Search

It is widely believed that information storage in neuronal circuits involves nanoscopic structural changes at synapses, resulting in the formation of synaptic engrams. However, direct evidence for this hypothesis is lacking. To test this conjecture, we combined chemical potentiation, functional analysis by paired pre-postsynaptic recordings, and structural analysis by electron microscopy (EM) and freeze-fracture replica labeling (FRL) at the rodent hippocampal mossy fiber synapse, a key synapse in the trisynaptic circuit of the hippocampus. Biophysical analysis of synaptic transmission revealed that forskolin-induced chemical potentiation increased the readily releasable vesicle pool size and vesicular release probability by 146% and 49%, respectively. Structural analysis of mossy fiber synapses by EM and FRL demonstrated an increase in the number of vesicles close to the plasma membrane and the number of clusters of the priming protein Munc13-1, indicating an increase in the number of both docked and primed vesicles. Furthermore, FRL analysis revealed a significant reduction of the distance between Munc13-1 and CaV2.1 Ca2+ channels, suggesting reconfiguration of the channel-vesicle coupling nanotopography. Our results indicate that presynaptic plasticity is associated with structural reorganization of active zones. We propose that changes in potential nanoscopic organization at synaptic vesicle release sites may be correlates of learning and memory at a plastic central synapse., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

PDZ domain mediated interactions with voltage-gated calcium (Ca V ) channel C-termini play important roles in localizing membrane Ca 2+ signaling. The first such interaction was described between the scaffolding protein Mint-1 and Ca V 2.2 in mammals. In this study, we show through various in silico analyses that Mint is an animal-specific gene with a highly divergent N-terminus but a strongly conserved C-terminus comprised of a phosphotyrosine binding domain, two tandem PDZ domains (PDZ-1 and PDZ-2), and a C-terminal auto-inhibitory element that binds and inhibits PDZ-1. In addition to Ca V 2 chanels, most genes that interact with Mint are also deeply conserved including amyloid precursor proteins, presenilins, neurexin, and CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast and bacterial 2-hybrid experiments, we show that Mint and Ca V 2 channels from cnidarians and placozoans interact in vitro, and in situ hybridization revealed co-expression in dissociated neurons from the cnidarian Nematostella vectensis. Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis strongly bound the divergent C-terminal ligands of cnidarian and placozoan Ca V 2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore Ca V 2 channel C-terminus. Altogether, our analyses suggest that the capacity of Mint to bind Ca V 2 channels predates bilaterian animals, and that evolutionary changes in Ca V 2 channel C-terminal sequences resulted in altered binding modalities with Mint., (© 2024. Crown.)

How G proteins inhibit N-type, voltage-gated, calcium-selective channels (Ca V 2.2) during presynaptic inhibition is a decades-old question. G proteins Gβγ bind to intracellular Ca V 2.2 regions, but the inhibition is voltage dependent. Using the hybrid electrophysiological and optical approach voltage-clamp fluorometry, we show that Gβγ acts by selectively inhibiting a subset of the four different Ca V 2.2 voltage-sensor domains (VSDs I to IV). During regular "willing" gating, VSD-I and -IV activations resemble pore opening, VSD III activation is hyperpolarized, and VSD II appears unresponsive to depolarization. In the presence of Gβγ, Ca V 2.2 gating is "reluctant": pore opening and VSD I activation are strongly and proportionally inhibited, VSD IV is modestly inhibited, while VSD III is not. We propose that Gβγ inhibition of VSDs I and IV underlies reluctant Ca V 2.2 gating and subsequent presynaptic inhibition.

Osteoarthritis (OA) represents a significant pain challenge globally, as current treatments are limited and come with substantial and adverse side effects. Voltage-gated calcium channels have proved to be pharmacologically effective targets, with multiple Food and Drug Administration-approved Ca V 2.2 modulators available for the treatment of pain. Although effective, drugs targeting Ca V 2.2 are complicated by the same obstacles facing other pain therapeutics-invasive routes of administration, narrow therapeutic windows, side effects, and addiction potential. We have identified a key regulator of Ca V 2.2 channels, collapsin response mediator protein 2, that allows us to indirectly regulate Ca V 2.2 expression and function. We previously developed a peptidomimetic modulator of collapsin response mediator protein 2, CBD3063, that effectively reverses neuropathic and inflammatory pain without negative side effects by reducing membrane expression of Ca V 2.2. The potent analgesic properties of CBD3063, combined with the lack of negative side effects, prompted us to assess the efficacy of CBD3063 in a rodent model of OA pain. Here, we demonstrate the intraperitoneal administration of CBD3063 alleviates both evoked and nonevoked behavioral hallmarks of OA pain. Further, we reveal that CBD3063 reduces OA-induced increased neural activity in the parabrachial nucleus, a key supraspinal site modulating the pain experience. Together, these studies suggest that CBD3063 is an effective analgesic for OA pain. PERSPECTIVE: Despite the high prevalence of OA pain worldwide, current treatment options remain limited. We demonstrate that CBD3063-mediated disruption of the Ca V 2.2-collapsin response mediator protein 2 interaction alleviates pain in a preclinical joint pain model, providing a promising basis for the development of new OA pain treatments., (Copyright © 2024 United States Association for the Study of Pain, Inc. Published by Elsevier Inc. All rights reserved.)

The abundance of CaV2 voltage-gated calcium channels is linked to presynaptic homeostatic plasticity (PHP), a process that recalibrates synaptic strength to maintain the stability of neural circuits. However, the molecular and cellular mechanisms governing PHP and CaV2 channels are not completely understood. Here, we uncover a previously not described form of PHP in Caenorhabditis elegans , revealing an inverse regulatory relationship between the efficiency of neurotransmitter release and the abundance of UNC-2/CaV2 channels. Gain-of-function unc-2SL(S240L) mutants, which carry a mutation analogous to the one causing familial hemiplegic migraine type 1 in humans, showed markedly reduced channel abundance despite increased channel functionality. Reducing synaptic release in these unc-2SL(S240L) mutants restored channel levels to those observed in wild-type animals. Conversely, loss-of-function unc-2DA(D726A) mutants, which harbor the D726A mutation in the channel pore, exhibited a marked increase in channel abundance. Enhancing synaptic release in unc-2DA mutants reversed this increase in channel levels. Importantly, this homeostatic regulation of UNC-2 channel levels is accompanied by the structural remodeling of the active zone (AZ); specifically, unc-2DA mutants, which exhibit increased channel abundance, showed parallel increases in select AZ proteins. Finally, our forward genetic screen revealed that WWP-1, a HECT family E3 ubiquitin ligase, is a key homeostatic mediator that removes UNC-2 from synapses. These findings highlight a self-tuning PHP regulating UNC-2/CaV2 channel abundance along with AZ reorganization, ensuring synaptic strength and stability., Competing Interests: Competing interests statement:The authors declare no competing interest.

NVA1309 is a non-brain penetrant next-generation gabapentinoid shown to bind Cavα2δ at R243 within a triple Arginine motif forming the binding site for gabapentin and pregabalin. In this study we have compared the effects of NVA1309 with Mirogabalin, a gabapentinoid drug with higher affinity for the voltage-gated calcium channel subunit Cavα2δ-1 than pregabalin which is approved for post-herpetic neuralgia in Japan, Korea and Taiwan. Both NVA1309 and mirogabalin inhibit Cav2.2 currents in vitro and decrease Cav2.2 plasma membrane expression with higher efficacy than pregabalin. Mutagenesis of the classical binding residue arginine R243 and the newly identified binding residue lysine K615 reverse the effect of mirogabalin on Cav2.2 current, but not that of NVA1309., (© 2024. The Author(s).)

