RESUMO
The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel ß subunit (CaVß), and the binding between CaVα1/CaVß appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVß. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVß independent promotion of dynamin-dependent endocytosis.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Dinaminas/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Substituição de Aminoácidos , Animais , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Dinaminas/metabolismo , Endocitose/genética , Feminino , Expressão Gênica , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Oócitos/citologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Transgenes , Xenopus laevisRESUMO
BACKGROUND: De novo variants in the voltage-gated calcium channel subunit α1 E gene (CACNA1E) have been described as causative of epileptic encephalopathy with contractures, macrocephaly and dyskinesias. METHODS: Following the observation of an index patient with developmental delay and autism spectrum disorder (ASD) without seizures who had a de novo deleterious CACNA1E variant, we screened GeneMatcher for other individuals with CACNA1E variants and neurodevelopmental phenotypes without epilepsy. The spectrum of pathogenic CACNA1E variants was compared to the mutational landscape of variants in the gnomAD control population database. RESULTS: We identified seven unrelated individuals with intellectual disability, developmental regression and ASD-like behavioral profile, and notably without epilepsy, who had de novo heterozygous putatively pathogenic variants in CACNA1E. Age of onset of clinical manifestation, presence or absence of regression and degree of severity were variable, and no clear-cut genotype-phenotype association could be recognized. The analysis of disease-associated variants and their comparison to benign variants from the control population allowed for the identification of regions in the CACNA1E protein that seem to be intolerant to substitutions and thus more likely to harbor pathogenic variants. As in a few reported cases with CACNA1E variants and epilepsy, one patient showed a positive clinical behavioral response to topiramate, a specific calcium channel modulator. LIMITATIONS: The significance of our study is limited by the absence of functional experiments of the effect of identified variants, the small sample size and the lack of systematic ASD assessment in all participants. Moreover, topiramate was given to one patient only and for a short period of time. CONCLUSIONS: Our results indicate that CACNA1E variants may result in neurodevelopmental disorders without epilepsy and expand the mutational and phenotypic spectrum of this gene. CACNA1E deserves to be included in gene panels for non-specific developmental disorders, including ASD, and not limited to patients with seizures, to improve diagnostic recognition and explore the possible efficacy of topiramate.
Assuntos
Transtorno do Espectro Autista , Canais de Cálcio Tipo R , Proteínas de Transporte de Cátions , Deficiência Intelectual , Transtorno do Espectro Autista/genética , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Criança , Deficiências do Desenvolvimento , Humanos , Deficiência Intelectual/genética , Fenótipo , Convulsões/genética , Cognição SocialRESUMO
The synaptic connection from medial habenula (MHb) to interpeduncular nucleus (IPN) is critical for emotion-related behaviors and uniquely expresses R-type Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates or inhibits transmitter release from MHb terminals depending on the IPN subnucleus, but the role of KCTDs is unknown. We therefore examined the localization and function of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3 currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b, and Cav2.3 co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3 with KCTDs therefore scales synaptic strength independent of GBR activation.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Habenula/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de GABA/metabolismo , Animais , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores de GABA/genética , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Sinapses/genética , Sinapses/metabolismoRESUMO
Central sensitization (CS) is characteristic of difficult to treat painful conditions, such as fibromyalgia and neuropathies and have sexual dimorphism involved. The calcium influx in nociceptive neurons is a key trigger for CS and the role of Cav2.1 and Cav2.2 voltage gated calcium channels (VGCC) in this role were evidenced with the use of ω-agatoxin IVA and ω-agatoxin MVIIA blockers, respectively. However, the participation of the α1 subunit of the voltage-gated channel Cav2.3, which conducts R-type currents, in CS is unknown. Furthermore, the role of sexual differences in painful conditions is still poorly understood. Thus, we investigated the role of Cav2.3 in capsaicin-induced secondary hyperalgesia in mice, which serve as a CS model predictive of the efficacy of novel analgesic drugs. Capsaicin injection in C57BL/6 mice caused secondary hyperalgesia from one to five hours after injection, and the effects were similar in male and female mice. In female but not male mice, intrathecal treatment with the Cav2.3 inhibitor SNX-482 partially and briefly reversed secondary hyperalgesia at a dose (300 pmol/site) that did not cause adverse effects. Moreover, Cav2.3 expression in the dorsal root ganglia (DRG) and spinal cord was reduced by intrathecal treatment with an antisense oligonucleotide (ASO) targeting Cav2.3 in female and male mice. However, ASO treatment was able to provide a robust and durable prevention of secondary hyperalgesia caused by capsaicin in female mice, but not in male mice. Thus, our results demonstrate that Cav2.3 inhibition, especially in female mice, has a relevant impact on a model of CS. Our results provide a proof of concept for Cav2.3 as a molecular target. In addition, the result associated to the role of differences in painful conditions linked to sex opens a range of possibilities to be explored and needs more attention. Thus, the relevance of testing Cav2.3 inhibition or knockdown in clinically relevant pain models is needed.
