High Voltage-activated Calcium Currents Research Articles

Human birth tissue products as a non-opioid medicine to inhibit post-surgical pain.

Pain after surgery causes significant suffering. Opioid analgesics cause severe side effects and accidental death. Therefore, there is an urgent need to develop non-opioid therapies for managing post-surgical pain. Local application of Clarix Flo (FLO), a human amniotic membrane (AM) product, attenuated established post-surgical pain hypersensitivity without exhibiting known side effects of opioid use in mice. This effect was achieved through direct inhibition of nociceptive dorsal root ganglion (DRG) neurons via CD44-dependent pathways. We further purified the major matrix component, the heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) from human AM that has greater purity and water solubility than FLO. HC-HA/PTX3 replicated FLO-induced neuronal and pain inhibition. Mechanistically, HC-HA/PTX3 induced cytoskeleton rearrangements to inhibit sodium current and high-voltage activated calcium current on nociceptive neurons, suggesting it is a key bioactive component mediating pain relief. Collectively, our findings highlight the potential of naturally derived biologics from human birth tissues as an effective non-opioid treatment for post-surgical pain. Moreover, we unravel the underlying mechanisms of pain inhibition induced by FLO and HC-HA/PTX3.

Role of primary sensory neurone cannabinoid type-1 receptors in pain and the analgesic effects of the peripherally acting agonist CB-13 in mice

BackgroundCannabinoid type-1 receptors (CB1Rs) are expressed in primary sensory neurones, but their role in pain modulation remains unclear. MethodsWe produced Pirt-CB1R conditional knockout (cKO) mice to delete CB1Rs in primary sensory neurones selectively, and used behavioural, pharmacological, and electrophysiological approaches to examine the influence of peripheral CB1R signalling on nociceptive and inflammatory pain. ResultsConditional knockout of Pirt-CB1R did not alter mechanical or heat nociceptive thresholds, complete Freund adjuvant-induced inflammation, or heat hyperalgesia in vivo. The intrinsic membrane properties of small-diameter dorsal root ganglion neurones were also comparable between cKO and wild-type mice. Systemic administration of CB-13, a peripherally restricted CB1/CB2R dual agonist (5 mg kg−1), inhibited nociceptive pain and complete Freund adjuvant-induced inflammatory pain. These effects of CB-13 were diminished in Pirt-CB1R cKO mice. In small-diameter neurones from wild-type mice, CB-13 concentration-dependently inhibited high-voltage activated calcium current (HVA-ICa) and induced a rightward shift of the channel open probability curve. The effects of CB-13 were significantly attenuated by AM6545 (a CB1R antagonist) and Pirt-CB1R cKO. ConclusionCB1R signalling in primary sensory neurones did not inhibit nociceptive or inflammatory pain, or the intrinsic excitability of nociceptive neurones. However, peripheral CB1Rs are important for the analgesic effects of systemically administered CB-13. In addition, HVA-ICa inhibition appears to be a key ionic mechanism for CB-13-induced pain inhibition. Thus, peripherally restricted CB1R agonists could have utility for pain treatment.

Spider Venom Peptide Pn3a Inhibition of Primary Afferent High Voltage-Activated Calcium Channels.

Despite potently inhibiting the nociceptive voltage-gated sodium (Nav) channel, Nav1.7, µ-theraphotoxin Pn3a is antinociceptive only upon co-administration with sub-therapeutic opioid agonists, or by itself at doses >3,000-fold greater than its Nav1.7 IC 50 by a yet undefined mechanism. Nav channels are structurally related to voltage-gated calcium (Cav) channels, Cav1 and Cav2. These channels mediate the high voltage-activated (HVA) calcium currents (I Ca) that orchestrate synaptic transmission in nociceptive dorsal root ganglion (DRG) neurons and are fine-tuned by opioid receptor (OR) activity. Using whole-cell patch clamp recording, we found that Pn3a (10 µM) inhibits ∼55% of rat DRG neuron HVA-I Ca and 60–80% of Cav1.2, Cav1.3, Cav2.1, and Cav2.2 mediated currents in HEK293 cells, with no inhibition of Cav2.3. As a major DRG I Ca component, Cav2.2 inhibition by Pn3a (IC 50 = 3.71 ± 0.21 µM) arises from an 18 mV hyperpolarizing shift in the voltage dependence of inactivation. We observed that co-application of Pn3a and µ-OR agonist DAMGO results in enhanced HVA-I Ca inhibition in DRG neurons whereas co-application of Pn3a with the OR antagonist naloxone does not, underscoring HVA channels as shared targets of Pn3a and opioids. We provide evidence that Pn3a inhibits native and recombinant HVA Cavs at previously reportedly antinociceptive concentrations in animal pain models. We show additive modulation of DRG HVA-I Ca by sequential application of low Pn3a doses and sub-therapeutic opioids ligands. We propose Pn3a's antinociceptive effects result, at least in part, from direct inhibition of HVA-I Ca at high Pn3a doses, or through additive inhibition by low Pn3a and mild OR activation.

