Ca(V)1 and Ca(V)2 channels engage distinct modes of Ca(2+) signaling to control CREB-dependent gene expression.

Activity-dependent gene expression triggered by Ca(2+) entry into neurons is critical for learning and memory, but whether specific sources of Ca(2+) act distinctly or merely supply Ca(2+) to a common pool remains uncertain. Here, we report that both signaling modes coexist and pertain to Ca(V)1 and Ca(V)2 channels, respectively, coupling membrane depolarization to CREB phosphorylation and gene expression. Ca(V)1 channels are advantaged in their voltage-dependent gating and use nanodomain Ca(2+) to drive local CaMKII aggregation and trigger communication with the nucleus. In contrast, Ca(V)2 channels must elevate [Ca(2+)](i) microns away and promote CaMKII aggregation at Ca(V)1 channels. Consequently, Ca(V)2 channels are ~10-fold less effective in signaling to the nucleus than are Ca(V)1 channels for the same bulk [Ca(2+)](i) increase. Furthermore, Ca(V)2-mediated Ca(2+) rises are preferentially curbed by uptake into the endoplasmic reticulum and mitochondria. This source-biased buffering limits the spatial spread of Ca(2+), further attenuating Ca(V)2-mediated gene expression.

Structure of human Cav2.2 channel blocked by the painkiller ziconotide.

The neuronal-type (N-type) voltage-gated calcium (Cav) channels, which are designated Cav2.2, have an important role in the release of neurotransmitters1-3. Ziconotide is a Cav2.2-specific peptide pore blocker that has been clinically used for treating intractable pain4-6. Here we present cryo-electron microscopy structures of human Cav2.2 (comprising the core α1 and the ancillary α2δ-1 and ß3 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 Cav2-unique intracellular segments and a phosphatidylinositol 4,5-bisphosphate molecule. Our studies reveal the molecular basis for Cav2.2-specific pore blocking by ziconotide and establish the framework for investigating electromechanical coupling in Cav channels.

RIM and RIM-BP Form Presynaptic Active-Zone-like Condensates via Phase Separation.

Both the timing and kinetics of neurotransmitter release depend on the positioning of clustered Ca2+ channels in active zones to docked synaptic vesicles on presynaptic plasma membranes. However, how active zones form is not known. Here, we show that RIM and RIM-BP, via specific multivalent bindings, form dynamic and condensed assemblies through liquid-liquid phase separation. Voltage-gated Ca2+ channels (VGCCs), via C-terminal-tail-mediated direct binding to both RIM and RIM-BP, can be enriched to the RIM and RIM-BP condensates. We further show that RIM and RIM-BP, together with VGCCs, form dense clusters on the supported lipid membrane bilayers via phase separation. Therefore, RIMs and RIM-BPs are plausible organizers of active zones, and the formation of RIM and RIM-BP condensates may cluster VGCCs into nano- or microdomains and position the clustered Ca2+ channels with Ca2+ sensors on docked vesicles for efficient and precise synaptic transmissions.

Synaptotagmin-11 facilitates assembly of a presynaptic signaling complex in post-Golgi cargo vesicles.

GABAB 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 Ca2+ 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.

Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics.

Increased cardiac contractility during the fight-or-flight response is caused by ß-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and ß subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of ß-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which ß-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or ß2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during ß-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for ß subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.

A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (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 Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 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 Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ 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 Cav2.2 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.

Active Zone Trafficking of CaV2/UNC-2 Channels Is Independent of ß/CCB-1 and α2δ/UNC-36 Subunits.

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 α1 subunit and two auxiliary ß and α2δ subunits. Although auxiliary subunits are critical for channel function, whether they are required for α1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans, we show that UNC-2/α1 localizes to AZs even in the absence of CCB-1/ß or UNC-36/α2δ, 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 α1 subunit and two auxiliary ß and α2δ 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 α1 and aid trafficking, they are recruited to α1 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.

Subcellular localization and ER-mediated cytotoxic function of α1A and α1ACT in spinocerebellar ataxia type 6.

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-polyQlong localized mainly to the Golgi apparatus, whereas a portion of α1ACT-polyQlong 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.