Assuntos
Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Sensibilização do Sistema Nervoso Central/genética , Hiperalgesia/genética , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo R/efeitos dos fármacos , Capsaicina , Proteínas de Transporte de Cátions/efeitos dos fármacos , Sensibilização do Sistema Nervoso Central/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Gânglios Espinais/metabolismo , Técnicas de Silenciamento de Genes , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos Antissenso/farmacologia , Caracteres Sexuais , Venenos de Aranha/farmacologia , Medula Espinal/metabolismoRESUMO
Eukaryote voltage-gated Ca2+ channels of the CaV2 channel family are hetero-oligomers formed by the pore-forming CaVα1 protein assembled with auxiliary CaVα2δ and CaVß subunits. CaVß subunits are formed by a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain connected through a HOOK domain. The GK domain binds a conserved cytoplasmic region of the pore-forming CaVα1 subunit referred as the "AID". Herein we explored the phylogenetic and functional relationship between CaV channel subunits in distant eukaryotic organisms by investigating the function of a MAGUK protein (XM_004990081) cloned from the choanoflagellate Salpingoeca rosetta (Sro). This MAGUK protein (Sroß) features SH3 and GK structural domains with a 25% primary sequence identity to mammalian CaVß. Recombinant expression of its cDNA with mammalian high-voltage activated Ca2+ channel CaV2.3 in mammalian HEK cells produced robust voltage-gated inward Ca2+ currents with typical activation and inactivation properties. Like CaVß, Sroß prevents fast degradation of total CaV2.3 proteins in cycloheximide assays. The three-dimensional homology model predicts an interaction between the GK domain of Sroß and the AID motif of the pore-forming CaVα1 protein. Substitution of AID residues Trp (W386A) and Tyr (Y383A) significantly impaired co-immunoprecipitation of CaV2.3 with Sroß and functional upregulation of CaV2.3 currents. Likewise, a 6-residue deletion within the GK domain of Sroß, similar to the locus found in mammalian CaVß, significantly reduced peak current density. Altogether our data demonstrate that an ancestor MAGUK protein reconstitutes the biophysical and molecular features responsible for channel upregulation by mammalian CaVß through a minimally conserved molecular interface.
Assuntos
Canais de Cálcio Tipo R/química , Proteínas de Transporte de Cátions/química , Guanilato Quinases/química , Proteínas de Protozoários/química , Substituição de Aminoácidos , Canais de Cálcio Tipo R/genética , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
So-called pharmacoresistant (R-type) voltage-gated Ca2+ channels are structurally only partially characterized. Most of them are encoded by the CACNA1E gene and are expressed as different Cav2.3 splice variants (variant Cav2.3a to Cav2.3e or f) as the ion conducting subunit. So far, no inherited disease is known for the CACNA1E gene but recently spontaneous mutations leading to early death were identified, which will be brought into focus. In addition, a short historical overview may highlight the development to understand that upregulation during aging, easier activation by spontaneous mutations or lack of bioavailable inorganic cations (Zn2+ and Cu2+) may lead to similar pathologies caused by cellular overexcitation.
