantar application into the foot pads of heathy rats and mice. Here, we investigated if epipregnanolone exerts an antinociceptive effect after intrathecal (i.t.) application in healthy rats, as well as an antihyperalgesic effect in a postsurgical pain model. We also studied if this endogenous neurosteroid blocks currents originating from high voltage-activated (HVA) calcium channels in rat sensory neurons. In in vivo studies, we found that epipregnanolone alleviated thermal and mechanical nociception in healthy rats after i.t. administration without affecting their sensory-motor abilities. Furthermore, epipregnanolone effectively reduced mechanical hyperalgesia after i.t application in rats after surgery. In subsequent in vitro studies, we found that epipregnanolone blocked isolated HVA currents in nociceptive sensory neurons with an IC50 of 3.3 μM in a G-protein-dependent fashion. We conclude that neurosteroids that have combined inhibitory effects on T-type and HVA calcium currents may be suitable for development of novel pain therapies during the perioperative period.

Structure-Activity Studies Reveal the Molecular Basis for GABAB-Receptor Mediated Inhibition of High Voltage-Activated Calcium Channels by α-Conotoxin Vc1.1.

α-Conotoxins are disulfide-bonded peptides from cone snail venoms and are characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several α-conotoxins with distinct selectivity for nAChR subtypes have been identified as potent analgesics in animal models of chronic pain. However, a number of α-conotoxins have been shown to inhibit N-type calcium channel currents in rodent dissociated dorsal root ganglion (DRG) neurons via activation of G protein-coupled GABAB receptors (GABABR). Therefore, it is unclear whether activation of GABABR or inhibition of α9α10 nAChRs is the analgesic mechanism. To investigate the mechanisms by which α-conotoxins provide analgesia, we synthesized a suite of Vc1.1 analogues where all residues, except the conserved cysteines, in Vc1.1 were individually replaced by alanine (A), lysine (K), and aspartic acid (D). Our results show that the amino acids in the first loop play an important role in binding of the peptide to the receptor, whereas those in the second loop play an important role for the selectivity of the peptide for the GABABR over α9α10 nAChRs. We designed a cVc1.1 analogue that is >8000-fold selective for GABABR-mediated inhibition of high voltage-activated (HVA) calcium channels over α9α10 nAChRs and show that it is analgesic in a mouse model of chronic visceral hypersensitivity (CVH). cVc1.1[D11A,E14A] caused dose-dependent inhibition of colonic nociceptors with greater efficacy in ex vivo CVH colonic nociceptors relative to healthy colonic nociceptors. These findings suggest that selectively targeting GABABR-mediated HVA calcium channel inhibition by α-conotoxins could be effective for the treatment of chronic visceral pain.

Characterization of ion channels on subesophageal ganglion neurons from Chinese tarantula Ornithoctonus huwena: Exploring the myth of the spider insensitive to its venom

Chinese tarantula Ornithoctonus huwena is one of the most venomous spiders distributing in the hilly areas of southern China. In this study, using whole-cell patch-clamp technique we investigated electrophysiological and pharmacological properties of ion channels from tarantula subesophageal ganglion neurons. It was found that the neurons express multiple kinds of ion channels at least including voltage-gated calcium channels, TTX-sensitive sodium channels and two types of potassium channels. They exhibit pharmacological properties similar to mammalian subtypes. Spider calcium channels were sensitive to ω-conotoxin GVIA and diltiazem, two well-known inhibitors of mammalian neuronal high-voltage-activated (HVA) subtypes. 4-Aminopyridine and tetraethylammonium could inhibit spider outward transient and delayed-rectifier potassium channels, respectively. Huwentoxin-I and huwentoxin-IV are two abundant toxic components in the venom of Ornithoctonus huwena. Interestingly, although in our previous work they inhibit HVA calcium channels and TTX-sensitive sodium channels from mammalian sensory neurons, respectively, they fail to affect the subtypes from spider neurons. Moreover, the crude venom has no effect on delayed-rectifier potassium channels and only slightly reduces transient outward potassium channels with an IC50 value of ∼51.3 mg/L. Therefore, our findings provide important evidence for ion channels from spiders having an evolution as self-defense and prey mechanism.

Functional segregation of voltage-activated calcium channels in motoneurons of the dorsal motor nucleus of the vagus.