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.

De Novo Pathogenic Variants in CACNA1E Cause Developmental and Epileptic Encephalopathy with Contractures, Macrocephaly, and Dyskinesias

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α1-subunit of the voltage-gated CaV2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

Dual Effect of Palmitate on Voltage-Gated Calcium Channels and Insulin Secretion in Pancreatic Beta Cells of Rats

A variety of signaling molecules modify voltage-gated calcium channels activity in pancreatic beta cells, among them are, free fatty acids (FFA). Acute exposure to FFA increases glucose-stimulated insulin secretion, while chronic exposure increases basal insulin secretion, but decreases glucose-stimulated (Olofsson et al., 2004; Zhou and Grill, 1994). Preliminary studies in our laboratory showed that pre-incubation with 1 mM palmitate during 48-72 hours reduced barium currents in beta cells. In this work, we analyze the effect of different palmitate concentrations on calcium currents and insulin secretion in beta cells of adult male Wistar rats. Electrophysiological recordings were performed using whole-cell voltage clamp technique. Reverse hemolytic plaque assay measured insulin secretion by isolated beta cells. Pre-incubation with 0.25 and 0.5 mM palmitate during 48-72 h increased the high voltage-activated (HVA) calcium current without affecting the LVA current. An acute 5 min pre-incubation with 0.5 mM palmitate also increased HVA current. In contrast, 24 h pre-incubation with 1 mM decreased both, LVA and HVA currents. A similar effect was observed during an acute incubation. Chronic pre-incubation with 0.25, 0.5 and 1 mM palmitate decreased the percentage of insulin secretory cells, immunoplaque area and insulin secretion index in a glucose-stimulated condition (15.6 mM). Chronic pre-incubation with 0.5 and 1 mM palmitate, also decreased the secretion index at basal glucose (5.6 mM). However, acute pre-incubation with 1 mM palmitate increased the immunoplaque area in 5.6 mM glucose. Together these results show that palmitate has a dual effect, depending on time and concentration on the calcium currents and insulin secretion in beta cells. Partially supported by DGAPA PAPIIT-UNAM Grants: IN213114, M Hiriart; IV100116 to A. Frank and M Hiriart, and IN211416 to M Velasco-Torres.

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.

The mechanisms of late-onset synaptic responses in a realistic model of Unipolar Brush Cells

Unipolar brush cells (UBCs) are excitatory glutamatergic interneurons of the cerebellar granular layer receiving both primary and secondary vestibular inputs through mossy fibers (excitatory input) and Golgi cell axon (inhibitory input). When injected with progressively increasing depolarizing currents from a negative membrane potential, the UBC generates a burst sustained by a calcium spike and then a protracted discharge with shorter latency and spike frequency adaptation. The intrinsic excitability of UBCs is determined by an H current and by Low Voltage activated and High Voltage activated calcium currents [2,3]. Fast inactivating T-type Calcium channels generate low-threshold spikes and L-type Calcium channel sustain tonic firing. The H current (activated between -60 mV and -80 mV) produces a slow hyperpolarization characterized by a “sag” in response to a hyperpolarizing step and an afterhyperpolarization at the end of a depolarizing step. Here we present a biologically realistic multi-compartmental mathematical model of the UBC realized with the NEURON-PYTHON simulator. According to literature [1-4], ionic channels are distributed among compartments (soma, dendrite, initial segment and axon). The model can reproduce the excitable properties of UBCs in currentclamp and voltage-clamp modes. The response to mossy fiber inputs was reproduced using synaptic models of AMPA and NMDA synaptic receptors. The model is also capable of reproducing the late onset response recently reported for this cellular type [5] by exploiting the interaction between cAMP, TRPC, and H current. This model, in addition to confirm the prim ary role of the aforementioned currents in UBC’s electroresponsiveness, will prove a valuable tool for investigating the UBC’s function in the cerebellar network.