Assuntos
Canais de Cálcio Tipo R/genética , Canais de Cálcio/genética , Mutação/genética , Envelhecimento/genética , Sequência de Aminoácidos , Animais , HumanosRESUMO
BACKGROUND/AIMS: Still in 1999 the first hints were published for the pharmacoresistant Cav2.3 calcium channel to be involved in the generation of epileptic seizures, as transcripts of alpha1E (Cav2.3) and alpha1G (Cav3.1) are changed in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Consecutively, the seizure susceptibility of mice lacking Cav2.3 was analyzed in great detail by using 4-aminopyridine, pentylene-tetrazol, N-methyl-D-aspartate and kainic acid to induce experimentally convulsive seizures. Further, γ-hydroxybutyrolactone was used for the induction of non-convulsive absence seizures. For all substances tested, Cav2.3-competent mice differed from their knockout counterparts in the sense that for convulsive seizures the deletion of the pharmacoresistant channel was beneficial for the outcome during experimentally induced seizures [1]. The antiepileptic drug lamotrigine reduces seizure activity in Cav2.3-competent but increases it in Cav2.3-deficient mice. In vivo, Cav2.3 must be under tight control by endogenous trace metal cations (Zn2+ and Cu2+). The dyshomeostasis of either of them, especially of Cu2+, may alter the regulation of Cav2.3 severely and its activity for Ca2+ conductance, and thus may change hippocampal and neocortical signaling to hypo- or hyperexcitation. METHODS: To investigate by telemetric EEG recordings the mechanism of generating hyperexcitation by kainate, mice were tested for their sensitivity of changes in neuronal (intracerebroventricular) concentrations of the trace metal cation Zn2+. As the blood-brain barrier limits the distribution of bioavailable Zn2+ or Cu2+ into the brain, we administered micromolar Zn2+ ions intracerebroventricularly in the presence of 1 mM histidine as carrier and compared the effects on behavior and EEG activity in both genotypes. RESULTS: Kainate seizures are more severe in Cav2.3-competent mice than in KO mice and histidine lessens seizure severity in competent but not in Cav2.3-deficient mice. Surprisingly, Zn2+ plus histidine resembles the kainate only control with more seizure severity in Cav2.3-competent than in deficient mice. CONCLUSION: Cav2.3 represents one important Zn2+-sensitive target, which is useful for modulating convulsive seizures.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Convulsões/tratamento farmacológico , Zinco/uso terapêutico , Animais , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Eletroencefalografia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Histidina/farmacologia , Íons/química , Ácido Caínico/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Convulsões/induzido quimicamente , Convulsões/patologia , Índice de Gravidade de Doença , Zinco/farmacologia , Ácido gama-Aminobutírico/metabolismoRESUMO
Degeneration of dopaminergic neurons in the substantia nigra causes the motor symptoms of Parkinson's disease. The mechanisms underlying this age-dependent and region-selective neurodegeneration remain unclear. Here we identify Cav2.3 channels as regulators of nigral neuronal viability. Cav2.3 transcripts were more abundant than other voltage-gated Ca2+ channels in mouse nigral neurons and upregulated during aging. Plasmalemmal Cav2.3 protein was higher than in dopaminergic neurons of the ventral tegmental area, which do not degenerate in Parkinson's disease. Cav2.3 knockout reduced activity-associated nigral somatic Ca2+ signals and Ca2+-dependent after-hyperpolarizations, and afforded full protection from degeneration in vivo in a neurotoxin Parkinson's mouse model. Cav2.3 deficiency upregulated transcripts for NCS-1, a Ca2+-binding protein implicated in neuroprotection. Conversely, NCS-1 knockout exacerbated nigral neurodegeneration and downregulated Cav2.3. Moreover, NCS-1 levels were reduced in a human iPSC-model of familial Parkinson's. Thus, Cav2.3 and NCS-1 may constitute potential therapeutic targets for combatting Ca2+-dependent neurodegeneration in Parkinson's disease.
Assuntos
Envelhecimento/genética , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Sobrevivência Celular/genética , Neurônios Dopaminérgicos/metabolismo , Proteínas Sensoras de Cálcio Neuronal/genética , Neuropeptídeos/genética , Doença de Parkinson/genética , Envelhecimento/metabolismo , Animais , Canais de Cálcio Tipo R/metabolismo , Sinalização do Cálcio , Proteínas de Transporte de Cátions/metabolismo , Neurônios Dopaminérgicos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas , Camundongos , Camundongos Knockout , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Substância Negra/metabolismo , Substância Negra/patologia , Regulação para Cima , Área Tegmentar Ventral/metabolismo , Área Tegmentar Ventral/patologiaRESUMO
Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α1-subunit of the voltage-gated CaV2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.
