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
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
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
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
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
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.
Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Microtúbulos/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Motivos de Aminoácidos , Animais , Transtorno do Espectro Autista/etiologia , Transtorno do Espectro Autista/genética , Transporte Axonal/genética , Transporte Axonal/fisiologia , Encéfalo/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo R/genética , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Espinhas Dendríticas/metabolismo , Feminino , Hipocampo/metabolismo , Masculino , Memória , Camundongos , Microtúbulos/metabolismo , RNA Mensageiro/metabolismo , Fatores Sexuais , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/metabolismoRESUMO
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: 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
RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane (PM) in mammalian cells. We had reported that RalA regulates biphasic insulin secretion, which we have now determined to be contributed by RalA direct interaction with voltage-gated calcium (Cav ) channels. RalA knockdown (KD) in INS-1 cells and primary rat ß-cells resulted in a reduction in Ca(2+) currents arising specifically from L-(Cav 1.2 and Cav 1.3) and R-type (Cav 2.3) Ca(2+) channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca(2+) currents. RalA co-immunoprecipitated with the Cav α2 δ-1 auxiliary subunit known to bind the three Cav s. Moreover, the functional molecular interactions between Cav α2 δ-1 and RalA on the PM shown by total internal reflection fluorescent microscopy/FRET analysis could be induced by glucose stimulation. KD of RalA inhibited trafficking of α2 δ-1 to insulin granules without affecting the localization of the other Cav subunits. Furthermore, we confirmed that RalA and α2 δ-1 functionally interact since RalA KD-induced inhibition of Cav currents could not be recovered by RalA when α2 δ-1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α2 δ-1 on insulin granules to tether these granules to PM Ca(2+) channels. This acts as a chaperoning step prior to and in preparation for sequential assembly of exocyst and excitosome complexes that mediate biphasic insulin secretion.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo R/metabolismo , Insulina/metabolismo , Subunidades Proteicas/metabolismo , Vesículas Secretórias/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo R/genética , Membrana Celular/metabolismo , Exocitose , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Células Secretoras de Insulina/metabolismo , Ligação Proteica , Subunidades Proteicas/genética , Transporte Proteico , RNA Interferente Pequeno , Ratos , Ratos Sprague-DawleyRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent intercellular signaling molecule involved in multiple homeostatic functions. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, making them a unique system to establish mechanisms underlying PACAP modulation of neuronal function. Calcium influx is required for the PACAP-increased cardiac neuron excitability, although the pathway is unknown. This study tested whether PACAP enhancement of calcium influx through either T-type or R-type channels contributed to the modulation of excitability. Real-time quantitative polymerase chain reaction analyses indicated transcripts for Cav3.1, Cav3.2, and Cav3.3 T-type isoforms and R-type Cav2.3 in cardiac neurons. These neurons often exhibit a hyperpolarization-induced rebound depolarization that remains when cesium is present to block hyperpolarization-activated nonselective cationic currents (Ih). The T-type calcium channel inhibitors, nickel (Ni(2+)) or mibefradil, suppressed the rebound depolarization, and treatment with both drugs hyperpolarized cardiac neurons by 2-4 mV. Together, these results are consistent with the presence of functional T-type channels, potentially along with R-type channels, in these cardiac neurons. Fifty micromolar Ni(2+), a concentration that suppresses currents in both T-type and R-type channels, blunted the PACAP-initiated increase in excitability. Ni(2+) also blunted PACAP enhancement of the hyperpolarization-induced rebound depolarization and reversed the PACAP-mediated increase in excitability, after being initiated, in a subset of cells. Lastly, low voltage-activated currents, measured under perforated patch whole cell recording conditions and potentially flowing through T-type or R-type channels, were enhanced by PACAP. Together, our results suggest that a PACAP-enhanced, Ni(2+)-sensitive current contributes to PACAP-induced modulation of neuronal excitability.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Níquel/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo R/genética , Canais de Cálcio Tipo R/metabolismo , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Feminino , Expressão Gênica , Cobaias , Masculino , Mibefradil/farmacologia , Microeletrodos , Miocárdio/citologia , Miocárdio/metabolismo , Neurônios/citologia , Técnicas de Patch-Clamp , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Análise de Célula ÚnicaRESUMO
