High Voltage-activated Calcium Channels Research Articles

Electrosome assembly: Structural insights from high voltage-activated calcium channel (CaV)-chaperone interactions.

Ion channels are multicomponent complexes (termed here as"electrosomes") that conduct the bioelectrical signals required for life. It has been appreciated for decades that assembly is critical for proper channel function, but knowledge of the factors that undergird this important process has been lacking. Although there are now exemplar structures of representatives of most major ion channel classes, there has been no direct structural information to inform how these complicated, multipart complexes are put together or whether they interact with chaperone proteins that aid in their assembly. Recent structural characterization of a complex of the endoplasmic membrane protein complex (EMC) chaperone and a voltage-gated calcium channel (CaV) assembly intermediate comprising the pore-forming CaVα1 and cytoplasmic CaVβ subunits offers the first structural view into the assembly of a member of the largest ion channel class, the voltagegated ion channel (VGIC) superfamily. The structure shows how the EMC remodels the CaVα1/CaVβ complex through a set of rigid body movements for handoff to the extracellular CaVα2δ subunit to complete channel assembly in a process that involves intersubunit coordination of a divalent cation and ordering of CaVα1 elements. These findings set a new framework for deciphering the structural underpinnings of ion channel biogenesis that has implications for understanding channel function, how drugs and disease mutations act, and for investigating how other membrane proteins may engage the ubiquitous EMC chaperone.

GABAB Receptor Modulation of Membrane Excitability in Human Pluripotent Stem Cell-Derived Sensory Neurons by Baclofen and α-Conotoxin Vc1.1.

GABAB receptor (GABABR) activation is known to alleviate pain by reducing neuronal excitability, primarily through inhibition of high voltage-activated (HVA) calcium (CaV2.2) channels and potentiating G protein-coupled inwardly rectifying potassium (GIRK) channels. Although the analgesic properties of small molecules and peptides have been primarily tested on isolated murine dorsal root ganglion (DRG) neurons, emerging strategies to develop, study, and characterise human pluripotent stem cell (hPSC)-derived sensory neurons present a promising alternative. In this study, hPSCs were efficiently differentiated into peripheral DRG-induced sensory neurons (iSNs) using a combined chemical and transcription factor-driven approach via a neural crest cell intermediate. Molecular characterisation and transcriptomic analysis confirmed the expression of key DRG markers such as BRN3A, ISLET1, and PRPH, in addition to GABABR and ion channels including CaV2.2 and GIRK1 in iSNs. Functional characterisation of GABABR was conducted using whole-cell patch clamp electrophysiology, assessing neuronal excitability under current-clamp conditions in the absence and presence of GABABR agonists baclofen and α-conotoxin Vc1.1. Both baclofen (100 μM) and Vc1.1 (1 μM) significantly reduced membrane excitability by hyperpolarising the resting membrane potential and increasing the rheobase for action potential firing. In voltage-clamp mode, baclofen and Vc1.1 inhibited HVA Ca2+ channel currents, which were attenuated by the selective GABABR antagonist CGP 55845. However, modulation of GIRK channels by GABABRs was not observed in the presence of baclofen or Vc1.1, suggesting that functional GIRK1/2 channels were not coupled to GABABRs in hPSC-derived iSNs. This study is the first to report GABABR modulation of membrane excitability in iSNs by baclofen and Vc1.1, highlighting their potential as a future model for studying analgesic compounds.