To achieve the functional polarization that underlies brain computation, neurons sort protein material into distinct compartments. Ion channel composition, for example, differs between axons and dendrites, but the molecular determinants for their polarized trafficking remain obscure. Here, we identify mechanisms that target voltage-gated Ca 2+ channels (Ca V s) to distinct subcellular compartments. In hippocampal neurons, Ca V 2s trigger neurotransmitter release at the presynaptic active zone, and Ca V 1s localize somatodendritically. After knockout of all three Ca V 2s, expression of Ca V 2.1, but not Ca V 1.3, restores neurotransmitter release. We find that chimeric Ca V 1.3s with Ca V 2.1 intracellular C-termini localize to the active zone, mediate synaptic vesicle exocytosis, and render release sensitive to Ca V 1 blockers. This dominant targeting function of the Ca V 2.1 C-terminus requires the first EF hand in its proximal segment, and replacement of the Ca V 2.1 C-terminus with that of Ca V 1.3 abolishes Ca V 2.1 active zone localization and function. We conclude that Ca V intracellular C-termini mediate compartment-specific targeting., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Neuropathic pain affects up to 10% of the global population and is caused by an injury or a disease affecting the somatosensory, peripheral, or central nervous system. NP is characterized by chronic, severe and opioid-resistant properties. Therefore, its clinical management remains very challenging. The N-type voltage-gated calcium channel, Ca v 2.2, is a validated target for therapeutic intervention in chronic and neuropathic pain. The conotoxin ziconotide (Prialt®) is an FDA-approved drug that blocks Ca v 2.2 channel but needs to be administered intrathecally. Thus, although being principally efficient, the required application route is very much in disfavour., Experimental Approach and Key Results: Here, we describe an orally available drug candidate, RD2, which competes with ziconotide binding to Ca v 2.2 at nanomolar concentrations and inhibits Ca v 2.2 almost completely reversible. Other voltage-gated calcium channel subtypes, like Ca v 1.2 and Ca v 3.2, were affected by RD2 only at concentrations higher than 10 μM. Data from sciatic inflammatory neuritis rat model demonstrated the in vivo proof of concept, as low-dose RD2 (5 mg·kg -1 ) administered orally alleviated neuropathic pain compared with vehicle controls. High-dose RD2 (50 mg·kg -1 ) was necessary to reduce pain sensation in acute thermal response assessed by the tail flick test., Conclusions and Implications: Taken together, these results demonstrate that RD2 has antiallodynic properties. RD2 is orally available, which is the most convenient application form for patients and caregivers. The surprising and novel result from standard receptor screens opens the room for further optimization into new promising drug candidates, which address an unmet medical need., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

GABA B receptors (GBRs), the G protein-coupled receptors for GABA, regulate synaptic transmission throughout the brain. A main synaptic function of GBRs is the gating of Cav2.2-type Ca 2+ channels. However, the cellular compartment where stable GBR/Cav2.2 signaling complexes form remains unknown. In this study, we demonstrate that the vesicular protein synaptotagmin-11 (Syt11) binds to both the auxiliary GBR subunit KCTD16 and Cav2.2 channels. Through these dual interactions, Syt11 recruits GBRs and Cav2.2 channels to post-Golgi vesicles, thus facilitating assembly of GBR/Cav2.2 signaling complexes. In addition, Syt11 stabilizes GBRs and Cav2.2 channels at the neuronal plasma membrane by inhibiting constitutive internalization. Neurons of Syt11 knockout mice exhibit deficits in presynaptic GBRs and Cav2.2 channels, reduced neurotransmitter release, and decreased GBR-mediated presynaptic inhibition, highlighting the critical role of Syt11 in the assembly and stable expression of GBR/Cav2.2 complexes. These findings support that Syt11 acts as a vesicular scaffold protein, aiding in the assembly of signaling complexes from low-abundance components within transport vesicles. This mechanism enables insertion of pre-assembled functional signaling units into the synaptic membrane., (© 2024. The Author(s).)

Although radiotherapy is the most effective treatment modality for brain tumors, it always injures the central nervous system, leading to potential sequelae such as cognitive dysfunction. Radiation induces molecular, cellular, and functional changes in neuronal and glial cells. The hippocampus plays a critical role in learning and memory; therefore, concerns about radiation-induced injury are widespread. Multiple studies have focused on this complex problem, but the results have not been fully elucidated. Naked mole rat brains were irradiated with 60 Co at a dose of 10 Gy. On 7 days, 14 days, and 28 days after irradiation, hippocampi in the control groups were obtained for next-generation sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were subsequently performed. Venn diagrams revealed 580 differentially expressed genes (DEGs) that were common at different times after irradiation. GO and KEGG analyses revealed that the 580 common DEGs were enriched in molecular transducer activity. In particular, CACNA1B mediated regulatory effects after irradiation. CACNA1B expression increased significantly after irradiation. Downregulation of CACNA1B led to a reduction in apoptosis and reactive oxygen species levels in hippocampal neurons. This was due to the interaction between CACNA1B and Nrf2, which disturbed the normal nuclear localization of Nrf2. In addition, CACNA1B downregulation led to a decrease in the cognitive functions of naked mole rats. These findings reveal the pivotal role of CACNA1B in regulating radiation-induced brain injury and will lead to the development of a novel strategy to prevent brain injury after irradiation., (© 2024 International Federation of Cell Biology.)

The Voltage-Gated Calcium Channel (VGCC) auxiliary subunit Cavα2δ-1 (CACNA2D1) is the target/receptor of gabapentinoids which are known therapeutics in epilepsy and neuropathic pain. Following damage to the peripheral sensory nervous system, Cavα2δ-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of chronic neuropathic pain. Gabapentinoids, such as gabapentin and pregabalin, engage with Cavα2δ-1 via binding an arginine residue (R241) within an RRR motif located at the N-terminus of human Cavα2δ-1. A novel, next generation gabapentinoid, engineered not to penetrate the brain, was able to generate a strong analgesic response in Chronic Constriction Injury animal model of chronic neuropathic pain and showed binding specificity for Cavα2δ-1 versus the Cavα2δ-2 subunit. This novel non-brain penetrant gabapentinoid, binds to R241 and a novel binding site on Cavα2δ-1, which is located within the VGCC_α2 domain, identified as a lysine residue within an IKAK amino acid motif (K634). The overall whole cell current amplitudes were diminished by the compound, with these inhibitory effects being diminished in R241A mutant Cavα2δ-1 subunits. The functional effects occurred at lower concentrations than those needed for inhibition by gabapentin or pregabalin, which apparently bound the Cavα2δ-1 subunit only on the R241 and not on the K634 residue. Our work sets the stage for the identification and characterisation of novel compounds with therapeutic properties in neuropathic pain and possibly in other disorders and conditions which require engagement of the Cavα2δ-1 target., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Neurons have the unique capacity to adapt output in response to changes in their environment. Within seconds, sensory nerve endings can become hypersensitive to stimuli in response to potentially damaging events. The underlying behavioral response is well studied, but several of the key signaling molecules that mediate sensory hypersensitivity remain unknown. We previously discovered that peripheral voltage-gated Ca V 2.2 channels in nerve endings in skin are essential for the rapid, transient increase in sensitivity to heat, but not to mechanical stimuli, that accompanies intradermal capsaicin. Here we report that the cytokine interleukin-1α (IL-1α), an alarmin, is necessary and sufficient to trigger rapid heat and mechanical hypersensitivity in skin. Of 20 cytokines screened, only IL-1α was consistently detected in hind paw interstitial fluid in response to intradermal capsaicin and, similar to behavioral sensitivity to heat, IL-1α levels were also dependent on peripheral Ca V 2.2 channel activity. Neutralizing IL-1α in skin significantly reduced capsaicin-induced changes in hind paw sensitivity to radiant heat and mechanical stimulation. Intradermal IL-1α enhances behavioral responses to stimuli and, in culture, IL-1α enhances the responsiveness of Trpv1-expressing sensory neurons. Together, our data suggest that IL-1α is the key cytokine that underlies rapid and reversible neuroinflammatory responses in skin., (© 2024. The Author(s).)