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.

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.

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.

Modeling UBC intrinsic excitability

Unipolar brush cells (UBCs) are excitatory glutamatergic interneurons of the cerebellar granular layer receiving both primary and secondary vestibular inputs through mossy fibers (excitatory input) and Golgi axon (inhibitory input). The brush like structure of the dendrite allows to form a giant synapses in the glomerulus and to produce an all or none post synaptic response with short delay and protracted kinetics. The excitable response of UBCs can be either a tonic discharge or a high-frequency burst of action potentials. When injected with progressively increasing depolarizing currents from a negative membrane potential, the UBC generates a burst sustained by a calcium spike (Figure ​(Figure1,1, +20pA) and then a protracted discharge with shorter latency and spike frequency adaption (Figure ​(Figure1,1, +25pA). The intrinsic excitability of UBCs is determined by an H current and by Low Voltage activated and High Voltage activated calcium currents [2,3]. Fast inactivating T-type Calcium channels generate low-threshold spikes and L-type Calcium channel sustain tonic firing. The H current (activated between -60mV and -80mV) produces a slow hyperpolarization characterized by a “sag” in response to a hyperpolarizing step (Figure ​(Figure1,1, -16pA) and an afterhyperpolarization at the end of a depolarizing step. Figure 1 Excitable response of the UBC model to step-current injection.

Altered profile and D2-dopamine receptor modulation of high voltage-activated calcium current in striatal medium spiny neurons from animal models of Parkinson's disease

In the present work we analyzed the profile of high voltage-activated (HVA) calcium (Ca2+) currents in freshly isolated striatal medium spiny neurons (MSNs) from rodent models of both idiopathic and familial forms of Parkinson's disease (PD). MSNs were recorded from reserpine-treated and 6-hydroxydopamine (6-OHDA)-lesioned rats, and from DJ-1 and PINK1 (PTEN induced kinase 1) knockout (−/−) mice. Our analysis showed no significant changes in total HVA Ca2+ current. However, we recorded a net increase in the L-type fraction of HVA Ca2+ current in dopamine-depleted rats, and of both N- and P-type components in DJ-1−/− mice, whereas no significant change in Ca2+ current profile was observed in PINK1−/− mice. Dopamine modulates HVA Ca2+ channels in MSNs, thus we also analyzed the effect of D1 and D2 receptor activation. The effect of the D1 receptor agonist SKF 83822 on Ca2+ current was not significantly different among MSNs from control animals or PD models. However, in both dopamine-depleted rats and DJ-1−/− mice the D2 receptor agonist quinpirole inhibited a greater fraction of HVA Ca2+ current than in the respective controls. Conversely, in MSNs from PINK1−/− mice we did not observe alterations in the effect of D2 receptor activation. Additionally, in both reserpine-treated and 6-OHDA-lesioned rats, the effect of quinpirole was occluded by the selective L-type Ca2+ channel blocker nifedipine, while in DJ-1−/− mice it was mostly occluded by ω-conotoxin GVIA, blocker of N-type channels. These results demonstrate that both dopamine depletion and DJ-1 deletion induce a rearrangement in the HVA Ca2+ channel profile, specifically involving those channels that are selectively modulated by D2 receptors.

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

Les chimiokines, de nouveaux acteurs dans le système dopaminergique

Previous neuroanatomical studies realized in our team allowed us to demonstrate the neuronal and glial expression of various chemokines and their receptors in central dopaminergic (DA) pathways. In the light of these original observations, we questioned the role of chemokines on the physiology of DA neuron and on the neurodegenerative process in the DA nigro-striatal pathway, which characterizes Parkinson's disease. We focused our attention on two particular chemokines, the Stromal cell-Derived Factor-1 (SDF-1/CXCL12) and the Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) and their cognate receptors CXCR4 and CCR2, as they are expressed constitutively in nearly all DA mesencephalic neurons. We demonstrated, by using in vivo and in vitro approaches, that SDF-1 and MCP-1 can modulate DA neurotransmission in the nigro-striatal pathway, modifying the electrophysiological state of the neuron and DA release, through their cognate receptors. These effects are produced through N-type high voltage-activated calcium currents for SDF-1 and potassium channels for MCP-1. We then discuss the possible implication of SDF-1 and its derivative SDF-1(5-67) in DA neurodegeneration.