Inhibition of T-Type Calcium Channels With TTA-P2 Reduces Chronic Neuropathic Pain Following Spinal Cord Injury in Rats

Spinal cord injury (SCI)-induced neuropathic pain (SCI-NP) develops in up to 60 to 70% of people affected by traumatic SCI, leading to a major decline in quality of life and increased risk for depression, anxiety, and addiction. Gabapentin and pregabalin, together with antidepressant drugs, are commonly prescribed to treat SCI-NP, but their efficacy is unsatisfactory. The limited efficacy of current pharmacological treatments for SCI-NP likely reflects our limited knowledge of the underlying mechanism(s) responsible for driving the maintenance of SCI-NP. The leading hypothesis in the field supports a major role for spontaneously active injured nociceptors in driving the maintenance of SCI-NP. Recent data from our laboratory provided additional support for this hypothesis and identified the T-type calcium channels as key players in driving the spontaneous activity of SCI-nociceptors, thus providing a rational pharmacological target to treat SCI-NP. To test whether T-type calcium channels contribute to the maintenance of SCI-NP, male and female SCI and sham rats were treated with TTA-P2 (a blocker of T-type calcium channels) to determine its effects on mechanical hypersensitivity (as measured with the von Frey filaments) and spontaneous ongoing pain (as measured with the conditioned place preference paradigm), and compared them to the effects of gabapentin, a blocker of high voltage–activated calcium channels. We found that both TTA-P2 and gabapentin reduced mechanical hypersensitivity in male and females SCI rats, but surprisingly only TTA-P2 reduced spontaneous ongoing pain in male SCI rats. PerspectivesSCI-induced neuropathic pain, and in particular the spontaneous ongoing pain component, is notoriously very difficult to treat. Our data provide evidence that inhibition of T-type calcium channels reduces spontaneous ongoing pain in SCI rats, supporting a clinically relevant role for T-type channels in the maintenance of SCI-induced neuropathic pain.

Selective posttranslational inhibition of CaVβ1-associated voltage-dependent calcium channels with a functionalized nanobody.

Ca2+ influx through high-voltage-activated calcium channels (HVACCs) controls diverse cellular functions. A critical feature enabling a singular signal, Ca2+ influx, to mediate disparate functions is diversity of HVACC pore-forming α1 and auxiliary CaVβ1-CaVβ4 subunits. Selective CaVα1 blockers have enabled deciphering their unique physiological roles. By contrast, the capacity to post-translationally inhibit HVACCs based on CaVβ isoform is non-existent. Conventional gene knockout/shRNA approaches do not adequately address this deficit owing to subunit reshuffling and partially overlapping functions of CaVβ isoforms. Here, we identify a nanobody (nb.E8) that selectively binds CaVβ1 SH3 domain and inhibits CaVβ1-associated HVACCs by reducing channel surface density, decreasing open probability, and speeding inactivation. Functionalizing nb.E8 with Nedd4L HECT domain yielded Chisel-1 which eliminated current through CaVβ1-reconstituted CaV1/CaV2 and native CaV1.1 channels in skeletal muscle, strongly suppressed depolarization-evoked Ca2+ influx and excitation-transcription coupling in hippocampal neurons, but was inert against CaVβ2-associated CaV1.2 in cardiomyocytes. The results introduce an original method for probing distinctive functions of ion channel auxiliary subunit isoforms, reveal additional dimensions of CaVβ1 signaling in neurons, and describe a genetically-encoded HVACC inhibitor with unique properties.

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.

Modeling analysis of subthreshold voltage signaling along hippocampal mossy fiber axons.