Calcium influx elevates mitochondrial oxidant stress (mOS) in dorsal motor nucleus of the vagus (DMV) neurons that are prone to Lewy body pathologies in presymptomatic Parkinson's disease (PD) patients. In experimental PD models, treatment with isradipine, the dihydropyridine with the highest affinity to Cav1.3 channels, prevents subthreshold calcium influx via Cav1.3 channels into midbrain dopamine neurons and protects them from mOS. In DMV neurons, isradipine is also effective in reducing mOS despite overwhelming evidence that subthreshold calcium influx is negligible compared with spike-triggered influx. To solve this conundrum we combined slice electrophysiology, two-photon laser scanning microscopy, mRNA profiling, and computational modeling. We find that the unusually depolarized subthreshold voltage trajectory of DMV neurons is positioned between the relatively hyperpolarized activation curve of Cav1.3 channels and that of other high-voltage activated (HVA) calcium channels, thus creating a functional segregation between Cav1.3 and HVA calcium channels. The HVA channels flux the bulk of calcium during spikes but can only influence pacemaking through their coupling to calcium-activated potassium currents. In contrast, Cav1.3 currents, which we show to be more than an order-of-magnitude smaller than the HVA calcium currents, are able to introduce sufficient inward current to speed up firing. However, Kv4 channels that are constitutively open in the subthreshold range guarantee slow pacemaking, despite the depolarizing action of Cav1.3 and other pacemaking currents. We propose that the efficacy of isradipine in preventing mOS in DMV neurons arises from its mixed effect on Cav1.3 channels and on HVA Cav1.2 channels.

The inhibition of high-voltage-activated calcium current by activation of MrgC11 involves phospholipase C-dependent mechanisms

High-voltage-activated (HVA) calcium channels play an important role in synaptic transmission. Activation of Mas-related G-protein-coupled receptor subtype C (MrgC; mouse MrgC11, rat homolog rMrgC) inhibits HVA calcium current (ICa) in small-diameter dorsal root ganglion (DRG) neurons, but the intracellular signaling cascade underlying MrgC agonist-induced inhibition of HVA ICa in native DRG neurons remains unclear. To address this question, we conducted patch-clamp recordings in MrgA3-eGFP-wild-type mice, in which most MrgA3-eGFP+ DRG neurons co-express MrgC11 and can be identified for recording. We found that the inhibition of HVA ICa by JHU58 (0.001–100nM, a dipeptide, MrgC-selective agonist) was significantly reduced by pretreatment with a phospholipase C (PLC) inhibitor (U73122, 1μM), but not by its inactive analog (U73343) or vehicle. Further, in rats that had undergone spinal nerve injury, pretreatment with intrathecal U73122 nearly abolished the inhibition of mechanical hypersensitivity by intrathecal JHU58. The inhibition of HVA ICa in MrgA3-eGFP+ neurons by JHU58 (100nM) was partially reduced by pretreatment with a Gβγ blocker (gallein, 100μM). However, applying a depolarizing prepulse and blocking the Gαi and Gαs pathways with pertussis toxin (PTX) (0.5μg/mL) and cholera toxin (CTX) (0.5μg/mL), respectively, had no effect. These findings suggest that activation of MrgC11 may inhibit HVA ICa in mouse DRG neurons through a voltage-independent mechanism that involves activation of the PLC, but not Gαi or Gαs, pathway.

AGAP, a new recombinant neurotoxic polypeptide, targets the voltage-gated calcium channels in rat small diameter DRG neurons

A previous study showed that antitumor-analgesic peptide (AGAP), a novel recombinant polypeptide, which had been expressed in Escherichia coli, exhibits analgesic and antitumor effects in mice. In the present study, we investigated the underlying analgesic mechanism of AGAP. The effect of AGAP on voltage-gated calcium channels (VGCCs) was assessed in acutely isolated rat dorsal root ganglia (DRG) neurons using the whole-cell patch clamp technique. The results showed that AGAP potently inhibited VGCCs, especially high-voltage activated (HVA) calcium channels. AGAP inhibited HVA and T-type calcium currents in a dose-dependent manner, but had no significant effect on their dynamic functions in rat small-diameter DRG neurons. AGAP inhibited N- and L-type calcium currents at 78.2% and 57.3%, respectively. Thus, the present study demonstrates that AGAP affects calcium currents through the inhibition of N-, L- and T-type channels in DRG neurons, explaining the potential mechanisms of antinociception.

Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels.

Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels.

Activation of MrgC receptor inhibits N-type calcium channels in small-diameter primary sensory neurons in mice

Mas-related G-protein-coupled receptor subtype C (mouse MrgC11 and rat rMrgC), expressed specifically in small-diameter primary sensory neurons, may constitute a novel pain inhibitory mechanism. We have shown previously that intrathecal administration of MrgC-selective agonists can strongly attenuate persistent pain in various animal models. However, the underlying mechanisms for MrgC agonist-induced analgesia remain elusive. Here, we conducted patch-clamp recordings to test the effect of MrgC agonists on high-voltage-activated (HVA) calcium current in small-diameter dorsal root ganglion (DRG) neurons. Using pharmacological approaches, we show for the first time that an MrgC agonist (JHU58) selectively and dose-dependently inhibits N-type, but not L- or P/Q-type, HVA calcium channels in mouse DRG neurons. Activation of HVA calcium channels is important to neurotransmitter release and synaptic transmission. Patch-clamp recordings in spinal cord slices showed that JHU58 attenuated the evoked excitatory postsynaptic currents in substantia gelatinosa (SG) neurons in wild-type mice, but not in Mrg knockout mice, after peripheral nerve injury. These findings indicate that activation of endogenously expressed MrgC receptors at central terminals of primary sensory fibers may decrease peripheral excitatory inputs onto SG neurons. Together, these results suggest potential cellular and molecular mechanisms that may contribute to intrathecal MrgC agonist-induced analgesia. Because MrgC shares substantial genetic homogeneity with human MrgX1, our findings may suggest a rationale for developing intrathecally delivered MrgX1 receptor agonists to treat pathological pain in humans and provide critical insight regarding potential mechanisms that may underlie its analgesic effects.

Identifying Key Amino Acid Residues That Affect α-Conotoxin AuIB Inhibition of α3β4 Nicotinic Acetylcholine Receptors

α-Conotoxin AuIB is a selective α3β4 nicotinic acetylcholine receptor (nAChR) subtype inhibitor. Its analgesic properties are believed to result from it activating GABAB receptors and subsequently inhibiting CaV2.2 voltage-gated calcium channels. The structural determinants that mediate diverging AuIB activity at these targets are unknown. We performed alanine scanning mutagenesis of AuIB and α3β4 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB·α3β4 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the α3β4 nAChR. NMR and CD spectroscopy studies demonstrated that [F9A]AuIB retains its native globular structure, so its activity loss is probably due to loss of specific toxin-receptor residue pairwise contacts. Compared with AuIB, the concentration-response curve for inhibition of α3β4 by [F9A]AuIB shifted rightward more than 10-fold, and its subtype selectivity profile changed. Homology modeling and molecular dynamics simulations suggest that Phe-9 of AuIB interacts with a two-residue binding pocket on the β4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the β4-Trp-59 and β4-Lys-61 residues of loop D, which form a putative binding pocket. AuIB analogues with Phe-9 substitutions corroborated the finding of a binding pocket on the β4 subunit and gave further insight into how AuIB Phe-9 interacts with the β4 subunit. In summary, we identified critical residues that mediate interactions between AuIB and its cognate nAChR subtype. These findings might help improve the design of analgesic conopeptides that selectively "avoid" nAChR receptors while targeting receptors involved with nociception.

Role of Low Voltage Activated Calcium Channels in Neuritogenesis and Active Migration of Embryonic Neural Progenitor Cells

The central role of calcium influx and electrical activity in embryonic development raises important questions about the role and regulation of voltage-dependent calcium influx. Using cultured neural progenitor cell (NPC) preparations, we recorded barium currents through voltage-activated channels using the whole-cell configuration of the patch-clamp technique and monitored intracellular free calcium concentrations with Fura-2 digital imaging. We found that NPCs as well as expressing high-voltage-activated (HVA) calcium channels express functional low-threshold voltage-dependent calcium channels in the very early stages of differentiation (5 h to 1 day). The size of the currents recorded at -50 versus -20 mV after 1 day in differentiation was dependent on the nature of the charge carrier. Peak currents measured at -20 mV in the presence 10 mM Ca2+ instead of 10 mM Ba2+ had a tendency to be smaller, whereas the nature of the divalent species did not influence the amplitude measured at -50 mV. The T-type channel blockers mibefradil and NNC 55-0396 significantly reduced the calcium responses elicited by depolarizing with extracellular potassium, while the overall effect of the HVA calcium channel blockers was small at differentiation day 1. At differentiation day 20, the calcium responses were effectively blocked by nifedipine. Time-lapse imaging of differentiating neurospheres cultured in the presence of low-voltage-activated (LVA) blockers showed a significant decrease in the number of active migrating neuron-like cells and neurite extensions. Together, these data provide evidence that LVA calcium channels are involved in the physiology of differentiating and migrating NPCs.

