High Voltage-gated Calcium Channels Research Articles

Peptides derived from high voltage-gated calcium channel β subunit reduce blood pressure in rats.

The β subunits of high voltage-gated calcium channels (HGCCs) are essential for optimal channel functions such as channel gating, activation-inactivation kinetics, and trafficking to the membrane. In this study, we report for the first time the potent blood pressure-reducing effects of peptide fragments derived from the β subunits in anesthetized and non-anesthetized rats. Intravenous administration of 16-mer peptide fragments derived from the interacting regions of the β1 [cacb1(344-359)], β2 [cacb2(392-407)], β3 [cacb3(292-307)], and β4 [cacb4(333-348)] subunits with the main α-subunit of HGCC decreased arterial blood pressure in a dose-dependent manner for 5-8 min in anesthetized rats. In contrast, the peptides had no effect on the peak amplitudes of voltage-activated Ca2+ current upon their intracellular application into the acutely isolated trigeminal ganglion neurons. Further, a single mutated peptide of cacb1(344-359)-cacb1(344-359)K357R-showed consistent and potent effects and was crippled by a two-amino acid-truncation at the N-terminal or C-terminal end. By conjugating palmitic acid with the second amino acid (lysine) of cacb1(344-359)K357R (named K2-palm), we extended the blood pressure reduction to several hours without losing potency. This prolonged effect on the arterial blood pressure was also observed in non-anesthetized rats. On the other hand, the intrathecal administration of acetylated and amidated cacb1(344-359)K357R peptide did not change acute nociceptive responses induced by the intradermal formalin injection in the plantar surface of rat hindpaw. Overall, these findings will be useful for developing antihypertensives.

Dynorphin promotes stress-induced depressive behaviors by inhibiting ventral pallidal neurons in rats.

Endogenous dynorphin signaling via kappa opioid receptors (KORs) plays a key role in producing the depressive and aversive consequences of stress. We investigated the behavioral effects of the dynorphin/KOR system in the ventral pallidum (VP) and studied the underlying mechanisms. To investigate the effects of dynorphin on the VP, we conducted behavioral experiments after microinjection of drugs or shRNA and brain-slice electrophysiological recordings. Histological tracing and molecular biological experiments were used to identify the distribution of KORs and the possible sources of dynorphin projections to the VP. An elevated dynorphin concentration and increased KOR activity were observed in the VP after acute stress. Infusion of dynorphin-A into the VP produced depressive-like phenotypes including anhedonia and despair and anxiety behaviors, but did not alter locomotor behavior. Mechanistically, dynorphin had an inhibitory effect on VP neurons-reducing their firing rate and inhibiting excitatory transmission-through direct activation of KORs and modulation of downstream G-protein-gated inwardly rectifying potassium (GIRK) channels and high-voltage gated calcium channels (VGCCs). Tracing revealed direct innervation of VP neurons by dynorphin-positive projections; potential sources of these dynorphinergic projections include the nucleus accumbens, amygdala, and hypothalamus. Blockade of dynorphin/KOR signaling in the VP by drugs or viral knock-down of KORs significantly reduced despair behavior in rats. Endogenous dynorphinergic modulation of the VP plays a critical role in mediating depressive reactions to stress.

A Review of Genetic and Physiological Disease Mechanisms Associated With Cav1 Channels: Implications for Incomplete Congenital Stationary Night Blindness Treatment.

Calcium channels are crucial to a number of cellular functions. The high voltage-gated calcium channel family comprise four heteromeric channels (Cav1.1-1.4) that function in a similar manner, but that have distinct expression profiles. Three of the pore-forming α1 subunits are located on autosomes and the forth on the X chromosome, which has consequences for the type of pathogenic mutation and the disease mechanism associated with each gene. Mutations in this family of channels are associated with malignant hyperthermia (Cav1.1), various QT syndromes (Cav1.2), deafness (Cav1.3), and incomplete congenital stationary night blindness (iCSNB; Cav1.4). In this study we performed a bioinformatic analysis on reported mutations in all four Cav α1 subunits and correlated these with variant frequency in the general population, phenotype and the effect on channel conductance to produce a comprehensive composite Cav1 mutation analysis. We describe regions of mutation clustering, identify conserved residues that are mutated in multiple family members and regions likely to cause a loss- or gain-of-function in Cav1.4. Our research highlights that therapeutic treatments for each of the Cav1 channels will have to consider channel-specific mechanisms, especially for the treatment of X-linked iCSNB.