Axons are classically thought of as electrically well isolated from other parts of the neurons due to the shape of a long cable-like structure. In contrast to this classical view on axonal compartmentalization, recent studies revealed that subthreshold depolarization of soma and dendrite passively propagates to the axons for a substantial distance, as demonstrated in some experimentally accessible axons including hippocampal mossy fibers and cortical pyramidal cell axons. Passive propagation of subthreshold dendritic EPSPs to the axons, defined as EPreSPs (excitatory presynaptic potentials), has been demonstrated to affect transmitter release from the axon terminals. To further characterize and explore the functional significance of passive subthreshold voltage signaling along the axons, the model of EPreSPs along hippocampal mossy fibers, proposed by Alle and Geiger, was reconstructed on the NEURON simulator. To test the effect of EPreSPs on action potentials and transmitter release from the axon terminals, additional conductances were incorporated into the previous passive propagation model. These include the axonal sodium, potassium, and leak channels as well as presynaptic calcium channels composed of P/Q-, N-, and R-types, which are reconstructed from the properties of those recorded from mossy fiber boutons experimentally. In this revised model, the preceding subthreshold EPreSPs slightly reduced the action potential-evoked presynaptic calcium currents by a decrease in the amplitude of action potentials due to the slow depolarization. It should be mentioned that EPreSPs by themselves elicited small calcium currents during subthreshold depolarization through these high-voltage activated calcium channels. Since the previous experimental study by simultaneous pre and postsynaptic recordings demonstrated that EPreSPs enhanced action potential-evoked transmitter release from the mossy fiber terminals, it has been suggested that different mechanisms from the enhancement of action potential-evoked presynaptic calcium entry may involve enhanced transmitter release by EPreSP. Small calcium entry by subthreshold EPreSPs may enhance transmitter release from the mossy fiber terminals by acting as high-affinity calcium sensors for enhancing transmitter release. Another form of axonal subthreshold voltage signaling, GABA-EPreSPs elicited by a spillover of GABA from surrounding interneurons, was also explored. Functional consequences of the two modes of axonal subthreshold voltage signaling were discussed with the simulation results.

Uncovering a new route to pain therapy

High-voltage-activated calcium channels (HVACCs) are promising targets for developing analgesics given their roles in controlling synaptic transmission, neuronal excitability and neuropeptide release in primary nociceptive neurons. Despite previous efforts in developing HVACCs inhibitors of various drug modalities, it remains undetermined whether targeting HVACCs directly by a gene therapy approach could lead to pain alleviation in vivo. To test this, Sun and colleagues adopted a post-translational ubiquitination-based knockdown method targeting HVACCs in primary sensory neurons. They showed ablation of HVACC currents in a subset of primary sensory neurons, dampened hyperexcitability of sensory neurons after nerve injury and reduced pain behavior with no apparent adverse effects [1]. The results open the possibility of targeting ion channels with ubiquitination-based knockdown as a promising gene therapy candidate for pain treatment in future clinical studies.

Neuronal Adenosine A1 Receptor is Critical for Olfactory Function but Unable to Attenuate Olfactory Dysfunction in Neuroinflammation.

Adenine nucleotides, such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), as well as the nucleoside adenosine are important modulators of neuronal function by engaging P1 and P2 purinergic receptors. In mitral cells, signaling of the G protein-coupled P1 receptor adenosine 1 receptor (A1R) affects the olfactory sensory pathway by regulating high voltage-activated calcium channels and two-pore domain potassium (K2P) channels. The inflammation of the central nervous system (CNS) impairs the olfactory function and gives rise to large amounts of extracellular ATP and adenosine, which act as pro-inflammatory and anti-inflammatory mediators, respectively. However, it is unclear whether neuronal A1R in the olfactory bulb modulates the sensory function and how this is impacted by inflammation. Here, we show that signaling via neuronal A1R is important for the physiological olfactory function, while it cannot counteract inflammation-induced hyperexcitability and olfactory deficit. Using neuron-specific A1R-deficient mice in patch-clamp recordings, we found that adenosine modulates spontaneous dendro-dendritic signaling in mitral and granule cells via A1R. Furthermore, neuronal A1R deficiency resulted in olfactory dysfunction in two separate olfactory tests. In mice with experimental autoimmune encephalomyelitis (EAE), we detected immune cell infiltration and microglia activation in the olfactory bulb as well as hyperexcitability of mitral cells and olfactory dysfunction. However, neuron-specific A1R activity was unable to attenuate glutamate excitotoxicity in the primary olfactory bulb neurons in vitro or EAE-induced olfactory dysfunction and disease severity in vivo. Together, we demonstrate that A1R modulates the dendro-dendritic inhibition (DDI) at the site of mitral and granule cells and impacts the processing of the olfactory sensory information, while A1R activity was unable to counteract inflammation-induced hyperexcitability.