Involvement of reactive oxygen species and high-voltage-activated calcium currents in nanoparticle zinc oxide-induced cytotoxicity in vitro

This study was to determine the possible neurotoxicity and mechanisms underlying the effects of nano-ZnO with sizes of 20–80 nm on central nervous system (CNS). The cytotoxicity of nano-ZnO was investigated in PC12 cells. The viability of cells was observed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the generation of reactive oxygen species (ROS) for cells was evaluated by a fluorometry assay. The apoptosis of cells was detected and analyzed by flow cytometry. In addition, effects of nano-ZnO on the properties of high-voltage-activated (HVA) calcium currents were studied in acutely isolated rat hippocampal pyramidal neurons using the whole-cell patch clamp technique. The results of MTT assay showed that nano-ZnO (10−4 g/mL) caused a significant decrease in cell viability (P < 0.05). Nano-ZnO induced intracellular accumulation of ROS and the apoptosis of PC12 cells with the increasing concentration of nano-ZnO in flow cytometric assay (P < 0.05). Further results of electrophysiological recording indicated that 10−4 g/mL nano-ZnO first altered the current–voltage curve and the peak amplitudes of HVA calcium currents at 10 min of the recording, and the peak current amplitudes were increased significantly at the end of 30 min (P < 0.05). All these results suggested that the increase of intracellular ROS was one of potential mechanisms of cellular apoptosis induced by nano-ZnO. Nano-ZnO could cause the elevation of cytosolic calcium levels by enhancement of HVA calcium currents, which would increase the generation of intracellular ROS, and consequently promote the neuronal apoptosis.

Blockade of β-cell KATP channels by the endocannabinoid, 2-arachidonoylglycerol

The endocannabinoid system has been demonstrated to be active in the pancreatic β-cell. However the effects of the endocannabinoids (ECs) on insulin secretion are not well defined and may vary depending on the metabolic state of the β-cell. Specifically it is not known whether the effects of the ECs occur by activation of the cannabinoid receptors or via their direct interaction with the ion channels of the β-cell. To begin to delineate the effects of ECs on β-cell function, we examined how the EC, 2-AG influences β-cell ion channels in the absence of glucose stimulation. The mouse insulinoma cell line R7T1 was used to survey the effects of 2-AG on the high voltage activated (HVA) calcium, the delayed rectifier (Kv), and the ATP-sensitive K (KATP) channels by whole cell patch clamp recording. At 2mM glucose, 2-AG inhibited the HVA calcium (the majority of which are L-type channels), Kv, and KATP channels. The channel exhibiting the most sensitivity to 2-AG blockade was the KATP channel, where the IC50 for 2-AG was 1μM. Pharmacological agents revealed that the blockade of all these channels was independent of cannabinoid receptors. Our results provide a mechanism for the previous observations that CB1R agonists increase insulin secretion at low glucose concentrations through CB1R independent blockade of the KATP channel.

Cav2 channels mediate low and high voltage‐activated calcium currents in Drosophila motoneurons