Assuntos
Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Contratura/genética , Discinesias/genética , Epilepsia/genética , Variação Genética/genética , Megalencefalia/genética , Espasmos Infantis/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Transtornos do Neurodesenvolvimento/genéticaRESUMO
Leptin acts on hypothalamic pro-opiomelanocortin (POMC) neurons to regulate glucose homeostasis, but the precise mechanisms remain unclear. Here, we demonstrate that leptin-induced depolarization of POMC neurons is associated with the augmentation of a voltage-gated calcium (CaV) conductance with the properties of the "R-type" channel. Knockdown of the pore-forming subunit of the R-type (CaV2.3 or Cacna1e) conductance in hypothalamic POMC neurons prevented sustained leptin-induced depolarization. In vivo POMC-specific Cacna1e knockdown increased hepatic glucose production and insulin resistance, while body weight, feeding, or leptin-induced suppression of food intake were not changed. These findings link Cacna1e function to leptin-mediated POMC neuron excitability and glucose homeostasis and may provide a target for the treatment of diabetes.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Glucose/metabolismo , Leptina/farmacologia , Fígado/metabolismo , Neurônios/metabolismo , Pró-Opiomelanocortina/metabolismo , Animais , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Células Cultivadas , Homeostase , Humanos , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacosRESUMO
OBJECTIVE: Most patients with Crohn's disease (CD) are diagnosed with the uncomplicated inflammatory form of the disease (Montreal stage B1). However, the majority of them will progress to complicated stricturing (B2) and penetrating (B3) CD during their lifetimes. The aim of our study was to identify the genetic factors associated with time to progression from uncomplicated to complicated CD. PATIENTS AND METHODS: Patients with an inflammatory phenotype at diagnosis were followed up for 10 years. Genotyping was carried out using Illumina ImmunoChip. After quality control, association analyses, Bonferroni's adjustments, linear and Cox's regression, and Kaplan-Meier analysis were carried out for 111 patients and Manhattan plots were constructed. RESULTS: Ten years after diagnosis, 39.1% of the patients still had the inflammatory form and 60.9% progressed to complicated disease, with an average time to progression of 5.91 years. Ileal and ileocolonic locations were associated with the complicated CD (P=1.08E-03). We found that patients with the AA genotype at single-nucleotide polymorphism rs16857259 near the gene CACNA1E progressed to the complicated form later (8.80 years) compared with patients with the AC (5.11 years) or CC (2.00 years) genotypes (P=3.82E-07). In addition, nine single-nucleotide polymorphisms (near the genes RASGRP1, SULF2, XPO1, ZBTB44, HLA DOA/BRD2, HLA DRB1/HLA DQA1, PPARA, PUDP, and KIAA1614) showed a suggestive association with disease progression (P<10). Multivariate Cox's regression analysis on the basis of clinical and genetic data confirmed the association of the selected model with disease progression (P=5.73E-16). CONCLUSION: Our study confirmed the association between the locus on chromosome 1 near the gene CACNA1E with time to progression from inflammatory to stricturing or penetrating CD. Predicting the time to progression is useful to the clinician in terms of individualizing patients' management.
Assuntos
Doença de Crohn/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Idoso , Produtos Biológicos/uso terapêutico , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Criança , Doença de Crohn/tratamento farmacológico , Doença de Crohn/patologia , DNA/genética , Progressão da Doença , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Glucocorticoides/uso terapêutico , Humanos , Imunossupressores/uso terapêutico , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Tempo , Adulto JovemRESUMO
INTRODUCTION: The relationship between blood metabolites and hemoglobin degradation products (BMHDPs) formed in the cerebrospinal fluid and the development of vasospasm and delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) has been the focus of several previous studies, but their molecular and cellular targets remain to be elucidated. METHODS: Because BMHDP-induced changes in Cav 2.3 channel function are thought to contribute to DCI after aSAH, we studied their modulation by unconjugated bilirubin (UCB) in an organotypical neuronal network from wild-type (WT) and Cav 2.3-deficient animals (KO). Murine retinae were isolated from WT and KO and superfused with nutrient solution. Electroretinograms were recorded before, during, and after superfusion with UCB. Transretinal signaling was analyzed as b-wave, implicit time, and area under the curve (AUC). RESULTS: Superfusion of UCB significantly attenuated the b-wave amplitude in the isolated retina from wild-type mice by 14.9% (P < 0.05), followed by gradual partial recovery (P = 0.09). Correspondingly, AUC decreased significantly with superfusion of UCB (P < 0.05). During washout, the b-wave amplitude returned to baseline (P = 0.2839). The effects of UCB were absent in Cav 2.3-deficient mice, lacking the expression of Cav 2.3 as proofed on the biochemical level. CONCLUSIONS: Ex vivo neuronal recording in the murine retina is able to detect transient impairment of transretinal signaling by UCB in WT, but not in KO. This new model may be useful to further clarify the role of calcium channels in neuronal signal alteration in the presence of BHMDPs.