Targeted ubiquitination of sensory neuron calcium channels reduces the development of neuropathic pain

Neuropathic pain caused by lesions to somatosensory neurons due to injury or disease is a widespread public health problem that is inadequately managed by small-molecule therapeutics due to incomplete pain relief and devastating side effects. Genetically encoded molecules capable of interrupting nociception have the potential to confer long-lasting analgesia with minimal off-target effects. Here, we utilize a targeted ubiquitination approach to achieve a unique posttranslational functional knockdown of high-voltage-activated calcium channels (HVACCs) that are obligatory for neurotransmission in dorsal root ganglion (DRG) neurons. CaV-aβlator comprises a nanobody targeted to CaV channel cytosolic auxiliary β subunits fused to the catalytic HECT domain of the Nedd4-2 E3 ubiquitin ligase. Subcutaneous injection of adeno-associated virus serotype 9 encoding CaV-aβlator in the hind paw of mice resulted in the expression of the protein in a subset of DRG neurons that displayed a concomitant ablation of CaV currents and also led to an increase in the frequency of spontaneous inhibitory postsynaptic currents in the dorsal horn of the spinal cord. Mice subjected to spare nerve injury displayed a characteristic long-lasting mechanical, thermal, and cold hyperalgesia underlain by a dramatic increase in coordinated phasic firing of DRG neurons as reported by in vivo Ca2+ spike recordings. CaV-aβlator significantly dampened the integrated Ca2+ spike activity and the hyperalgesia in response to nerve injury. The results advance the principle of targeting HVACCs as a gene therapy for neuropathic pain and demonstrate the therapeutic potential of posttranslational functional knockdown of ion channels achieved by exploiting the ubiquitin-proteasome system.

Direct inhibition of CaV2.3 by Gem is dynamin dependent and does not require a direct alfa/beta interaction

The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel β subunit (CaVβ), and the binding between CaVα1/CaVβ appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVβ. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVβ independent promotion of dynamin-dependent endocytosis.

ɑO-Conotoxin GeXIVA isomers modulate N-type calcium (CaV 2.2) channels and inwardly-rectifying potassium (GIRK) channels via GABAB receptor activation.

αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABAB receptors (GABAB R) inhibiting HVA N-type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba2+ currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABAB R antagonist CGP 55845. In transfected HEK293T cells co-expressing human GABAB R1 and R2subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization-activated Ba2+ currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABAB R-coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly-rectifying K+ currents mediated by human GIRK1/2 channels co-expressed with GABAB R in HEK293T cells. This study highlights GABAB R as a potentially important receptor target for the activity of αO-conotoxin GeXIVA to mediate analgesia.

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.

Expression of CaV3.1 T-type Calcium Channels in Acutely Isolated Adult Rat Odontoblasts

Odontoblasts, which consist the outermost compartment of the dental pulp, are primarily engaged in dentin formation. Earlier evidence suggests that voltage-gated calcium channels, such as the high voltage-activated L-type calcium channels, serve as a calcium entry route to mediate dentin formation in odontoblasts. However, the involvement of other voltage-gated calcium channels in regulating intracellular Ca2+ remain unanswered. The expression of voltage-gated calcium channel subtypes of the P/Q- (CaV2.1), N-(CaV2.2), R- (CaV2.3), and T- (CaV3.1-3.3) type were screened in adult rat odontoblasts by single cell RT-PCR. Among these candidates, immunopositivity against CaV3.1 was examined in the odontoblastic layer in teeth sections and dissociated odontoblasts. To confirm the functional expression of CaV3.1 in odontoblasts, intracellular Ca2+ increase in response to membrane depolarization was monitored with Fura-2-based ratiometric calcium imaging. Among the candidate calcium channels, we found that mRNA for CaV3.1 is mainly detected in odontoblasts, with its expression being detected in the odontoblastic layer and dissociated odontoblasts. High extracellular K+-induced membrane depolarization was inhibited by pharmacological blockers for T-type calcium channels such as amiloride or ML218. Our results demonstrate that among P/Q-, N-, R-, and T-type calcium channels, CaV3.1 is mainly expressed in odontoblasts to mediate intracellular Ca2+ signaling in response to membrane depolarization. These findings suggest that CaV3.1 may facilitate intracellular Ca2+ dynamics especially in the range of subliminal depolarizations near resting membrane potentials where other high voltage-gated calcium channels such as the L-type are likely to be inactive.