Aim: Our study aims to identify novel non-coding RNA-mRNA regulatory networks associated with β-cell dysfunction and compensatory responses in obesity-related diabetes., Methods: Glucose metabolism, islet architecture and secretion, and insulin sensitivity were characterized in C57BL/6J mice fed on a 60% high-fat diet (HFD) or control for 24 weeks. Islets were isolated for whole transcriptome sequencing to identify differentially expressed (DE) mRNAs, miRNAs, IncRNAs, and circRNAs. Regulatory networks involving miRNA-mRNA, lncRNA-mRNA, and lncRNA-miRNA-mRNA were constructed and functions were assessed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses., Results: Despite compensatory hyperinsulinemia and a significant increase in β-cell mass with a slow rate of proliferation, HFD mice exhibited impaired glucose tolerance. In isolated islets, insulin secretion in response to glucose and palmitic acid deteriorated after 24 weeks of HFD. Whole transcriptomic sequencing identified a total of 1324 DE mRNAs, 14 DE miRNAs, 179 DE lncRNAs, and 680 DE circRNAs. Our transcriptomic dataset unveiled several core regulatory axes involved in the impaired insulin secretion in HFD mice, such as miR-6948-5p/Cacna1c, miR-6964-3p/Cacna1b, miR-3572-5p/Hk2, miR-3572-5p/Cckar and miR-677-5p/Camk2d. Additionally, proliferative and apoptotic targets, including miR-216a-3p/FKBP5, miR-670-3p/Foxo3, miR-677-5p/RIPK1, miR-802-3p/Smad2 and ENSMUST00000176781/Caspase9 possibly contribute to the increased β-cell mass in HFD islets. Furthermore, competing endogenous RNAs (ceRNA) regulatory network involving 7 DE miRNAs, 15 DE lncRNAs and 38 DE mRNAs might also participate in the development of HFD-induced diabetes., Conclusions: The comprehensive whole transcriptomic sequencing revealed novel non-coding RNA-mRNA regulatory networks associated with impaired insulin secretion and increased β-cell mass in obesity-related diabetes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

High-voltage-gated calcium channels have pivot role in the cellular and molecular mechanisms of various neurological disorders, including epilepsy. Similar to other calcium channels, P/Q-type calcium channels (Ca v 2.1) are also responsible for vesicle release at synaptic terminals. Up to date, there are very limited reports showing the mechanisms of Ca v 2.1 in epileptogenesis. In the present study, we investigated the anticonvulsive and neuroprotective effects of ω-agatoxin IVA, a specific Ca v 2.1 blocker, in a chemical kindling model of epileptogenesis. Righting reflex and inclined plane tests were used to assess motor coordination. Electroencephalography was recorded for electrophysiological monitoring of seizure activity in freely moving rats. Immunohistochemical analyses were performed for brain-derived neurotrophic factor (BDNF) and cleaved caspase-3 expressions in the prefrontal cortex, striatum, hippocampus, and thalamic nucleus. ω-Agatoxin IVA injected into the right lateral ventricle significantly prolonged the onset of seizures in a dose-dependent manner. In addition, repeated intraperitoneal administrations of ω-agatoxin IVA significantly suppressed the development of kindling and epileptic discharges without altering motor coordination. In addition, ω-agatoxin IVA significantly increased BDNF expressions, and decreased cleaved caspase-3 expressions in the brain when compared to PTZ + saline group. Our current study emphasizes the significance of the inhibition of P/Q type calcium channels by ω-agatoxin IVA, which suppresses the development of epileptogenesis and provides a new potential pathway for epilepsy treatment., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Spinocerebellar ataxia type 6 (SCA6) is a polyglutamine (polyQ) disease, which is caused by the elongation of CAG repeats encoding polyQ in the CACNA1A gene. The CACNA1A gene encodes two proteins, namely, α1A (a subunit of the plasma membrane calcium channel), which is translated in its entire length, and α1ACT, which is translated from the second cistron, and both proteins have a polyQ tract. The α1A-polyQ and α1ACT-polyQ proteins with an elongated polyQ stretch have been reported to form aggregates in cells and induce neuronal cell death, but the subcellular localization of these proteins and their cytotoxic properties remain unclear. In this study, we first analyzed SCA6 model mice and found that α1A-polyQ long localized mainly to the Golgi apparatus, whereas a portion of α1ACT-polyQ long localized to the nucleus. Analysis using Neuro2a cells also showed similar subcellular localizations of these proteins, and a proportion of both proteins localized to the endoplasmic reticulum (ER). Cytotoxic studies demonstrated that both proteins induce both the ER stress response and apoptosis, indicating that they are able to induce ER stress-induced apoptosis., Competing Interests: Declaration of competing interest We do not have any conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Transmembrane Ca v 2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Ca v 2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Ca v 2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Ca v 2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A 1 R 2 dipeptide in CBD3 as the anchoring Ca v 2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Ca v 2.2 from CRMP2, reduced membrane Ca v 2.2 expression and Ca 2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Ca v 2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

The CaV2 voltage-gated calcium channel is the major conduit of calcium ions necessary for neurotransmitter release at presynaptic active zones (AZs). The CaV2 channel is a multimeric complex that consists of a pore-forming α 1 subunit and two auxiliary β and α 2 δ subunits. Although auxiliary subunits are critical for channel function, whether they are required for α 1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans , we show that UNC-2/α 1 localizes to AZs even in the absence of CCB-1/β or UNC-36/α 2 δ, albeit at low levels. When UNC-2 is manipulated to be trapped in the endoplasmic reticulum (ER), CCB-1 and UNC-36 fail to colocalize with UNC-2 in the ER, indicating that they do not coassemble with UNC-2 in the ER. Moreover, blocking ER-associated degradation does not further increase presynaptic UNC-2 channels in ccb-1 or unc-36 mutants, indicating that UNC-2 levels are not regulated in the ER. An unc-2 mutant lacking C-terminal AZ protein interaction sites with intact auxiliary subunit binding sites displays persistent presynaptic UNC-2 localization and a prominent increase of UNC-2 channels in nonsynaptic axonal regions, underscoring a protective role of auxiliary subunits against UNC-2 degradation. In the absence of UNC-2, presynaptic CCB-1 and UNC-36 are profoundly diminished to barely detectable levels, indicating that UNC-2 is required for the presynaptic localization of CCB-1 and UNC-36. Together, our findings demonstrate that although the pore-forming subunit does not require auxiliary subunits for its trafficking and transport to AZs, it recruits auxiliary subunits to stabilize and expand calcium channel signalosomes. SIGNIFICANCE STATEMENT Synaptic transmission in the neuron hinges on the coupling of synaptic vesicle exocytosis with calcium influx. This calcium influx is mediated by CaV2 voltage-gated calcium channels. These channels consist of one pore-forming α 1 subunit and two auxiliary β and α 2 δ subunits. The auxiliary subunits enhance channel function and regulate the overall level of channels at presynaptic terminals. However, it is not settled how these auxiliary subunits regulate the overall channel level. Our study in C. elegans finds that although the auxiliary subunits do not coassemble with α 1 and aid trafficking, they are recruited to α 1 and stabilize the channel complex at presynaptic terminals. Our study suggests that drugs that target the auxiliary subunits can directly destabilize and have an impact on CaV2 channels., (Copyright © 2023 the authors.)

Some people living with HIV present painful sensory neuropathy (HIV-SN) that is pharmacoresistant, sex-associated, and a major source of morbidity. Since the specific mechanisms underlying HIV-SN are not well understood, the aim of our study was to characterize a novel model of painful HIV-SN by combining the HIV-1 gp120 protein and the antiretroviral stavudine (d4T) in mice and to investigate the pronociceptive role of the family 2 voltage-gated calcium channel (VGCC) α1 subunit (Cav2.X channels) in such a model. HIV-SN was induced in male and female C57BL/6 mice by administration of gp120 and/or d4T and detected by a battery of behavior tests and by immunohistochemistry. The role of Cav2.X channels was assessed by the treatment with selective blockers and agonists as well as by mRNA detection. Repeated administration with gp120 and/or d4T produced long-lasting touch-evoked painful-like behaviors (starting at 6 days, reaching a maximum on day 13, and lasting up to 28 days after treatment started), with a greater intensity in female mice treated with the combination of gp120 + d4T. Moreover, gp120 + d4T treatment reduced the intraepidermal nerve fibers and well-being of female mice, without altering other behaviors. Mechanistically, gp120 + d4T treatment induced Cav2.1, 2.2, and 2.3 transcriptional increases in the dorsal root ganglion and the Cav2.X agonist-induced nociception. Accordingly, intrathecal selective Cav2.2 blockade presented longer and better efficacy in reversing the hyperalgesia induced by gp120 + d4T treatment compared with Cav2.1 or Cav2.3, but also presented the worst safety (inducing side effects at effective doses). We conclude that the family 2 calcium channels (Cav2.X) exert a critical pronociceptive role in a novel mouse model of HIV-SN., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: As a peptide originally discovered from Conus achates by mass spectrometry and cDNA sequencing, Ac6.4 contains 25 amino acid residues and three disulfide bridges. Our previous study found that this peptide possesses 80% similarity to MVIIA by BLAST and that MVIIA is a potent and selective blocker of N-type voltage-sensitive calcium channels in neurons., Objective: To recognize the target protein and analgesic activity of Ac6.4 from Conus achates ., Methods: In the present study, we synthesized Ac6.4, expressed the Trx-Ac6.4 fusion protein, tested Ac6.4 for its inhibitory activity against Cav2.2 in CHO cells and investigated Ac6.4 and Trx-Ac6.4 for their analgesic activities in mice., Results: Data revealed that Ac6.4 had strong inhibitory activity against Cav2.2 (IC 50 = 43.6 nM). After intracranial administration of Ac6.4 (5, 10, 20 μg/kg) and Trx-Ac6.4 (20, 40, 80 μg/kg), significant analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent., Conclusion: This study expands our knowledge of the peptide Ac6.4 and provides new possibilities for developing Cav2.2 inhibitors and analgesic drugs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