Calcium channels blockers toxins attenuate abdominal hyperalgesia and inflammatory response associated with the cerulein-induced acute pancreatitis in rats

Agents that modulate the activity of high-voltage gated calcium channels (HVCCs) exhibit experimentally and clinically significant effect by relieving visceral pain. Among these agents, the toxins Phα1β and ω-conotoxin MVIIA effectively reduce chronic pain in rodent models. The molecular mechanisms underlying the chronic pain associated with acute pancreatitis (AP) are poorly understood. Hypercalcemia is a risk factor; the role of cytosolic calcium is considered to be a modulator of pancreatitis. Blockade of Ca2+ signals may be useful as a prophylactic treatment of pancreatitis. We explored the pathophysiological roles of three peptide toxins: Phα1β and its recombinant form CTK 01512-2—blockers of TRPA1 receptor and HVCCs and ω-conotoxin MVIIA, a specific blocker of N-type calcium channels in cerulein-induced AP. Cerulein injection elicits AP in rats, evidenced by an increase in hyperalgesic pain, inflammatory infiltration, amylase and lipase secretion, and reactive oxygen species, TNF-α, and p65 NF-κB levels. These effects of cerulein-induced AP were abolished by Phα1β and its recombinant form CTK 01512-2, whereas ω-conotoxin MVIIA had no effect on the induced increase in pancreatic enzyme secretion. Our results demonstrate that Phα1β and CTK 01512-2 toxins—antagonists of HVCCs and TRPA1 receptor presented an effective response profile, in the control of nociception and inflammatory process in the AP model in rats, without causing changes in spontaneous locomotion of the rats.

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

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

Selected Ionotropic Receptors and Voltage-Gated Ion Channels: More Functional Competence for Human Induced Pluripotent Stem Cell (iPSC)-Derived Nociceptors.

Preclinical research using different rodent model systems has largely contributed to the scientific progress in the pain field, however, it suffers from interspecies differences, limited access to human models, and ethical concerns. Human induced pluripotent stem cells (iPSCs) offer major advantages over animal models, i.e., they retain the genome of the donor (patient), and thus allow donor-specific and cell-type specific research. Consequently, human iPSC-derived nociceptors (iDNs) offer intriguingly new possibilities for patient-specific, animal-free research. In the present study, we characterized iDNs based on the expression of well described nociceptive markers and ion channels, and we conducted a side-by-side comparison of iDNs with mouse sensory neurons. Specifically, immunofluorescence (IF) analyses with selected markers including early somatosensory transcription factors (BRN3A/ISL1/RUNX1), the low-affinity nerve growth factor receptor (p75), hyperpolarization-activated cyclic nucleotide-gated channels (HCN), as well as high voltage-gated calcium channels (VGCC) of the CaV2 type, calcium permeable TRPV1 channels, and ionotropic GABAA receptors, were used to address the characteristics of the iDN phenotype. We further combined IF analyses with microfluorimetric Ca2+ measurements to address the functionality of these ion channels in iDNs. Thus, we provide a detailed morphological and functional characterization of iDNs, thereby, underpinning their enormous potential as an animal-free alternative for human specific research in the pain field for unveiling pathophysiological mechanisms and for unbiased, disease-specific personalized drug development.

α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels.