Assuntos
Bilirrubina/metabolismo , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Neurônios/metabolismo , Retina/metabolismo , Transmissão Sináptica/fisiologia , Animais , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Masculino , Camundongos Transgênicos , Técnicas de Cultura de TecidosRESUMO
BACKGROUND/AIMS: Lamotrigine (LTG) is a popular modern antiepileptic drug (AED), however, its mechanism of action has yet to be fully understood, as it is known to modulate many members of several ion channel families. In heterologous systems, LTG inhibits Cav2.3 (R-type) calcium currents, which contribute to kainic-acid- (KA) induced epilepsy in vivo. To gain insight into the role of R-type currents in LTG drug action in vivo, we compared the effects of LTG to topiramate and lacosamide in Cav2.3-deficient mice and controls on KA-induced seizures. METHODS: Behavioral seizure rating and quantitative electrocorticography were performed after injection of 20 mg/kg [and 30 mg/kg] KA. One hour before KA injection, mice were pretreated with either 30 mg/kg LTG, 50 mg/kg topiramate (TPM) or 30 mg/kg lacosamide (LSM). RESULTS: Ablation of Cav2.3 reduced total seizure scores by 28.6% (p=0.0012) and pretreatment with LTG reduced seizure activity of control mice by 23.2% (p=0.02). In Cav2.3-deficient mice LTG pretreatment increased seizure activity by 22.1% (p=0.018) and increased the percentage of degenerated CA1 pyramidal neurons (p=0.02). All three tested AEDs reduced seizure activity in control mice, however only the non-calcium channel modulating AED, LSM had an anticonvulsive effect in Cav2.3-deficient mice. Furthermore LTG altered electrocorticographic parameters differently in the two genotypes, decreasing relative power of ictal spikes in control mice compared to Cav2.3-defcient mice. CONCLUSION: These findings give first in vivo evidence for an essential role for Cav2.3 in LTG pharmacology and shed light on a paradoxical effect of LTG in their absence. Furthermore, LTG appears to promote ictal activity in Cav2.3-deficient mice resulting in increased neurotoxicity in the CA1 region. This paradoxical mechanism, possibly reflecting rebound hyperexcitation of pyramidal CA1 neurons after increased inhibition, may be key in understanding LTG-induced seizure aggravation, observed in clinical practice.
Assuntos
Anticonvulsivantes/farmacologia , Comportamento Animal/efeitos dos fármacos , Canais de Cálcio Tipo R/genética , Epilepsia/patologia , Fármacos Neuroprotetores/farmacologia , Triazinas/farmacologia , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Animais , Anticonvulsivantes/uso terapêutico , Canais de Cálcio Tipo R/deficiência , Eletrocorticografia , Epilepsia/induzido quimicamente , Epilepsia/prevenção & controle , Frutose/análogos & derivados , Frutose/farmacologia , Frutose/uso terapêutico , Genótipo , Imuno-Histoquímica , Ácido Caínico/toxicidade , Lacosamida , Lamotrigina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fármacos Neuroprotetores/uso terapêutico , Células Piramidais/efeitos dos fármacos , Células Piramidais/patologia , Topiramato , Triazinas/uso terapêuticoRESUMO
In regulatory toxicology, the dose-response relationship is a key element towards fulfilling safety assessments and satisfying regulatory authorities. Conventionally, the larger the dose, the greater the response, following the dogma "the dose makes the poison". Many endocrine disrupting chemicals, including bisphenol-A (BPA), induce non-monotonic dose response (NMDR) relationships, which are unconventional and have tremendous implications in risk assessment. Although several molecular mechanisms have been proposed to explain NMDR relationships, they are largely undemonstrated. Using mouse pancreatic ß-cells from wild-type and oestrogen receptor ERß-/- mice, we found that exposure to increasing doses of BPA affected Ca2+ entry in an NMDR manner. Low doses decreased plasma membrane Ca2+ currents after downregulation of Cav2.3 ion channel expression, in a process involving ERß. High doses decreased Ca2+ currents through an ERß-mediated mechanism and simultaneously increased Ca2+ currents via oestrogen receptor ERα. The outcome of both molecular mechanisms explains the NMDR relationship between BPA and Ca2+ entry in ß-cells.