α-conotoxin AuIB is the only one of the 4/6 type α-conotoxins (α-CTxs) that inhibits the γ-aminobutyric acid receptor B (GABA B R)-coupled N-type calcium channel (Ca V 2.2). To improve its inhibitory activity, a series of variants were synthesized and evaluated according to the structure-activity relationships of 4/7 type α-CTxs targeting GABA B R-coupled Ca V 2.2. Surprisingly, only the substitution of Pro7 with Arg results in a 2-3-fold increase in the inhibition of GABA B R-coupled Ca V 2.2 (IC 50 is 0.74 nM); substitutions of position 9-12 with basic or hydrophobic amino acid and the addition of hydrophobic amino acid Leu or Ile at the second loop to mimic 4/7 type α-CTxs all failed to improve the inhibitory activity of AuIB against GABA B R-coupled Ca V 2.2. Interestingly, the most potent form of AuIB[P7R] has disulfide bridges of "1-4, 2-3" (ribbon), which differs from the "1-3, 2-4" (globular) in the isoforms of wildtype AuIB. In addition, AuIB[P7R](globular) displays potent analgesic activity in the acetic acid writhing model and the partial sciatic nerve injury (PNL) model. Our study demonstrated that 4/6 type α-CTxs, with the disulfide bridge connectivity "1-4, 2-3," are also potent inhibitors for GABA B R-coupled Ca V 2.2, exhibiting potent analgesic activity.

The N-type calcium channel, Ca V 2.2 is key to neurotransmission from the primary afferent terminals of dorsal root ganglion (DRG) neurons to their postsynaptic targets in the spinal cord. In this study, we have utilized Ca V 2.2_HA knock-in mice, because the exofacial epitope tag in Ca V 2.2_HA enables accurate detection and localization of endogenous Ca V 2.2. Ca V 2.2_HA knock-in mice were used as a source of DRGs to exclusively study the presynaptic expression of N-type calcium channels in co-cultures between DRG neurons and wild-type spinal cord neurons. Ca V 2.2_HA is strongly expressed on the cell surface, particularly in TRPV1-positive small and medium DRG neurons. Super-resolution images of the presynaptic terminals revealed an increase in Ca V 2.2_HA expression and increased association with the postsynaptic marker Homer over time in vitro. Brief application of the TRPV1 agonist, capsaicin, resulted in a significant down-regulation of cell surface Ca V 2.2_HA expression in DRG neuron somata. At their presynaptic terminals, capsaicin caused a reduction in Ca V 2.2_HA proximity to and co-localization with the active zone marker RIM 1/2, as well as a lower contribution of N-type channels to single action potential-mediated Ca 2+ influx. The mechanism of this down-regulation of Ca V 2.2_HA involves a Rab11a-dependent trafficking process, since dominant-negative Rab11a (S25N) occludes the effect of capsaicin on presynaptic Ca V 2.2_HA expression, and also prevents the effect of capsaicin on action potential-induced Ca 2+ influx. Taken together, these data suggest that capsaicin causes a decrease in cell surface Ca V 2.2_HA expression in DRG terminals via a Rab11a-dependent endosomal trafficking pathway., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Physiological Society.)

High-voltage-activated Ca 2+ (Ca V ) channels that adjust Ca 2+ influx upon membrane depolarization are differentially regulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in an auxiliary Ca V β subunit-dependent manner. However, the molecular mechanism by which the β subunits control the PIP 2 sensitivity of Ca V channels remains unclear. By engineering various α1B and β constructs in tsA-201 cells, we reported that at least two PIP 2 -binding sites, including the polybasic residues at the C-terminal end of I-II loop and the binding pocket in S4 II domain, exist in the Ca V 2.2 channels. Moreover, they were distinctly engaged in the regulation of channel gating depending on the coupled Ca V β2 subunits. The membrane-anchored β subunit abolished the PIP 2 interaction of the phospholipid-binding site in the I-II loop, leading to lower PIP 2 sensitivity of Ca V 2.2 channels. By contrast, PIP 2 interacted with the basic residues in the S4 II domain of Ca V 2.2 channels regardless of β2 isotype. Our data demonstrated that the anchoring properties of Ca V β2 subunits to the plasma membrane determine the biophysical states of Ca V 2.2 channels by regulating PIP 2 coupling to the nonspecific phospholipid-binding site in the I-II loop., Competing Interests: CP, WY, BS No competing interests declared, (© 2022, Park et al.)

The analgesic α -conotoxins Vc1.1, RgIA, and PeIA attenuate nociceptive transmission via activation of G protein-coupled GABA B receptors (GABA B Rs) to modulate N-type calcium channels in primary afferent neurons and recombinantly coexpressed human GABA B R and Cav2.2 channels in human embryonic kidney 293T cells. Here, we investigate the effects of analgesic α -conotoxins following the mutation of amino acid residues in the Venus flytrap (VFT) domains of the GABA B R subunits predicted through computational peptide docking and molecular dynamics simulations. Our docking calculations predicted that all three of the α -conotoxins form close contacts with VFT residues in both B1 and B2 subunits, comprising a novel GABA B R ligand-binding site. The effects of baclofen and α -conotoxins on the peak Ba 2+ current (I Ba ) amplitude were investigated on wild-type and 15 GABA B R mutants individually coexpressed with human Cav2.2 channels. Mutations at the interface of the VFT domains of both GABA B R subunits attenuated baclofen-sensitive I Ba inhibition by the analgesic α -conotoxins. In contrast, mutations located outside the putative peptide-binding site (D380A and R98A) did not. The key GABA B R residues involved in interactions with the α -conotoxins are K168 and R207 on the B2 subunit and S130, S153, R162, E200, F227, and E253 on the B1 subunit. The double mutant, S130A + S153A, abolished inhibition by both baclofen and the α -conotoxins. Depolarization-activated I Ba mediated by both wild-type and all GABA B R mutants were inhibited by the selective GABA B R antagonist CGP 55845. This study identifies specific residues of GABA B R involved in the binding of the analgesic α -conotoxins to the VFT domains of the GABA B R. SIGNIFICANCE STATEMENT: This study defines the binding site of the analgesic α -conotoxins Vc1.1, RgIA, and PeIA on the human GABA B receptor to activate Gi/o proteins and inhibit Cav2.2 channels. Computational docking and molecular dynamics simulations of GABA B R identified amino acids of the Venus flytrap (VFT) domains with which the α -conotoxins interact. GABA B R alanine mutants attenuated baclofen-sensitive Cav2.2 inhibition by the α -conotoxins. We identify an allosteric binding site at the interface of the VFT domains of the GABA B R subunits for the analgesic α -conotoxins., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Distant metastasis is the main cause of mortality in breast cancer patients. Using the breast cancer genomic data from The Cancer Genome Atlas (TCGA), we identified brain specific Ca v 2.2 as a critical regulator of metastasis. Ca v 2.2 expression is significantly upregulated in breast cancer and its higher expression is inversely correlated with survival suggesting a previously unappreciated role of Ca v 2.2 in breast cancer. Ca v 2.2 is required for breast cancer migration, invasion, and metastasis. Interestingly, Ca v 2.2 promotes invadopodia formation and extracellular matrix (ECM) degradation through the stabilization of invadopodia component cortactin in a proteosome-dependent manner. Moreover, deubiquitinating enzyme USP43 mediated the functions of Ca v 2.2 in cortactin stabilization, invadopodia formation, ECM degradation, and metastasis. Interestingly, Ca v 2.2 upregulates USP43 expression through NFAT2 dephosphorylation and nuclear localization. Our study uncovered a novel pathway that regulates cortactin expression and invadopodia formation in breast cancer metastasis., (© 2022. The Author(s).)