Action potential-evoked neurotransmitter release is impaired in knock-out neurons lacking synaptic cell-adhesion molecules α-neurexins (αNrxns), the extracellularly longer variants of the three vertebrate Nrxn genes. Ca2+ influx through presynaptic high-voltage gated calcium channels like the ubiquitous P/Q-type (CaV2.1) triggers release of fusion-ready vesicles at many boutons. α2δ Auxiliary subunits regulate trafficking and kinetic properties of CaV2.1 pore-forming subunits but it has remained unclear if this involves αNrxns. Using live cell imaging with Ca2+ indicators, we report here that the total presynaptic Ca2+ influx in primary hippocampal neurons of αNrxn triple knock-out mice of both sexes is reduced and involved lower CaV2.1-mediated transients. This defect is accompanied by lower vesicle release, reduced synaptic abundance of CaV2.1 pore-forming subunits, and elevated surface mobility of α2δ-1 on axons. Overexpression of Nrxn1α in αNrxn triple knock-out neurons is sufficient to restore normal presynaptic Ca2+ influx and synaptic vesicle release. Moreover, coexpression of Nrxn1α together with α2δ-1 subunits facilitates Ca2+ influx further but causes little augmentation together with a different subunit, α2δ-3, suggesting remarkable specificity. Expression of defined recombinant CaV2.1 channels in heterologous cells validates and extends the findings from neurons. Whole-cell patch-clamp recordings show that Nrxn1α in combination with α2δ-1, but not with α2δ-3, facilitates Ca2+ currents of recombinant CaV2.1 without altering channel kinetics. These results suggest that presynaptic Nrxn1α acts as a positive regulator of Ca2+ influx through CaV2.1 channels containing α2δ-1 subunits. We propose that this regulation represents an important way for neurons to adjust synaptic strength.SIGNIFICANCE STATEMENT Synaptic transmission between neurons depends on the fusion of neurotransmitter-filled vesicles with the presynaptic membrane, which subsequently activates postsynaptic receptors. Influx of calcium ions into the presynaptic terminal is the key step to trigger vesicle release and involves different subtypes of voltage-gated calcium channels. We study the regulation of calcium channels by neurexins, a family of synaptic cell-adhesion molecules that are essential for many synapse properties. Using optical measurements of calcium influx in cultured neurons and electrophysiological recordings of calcium currents from recombinant channels, we show that a major neurexin variant facilitates calcium influx through P/Q-type channels by interacting with their α2δ-1 auxiliary subunits. These results propose a novel way how neurons can modulate the strength of distinct synapses.

DDX1 regulates alternative splicing and insulin secretion in pancreatic β cells

DEAD-box helicase 1 (DDX1) is a multifunction protein involved in diverse cellular processes including transcription, viral replication, mRNA/miRNA processing, and tRNA splicing. Here, we report a novel function of DDX1 in mRNA alternative splicing in pancreatic β cells. By performing integrated data analysis of high-throughput RNA sequencing (RNA-Seq), and cross-linking and immunoprecipitation coupled with deep sequencing (CLIP-Seq), we identify hundreds of alternative splicing genes that are targeted by DDX1. These DDX1-targeted alternative splicing genes are mainly associated with calcium ion binding, high voltage-gated calcium channel, and transmembrane transporter. Functionally, silencing DDX1 impairs calcium influx and insulin secretion in the pancreatic β cells. These results reveal an important role for DDX1 in the regulation of gene alternative splicing and insulin secretion in pancreatic β cells.

Trauma exposure interacts with the genetic risk of bipolar disorder in alcohol misuse of US soldiers.

To investigate whether trauma exposure moderates the genetic correlation between substance use disorders and psychiatric disorders, we tested whether trauma exposure modifies the association of genetic risks for mental disorders with alcohol misuse and nicotine dependence (ND) symptoms. High-resolution polygenic risk scores (PRSs) were calculated for 10 732 US Army soldiers (8346 trauma-exposed and 2386 trauma-unexposed) based on genome-wide association studies of bipolar disorder (BD), major depressive disorder, and schizophrenia. The main finding was a significant BD PRS-by-trauma interaction with respect to alcohol misuse (P = 6.07 × 10-3 ). We observed a positive correlation between BD PRS and alcohol misuse in trauma-exposed soldiers (r = 0.029, P = 7.5 × 10-3 ) and a negative correlation in trauma-unexposed soldiers (r = -0.071, P = 5.61 × 10-4 ). Consistent (nominally significant) result with concordant effect, directions were observed in the schizophrenia PRS-by-trauma interaction analysis. The variants included in the BD PRS-by-trauma interaction showed significant enrichments for gene ontologies related to high voltage-gated calcium channel activity (GO:0008331, P = 1.51 × 10-5 ; GO:1990454, P = 4.49 × 10-6 ; GO:0030315, P = 2.07 × 10-6 ) and for Beta1/Beta2 adrenergic receptor signaling pathways (P = 2.61 × 10-4 ). These results indicate that the genetic overlap between alcohol misuse and BD is significantly moderated by trauma exposure. This provides molecular insight into the complex mechanisms that link substance abuse, psychiatric disorders, and trauma exposure.