Assuntos
Compostos Benzidrílicos/toxicidade , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Células Secretoras de Insulina/metabolismo , Fenóis/toxicidade , Animais , Canais de Cálcio Tipo R/biossíntese , Canais de Cálcio Tipo R/genética , Sinalização do Cálcio/genética , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Transporte de Cátions/genética , Relação Dose-Resposta a Droga , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Camundongos KnockoutRESUMO
Pathophysiological mechanisms underlying pain associated with cancer are poorly understood. microRNAs (miRNAs) are a class of noncoding RNAs with emerging functional importance in chronic pain. In a genome-wide screen for miRNAs regulated in dorsal root ganglia (DRG) neurons in a mouse model of bone metastatic pain, we identified miR-34c-5p as a functionally important pronociceptive miRNA. Despite these functional insights and therapeutic potential for miR-34c-5p, its molecular mechanism of action in peripheral sensory neurons remains unknown. Here, we report the identification and validation of key target transcripts of miRNA-34c-5p. In-depth bioinformatics analyses revealed Cav2.3, P2rx6, Oprd1, and Oprm1 as high confidence putative targets for miRNA-34c-5p. Of these, canonical and reciprocal regulation of miR-34c-5p and Cav2.3 was observed in cultured sensory neurons as well as in DRG in vivo in mice with cancer pain. Coexpression of miR-34c-5p and Cav2.3 was observed in peptidergic and nonpeptidergic nociceptors, and luciferase reporter assays confirmed functional binding of miR-34c-5p to the 3' UTR of Cav2.3 transcripts. Importantly, knocking down the expression of Cav2.3 specifically in DRG neurons led to hypersensitivity in mice. In summary, these results show that Cav2.3 is a novel mechanistic target for a key pronociceptive miRNA, miR-34c-5p, in the context of cancer pain and indicate an antinociceptive role for Cav2.3 in peripheral sensory neurons. The current study facilitates a deeper understanding of molecular mechanisms underlying cancer pain and suggests a potential for novel therapeutic strategies targeting miR-34c-5p and Cav2.3 in cancer pain.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Dor do Câncer/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Gânglios Espinais/patologia , MicroRNAs/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Neoplasias Ósseas/complicações , Canais de Cálcio Tipo R/genética , Dor do Câncer/etiologia , Dor do Câncer/patologia , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/genética , Dependovirus/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Ontologia Genética , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução Genética , TransfecçãoRESUMO
OBJECTIVE: To search for differences in prevalence of a CACNA1E variant between migraine without aura, various phenotypes of migraine with aura, and healthy controls. BACKGROUND: Familial hemiplegic migraine type 1 (FHM1) is associated with mutations in the CACNA1A gene coding for the alpha 1A (Cav 2.1) pore-forming subunit of P/Q voltage-dependent Ca2+ channels. These mutations are not found in the common forms of migraine with or without aura. The alpha 1E subunit (Cav 2.3) is the counterpart of Cav 2.1 in R-type Ca2+ channels, has different functional properties, and is encoded by the CACNA1E gene. METHODS: First, we performed a total exon sequencing of the CACNA1E gene in three probands selected because they had no abnormalities in the three FHM genes. In a patient suffering from basilar-type migraine, we identified a single nucleotide polymorphism (SNP) in exon 20 of the CACNA1E gene (Asp859Glu - rs35737760; Minor Allele Frequency 0.2241) hitherto not studied in migraine. In a second step, we determined its occurrence in four groups by direct sequencing on blood genomic DNA: migraine patients without aura (N = 24), with typical aura (N = 55), complex neurological auras (N = 19; hemiplegic aura: N = 15; brain stem aura: N = 4), and healthy controls (N = 102). RESULTS: The Asp859Glu - rs35737760 SNP of the CACNA1E gene was present in 12.7% of control subjects and in 20.4% of the total migraine group. In the migraine group it was significantly over-represented in patients with complex neurological auras (42.1%), OR 4.98 (95% CI: 1.69-14.67, uncorrected P = .005, Bonferroni P = .030, 2-tailed Fisher's exact test). There was no significant difference between migraine with typical aura (10.9%) and controls. CONCLUSIONS: We identified a polymorphism in exon 20 of the CACNA1E gene (Asp859Glu - rs35737760) that is more prevalent in hemiplegic and brain stem aura migraine. This missense variant causes a change from aspartate to glutamate at position 859 of the Cav 2.3 protein and might modulate the function of R-type Ca2+ channels. It could thus be relevant for migraine with complex neurological aura, although this remains to be proven.