Transcription factor 21 (TCF21) directly binds and regulates SF1 mRNA expression in tumor and normal adrenocortical cells, and both are involved in the development and steroidogenesis of the adrenal cortex. TCF21 is a tumor suppressor gene and its expression is reduced in malignant tumors. In adrenocortical tumors, it is less expressed in adrenocortical carcinomas (ACC) than in adrenocortical adenomas (ACA) and normal tissues. However, a comprehensive analysis to identify TCF21 targets has not yet been conducted in any type of cancer. In this study, we performed Chromatin Immunoprecipitation and Sequencing (ChIP-Seq) in an adrenocortical carcinoma cell line (NCI-H295R) overexpressing TCF21, with the aim of identifying TCF21 new targets. The five most frequently identified sequences corresponded to the PRDM7, CNTNAP2, CACNA1B, PTPRN2, and KCNE1B genes. Validation experiments showed that, in NCI-H295R cells, TCF21 negatively regulates the expression of the CACNA1B gene. Recently, it was observed that the N-type calcium channel v2.2 (Cav2.2) encoded by the CACNA1B gene is important in Angiotensin II signal transduction for corticosteroid biosynthesis in NCI-H295R adrenocortical carcinoma cells. Indeed, TCF21 inhibits CACNA1B and Cav2.2 expression in NCI-H295R. In addition, in a cohort of 55 adult patients with adrenocortical tumors, CACNA1B expression was higher in ACC than ACA and was related to poor disease-free survival in ACC patients. These results suggest a mechanism of steroidogenesis control by TCF21 in adrenocortical tumor cells, in addition to the control observed through SF1 inhibition. Importantly, steroid production could impair tumor immunogenicity, contributing to the immune resistance described in adrenal cancer.

Inclinicalpractice, high-voltage, long-duration pulsed radiofrequency (HL-PRF) is effective for several types of intractable neuropathic pain (NP), but the mechanisms have not been well explored. Cav2.2 channels could increase neuronal excitability and neurotransmission accompanying NP. This study investigated the relationship of the efficacy of HL-PRF on NP with the levels of Cav2.2 in the spinal dorsal horn (SDH) and dorsal root ganglions (DRGs) of chronic constriction injury (CCI) in rats. Sham HL-PRF, GVIA (a specific Cav2.2 channel blocker), HL-PRF, or GVIA + HL-PRF was applied to CCI rats. The results showed: compared with the sham group, the PWT and PWL of CCI rats decreased significantly (P < 0.05), and Cav2.2 expression was elevated significantly in the SDH and DRGs (P < 0.05). Compared with the CCI group, both HL-PRF and ω-conotoxin GVIA treatment reversed the increased PWT and PWL (P < 0.05) and downregulated the overexpression of Cav2.2 in the SDH and DRGs (P < 0.05). Furthermore, PWT, PWL, and the expression of Cav2.2 in the SDH and DRGs were not significantly different among the 3 treatment groups. HL-PRF on L 5 DRG reversed the hyperalgesia behavior of NP and reduced the levels of Cav2.2 in the ipsilateral SDH and DRGs in CCI rats. Moreover, the underlying mechanism may be related to the downregulation of CaV2.2 protein levels in both SDH and DRG., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Voltage-gated calcium channel Ca v 2.1 undergoes Ca 2+ -dependent facilitation and inactivation, which are important in short-term synaptic plasticity. In presynaptic terminals, Ca v 2.1 forms large protein complexes that include synaptotagmins. Synaptotagmin-7 (Syt-7) is essential to mediate short-term synaptic plasticity in many synapses. Here, based on evidence that Ca v 2.1 and Syt-7 are both required for short-term synaptic facilitation, we investigated the direct interaction of Syt-7 with Ca v 2.1 and probed its regulation of Ca v 2.1 function. We found that Syt-7 binds specifically to the α 1A subunit of Ca v 2.1 through interaction with the synaptic-protein interaction (synprint) site. Surprisingly, this interaction enhances facilitation in paired-pulse protocols and accelerates the onset of facilitation. Syt-7α induces a depolarizing shift in the voltage dependence of activation of Ca v 2.1 and slows Ca 2+ -dependent inactivation, whereas Syt-7β and Syt-7γ have smaller effects. Our results identify an unexpected, isoform-specific interaction between Ca v 2.1 and Syt-7 through the synprint site, which enhances Ca v 2.1 facilitation and modulates its inactivation., (Copyright © 2022 Djillani et al.)

The striatum is innervated by histaminergic fibers and expresses a high density of histamine H 3 receptors (H 3 Rs), present on medium spiny neurons (MSNs) and corticostriatal afferents. In this study, in sagittal slices from the rat dorsal striatum, excitatory postsynaptic potentials (EPSPs) were recorded in MSNs after electrical stimulation of corticostriatal axons. The effect of H 3 R activation and blockers of calcium and potassium channels was evaluated with the paired-pulse facilitation protocol. In the presence of the H 3 R antagonist/inverse agonist clobenpropit (1 μM), the H 3 R agonist immepip (1 μM) had no effect on the paired-pulse ratio (PPR), but in the absence of clobenpropit, immepip induced a significant increase in PPR, accompanied by a reduction in EPSP amplitude, suggesting presynaptic inhibition. The blockade of Ca V 2.1 (P/Q-type) channels with ω-agatoxin TK (400 nM) increased PPR and prevented the effect of immepip. The Ca V 2.2 (N-type) channel blocker ω-conotoxin GVIA (1 μM) also increased PPR, but did not occlude the immepip action. Functional K IR 3 channels are present in corticostriatal terminals, and in experiments in which immepip increased PPR, the K IR 3 blocker tertiapin-Q (30 nM) prevented the effect of the H 3 R agonist. These results indicate that the presynaptic modulation by H 3 Rs of corticostriatal synapses involves the inhibition of Ca v 2.1 calcium channels and the activation of K IR 3 potassium channels., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Voltage-gated calcium channel (VGCC) subunits have been genetically associated with autism spectrum disorders (ASD). The properties of the pore-forming VGCC subunit are modulated by auxiliary β-subunits, which exist in four isoforms (Ca V β 1-4 ). Our previous findings suggested that activation of L-type VGCCs is a common feature of Ca V β 2 subunit mutations found in ASD patients. In the current study, we functionally characterized a novel Ca V β 1b variant (p.R296C) identified in an ASD patient. We used whole-cell and single-channel patch clamp to study the effect of Ca V β 1b&#95;R296C on the function of L- and N-type VGCCs. Furthermore, we used co-immunoprecipitation followed by Western blot to evaluate the interaction of the Ca V β 1b -subunits with the RGK-protein Gem. Our data obtained at both, whole-cell and single-channel levels, show that compared to a wild-type Ca V β 1b , the Ca V β 1b&#95;R296C variant inhibits L- and N-type VGCCs. Interaction with and modulation by the RGK-protein Gem seems to be intact. Our findings indicate functional effects of the Ca V β 1b&#95;R296C variant differing from that attributed to Ca V β 2 variants found in ASD patients. Further studies have to detail the effects on different VGCC subtypes and on VGCC expression., (© 2022. The Author(s).)

Ca V 2.3 channels are subthreshold voltage-gated calcium channels that play crucial roles in neurotransmitter release and regulation of membrane excitability, yet modulation of these channels with endogenous molecules and their role in pain processing is not well studied. Here, we hypothesized that an endogenous amino acid l-cysteine could be a modulator of these channels and may affect pain processing in mice. To test this hypothesis, we employed conventional patch-clamp technique in the whole-cell configuration using recombinant Ca V 2.3 subunit stably expressed in human embryonic kidney (HEK-293) cells. We found in our in vitro experiments that l-cysteine facilitated gating and increased the amplitudes of recombinant Ca V 2.3 currents likely by chelating trace metals that tonically inhibit the channel. In addition, we took advantage of mouse genetics in vivo using the acetic acid visceral pain model that was performed on wildtype and homozygous Cacna1e knockout male littermates. In ensuing in vivo experiments, we found that l-cysteine administered both subcutaneously and intraperitoneally evoked more prominent pain responses in the wildtype mice, while the effect was completely abolished in knockout mice. Conversely, intrathecal administration of l-cysteine lowered visceral pain response in the wildtype mice, and again the effect was completely abolished in the knockout mice. Our study strongly suggests that l-cysteine-mediated modulation of Ca V 2.3 channels plays an important role in visceral pain processing. Furthermore, our data are consistent with the contrasting roles of Ca V 2.3 channels in mediating visceral nociception in the peripheral and central pain pathways., (© 2022. The Author(s).)