Dynamic association of calcium channel subunits at the cellular membrane.

High voltage gated calcium channels (VGCCs) are composed of at least three subunits, one pore forming [Formula: see text]-subunit, an intracellular [Formula: see text]-variant, and a mostly extracellular [Formula: see text]-variant. Interactions between these subunits determine the kinetic properties of VGCCs. It is unclear whether these interactions are stable over time or rather transient. Here, we used single-molecule tracking to investigate the surface diffusion of [Formula: see text]- and [Formula: see text]-subunits at the cell surface. We found that [Formula: see text]-subunits show higher surface mobility than [Formula: see text]-subunits, and that they are only transiently confined together, suggesting a weak association between [Formula: see text]- and [Formula: see text]-subunits. Moreover, we observed that different [Formula: see text]-subunits engage in different degrees of association with the [Formula: see text]-subunit, revealing the tighter interaction of [Formula: see text] with [Formula: see text]. These data indicate a distinct regulation of the [Formula: see text] interaction in VGCC subtypes. We modeled their membrane dynamics in a Monte Carlo simulation using experimentally determined diffusion constants. Our modeling predicts that the ratio of associated [Formula: see text]- and [Formula: see text]-subunits mainly depends on their expression density and confinement in the membrane. Based on the different motilities of particular [Formula: see text]-subunit combinations, we propose that their dynamic assembly and disassembly represent an important mechanism to regulate the signaling properties of VGCC.

Modulation of the Ca2 + signaling pathway by celangulin I in the central neurons of Spodoptera exigua

Celangulin I is an insecticidal component isolated from Chinese bittersweet Celastrus angulatus. The present study explored the possible effects of celangulin I on the calcium signaling pathway, especially on the L-type Ca2+ channel and the calcium channels in the endoplasmic reticulum in the central neurons isolated from the third instar larvae of Spodoptera exigua using whole-cell patch-clamp and calcium imaging technique. The results showed that celangulin I could activate the high voltage-gated calcium channel at the concentration of 150μM. The peak currents were increased by 17% of the initial value at the end of the 10-min recording after treated with celangulin I. The rises of intracellular calcium ion concentration ([Ca2+]i) in neurons treated by celangulin I showed that the effects of celangulin I were concentration-dependent. Activation of the RyRs by ryanodine decreased the calcium release induced by celangulin I, indicating that celangulin I exerts effect on insect RyRs. Furthermore, we also provided evidence for the first time that celangulin I activates inositol 1,4,5-trisphosphate (IP3) sensitive intracellular calcium release channels in the endoplasmic reticulum third instar larvae neurons of S. exigua. Plausibly, these experimental results can explain the characteristic symptoms of anesthesia and paralysis in celangulin I treated insects.

Homologous Serine/Threonine in the CaV 2.2α1 and 2.3α1 Subunits Behave Similarly, as Stimulatory and Inhibitory PKC Sites

High voltage-gated calcium (Cav) channels are regulated by PKC isozymes. These isozymes target selected serine/threonine (Ser/Thr) PKC phosphorylation sites in the intracellular regions of Cavα1 subunits of these channels. It has been found earlier using Cav2.2α1 subunits that stimulatory (Thr-422, Ser-2108 and Ser-2132) and inhibitory (Ser-425) PKC sites exist and their activation with PKC isozymes led to potentiation and depression of calcium currents (ICa) respectively. Based on the above report, it was planned to examine if the homologous sites in the Cav2.3α1 subunits behave similarly. In this regard the WT Cav2.3α1 or Ser/Thr Ala mutants of stimulatory (Thr-365, Ser-1995 and Ser-2011) or inhibitory (Ser-369) sites were expressed along with β1b and 2/δ cDNA subunits in Xenopus oocytes and the barium currents (IBa) were studied. Intracellular injection of PKC isozymes βII or e potentiated WT Cav2.3 currents. While both PKC βII and e potentiated IBa through Thr-365 (T365/S369A/S1995A/S2011A), only PKC e increased IBa through Ser-1995 (T365A/S369A/S1995/S2011A) channels. Ser-2011 failed to act as a stimulatory site contrary to its homologous site, Ser-2132 in the Cav2.2α1 subunits. However, Ser-369 acted as inhibitory site as its homolog Ser-425 in the Cav2.2α1 subunits. Both PKC βII and e inhibited IBa through Ser-369 (T365A/S369/S1995A/S2011A) channels. When both Thr-365 and Ser-369 were present (T365/S369/S1995A/S2011A), IBa was neither stimulated nor inhibited. However, stimulation was dominant when two stimulatory sites (Thr-365 & Ser-1995) were present along with Ser-369. Experiments with other mutants, including Ser/Thr Asp constructs are being studied and will be discussed.