Assuntos
Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Ataxia Cerebelar/genética , Predisposição Genética para Doença/genética , Transtornos de Enxaqueca/genética , Polimorfismo de Nucleotídeo Único/genética , Ácido Aspártico/genética , Estudos de Casos e Controles , Análise Mutacional de DNA , Éxons/genética , Feminino , Frequência do Gene , Ácido Glutâmico/genética , Humanos , Masculino , Transtornos de Enxaqueca/classificação , Fenótipo , Estudos Retrospectivos , Estatísticas não ParamétricasRESUMO
OBJECTIVE: α-Conotoxin Vc1.1 is a small disulfide-bonded peptide from the venom of the marine cone snail Conus victoriae. Vc1.1 has antinociceptive actions in animal models of neuropathic pain, but its applicability to inhibiting human dorsal root ganglion (DRG) neuroexcitability and reducing chronic visceral pain (CVP) is unknown. DESIGN: We determined the inhibitory actions of Vc1.1 on human DRG neurons and on mouse colonic sensory afferents in healthy and chronic visceral hypersensitivity (CVH) states. In mice, visceral nociception was assessed by neuronal activation within the spinal cord in response to noxious colorectal distension (CRD). Quantitative-reverse-transcription-PCR, single-cell-reverse-transcription-PCR and immunohistochemistry determined γ-aminobutyric acid receptor B (GABABR) and voltage-gated calcium channel (CaV2.2, CaV2.3) expression in human and mouse DRG neurons. RESULTS: Vc1.1 reduced the excitability of human DRG neurons, whereas a synthetic Vc1.1 analogue that is inactive at GABABR did not. Human DRG neurons expressed GABABR and its downstream effector channels CaV2.2 and CaV2.3. Mouse colonic DRG neurons exhibited high GABABR, CaV2.2 and CaV2.3 expression, with upregulation of the CaV2.2 exon-37a variant during CVH. Vc1.1 inhibited mouse colonic afferents ex vivo and nociceptive signalling of noxious CRD into the spinal cord in vivo, with greatest efficacy observed during CVH. A selective GABABR antagonist prevented Vc1.1-induced inhibition, whereas blocking both CaV2.2 and CaV2.3 caused inhibition comparable with Vc1.1 alone. CONCLUSIONS: Vc1.1-mediated activation of GABABR is a novel mechanism for reducing the excitability of human DRG neurons. Vc1.1-induced activation of GABABR on the peripheral endings of colonic afferents reduces nociceptive signalling. The enhanced antinociceptive actions of Vc1.1 during CVH suggest it is a novel candidate for the treatment for CVP.
Assuntos
Colo/fisiologia , Conotoxinas/farmacologia , Gânglios Espinais/fisiologia , Neurônios Aferentes/fisiologia , Nociceptividade/efeitos dos fármacos , Receptores de GABA-B/análise , Receptores de GABA-B/genética , Animais , Baclofeno/farmacologia , Canais de Cálcio Tipo N/análise , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo R/análise , Canais de Cálcio Tipo R/genética , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/análise , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Células Cultivadas , Dor Crônica/prevenção & controle , Modelos Animais de Doenças , Eletrofisiologia , Feminino , Agonistas dos Receptores de GABA-B/farmacologia , Antagonistas de Receptores de GABA-B/farmacologia , Gânglios Espinais/química , Gânglios Espinais/efeitos dos fármacos , Expressão Gênica , Humanos , Masculino , Camundongos , Neurônios Aferentes/química , Neurônios Aferentes/efeitos dos fármacos , Receptores de GABA-B/metabolismo , Regulação para Cima , Dor Visceral/prevenção & controle , Adulto JovemRESUMO
OBJECTIVE: To observe the regulation of electroacupuncture on gene expression at calcium signaling pathways in mice with cerebral ischemia reperfusion. METHODS: Sixty male, inbred Kunming mice were randomly assigned to three groups: repeated cerebral ischemia reperfusion group (RG, n = 24), sham-operated group (SG, n = 12), and electroacupuncture group (EG, n = 24). Mice in RG and EG groups were modeled by repeated cerebral ischemia reperfusion surgery, and EG mice were treated with electroacupuncture for 30 min after recovery from anesthesia. Changes in gene expression profile of mice hippocampi were analyzed by global expression profile microarray. Genes that were up-regulated or down-regulated greater than 1.5 folds were considered to be biologically meaningful. Real-time quantitative polymerase chain reaction (q-PCR) method was used to verify the expression of selected genes based on the algorithm [2^ (ΔΔCt)]. RESULTS: Compared with SG mice, 242 genes showed different in expressions in RG mice: 107 down-regulated and 135 up-regulated. Compared with RG mice, 609 genes showed a difference of expression in EG mice: 315 down-regulated and 375 up-regulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated two pathways: calcium signaling and long-term potentiation in which 11 differentially expressed genes selected. Six of the 11 genes in the calcium signaling pathway were verified after real-time q-PCR testing. CONCLUSION: Electroacupuncture treatment of cerebral ischemia reperfusion appears to regulate Atp2a2, Cacna1e, Camk2a, Gnas, Grm1, Rapgef3 genes in the calcium signaling pathway.