Voltage-gated, CaV2.3 calcium channels and neurokinin-1 (NK1) receptors are both present in nuclei of the central nervous system. When transiently coexpressed in human embryonic kidney (HEK) 293 cells, CaV2.3 is primarily inhibited during strong, agonist-dependent activation of NK1 receptors. NK1 receptors localize to plasma membrane rafts, and their modulation by Gq/11 protein-coupled signaling is sensitive to plasma membrane cholesterol. Here, we show that inhibition of CaV2.3 by NK1 receptors is attenuated following methyl-ß-cyclodextrin (MBCD)-mediated depletion of membrane cholesterol. By contrast, inhibition of CaV2.3 was unaffected by intracellular diffusion of caveolin-1 scaffolding peptide or by overexpression of caveolin-1. Interestingly, MΒCD treatment had no effect on the macroscopic biophysical properties of CaV2.3, though it significantly decreased whole-cell membrane capacitance. Our data indicate that (1) cholesterol supports at least one component of the NK1 receptor-linked signaling pathway that inhibits CaV2.3 and (2) caveolin-1 is dispensable within this pathway. Our findings suggest that NK1 receptors reside within non-caveolar membrane rafts and that CaV2.3 resides nearby but outside the rafts. Raft-dependent modulation of CaV2.3 could be important in the physiological and pathophysiological processes in which these channels participate, including neuronal excitability, synaptic plasticity, epilepsy, and chronic pain.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Caveolina 1/metabolismo , Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Receptor Cross-Talk , Receptores da Neurocinina-1/metabolismo , Animais , Canais de Cálcio Tipo R/genética , Caveolina 1/genética , Células HEK293 , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Potenciais da Membrana , Coelhos , Ratos , Receptores da Neurocinina-1/genética , Transfecção , beta-Ciclodextrinas/farmacologiaRESUMO
Backfat thickness (BFT) and average daily gain (ADG) are two important economic traits in commercial swine production. Identifying QTLs and uncovering the molecular mechanism for BFT and ADG would greatly help to speed up the breeding progress. In current breeding program, EBV for these two traits are calculated and formulated a comprehensive breeding index, which then be used to improve pig performance. Using Illumina PorcineSNP60 BeadChip, a pilot genomewide association studies (GWAS) for BFT and ADG in 83 Duroc pigs were performed. A total of 31 genome-wise significant SN Ps were detected to be associated with BFT on SSC 4, 9, 11, 12 and 14, ten of which were coincident with previously reported QTL regions. There are two genome-wise loci prominently associated with ADG on SSC2 and SSC13, respectively. The two loci on SSC2 are well overlapped with the QTL regions previously reported. All the 31 significant SNPs associated with BFT are verified on 219 outbreed pigs, six SN Ps reach an extreme significant level and seven SNP reaches a significant level, CACNA1E and ACBD6 are chosen as positional candidate genes. Our findings not only confirmed previously findings, but also revealed a number of novel SNPs associated with BFT and ADG. Two positional candidate genes CACNA1E and ACBD6 were identified for further study. These results would facilitate the identification of causative genes for BFT and ADG.
Assuntos
Adiposidade/genética , Loci Gênicos , Polimorfismo de Nucleotídeo Único , Característica Quantitativa Herdável , Suínos/genética , Aumento de Peso/genética , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Canais de Cálcio Tipo R/genética , Feminino , MasculinoRESUMO
Pancreatic islets have a central role in blood glucose homeostasis. In addition to insulin-producing beta-cells and glucagon-secreting alpha-cells, the islets contain somatostatin-releasing delta-cells. Somatostatin is a powerful inhibitor of insulin and glucagon secretion. It is normally secreted in response to glucose and there is evidence suggesting its release becomes perturbed in diabetes. Little is known about the control of somatostatin release. Closure of ATP-regulated K(+)-channels (K(ATP)-channels) and a depolarization-evoked increase in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) have been proposed to be essential. Here, we report that somatostatin release evoked by high glucose (>or=10 mM) is unaffected by the K(ATP)-channel activator diazoxide and proceeds normally in K(ATP)-channel-deficient islets. Glucose-induced somatostatin secretion is instead primarily dependent on Ca(2+)-induced Ca(2+)-release (CICR). This constitutes a novel mechanism for K(ATP)-channel-independent metabolic control of pancreatic hormone secretion.