Analysis of tetrodotoxin-sensitive sodium and low voltage-activated calcium channels in developing mouse retinal horizontal cells

Expression patterns of voltage-gated ion channels determine the spatio-temporal dynamics of ion currents that supply excitable neurons in developing tissue with proper electrophysiological properties. The purpose of the study was to identify fast cationic inward currents in mouse retinal horizontal cells (HCs) and describe their biophysical properties at different developmental stages. We also aimed to reveal their physiological role in shaping light responses (LRs) in adult HCs. HCs were recorded in horizontal slices of wild-type mouse retina at postnatal stages ranging from p8 through p60. Voltage-dependent inward currents were isolated with appropriate voltage protocols and blockers specific for sodium and T-type calcium channels. LRs were evoked with full-field flashes (130 μW/cm2). Transient and steady inward currents were identified at all developmental stages. Transient currents were mediated by T-type calcium and TTX-sensitive sodium channels, whereas steady currents were blocked by cadmium, indicating the presence of high voltage-activated calcium channels. Activation and steady-state inactivation kinetics of T-type calcium channels revealed a contribution to the resting membrane potential during postnatal development. Additionally, both sodium and T-type calcium channels had an impact on HC LRs at light offset in adult animals. Our results showed that the voltage-dependent inward currents of postnatally developing mouse HCs consist of T-type calcium, TTX-sensitive sodium, and high voltage-activated calcium channels, and that transient ionic currents contributed to light-evoked responses of adult HCs, suggesting a role in HC information processing.

The TCF7L2-dependent high-voltage activated calcium channel subunit α2δ-1 controls calcium signaling in rodent pancreatic beta-cells

The transcription factor TCF7L2 remains the most important diabetes gene identified to date and genetic risk carriers exhibit lower insulin secretion. We show that Tcf7l2 regulates the auxiliary subunit of voltage-gated Ca2+ channels, Cacna2d1 gene/α2δ-1 protein levels. Furthermore, suppression of α2δ-1 decreased voltage-gated Ca2+ currents and high glucose/depolarization-evoked Ca2+ signaling which mimicked the effect of silencing of Tcf7l2. This appears to be the result of impaired voltage-gated Ca2+ channel trafficking to the plasma membrane, as Cav1.2 channels accumulated in the recycling endosomes after α2δ-1 suppression, in clonal as well as primary rodent beta-cells. This impaired the capacity for glucose-induced insulin secretion in Cacna2d1-silenced cells. Overexpression of α2δ-1 increased high-glucose/K+-stimulated insulin secretion. Furthermore, overexpression of α2δ-1 in Tcf7l2-silenced cells rescued the Tcf7l2-dependent impairment of Ca2+ signaling, but not the reduced insulin secretion. Taken together, these data clarify the connection between Tcf7l2, α2δ-1 in Ca2+-dependent insulin secretion.

Structure, function and regulation of CaV 2.2 N-type calcium channels.

N-type or CaV2.2 high-voltage activated calcium channels are distinguished by exclusively neuronal tissue distribution, sensitivity to ω-conotoxins, prominent inhibition by G-proteins, and a unique role in nociception. Most investigated modulatory pathway regulating the CaV2.2 channels is G-protein-coupled receptor-activated pathway leading to current inhibition by Gβγ subunit of G-protein. Binding of Gβγ dimer to α1 subunit of the CaV2.2 channel transfers the channel form "willing" to "reluctant" gating state. Channel phosphorylation by protein kinase C potentiates N-type calcium current. CaV2.2 channels could be functionally regulated also by a number of protein-protein interactions. Ca V2.2 null mice are hyposensitive to inflammatory and neuropathic pain, otherwise they have a mild phenotype. Consistent with the mild phenotype of the CaV2.2-/- mice, reports on mutations linked to a disease phenotype are scarce. Only one mutation related to human heritable diseases was identified until now. Pharmaceutical inhibition of CaV2.2 channels either by direct inhibition of the channel, by an activation of G-protein coupled receptors, or by inhibition of membrane targeting of the channel protein are promising strategies for treatment of severe chronic and/or neuropathic pain.