Different blends of membrane currents underlie distinct functions of neurons in the brain. A major step towards understanding neuronal function, therefore, is to identify the genes that encode different ionic currents. This study combined in situ patch clamp recordings of somatodendritic calcium currents in an identified adult Drosophila motoneuron with targeted genetic manipulation. Voltage clamp recordings revealed transient low voltage-activated (LVA) currents with activation between –60 mV and –70 mV as well as high voltage-activated (HVA) current with an activation voltage around –30 mV. LVA could be fully inactivated by prepulses to –50 mV and was partially amiloride sensitive. Recordings from newly generated mutant flies demonstrated that DmαG (Ca(v)3 homolog) encoded the amiloride-sensitive portion of the transient LVA calcium current. We further demonstrated that the Ca(v)2 homolog, Dmca1A, mediated the amiloride-insensitive component of LVA current. This novel role of Ca(v)2 channels was substantiated by patch clamp recordings from conditional mutants, RNAi knock-downs, and following Dmca1A overexpression. In addition, we show that Dmca1A underlies the HVA somatodendritic calcium currents in vivo. Therefore, the Drosophila Ca(v)2 homolog, Dmca1A, underlies HVA and LVA somatodendritic calcium currents in the same neuron. Interestingly, DmαG is required for regulating LVA and HVA derived from Dmca1A in vivo. In summary, each vertebrate gene family for voltage-gated calcium channels is represented by a single gene in Drosophila, namely Dmca1D (Ca(v)1), Dmca1A (Ca(v)2) and DmαG (Ca(v)3), but the commonly held view that LVA calcium currents are usually mediated by Ca(v)3 rather than Ca(v)2 channels may require reconsideration.

Gem Stabilizes Voltage-Gated Calcium Channels in the Inactivated State: Implications for Human Disease

RGK proteins (Rad, Rem1, Rem2 and Gem/Kir) are small GTPases that often colocalize with and strongly inhibit high-voltage activated (HVA) calcium channels (L-, N-, P/Q- and R-type). The mechanism of inhibition is largely unclear. The pore-forming alpha-1 subunit of HVA calcium channels requires the beta subunit for cell surface trafficking and gating. We previously showed direct binding between alpha-1 and Gem and obtained a Gem-insensitive chimeric P/Q alpha-1containing the IIS1-S3 region of a Gem-resistant, low-voltage activated T-type channel (Fan et al., PNAS, 2010). In this study, we investigated the cause of this insensitivity. Since the IIS1-S3 region of P/Q alpha-1 has too few cytosolic amino acids to be a Gem binding site, and since we had identified a different alpha-1 region that binds Gem, we focused on altered biophysical properties of the chimera. We observed that the chimeric channel inactivated much slower than WT P/Q channel did; we therefore examined whether slow inactivation was responsible for Gem insensitivity. Mutating residues known to increase channel inactivation enhanced Gem inhibition, whereas mutating residues known to slow channel inactivation weakened or abolished Gem inhibition. Replacing beta3 with beta2a, an auxiliary subunit known to slow channel inactivation, also weakened Gem inhibition. Furthermore, recovery from inactivation of WT P/Q channels in the presence of Gem was markedly slower than without Gem. Thus, Gem appeared to stabilize the channel in the inactivated state. These results shed light on a new factor - altered regulation by RGK proteins - that may contribute to HVA calcium channel-related channelopathies. Indeed, we found that familial hemiplegic migraine mutations that speed P/Q channel inactivation strengthened Gem inhibition, whereas Timothy syndrome mutations that slow channel inactivation greatly weakened Gem inhibition.

Amitriptyline modulates calcium currents and intracellular calcium concentration in mouse trigeminal ganglion neurons

Migraine is increasingly recognized as a channelopathy, and abnormalities of voltage-activated ionic channels could represent the molecular basis for the altered neuronal functioning. The high-voltage-activated (HVA) Ca2+ channels in the trigeminovascular system play a role in the pathophysiology of migraine. In the present study, effects of amitriptyline (AMT), a commonly used migraine prophylactic drug, on the HVA calcium currents (ICa) were examined in mouse trigeminal ganglion neurons using whole-cell patch clamp technique. AMT produced concentration- and use-dependent inhibition of HVA ICa. Bath application of GÖ-6983 (a selective protein kinase C inhibitor) or H89 (a protein kinase A inhibitor) did not reduce the AMT-induced inhibition of HVA ICa. A similar inhibition was observed when calcium imaging was used to directly monitor the effects of AMT on KCl-induced increments of intracellular Ca2+ concentration ([Ca2+]i). By blocking HVA Ca2+ channels and Ca2+ entry into cells, AMT could prevent the release of neurotransmitters and help restore the neuronal threshold for excitation. Our findings suggest interesting therapeutic mechanisms for AMT in migraine prevention.