Assuntos
Canais de Cálcio Tipo R/fisiologia , Cálcio/metabolismo , Glucose/farmacologia , Somatostatina/metabolismo , Animais , Cálcio/farmacologia , Canais de Cálcio Tipo R/genética , Citofotometria , Diazóxido/farmacologia , Relação Dose-Resposta a Droga , Eletrofisiologia , Imuno-Histoquímica , Técnicas In Vitro , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Isradipino/farmacologia , Compostos Macrocíclicos/farmacologia , Manoeptulose/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Microscopia Confocal , Oxazóis/farmacologia , Potássio/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/fisiologia , Rianodina/farmacologia , Células Secretoras de Somatostatina/efeitos dos fármacos , Células Secretoras de Somatostatina/metabolismoRESUMO
Besides opening and closing, high voltage-activated calcium channels transit to a nonconducting inactivated state from which they do not re-open unless the plasma membrane is repolarized. Inactivation is critical for temporal regulation of intracellular calcium signaling and prevention of a deleterious rise in calcium concentration. R-type high voltage-activated channels inactivate fully in a few hundred milliseconds when expressed alone. However, when co-expressed with a particular ß-subunit isoform, ß(2a), inactivation is partial and develops in several seconds. Palmitoylation of a unique di-cysteine motif at the N terminus anchors ß(2a) to the plasma membrane. The current view is that membrane-anchored ß(2a) immobilizes the channel inactivation machinery and confers slow inactivation phenotype. ß-Subunits contain one Src homology 3 and one guanylate kinase domain, flanked by variable regions with unknown structures. Here, we identified a short polybasic segment at the boundary of the guanylate kinase domain that slows down channel inactivation without relocating a palmitoylation-deficient ß(2a) to the plasma membrane. Substitution of the positively charged residues within this segment by alanine abolishes its slow inactivation-conferring phenotype. The linker upstream from the polybasic segment, but not the N- and C-terminal variable regions, masks the effect of this determinant. These results reveal a novel mechanism for inhibiting voltage-dependent inactivation of R-type calcium channels by the ß(2a)-subunit that might involve electrostatic interactions with an unknown target on the channel's inactivation machinery or its modulatory components. They also suggest that intralinker interactions occlude the action of the polybasic segment and that its functional availability is regulated by the palmitoylated state of the ß(2a)-subunit.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Ativação do Canal Iônico/fisiologia , Lipoilação/fisiologia , Subunidades Proteicas/metabolismo , Substituição de Aminoácidos , Animais , Canais de Cálcio Tipo R/genética , Humanos , Mapeamento de Peptídeos , Subunidades Proteicas/genética , Xenopus laevis , Domínios de Homologia de srcRESUMO
Ca(V)ß subunits are formed by a Src homology 3 domain and a guanylate kinase-like (GK) domain connected through a variable HOOK domain. Complete deletion of the Src homology 3 domain (75 residues) as well as deletion of the HOOK domain (47 residues) did not alter plasma membrane density of Ca(V)2.3 nor its typical activation gating. In contrast, six-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of Ca(V)2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of Ca(V)ß (P175A, P179A, M195A, M196A, K198A, S295A, R302G, R307A, E339G, N340G, and A345G) did not significantly alter cell surface targeting or gating modulation of Ca(V)2.3. Nonetheless, mutations of a quartet of leucine residues (either single or multiple mutants) in the α3, α6, ß10, and α9 regions of the GK domain were found to significantly impair cell surface density of Ca(V)2.3 channels. Furthermore, the normalized protein density of Ca(V)2.3 was nearly abolished with the quadruple Ca(V)ß3 Leu mutant L200G/L303G/L337G/L342G. Altogether, our observations suggest that the four leucine residues in Ca(V)ß3 form a hydrophobic pocket surrounding key residues in the α-interacting domain of Ca(V)2.3. This interaction appears to play an essential role in conferring Ca(V)ß-induced modulation of the protein density of Ca(V)α1 subunits in Ca(V)2 channels.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Animais , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Membrana Celular/genética , Células HEK293 , Humanos , Leucina/genética , Leucina/metabolismo , Estrutura Secundária de Proteína , Ratos , Domínios de Homologia de srcRESUMO
Ca(v)2.3 containing voltage-activated Ca(2+) channels are expressed in excitable cells and trigger neurotransmitter and peptide-hormone release. Their expression remote from the fast release sites leads to the accumulation of presynaptic Ca(2+) which can both, facilitate and inhibit the influx of Ca(2+) ions through Ca(v)2.3. The facilitated Ca(2+) influx was recently related to hippocampal postsynaptic facilitation and long term potentiation. To analyze Ca(2+) mediated modulation of cellular processes more in detail, protein partners of the carboxy terminal tail of Ca(v)2.3 were identified by yeast-2-hybrid screening, leading in two human cell lines to the detection of a novel, extended and rarely occurring splice variant of calmodulin-2 (CaM-2), called CaM-2-extended (CaM-2-ext). CaM-2-ext interacts biochemically with the C-terminus of Ca(v)2.3 similar to the classical CaM-2 as shown by co-immunoprecipitation. Functionally, only CaM-2-ext reduces whole cell inward currents significantly. The insertion of the novel 46 nts long exon and the consecutive expression of CaM-2-ext must be dependent on a new upstream translation initiation site which is only rarely used in the tested human cell lines. The structure of the N-terminal extension is predicted to be more hydrophobic than the remaining CaM-2-ext protein, suggesting that it may help to dock it to the lipophilic membrane surrounding.