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.

A novel \u03b1-conopeptide Eu1.6 inhibits N-type (CaV2.2) calcium channels and exhibits potent analgesic activity

We here describe a novel α-conopeptide, Eu1.6 from Conus eburneus, which exhibits strong anti-nociceptive activity by an unexpected mechanism of action. Unlike other α-conopeptides that largely target nicotinic acetylcholine receptors (nAChRs), Eu1.6 displayed only weak inhibitory activity at the α3β4 and α7 nAChR subtypes and TTX-resistant sodium channels, and no activity at TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons, or opiate receptors, VR1, KCNQ1, L- and T-type calcium channels expressed in HEK293 cells. However, Eu1.6 inhibited high voltage-activated N-type calcium channel currents in isolated mouse DRG neurons which was independent of GABAB receptor activation. In HEK293 cells expressing CaV2.2 channels alone, Eu1.6 reversibly inhibited depolarization-activated Ba2+ currents in a voltage- and state-dependent manner. Inhibition of CaV2.2 by Eu1.6 was concentration-dependent (IC50 ~1 nM). Significantly, systemic administration of Eu1.6 at doses of 2.5–5.0 μg/kg exhibited potent analgesic activities in rat partial sciatic nerve injury and chronic constriction injury pain models. Furthermore, Eu1.6 had no significant side-effect on spontaneous locomotor activity, cardiac and respiratory function, and drug dependence in mice. These findings suggest α-conopeptide Eu1.6 is a potent analgesic for the treatment of neuropathic and chronic pain and opens a novel option for future analgesic drug design.

Nifedipine Inhibition of High-Voltage Activated Calcium Channel Currents in Cerebral Artery Myocytes Is Influenced by Extracellular Divalent Cations.

Voltage-dependent calcium channels (VDCCs) play an essential role in regulating cerebral artery diameter and it is widely appreciated that the L-type VDCC, CaV1.2, encoded by the CACNA1C gene, is a principal Ca2+ entry pathway in vascular myocytes. However, electrophysiological studies using 10 mM extracellular barium ([Ba2+]o) as a charge carrier have shown that ~20% of VDCC currents in cerebral artery myocytes are insensitive to 1,4-dihydropyridine (1,4-DHP) L-type VDDC inhibitors such as nifedipine. Here, we investigated the hypothesis that the concentration of extracellular divalent cations can influence nifedipine inhibition of VDCC currents. Whole-cell VDCC membrane currents were obtained from freshly isolated rat cerebral artery myocytes in extracellular solutions containing Ba2+ and/or Ca2+. In the absence of [Ca2+]o, both nifedipine-sensitive and -insensitive calcium currents were observed in 10 mM [Ba2+]o. However, VDCC currents were abolished by nifedipine when using a combination of 10 mM [Ba2+]o and 100 μM [Ca2+]o. VDCC currents were also completely inhibited by nifedipine in either 2 mM [Ba2+]o or 2 mM [Ca2+]o. The biophysical characteristics of all recorded VDCC currents were consistent with properties of a high-voltage activated VDCC, such as CaV1.2. Further, VDCC currents recorded in 10 mM [Ba2+]o ± 100 μM [Ca2+]o or 2 mM [Ba2+]o exhibited similar sensitivity to the benzothiazepine L-type VDCC blocker, diltiazem, with complete current inhibition at 100 μM. These data suggest that nifedipine inhibition is influenced by both Ca2+ binding to an external site(s) on these channels and surface charge effects related to extracellular divalent cations. In sum, this work demonstrates that the extracellular environment can profoundly impact VDCC current measurements.