Combining cell type-specific optogenetics and whole cell recordings on mouse acute hippocampal slices, we compared GABA release from cholecystokinin-expressing (CCK) and parvalbumin-expressing (PV) interneurons onto CA1 pyramidal neurons. Baclofen, a selective GABA B receptor agonist, inhibited GABAergic synaptic transmission greater from CCK terminals, compared to that from PV terminals. The N-type calcium channels on CCK and P/Q-type calcium channels on PV terminals contributed to the GABA B receptor-mediated inhibition, respectively. Our data thus provide direct evidence that GABA B receptors differentially modulate GABA release from CCK and PV interneurons, adding to an increasing list of differences between these two interneuron subtypes in modulating hippocampal pyramidal neurons., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Purpose: This study mainly aimed to explore the influences of Calcium Voltage-Gated Channel Subunit Alpha1 B (CACNA1B) on the development of breast cancer and the related mechanism., Materials and Methods: The information of patients with breast cancer from TCGA database was used for analyses of CACNA1B expression and its prognostic value. Loss- and gain- of functions of CACNA1B were conducted in MCF7 and Bcap-37 cells, respectively. CCK-8, colony formation and transwell assays were applied for evaluating the cell viability and motility. Western blot was used for protein expression detection., Results: We revealed that highly expressed CACNA1B in breast cancer tissues was related to poor prognosis according to the data gained from TCGA database. The outcomes of functional assays showed that depletion of CACNA1B restrained MCF7 cell growth, invasion and migration and high-expression of CACNA1B fortified the growth, invasion and migration in Bcap-37 cells. Finally, we manifested that silencing CACNA1B obviously raised the protein expression level of E-cadherin and reduced the protein levels of Cyclin D1, N-cadherin and Snail in MCF7 cells, whilst, over-expression of CACNA1B reduced the level of E-cadherin and increased the expression of Cyclin D1, N-cadherin and Snail in Bcap-37 cells., Conclusions: These results identified CACNA1B as a forwarder of the growth, invasion and migration in breast cancer cells.

The differentiation of adipose tissue-derived stem cells (ASCs) to neuronal cells is greatly promoted by valproic acid (VPA), and is synergistically enhanced by the following treatment with neuronal induction medium (NIM) containing cAMP-elevating agents. In the present study, we investigated the synergism between VPA and NIM in neuronal differentiation of ASCs, assessed by the expression of neurofilament medium polypeptide (NeFM), with respect to Ca 2+ entry. VPA (2 mM) treatment for 3 days followed by NIM for 2 h synergistically increased the incidence of neuronal cells differentiated from ASCs to an extent more than VPA alone treatment for 6 days, shortening the time required for the differentiation. VPA increased intracellular Ca 2+ and the mRNAs of voltage-gated Ca 2+ channels, Cacna1b (Cav2.2) and Cacna1h (Cav3.2), in ASCs. Inward currents through Ca 2+ channels were evoked electrophysiologically at high voltage potential in ASCs treated with VPA. NIM reduced the mRNAs of NeFM and Cacna1b in VPA-promoted neuronal differentiation of ASCs. It was concluded that functional N-type voltage-gated Ca 2+ channels (Cav2.2) are selectively expressed in VPA-promoted neuronal differentiation of ASCs. NIM seems to enhance the mRNA translation of molecules required for the differentiation. Neuronal cells obtained from ASCs by this protocol will be used as a cell source for regenerative therapy of neurological disorders associated with altered Cav2.2 activity., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 GABA B receptors (GABA B R) inhibiting HVA N-type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba 2+ currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABA B R antagonist CGP 55845. In transfected HEK293T cells co-expressing human GABA B R1 and R2 subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization-activated Ba 2+ 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 GABA B 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 GABA B R in HEK293T cells. This study highlights GABA B R as a potentially important receptor target for the activity of αO-conotoxin GeXIVA to mediate analgesia., (© 2021 International Society for Neurochemistry.)

Background and Purpose: Activation of GIRK channels via G protein-coupled GABA B receptors has been shown to attenuate nociceptive transmission. The analgesic α-conotoxin Vc1.1 activates GABA B receptors resulting in inhibition of Ca v 2.2 and Ca v 2.3 channels in mammalian primary afferent neurons. Here, we investigated the effects of analgesic α-conotoxins on recombinant and native GIRK-mediated K + currents and on neuronal excitability., Experimental Approach: The effects of analgesic α-conotoxins, Vc1.1, RgIA, and PeIA, were investigated on inwardly-rectifying K + currents in HEK293T cells recombinantly co-expressing either heteromeric human GIRK1/2 or homomeric GIRK2 subunits, with GABA B receptors. The effects of α-conotoxin Vc1.1 and baclofen were studied on GIRK-mediated K + currents and the passive and active electrical properties of adult mouse dorsal root ganglion neurons., Key Results: Analgesic α-conotoxins Vc1.1, RgIA, and PeIA potentiate inwardly-rectifying K + currents in HEK293T cells recombinantly expressing human GIRK1/2 channels and GABA B receptors. GABA B receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen occurs via a pertussis toxin-sensitive G protein and is inhibited by the selective GABA B receptor antagonist CGP 55845. In adult mouse dorsal root ganglion neurons, GABA B receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen hyperpolarizes the cell membrane potential and reduces excitability., Conclusions and Implications: This is the first report of GIRK channel potentiation via allosteric α-conotoxin Vc1.1-GABA B receptor agonism, leading to decreased neuronal excitability. Such action potentially contributes to the analgesic effects of Vc1.1 and baclofen observed in vivo., (© 2021 The British Pharmacological Society.)

CACNA1A deletions cause epilepsy, ataxia, and a range of neurocognitive deficits, including inattention, impulsivity, intellectual deficiency and autism. To investigate the underlying mechanisms, we generated mice carrying a targeted Cacna1a deletion restricted to parvalbumin-expressing (PV) neurons (PV Cre ;Cacna1a c/+ ) or to cortical pyramidal cells (PC) (Emx1 Cre ;Cacna1a c/+ ). GABA release from PV-expressing GABAergic interneurons (PV-INs) is reduced in PV Cre ;Cacna1a c/+ mutants, resulting in impulsivity, cognitive rigidity and inattention. By contrast, the deletion of Cacna1a in PCs does not impact cortical excitability or behaviour in Emx1 Cre ;Cacna1a c/+ mutants. A targeted Cacna1a deletion in the orbitofrontal cortex (OFC) results in reversal learning deficits while a medial prefrontal cortex (mPFC) deletion impairs selective attention. These deficits can be rescued by the selective chemogenetic activation of cortical PV-INs in the OFC or mPFC of PV Cre ;Cacna1a c/+ mutants. Thus, Cacna1a haploinsufficiency disrupts perisomatic inhibition in frontal cortical circuits, leading to a range of potentially reversible neurocognitive deficits., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Structural studies with an α subunit fragment of voltage-gated calcium (CaV) channels in complex with the CaVβ subunits revealed a high homology between the various CaVα-β subunits, predicting that targeting of this interface would result in nonselective compounds. Despite this likelihood, my laboratory initiated a rational structure-based screening campaign focusing on "hot spots" on the alpha interacting domain (AID) of the CaVβ2a subunits and identified the small molecule 2-(3,5-dimethylisoxazol-4-yl)-N-((4-((3-phenylpropyl)amino)quinazolin-2-yl)methyl)acetamide ( IPPQ ) which selectively targeted the interface between the N-type calcium (CaV2.2) channel and CaVβ. IPPQ (i) specifically bound to CaVβ2a; (ii) inhibited CaVβ2 's interaction with CaV.2-AID; (iii) inhibited CaV2.2 currents in sensory neurons; (iv) inhibited pre-synaptic localization of CaV2.2 in vivo ; and (v) inhibited spinal neurotransmission, which resulted in decreased neurotransmitter release. IPPQ was anti-nociceptive in naïve rats and reversed mechanical allodynia and thermal hyperalgesia in rodent models of acute, neuropathic, and genetic pain. In structure-activity relationship (SAR) studies focused on improving binding affinity of IPPQ , another compound (BTT-369), a benzoyl-3,4-dihydro-1'H,2 H-3,4'-bipyrazole class of compounds, was reported by Chen and colleagues, based on work conducted in my laboratory beginning in 2008. BTT-369 contains tetraaryldihydrobipyrazole scaffold - a chemotype featuring phenyl groups known to be significantly metabolized, lower the systemic half-life, and increase the potential for toxicity. Furthermore, the benzoylpyrazoline skeleton in BTT-369 is patented across multiple therapeutic indications. Prior to embarking on an extensive optimization campaign of IPPQ , we performed a head-to-head comparison of the two compounds. We conclude that IPPQ is superior to BTT-369 for on-target efficacy, setting the stage for SAR studies to improve on IPPQ for the development of novel pain therapeutics.