Assuntos
Isquemia Encefálica/genética , Isquemia Encefálica/terapia , Sinalização do Cálcio , Eletroacupuntura , Hipocampo/metabolismo , Pontos de Acupuntura , Animais , Isquemia Encefálica/metabolismo , Canais de Cálcio Tipo R/genética , Canais de Cálcio Tipo R/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cromograninas/genética , Cromograninas/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Masculino , Camundongos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , TranscriptomaRESUMO
UNLABELLED: Cav2.3 (R-type) voltage-activated Ca(2+) channels (VACCs), encoded by the calcium channel, voltage-dependent, R-type, α1E subunit (CACNA1E) gene, are responsible for transmission of somatic inflammatory pain, and activation of antinociception elicited by visceral inflammatory pain stimuli. Carriers of the minor G allele of the rs3845446 single-nucleotide polymorphism (SNP) of the CACNA1E gene reportedly exhibit a decrease in opioid requirements to control typical somatic inflammatory pain after orthognathic surgery (ie, a painful cosmetic surgery), suggesting the downregulation of Cav2.3 VACC function that is responsible for transmission of somatic inflammatory pain in these carriers. Gastrointestinal surgery involves somatic and visceral inflammatory pain, in which visceral inflammatory pain stimuli activate Cav2.3 VACC-mediated antinociception. Unknown is whether pain-related phenotypes after gastrointestinal surgery are affected in these carriers. The present study used a correlational design to examine the effect of the rs3845446 SNP on postoperative pain-related phenotypes in 2 groups of patients who underwent gastrointestinal surgery. Carriers of the minor G allele had greater opioid requirements after laparoscopic colectomy when intravenous patient-controlled analgesia was used, and reported higher pain scores after open gastrointestinal surgery when postoperative analgesia was managed with continuous epidural analgesia and rescue analgesics. These results suggest that pain-related phenotypes after gastrointestinal surgery are enhanced in carriers of the minor G allele of the rs3845446 SNP, possibly through impairment of Cav2.3 VACC function that is responsible for the activation of visceral inflammatory pain stimulus-elicited antinociception. PERSPECTIVE: Carriers of the minor allele of the rs3845446 SNP of the CACNA1E gene required more opioid or reported higher pain scores after gastrointestinal surgery, and required less opioid after orthognathic surgery. The difference may result from the presence of visceral inflammatory pain stimulus that activates Cav2.3 VACCs-mediated antinociception.
Assuntos
Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Procedimentos Cirúrgicos do Sistema Digestório , Cirurgia Ortognática , Dor Pós-Operatória/genética , Polimorfismo de Nucleotídeo Único , Fatores Etários , Analgésicos Opioides/uso terapêutico , Feminino , Técnicas de Genotipagem , Heterozigoto , Humanos , Laparoscopia , Masculino , Pessoa de Meia-Idade , Fenótipo , Análise de RegressãoRESUMO
Activity-dependent neuroprotective protein (ADNP), essential for brain formation, is a frequent autism spectrum disorder (ASD)-mutated gene. ADNP associates with microtubule end-binding proteins (EBs) through its SxIP motif, to regulate dendritic spine formation and brain plasticity. Here, we reveal SKIP, a novel four-amino-acid peptide representing an EB-binding site, as a replacement therapy in an outbred Adnp-deficient mouse model. We discovered, for the first time, axonal transport deficits in Adnp(+/-) mice (measured by manganese-enhanced magnetic resonance imaging), with significant male-female differences. RNA sequencing evaluations showed major age, sex and genotype differences. Function enrichment and focus on major gene expression changes further implicated channel/transporter function and the cytoskeleton. In particular, a significant maturation change (1 month-five months) was observed in beta1 tubulin (Tubb1) mRNA, only in Adnp(+/+) males, and sex-dependent increase in calcium channel mRNA (Cacna1e) in Adnp(+/+) males compared with females. At the protein level, the Adnp(+/-) mice exhibited impaired hippocampal expression of the calcium channel (voltage-dependent calcium channel, Cacnb1) as well as other key ASD-linked genes including the serotonin transporter (Slc6a4), and the autophagy regulator, BECN1 (Beclin1), in a sex-dependent manner. Intranasal SKIP treatment normalized social memory in 8- to 9-month-old Adnp(+/-)-treated mice to placebo-control levels, while protecting axonal transport and ameliorating changes in ASD-like gene expression. The control, all d-amino analog D-SKIP, did not mimic SKIP activity. SKIP presents a novel prototype for potential ASD drug development, a prevalent unmet medical need.