Calcium Channel α2δ-1 Subunit Knockout Causes Diabetes Due to Impaired Insulin Release

Pancreatic β-cells, the major cellular component of the island of Langerhans, are responsible for the synthesis of insulin and its secretion in response to elevated blood glucose. High voltage-gated calcium channels (HVCC) are intimately involved in excitation-secretion coupling in pancreatic islet cells and calcium entering through HVCC is an important regulator of insulin synthesis. HVCC are multi-subunit protein complexes comprised of the main pore-forming α1 subunit and auxiliary extracellular α2δ and intracellular β subunits. Here we show that genetic ablation of the α2δ-1 subunit (the main pancreatic α2δ isoform) results in the postnatal development of diabetes. Homozygous α2δ-1 KO mice show highly elevated urine production and develop ∼9-fold higher blood glucose levels compared to WT littermates. Morphological analysis of the pancreas shows a reduction in the number of islets and their size due to a dramatic decrease in β-cell mass in an age-dependant manner. The reduced β-cell mass is not caused by an islet-specific autoimmune reaction, but might result from prolonged hyperglycaemia toxicity. Voltage-clamp recording in dissociated pancreatic β-cells shows a more than two-fold decrease in calcium current amplitude. Glucose stimulated calcium oscillations in whole isolated pancreatic islets shows a strong decrease in amplitude of both the first and second phase of insulin release, and an increased oscillation frequency of the second phase. On-going pharmacological experiments will identify which pore-forming α1 subunits are primarily affected by α2δ-1 deletion and how this effects insulin secretion. These findings indicate that α2δ-1 is an important determinant of normal β-cell physiology, critical for insulin release. Support: FWF W1101, P23479, LFU-P7400-027-011.

Knockout of the α2δ-1 Calcium Channel Subunit Alters Calcium Homeostasis and Electrical Activity in Pancreatic Islet Cells

Mouse pancreatic islets contain approximately 80% β-cells, 15% α-cells and 5% δ- and F-cells. High voltage-gated calcium channels (HVCC) regulate the biosynthesis and release of pancreatic hormones. Based on their pharmacological properties L-type calcium channels (LTCC) (Cav1.2 or Cav1.3) represent the predominant current component in islet cells. Insulin release depends primarily on LTCC and Cav2.3, whereas glucagon release relies on Cav2.1 or Cav2.2. All these α1 subunits form functional complexes with auxiliary channel subunits likely including α2δ-1. Here we investigate the role of the α2δ-1 subunit in calcium homeostasis and electrical activity of pancreatic α- and β-cells using α2δ-1 knockout mice. Voltage clamp experiments of freshly dissociated α- and β-cells show dramatically (2.5-fold) decreased calcium currents in α2δ-1-/- compared to wild-type cells. Previously we demonstrated that Cav1.2 lacking α2δ-1 subunits displays left-shifted voltage-dependence of activation and slowed calcium current kinetics. α- and β-cells of α2δ-1-/- mice showed little to no changes in the voltage-dependence of activation or kinetic properties of calcium influx compared to wild-type cells. Interestingly, the remaining current is almost completely blocked by the LTCC antagonist isradipine (2µM). Fluorescent calcium transients in response to high glucose (16.7 mM) showed a decreased amplitude and increased frequency of oscillations in isolated islets of α2δ-1-/- mice compared to wild-type. Analysis of low glucose-induced calcium oscillations in α-cells, and of the electrical activity of both cell types in intact islets are currently under way. Together, our results indicate that α2δ-1 is critical for membrane incorporation of calcium channels but does not affect LTCC gating properties in pancreatic endocrine cells. The reduced calcium current in response to glucose stimulus results in altered calcium transients and hormone release in pancreatic islets. Support: FWF W1101, P23479, LFU-P7400-027-011.