N-type voltage-gated calcium (Ca V ) channels mediate Ca 2+ influx at presynaptic terminals in response to action potentials and play vital roles in synaptogenesis, release of neurotransmitters, and nociceptive transmission. Here, we elucidate a cryo-electron microscopy (cryo-EM) structure of the human Ca V 2.2 complex in apo, ziconotide-bound, and two Ca V 2.2-specific pore blockers-bound states. The second voltage-sensing domain (VSD) is captured in a resting-state conformation, trapped by a phosphatidylinositol 4,5-bisphosphate (PIP 2 ) molecule, which is distinct from the other three VSDs of Ca V 2.2, as well as activated VSDs observed in previous structures of Ca V channels. This structure reveals the molecular basis for the unique inactivation process of Ca V 2.2 channels, in which the intracellular gate formed by S6 helices is closed and a W-helix from the domain II-III linker stabilizes closed-state inactivation. The structures of this inactivated, drug-bound complex lay a solid foundation for developing new state-dependent blockers for treatment of chronic pain., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Peptides isolated from spider venoms are of pharmacological interest due to their neurotoxic activity, acting on voltage-dependent ion channels present in different types of human body tissues. Three peptide toxins titled as Ap2, Ap3 and Ap5 were purified by RP-HPLC from Acanthoscurria paulensis venom. They were partially sequenced by MALDI In-source Decay method and their sequences were completed and confirmed by transcriptome analysis of the venom gland. The Ap2, Ap3 and Ap5 peptides have, respectively, 42, 41 and 46 amino acid residues, and experimental molecular masses of 4886.3, 4883.7 and 5454.7 Da, with the Ap2 peptide presenting an amidated C-terminus. Amongst the assayed channels - NaV1.1, NaV1.5, NaV1.7, CaV1.2, CaV2.1 and CaV2.2 - Ap2, Ap3 and Ap5 inhibited 20-30 % of CaV2.1 current at 1 μM concentration. Ap3 also inhibited sodium current in NaV1.1, Nav1.5 and Nav1.7 channels by 6.6 ± 1.91 % (p = 0.0276), 4.2 ± 1.09 % (p = 0.0185) and 16.05 ± 2.75 % (p = 0.0282), respectively. Considering that Ap2, Ap3 and Ap5 belong to the 'U'-unknown family of spider toxins, which has few descriptions of biological activity, the present work contributes to the knowledge of these peptides and demonstrates this potential as channel modulators., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Voltage-gated Ca V 2.2 calcium channels are expressed in nociceptors at presynaptic terminals, soma, and axons. Ca V 2.2 channel inhibitors applied to the spinal cord relieve pain in humans and rodents, especially during pathologic pain, but a biological function of nociceptor Ca V 2.2 channels in processing of nociception, outside presynaptic terminals in the spinal cord, is underappreciated. Here, we demonstrate that functional Ca V 2.2 channels in peripheral axons innervating skin are required for capsaicin-induced heat hypersensitivity in male and female mice. We show that Ca V 2.2 channels in TRPV1-nociceptor endings are activated by capsaicin-induced depolarization and contribute to increased intracellular calcium. Capsaicin induces hypersensitivity of both thermal nociceptors and mechanoreceptors, but only heat hypersensitivity depends on peripheral Ca V 2.2 channel activity, and especially a cell-type-specific Ca V 2.2 splice isoform. Ca V 2.2 channels at peripheral nerve endings might be important therapeutic targets to mitigate certain forms of chronic pain. SIGNIFICANCE STATEMENT It is generally assumed that nociceptor termini in the spinal cord dorsal horn are the functionally significant sites of Ca V 2.2 channel in control of transmitter release and the transmission of sensory information from the periphery to central sites. We show that peripheral Ca V 2.2 channels are essential for the classic heat hypersensitivity response to develop in skin following capsaicin exposure. This function of Ca V 2.2 is highly selective for heat, but not mechanical hypersensitivity induced by capsaicin exposure, and is not a property of closely related Ca V 2.1 channels. Our findings suggest that interrupting Ca V 2.2-dependent calcium entry in skin might reduce heat hypersensitivity that develops after noxious heat exposure and may limit the degree of heat hypersensitivity associated with certain other forms of pain., (Copyright © 2021 DuBreuil, Lopez Soto et al.)

Although quiescent hepatic stellate cells (HSCs) have been suggested to regulate hepatic blood flow, there is no direct evidence that quiescent HSCs display contractile abilities. Here, we developed a new method to quantitatively measure the contraction of single isolated HSCs and evaluated whether endothelin-1 (ET-1) induced contraction of HSCs in a non-activated state. HSCs isolated from mice were seeded on collagen gel containing fluorescent beads. The beads around a single HSC were observed gravitating toward the cell upon contraction. By recording the movement of each bead by fluorescent microscopy, the real-time contraction of HSCs was quantitatively evaluated. ET-1 induced a slow contraction of non-activated HSCs, which was inhibited by the non-muscle myosin II inhibitor blebbistatin, the calmodulin inhibitor W-7, and the ETA receptor antagonist ambrisentan. ET-1-induced contraction was also largely reduced in Ca2+-free conditions, but sustained contraction still remained. The tonic contraction was further diminished by the Rho-kinase inhibitor H-1152. The mRNA expression of P/Q-type voltage-dependent Ca2+ channels (VDCC), as well as STIM and Orai, constituents of store-operated channels (SOCs), was observed in mouse non-activated HSCs. ET-1-induced contraction was not affected by amlodipine, a VDCC blocker, whereas it was partly reduced by Gd3+ and amiloride, non-selective cation channel blockers. However, neither YM-58483 nor SKF-96365, which inhibit SOCs, had any effects on the contraction. These results suggest that ET-1 leads to Ca2+-influx through cation channels other than SOCs and produces myosin II-mediated contraction of non-activated HSCs via ETA receptors, as well as via mechanisms involving Ca2+-calmodulin and Rho kinase., Competing Interests: The authors have declared that no competing interests exist.

The neuronal-type (N-type) voltage-gated calcium (Ca v ) channels, which are designated Ca v 2.2, have an important role in the release of neurotransmitters 1-3 . Ziconotide is a Ca v 2.2-specific peptide pore blocker that has been clinically used for treating intractable pain 4-6 . Here we present cryo-electron microscopy structures of human Ca v 2.2 (comprising the core α1 and the ancillary α2δ-1 and β3 subunits) in the presence or absence of ziconotide. Ziconotide is thoroughly coordinated by helices P1 and P2, which support the selectivity filter, and the extracellular loops (ECLs) in repeats II, III and IV of α1. To accommodate ziconotide, the ECL of repeat III and α2δ-1 have to tilt upward concertedly. Three of the voltage-sensing domains (VSDs) are in a depolarized state, whereas the VSD of repeat II exhibits a down conformation that is stabilized by Ca v 2-unique intracellular segments and a phosphatidylinositol 4,5-bisphosphate molecule. Our studies reveal the molecular basis for Ca v 2.2-specific pore blocking by ziconotide and establish the framework for investigating electromechanical coupling in Ca v channels., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Objectives: Neuropathic pain (NP) is a chronic inflammation of the sciatic nerve, associated with complex pathophysiological events like neuronal ectopic discharge with changes in neurotransmitters, growth factors, receptors/ion channels including N-methyl-d-aspartate receptors, Transient receptor cation channels, Voltage-gated calcium channels. All these events eventually lead to inflammation and apoptosis of the sciatic nerve in NP. Icariin (ICA), a natural flavonoid is well known for its anti-inflammatory potential. Hence, the present study is designed to evaluate its anti-inflammatory potential against neuropathic pain using in silico and in vivo studies., Methods: In silico studies were conducted using targets of N-methyl-D-aspartate receptor subtype-2B (NR2B), The capsaicin receptor transient receptor cation channel subfamily-V member-1 (TRPV1), N-type voltage-gated calcium (CaV2.2) channels. In in vivo studies, after partial sciatic nerve ligation surgery to animals, received their respective treatment for 21 days, further TNF-α, IL-6, Bax (proapoptotic) and Bcl-2 (antiapoptotic) expressions were estimated., Key Findings: ICA decreased the expressions of TNF-α, IL-6, Bax and increased expression of Bcl-2. In silico studies revealed a good energy binding score towards NR2B, TRPV1 receptors and CaV2.2 ion Channel., Conclusions: ICA could be a promising agent in alleviating neuropathic pain by inhibiting NR2B, TRPV1 receptors and Cav2.2 channels, which induces anti-apoptotic potential and inhibits inflammation., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Zinc, an abundant transition metal, serves as a signalling molecule in several biological systems. Zinc transporters are genetically associated with cardiovascular diseases but the function of zinc in vascular tone regulation is unknown. We found that elevating cytoplasmic zinc using ionophores relaxed rat and human isolated blood vessels and caused hyperpolarization of smooth muscle membrane. Furthermore, zinc ionophores lowered blood pressure in anaesthetized rats and increased blood flow without affecting heart rate. Conversely, intracellular zinc chelation induced contraction of selected vessels from rats and humans and depolarized vascular smooth muscle membrane potential. We demonstrate three mechanisms for zinc-induced vasorelaxation: (1) activation of transient receptor potential ankyrin 1 to increase calcitonin gene-related peptide signalling from perivascular sensory nerves; (2) enhancement of cyclooxygenase-sensitive vasodilatory prostanoid signalling in the endothelium; and (3) inhibition of voltage-gated calcium channels in the smooth muscle. These data introduce zinc as a new target for vascular therapeutics.

Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER) chaperones implicated in neuropathic pain. Here we examine if the Sig-1R may relate to neuropathic pain at the level of dorsal root ganglia (DRG). We focus on the neuronal excitability of DRG in a "spare nerve injury" (SNI) model of neuropathic pain in rats and find that Sig-1Rs likely contribute to the genesis of DRG neuronal excitability by decreasing the protein level of voltage-gated Cav2.2 as a translational inhibitor of mRNA. Specifically, during SNI, Sig-1Rs translocate from ER to the nuclear envelope via a trafficking protein Sec61β. At the nucleus, the Sig-1R interacts with cFos and binds to the promoter of 4E-BP1, leading to an upregulation of 4E-BP1 that binds and prevents eIF4E from initiating the mRNA translation for Cav2.2. Interestingly, in Sig-1R knockout HEK cells, Cav2.2 is upregulated. In accordance with those findings, we find that intra-DRG injection of Sig-1R agonist (+)pentazocine increases frequency of action potentials via regulation of voltage-gated Ca2+ channels. Conversely, intra-DRG injection of Sig-1R antagonist BD1047 attenuates neuropathic pain. Hence, we discover that the Sig-1R chaperone causes neuropathic pain indirectly as a translational inhibitor.

Glutamate is the major excitatory neurotransmitter in the brain and is involved in many brain functions. In this study, we investigated whether typhaneoside, a flavonoid from Typhae angustifolia pollen, affects endogenous glutamate release from rat cortical synaptosomes. Using a one-line enzyme-coupled fluorometric assay, glutamate release stimulated by the K + channel blocker 4-aminopyridine was monitored to explore the possible underlying mechanisms. The vesicular transporter inhibitor bafilomycin A1 and chelation of extracellular Ca 2+ ions with EGTA suppressed the effect of typhaneoside on the induced glutamate release. Nevertheless, the typhaneoside activity has not been affected by the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate. The synaptosomal plasma membrane potential was assayed using a membrane potential-sensitive dye DiSC 3 (5), and cytosolic Ca 2+ concentrations ([Ca 2+ ] C ) was monitored by a Ca 2+ indicator Fura-2. Results showed that typhaneoside did not alter the synaptosomal membrane potential but lowered 4-aminopyridine-induced increases in [Ca 2+ ] C . Furthermore, the Ca v 2.2 (N-type) channel blocker ω-conotoxin GVIA blocked Ca 2+ entry and inhibited the effect of typhaneoside on 4-aminopyridine-induced glutamate release. However, the inhibitor of intracellular Ca 2+ release dantrolene and the mitochondrial Na + /Ca 2+ exchanger blocker 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one have no effect on the suppression of glutamate release mediated by typhaneoside. Moreover, inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) prevented the inhibitory effect of typhaneoside on induced glutamate release. Typhaneoside reduced 4-aminopyridine-induced phosphorylation of ERK1/2 and the major presynaptic ERK target synapsin I, which is a synaptic vesicle-associated protein. In conclusion, these findings suggest a role for typhaneoside in modulating glutamate release by suppressing voltage-dependent Ca 2+ channel mediated presynaptic Ca 2+ influx and the MAPK/ERK/synapsin I signaling cascade.

N-type voltage-gated calcium channels (Ca V 2.2) are predominantly expressed at presynaptic terminals, and their function is regulated by auxiliary α 2 δ and β subunits. All four mammalian α 2 δ subunits enhance calcium currents through Ca V 1 and Ca V 2 channels, and this increase is attributed, in part, to increased Ca V expression at the plasma membrane. In the present study we provide evidence that α 2 δ-1, like α 2 δ-2, is recycled to the plasma membrane through a Rab11a-dependent endosomal recycling pathway. Using a dominant-negative Rab11a mutant, Rab11a(S25N), we show that α 2 δ-1 increases plasma membrane Ca V 2.2 expression by increasing the rate and extent of net forward Ca V 2.2 trafficking in a Rab11a-dependent manner. Dominant-negative Rab11a also reduces the ability of α 2 δ-1 to increase Ca V 2.2 expression on the cell-surface of hippocampal neurites. In contrast, α 2 δ-3 does not enhance rapid forward Ca V 2.2 trafficking, regardless of whether Rab11a(S25N) is present. In addition, whole-cell Ca V 2.2 currents are reduced by co-expression of Rab11a(S25N) in the presence of α 2 δ-1, but not α 2 δ-3. Taken together these data suggest that α 2 δ subtypes participate in distinct trafficking pathways which in turn influence the localisation and function of Ca V 2.2.

Cardiac sympathetic overactivation is involved in arrhythmogenesis in patients with chronic heart failure (CHF). Inflammatory infiltration in the stellate ganglion (SG) is a critical factor for cardiac sympathoexcitation in patients with ventricular arrhythmias. This study aims to investigate if macrophage depletion in SGs decreases cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF. Surgical ligation of the coronary artery was used for induction of CHF. Clodronate liposomes were microinjected into bilateral SGs of CHF rats for macrophage depletion. Using cytokine array, immunofluorescence staining, and Western blot analysis, we found that macrophage expansion and expression of TNFα and IL-1β in SGs were markedly increased in CHF rats. Flow cytometry data confirmed that the percentage of macrophages in SGs was higher in CHF rats than that in sham rats. Clodronate liposomes significantly reduced CHF-elevated proinflammatory cytokine levels and macrophage expansion in SGs. Clodronate liposomes also reduced CHF-increased N-type Ca 2+ currents and excitability of cardiac sympathetic postganglionic neurons and inhibited CHF-enhanced cardiac sympathetic nerve activity. ECG data from 24-h, continuous telemetry recording in conscious rats demonstrated that clodronate liposomes not only restored CHF-induced heterogeneity of ventricular electrical activities, but also decreased the incidence and duration of ventricular tachycardia/fibrillation in CHF. Macrophage depletion with clodronate liposomes attenuated CHF-induced cardiac sympathetic overactivation and ventricular arrhythmias through reduction of macrophage expansion and neuroinflammation in SGs.

Junctions between the endoplasmic reticulum and plasma membrane that are induced by the neuronal junctophilins are of demonstrated importance, but their molecular architecture is still poorly understood and challenging to address in neurons. This is due to the small size of the junctions and the multiple isoforms of candidate junctional proteins in different brain areas. Using colocalization of tagged proteins expressed in tsA201 cells, and electrophysiology, we compared the interactions of JPH3 and JPH4 with different calcium channels. We found that JPH3 and JPH4 caused junctional accumulation of all the tested high-voltage-activated Ca V isoforms, but not a low-voltage-activated Ca V . Also, JPH3 and JPH4 noticeably modify Ca V 2.1 and Ca V 2.2 inactivation rate. RyR3 moderately colocalized at junctions with JPH4, whereas RyR1 and RyR2 did not. By contrast, RyR1 and RyR3 strongly colocalized with JPH3, and RyR2 moderately. Likely contributing to this difference, JPH3 binds to cytoplasmic domain constructs of RyR1 and RyR3, but not of RyR2., Competing Interests: SP No competing interests declared, KB Reviewing editor, eLife, (© 2021, Perni and Beam.)