T-Type Calcium Channel Inhibition Underlies the Analgesic Effects of the Endogenous Lipoamino Acids

Lipoamino acids are anandamide-related endogenous molecules that induce analgesia via unresolved mechanisms. Here, we provide evidence that the T-type/Cav3 calcium channels are important pharmacological targets underlying their physiological effects. Various lipoamino acids, including N-arachidonoyl glycine (NAGly), reversibly inhibited Cav3.1, Cav3.2, and Cav3.3 currents, with potent effects on Cav3.2 [EC(50) approximately 200 nm for N-arachidonoyl 3-OH-gamma-aminobutyric acid (NAGABA-OH)]. This inhibition involved a large shift in the Cav3.2 steady-state inactivation and persisted during fatty acid amide hydrolase (FAAH) inhibition as well as in cell-free outside-out patch. In contrast, lipoamino acids had weak effects on high-voltage-activated (HVA) Cav1.2 and Cav2.2 calcium currents, on Nav1.7 and Nav1.8 sodium currents, and on anandamide-sensitive TRPV1 and TASK1 currents. Accordingly, lipoamino acids strongly inhibited native Cav3.2 currents in sensory neurons with small effects on sodium and HVA calcium currents. In addition, we demonstrate here that lipoamino acids NAGly and NAGABA-OH produced a strong thermal analgesia and that these effects (but not those of morphine) were abolished in Cav3.2 knock-out mice. Collectively, our data revealed lipoamino acids as a family of endogenous T-type channel inhibitors, suggesting that these ligands can modulate multiple cell functions via this newly evidenced regulation.

Dissociation between Rat Hippocampal CA1 and Dentate Gyrus Cells in Their Response to Corticosterone: Effects on Calcium Channel Protein and Current

Stress and corticosterone affect, via glucocorticoid receptors, cellular physiology in the rodent brain. A well-documented example concerns corticosteroid effects on high-voltage activated (L type) calcium currents in the hippocampal CA1 area. We tested whether corticosterone also affects calcium currents in another hippocampal area that highly expresses glucocorticoid receptors, i.e. the dentate gyrus (DG). Remarkably, corticosterone (100 nm, given for 20 min, 1-4.5 hr before recording) did not change high-voltage activated calcium currents in the DG, whereas currents in the CA1 area of the same rats were increased. Follow-up studies revealed that no apparent dissociation between the two areas was observed with respect to transcriptional regulation of calcium channel subunits; thus, in both areas corticosterone increased mRNA levels of the calcium channel-beta4 but not the (alpha) Ca(v)1.2 subunit. At the protein level, however, beta4 and Ca(v)1.2 levels were significantly up-regulated by corticosterone in the CA1 but not the DG area. These data suggest that stress-induced elevations in the level of corticosterone result in a regionally differentiated physiological response that is not simply determined by the glucocorticoid receptor distribution and that the observed regional differentiation may be caused by a gene involved in the translational machinery or in mechanisms regulating mRNA or protein stability.

A Small Peptide Inhibitor of the Low Voltage-Activated Calcium Channel Cav3.1

The calcium channel gamma(6) subunit modulates low voltage-activated (LVA) calcium current in both human embryonic kidney (HEK) cells and cardiomyocytes, although the mechanism of modulation is unknown. We recently showed that gamma(6) contains a critical GxxxA motif in the first transmembrane domain (TM1) that is essential for its inhibition of the Cav3.1 (LVA) calcium current. In this study, we tested the hypothesis that an eight-amino acid peptide that contains the GxxxA motif from gamma(6) TM1 can act as a novel pharmacological inhibitor of the Cav3.1 calcium current by performing whole-cell electrophysiology. Our results demonstrate that the peptide inhibits Cav3.1 current by dynamically binding and dissociating from the Cav3.1 channel in a concentration-dependent but largely voltage-independent manner. By selectively substituting residues within the peptide, we show that both the GxxxA framework and surrounding aliphatic side-chains contribute to the presumably interhelical interactions between gamma(6) TM1 and the Cav3.1 channel. The fast kinetics of the interaction supports the view that gamma(6) acts as an endogenous LVA channel antagonist within the plasma membrane, suggesting a mechanism other than regulation of surface expression or membrane trafficking of the pore-forming subunit of the channel. We also demonstrate that the peptide has different affinities for Cav3.1 and Cav1.2 calcium currents, which is consistent with the selective effect of gamma(6) on LVA and high voltage-activated calcium currents in vivo.