Exposure to extremely low-frequency electromagnetic fields inhibits T-type calcium channels via AA/LTE4 signaling pathway

Extremely low-frequency electromagnetic fields (ELF-EMF) causes various biological effects through altering intracellular calcium homeostasis. The role of high voltage-gated (HVA) calcium channels in ELF-EMF induced effects has been extensively studied. However, the effect of ELF-EMF on low-voltage-gated (LVA) T-type calcium channels has not been reported. In this study, we test the effect of ELF-EMF (50Hz) on human T-type calcium channels transfected in HEK293 cells. Conversely to its stimulant effects on HVA channels, ELF-EMF exposure inhibited all T-type (Cav3.1, Cav3.2 and Cav3.3) channels. Neither the protein expression nor the steady-state activation and inactivation kinetics of Cav3.2 channels were altered by ELF-EMF (50Hz, 0.2mT) exposure. Exposure to ELF-EMF increased both arachidonic acid (AA) and leukotriene E4 (LTE4) levels in HEK293 cells. CAY10502 and bestatin, which block the increase of AA and LTE4 respectively, abrogated the ELF-EMF inhibitory effect on Cav3.2 channels. Exogenous LTE4 mimicked the ELF-EMF inhibition of T-type calcium channels. ELF-EMF (50Hz) inhibits native T-type calcium channels in primary cultured mouse cortical neurons via LTE4. We conclude that 50Hz ELF-EMF inhibits T-type calcium channels through AA/LTE4 signaling pathway.

Synthesis and insecticidal activities of 2,3-dihydroquinazolin-4(1H)-one derivatives targeting calcium channel

A series of compounds containing dihydroquinazolinone moiety was designed and synthesized. Amine bridge part was changed in comparison with known anthranilic diamides insecticides. Their insecticidal activities against oriental armyworm (Mythimna separata) indicated that most of the compounds showed moderate to high activities at the tested concentrations. In particular, compounds 5a and 5k showed 80% larvicidal activities against oriental armyworm at the concentration of 5mg/L. The present study also explored the possible effects of target compounds on the high voltage-gated calcium channel and the calcium channels in the endoplasmic reticulum in the central neurons isolated from the third instar larvae of Spodoptera exigua using whole-cell patch clamp and calcium imaging technique. The results showed that compound 5a activated the high voltage-gated calcium channel in the central neurons of S. exigua weakly. The peak currents only increased by 6% of the initial value at the end of the 10-min recording after treated with 0.22μM 5a, while chlorantraniliprole has an opposite effect. The effects of 5a on the intracellular calcium ion concentration ([Ca2+]i) in neurons were well investigated. The experimental results indicated that these novel compounds have different mechanism compared with chlorantraniliprole.

Early infantile epileptic encephalopathy associated with a high voltage gated calcium channelopathy

BackgroundEarly infantile epileptic encephalopathies usually manifest as severely impaired cognitive and motor development and often result in a devastating permanent global developmental delay and intellectual disability. A large set of...

P2‐083: Age‐related reductions in T‐type calcium channels and their contribution to Alzheimer's disease pathogenesis