CACNA1B protects naked mole-rat hippocampal neuron from apoptosis via altering the subcellular localization of Nrf2 after 60Co irradiation.

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 60Co 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.

Mechanism of local and global Ca2+ sensing by calmodulin in complex with a Ca2+ channel.

Calmodulin (CaM) in complex with Ca(2+) channels constitutes a prototype for Ca(2+) sensors that are intimately colocalized with Ca(2+) sources. The C-lobe of CaM senses local, large Ca(2+) oscillations due to Ca(2+) influx from the host channel, and the N-lobe senses global, albeit diminutive Ca(2+) changes arising from distant sources. Though biologically essential, the mechanism underlying global Ca(2+) sensing has remained unknown. Here, we advance a theory of how global selectivity arises, and we experimentally validate this proposal with methodologies enabling millisecond control of Ca(2+) oscillations seen by the CaM/channel complex. We find that global selectivity arises from rapid Ca(2+) release from CaM combined with greater affinity of the channel for Ca(2+)-free versus Ca(2+)-bound CaM. The emergence of complex decoding properties from the juxtaposition of common elements, and the techniques developed herein, promise generalization to numerous molecules residing near Ca(2+) sources.

Primary and secondary motoneurons use different calcium channel types to control escape and swimming behaviors in zebrafish.

The escape response and rhythmic swimming in zebrafish are distinct behaviors mediated by two functionally distinct motoneuron (Mn) types. The primary (1°Mn) type depresses and has a large quantal content (Qc) and a high release probability (Pr). Conversely, the secondary (2°Mn) type facilitates and has low and variable Qc and Pr. This functional duality matches well the distinct associated behaviors, with the 1°Mn providing the strong, singular C bend initiating escape and the 2°Mn conferring weaker, rhythmic contractions. Contributing to these functional distinctions is our identification of P/Q-type calcium channels mediating transmitter release in 1°Mns and N-type channels in 2°Mns. Remarkably, despite these functional and behavioral distinctions, all ∼15 individual synapses on each muscle cell are shared by a 1°Mn bouton and at least one 2°Mn bouton. This blueprint of synaptic sharing provides an efficient way of controlling two different behaviors at the level of a single postsynaptic cell.

Heat But Not Mechanical Hypersensitivity Depends on Voltage-Gated CaV2.2 Calcium Channel Activity in Peripheral Axon Terminals Innervating Skin.

Voltage-gated CaV2.2 calcium channels are expressed in nociceptors at presynaptic terminals, soma, and axons. CaV2.2 channel inhibitors applied to the spinal cord relieve pain in humans and rodents, especially during pathologic pain, but a biological function of nociceptor CaV2.2 channels in processing of nociception, outside presynaptic terminals in the spinal cord, is underappreciated. Here, we demonstrate that functional CaV2.2 channels in peripheral axons innervating skin are required for capsaicin-induced heat hypersensitivity in male and female mice. We show that CaV2.2 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 CaV2.2 channel activity, and especially a cell-type-specific CaV2.2 splice isoform. CaV2.2 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 CaV2.2 channel in control of transmitter release and the transmission of sensory information from the periphery to central sites. We show that peripheral CaV2.2 channels are essential for the classic heat hypersensitivity response to develop in skin following capsaicin exposure. This function of CaV2.2 is highly selective for heat, but not mechanical hypersensitivity induced by capsaicin exposure, and is not a property of closely related CaV2.1 channels. Our findings suggest that interrupting CaV2.2-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.

Analgesic α-Conotoxin Binding Site on the Human GABAB Receptor.