Differential regulation of low and high voltage-activated calcium channels in neonatal rat myocytes following chronic PKA modulation

The biophysical properties of voltage-dependent cardiac calcium channels (VDCC) can be modulated by protein kinases. In this study, we investigate whether long-term treatment with protein kinase A (PKA) modulators alters the VDCC activity in neonatal ventricular myocytes. Using whole-cell patch-clamp recordings, we found an increase in high-voltage activated (HVA) current density and a corresponding decrease in low-voltage activated (LVA) current density in neonatal rat ventricular myocytes up to 6 days in culture. Long-term exposure to 8Br-cAMP, a PKA stimulator, increased the HVA current density at 7 and 24 hours. In contrast, H89, a PKA inhibitor, caused a biphasic change in the HVA, an initial reduction at 7 hours exposure followed by an increase up to 4 days. In addition, H89 caused a sustained increase in LVA currents from 7 hours to 4 days. These findings suggest that chronic exposure to H89 changes LVA and HVA calcium current activities in cardiac myocytes. PKA is a key target of beta-adrenoceptor activiation, thus, our findings suggest long-term repeated use of beta-adrenergic drugs may induce unexpected functional alteration of VDCCs.

Effects of gabapentin on high-voltage-activated calcium current in dorsal root ganglion neurons in rats

To compare the effects of gabapentin on high-voltage-activated calcium (HVA) current in dorsal root ganglion (DRG) neurons in normal and nerve-injured rats and understand the reasons of their differences. Pathogen-free male SD rats (weight 180 - 220 mg) aged 4 - 6 weeks were used. The animals were anesthetized with intraperitoneal pentobarbital sodium 50 mg/kg. L(5) spinal nerve was ligated between DRG and sciatic nerve and cut distal to the ligature. The animals were decapitated at Day 14 post-operation. L(5) (SNL-L(5) group) and L(4) DRGs (SNL-L(4) group) were respectively isolated and the ganglionic neurons enzymatically dissociated. The control group of rats was not operated. The lumbar DRG neurons of normal rats were treated similarly. The HVA-Ca(2+) current was recorded by the technique of whole cell patch clamp. Compared with the SNL-L(4) group [(16.0 ± 1.9)%, (26.9 ± 2.0)%, (27.4 ± 2.3)%] and the control group, gabapentin inhibited the peak calcium current highlier at 10, 100 and 300 µmol/L in the SNL-L(5) group [(18.5 ± 1.7)%, (32.0 ± 2.6)%, (32.7 ± 2.8)%] (P < 0.05). The steady-state inactivation curves shifted to more hyperpolarized potentials in the SNL-L(5) group. The N-type relative contribution to the gabapentin-sensitive HVA-Ca(2+) current was markedly elevated in the SNL-L(5) group compared with that in other two groups (P < 0.05). Gabapentin enhances the inhibition of HVA-Ca(2+) current in injured DRG neurons following spinal nerve ligation in rats. The alteration in the activation of electrophysiological properties and the increase of N-type relative contribution to the total HVA-Ca(2+) current may be involved in the mechanism.

Changes in high voltage-activated calcium current in dorsal root ganglion neurons isolated from rats with neuropathic pain

Objective To investigate the changes in high voltage-activated （HVA） calcium current in dorsal root ganglion （DRG） neurons isolated from rats with neuropathic pain. Methods Pathogen-free male SD rats aged 4-6 weeks weighing 180-220 g were used in this study. The animals were anesthetized with intraperitoneal pentobarbital sodium 50 mg/kg. Neuropathic pain was induced by ligation of L5 spinal nerve between DRG and sciatic nerve. The nerve was transected distal to the ligature. The animals which showed positive signs of neuropathic pain were decapitated on the 14th postoperative day. L5 and L4 DRGs were isolated and the neurons in the ganglia were enzymatically dissociated （group L5 and L4）. The control group received no surgery （group C）. The HVA Ca2＋ current was recorded using whole-cell patch clamp technique. Results Peak calcium current density was significantly lower in group L5 and L4 than in group C, and was significantly lower in group L5 than in group L4 . Halfactivation value （Va 1/2） was also significantly lower in group L5 than in group C and L4 （P 〈 0.05）. The relative contribution of N-type to the total HVA Ca2＋ current was significantly greater in group L5 than in group C and L4（P 〈 0.05）. There was no significant difference in the steady-state inactivation curves among the 3 groups. Conclusion In rats with neuropathic pain, the HVA Ca2＋ current in the injured DRG neurons may play a key role in the induction of neuropathic pain. Key words: Calcium channels; Neuralgia; Ganglia,spinal; Neurons