Over the past 20 years aberrant calcium dysregulation has been consistently implicated in Alzheimer's disease (AD), either in the initiation of AD, the progression of AD-related pathologies, or in mediating the neuronal and synaptic loss resulting from AD pathology. While many studies have implicated high voltage-gated calcium channels in the calcium dyshomeostasis frequently observed in AD, low voltage-gated T-type calcium channels remained to be investigated. We therefore assessed steady state protein levels of CaV 3.1 in 3, 18, and 24 month-old wild-type mice and found that the channels were robustly reduced with age. To understand the effects of reduced T-type channel levels in the aged brain on the pathogenesis of AD, we treated 3xTg-AD mice with a specific T-type channel blocker, NNC-55-0396, to mimic the age-related reductions. Two groups of female 3xTg-AD mice between the ages of 14 and 16 months were injected intraperitoneally every other day for two weeks with either NNC-55-0396 (20 mg/kg) or vehicle and then sacrificed. Despite an experimental period of just two weeks, in mice with established pathology, we found significant increases in soluble levels of Aβ1-42 with T-type channel inhibition. In addition, insoluble levels of Aβ 1-42 trended toward an increase. Accordingly, Western blotting revealed decreases in C83 and the constitutive α -secretase ADAM10, suggesting that blockade of T-type channels downregulates the non-amyloidogenic processing pathway of APP. Notably, no significant changes in tau pathology were observed. However, the cleavage of p35 to p25, which is carried out by calpains, was dramatically reduced with treatment. Further investigation revealed that calpain 1 and 2 protein levels were decreased with drug treatment. Another substrate of calpains, spectrin, was also analyzed in these samples and was found to be similarly decreased in drug-treated mice compared to controls, suggesting that T-type calcium currents contribute substantially to the regulation of calpains. From our results, we conclude that age-related reductions in T-type calcium channels may have the potential to push APP processing toward the amyloidogenic pathway. This could, in turn, predispose the aging brain to develop Alzheimer's disease.

Oxytocin Inhibits the Membrane Depolarization-Induced Increase in Intracellular Calcium in Capsaicin Sensitive Sensory Neurons

Lumbar intrathecal injection of oxytocin produces antinociception in rats and analgesia in humans. Classically, oxytocin receptors couple to stimulatory G proteins, increase inositol-3-phosphate production, and result in neuronal excitation. Most work to date has focused on a spinal site of oxytocin to excite γ-aminobutyric acid interneurons to produce analgesia. Here we ask whether oxytocin might also affect primary sensory afferents by modulating high voltage-gated calcium channels, such as it does in the brain. Dorsal root ganglion cells from adult rats were acutely dissociated and cultured, and changes in intracellular calcium determined by fluorescent microscopy using an indicator dye. The effects of oxytocin alone and in the presence of transient depolarization from increased extracellular KCl concentration were determined, and the pharmacology of these effects were studied. Cells from injured dorsal root ganglion cells after spinal nerve ligation were also studied. Oxytocin produced a concentration-dependent inhibition of the increase in intracellular calcium from membrane depolarization, an effect blocked more efficiently by oxytocin- than vasopressin-receptor selective antagonists. Oxytocin-induced inhibition was present in cells responding to capsaicin, and when internal stores of calcium were depleted with thapsigargin. Oxytocin produced similar inhibition in cells from animals with spinal nerve ligation. These data suggest that oxytocin produces antinociception after intrathecal delivery in part by reducing excitatory neurotransmitter release from the central terminals of nociceptors.

Islet amyloid polypeptide acts on glucose‐ stimulated beta cells to reduce voltage‐gated calcium channel activation, intracellular Ca2+ concentration, and insulin secretion

the mechanism by which islet amyloid polypeptide (IAPP) inhibits insulin release is unclear. We hypothesized that reduced voltage-gated calcium channel activity and intracellular Ca(2+) concentration might contribute to IAPP-mediated inhibition of glucose-stimulated insulin release. rat islet beta cells were cultured and treated with various extracellular concentrations of IAPP, and insulin release was stimulated via addition of glucose. Activation voltage, high voltage-gated calcium channel currents, intracellular Ca(2+) concentration, and insulin secretion were detected by patch clamp electrophysiology, fluorescent digital imaging microscopy using calcium-sensitive fluorescent dye, and radioimmunoassay, respectively. high voltage-gated calcium channel currents, intracellular Ca(2+) concentration, and insulin secretion increased in a dose-dependent manner when rat beta cells were exposed to glucose. After short-term IAPP treatment (5 or 10 µM), these parameters decreased significantly in glucose-stimulated beta cells. However, no significant changes were observed with lower doses of IAPP. glucose-stimulated islet beta-cell high voltage-gated calcium channels were activated in conjunction with insulin secretion, while high extracellular concentrations of IAPP inhibited beta-cell high voltage-gated calcium channel activation and insulin secretion in a dose-dependent manner.