The analgesic α-conotoxins Vc1.1, RgIA, and PeIA attenuate nociceptive transmission via activation of G protein-coupled GABAB receptors (GABABRs) to modulate N-type calcium channels in primary afferent neurons and recombinantly coexpressed human GABABR 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 GABABR 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 GABABR ligand-binding site. The effects of baclofen and α-conotoxins on the peak Ba2+ current (IBa) amplitude were investigated on wild-type and 15 GABABR mutants individually coexpressed with human Cav2.2 channels. Mutations at the interface of the VFT domains of both GABABR subunits attenuated baclofen-sensitive IBa inhibition by the analgesic α-conotoxins. In contrast, mutations located outside the putative peptide-binding site (D380A and R98A) did not. The key GABABR 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 IBa mediated by both wild-type and all GABABR mutants were inhibited by the selective GABABR antagonist CGP 55845. This study identifies specific residues of GABABR involved in the binding of the analgesic α-conotoxins to the VFT domains of the GABABR. SIGNIFICANCE STATEMENT: This study defines the binding site of the analgesic α-conotoxins Vc1.1, RgIA, and PeIA on the human GABAB receptor to activate Gi/o proteins and inhibit Cav2.2 channels. Computational docking and molecular dynamics simulations of GABABR identified amino acids of the Venus flytrap (VFT) domains with which the α-conotoxins interact. GABABR 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 GABABR subunits for the analgesic α-conotoxins.

A high-affinity, partial antagonist effect of 3,4-diaminopyridine mediates action potential broadening and enhancement of transmitter release at NMJs.

3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach ∼1 µM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert-Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype ("L-type") of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP's mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1-10 µM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1-1 mM) antagonist activity. We also showed that 1.5-µM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-µM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-µM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.

L-cysteine modulates visceral nociception mediated by the CaV2.3 R-type calcium channels.

CaV2.3 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 CaV2.3 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 CaV2.3 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 CaV2.3 channels plays an important role in visceral pain processing. Furthermore, our data are consistent with the contrasting roles of CaV2.3 channels in mediating visceral nociception in the peripheral and central pain pathways.

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 R2 subunits 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.

Valproic acid promotes differentiation of adipose tissue-derived stem cells to neuronal cells selectively expressing functional N-type voltage-gated Ca2+ channels.

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 Ca2+ 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 Ca2+ and the mRNAs of voltage-gated Ca2+ channels, Cacna1b (Cav2.2) and Cacna1h (Cav3.2), in ASCs. Inward currents through Ca2+ 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 Ca2+ 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.

CACNA1B facilitates breast cancer cell growth and migration by regulating cyclin D1 and EMT: the implication of CACNA1B in breast cancer.

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.

Histamine H3 Receptor Activation Modulates Glutamate Release in the Corticostriatal Synapse by Acting at CaV2.1 (P/Q-Type) Calcium Channels and GIRK (KIR3) Potassium Channels.

The striatum is innervated by histaminergic fibers and expresses a high density of histamine H3 receptors (H3Rs), 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 H3R activation and blockers of calcium and potassium channels was evaluated with the paired-pulse facilitation protocol. In the presence of the H3R antagonist/inverse agonist clobenpropit (1 µM), the H3R 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 CaV2.1 (P/Q-type) channels with ω-agatoxin TK (400 nM) increased PPR and prevented the effect of immepip. The CaV2.2 (N-type) channel blocker ω-conotoxin GVIA (1 µM) also increased PPR, but did not occlude the immepip action. Functional KIR3 channels are present in corticostriatal terminals, and in experiments in which immepip increased PPR, the KIR3 blocker tertiapin-Q (30 nM) prevented the effect of the H3R agonist. These results indicate that the presynaptic modulation by H3Rs of corticostriatal synapses involves the inhibition of Cav2.1 calcium channels and the activation of KIR3 potassium channels.

Reversing frontal disinhibition rescues behavioural deficits in models of CACNA1A-associated neurodevelopment disorders.

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 (PVCre;Cacna1ac/+) or to cortical pyramidal cells (PC) (Emx1Cre;Cacna1ac/+). GABA release from PV-expressing GABAergic interneurons (PV-INs) is reduced in PVCre;Cacna1ac/+ mutants, resulting in impulsivity, cognitive rigidity and inattention. By contrast, the deletion of Cacna1a in PCs does not impact cortical excitability or behaviour in Emx1Cre;Cacna1ac/+ 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 PVCre;Cacna1ac/+ mutants. Thus, Cacna1a haploinsufficiency disrupts perisomatic inhibition in frontal cortical circuits, leading to a range of potentially reversible neurocognitive deficits.