Assuntos
Processamento Alternativo , Canais de Cálcio Tipo R/metabolismo , Calmodulina/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/genética , Calmodulina/química , Calmodulina/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Células HEK293 , Humanos , Imunoprecipitação , Dados de Sequência MolecularRESUMO
BACKGROUND: Visceral pain is common symptom involved in many gastrointestinal disorders such as inflammatory bowel disease. The underlying molecular mechanisms remain elusive. We investigated the molecular mechanisms and the role for voltage gated calcium channel (VGCC) in the pathogenesis in a rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS) induced visceral inflammatory hypersensitivity. RESULTS: Using Agilent cDNA arrays, we found 172 genes changed significantly in dorsal root ganglia (DRG) of TNBS treated rats. Among these changed genes, Cav1.2 and Cav2.3 were significantly up-regulated. Then the RT-PCR and Western blot further confirmed the up-regulation of Cav1.2 and Cav2.3. The whole cell patch clamp recording of acutely dissociated colonic specific DRG neurons showed that the peak IBa density was significantly increased in colonic neurons of TNBS treated rats compared with control rats (-127.82 ± 20.82 pA/pF Vs -91.67 ± 19.02 pA/pF, n = 9, *P < 0.05). To distinguish the different type of calcium currents with the corresponding selective channel blockers, we found that L-type (-38.56 ± 3.97 pA/pF Vs -25.75 ± 3.35 pA/pF, n = 9, * P < 0.05) and R-type (-13.31 ± 1.36 pA/pF Vs -8.60 ± 1.25 pA/pF, n = 9, * P < 0.05) calcium current density were significantly increased in colonic DRG neurons of TNBS treated rats compared with control rats. In addition, pharmacological blockade with L-type antagonist (nimodipine) and R-type antagonist (SNX-482) with intrathecal injection attenuates visceral pain in TNBS induced inflammatory visceral hypersensitivity. CONCLUSION: Cav1.2 and Cav2.3 in colonic primary sensory neurons play an important role in visceral inflammatory hyperalgesia, which maybe the potential therapeutic targets.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Hipersensibilidade/patologia , Vísceras/patologia , Animais , Western Blotting , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo R/genética , Carbocianinas/metabolismo , Proteínas de Transporte de Cátions/genética , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Perfilação da Expressão Gênica , Hipersensibilidade/genética , Injeções Espinhais , Masculino , Nimodipina/administração & dosagem , Nimodipina/farmacologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Venenos de Aranha/administração & dosagem , Venenos de Aranha/farmacologia , Ácido Trinitrobenzenossulfônico , Vísceras/efeitos dos fármacos , Vísceras/metabolismoRESUMO
Voltage-gated Ca(2+) channels regulate cardiac automaticity, rhythmicity and excitation-contraction coupling. Whereas L-type (Cav 1·2, Cav 1·3) and T-type (Cav 3·1, Cav 3·2) channels are widely accepted for their functional relevance in the heart, the role of Cav 2·3 Ca(2+) channels expressing R-type currents remains to be elucidated. We have investigated heart rate dynamics in control and Cav 2·3-deficient mice using implantable electrocardiogram radiotelemetry and pharmacological injection experiments. Autonomic block revealed that the intrinsic heart rate does not differ between both genotypes. Systemic administration of isoproterenol resulted in a significant reduction in interbeat interval in both genotypes. It remained unaffected after administering propranolol in Cav 2·3(-|-) mice. Heart rate from isolated hearts as well as atrioventricular conduction for both genotypes differed significantly. Additionally, we identified and analysed the developmental expression of two splice variants, i.e. Cav 2·3c and Cav 2·3e. Using patch clamp technology, R-type currents could be detected in isolated prenatal cardiomyocytes and be related to R-type Ca(2+) channels. Our results indicate that on the systemic level, the pharmacologically inducible heart rate range and heart rate reserve are impaired in Cav 2·3 (-|-) mice. In addition, experiments on Langendorff perfused hearts elucidate differences in basic properties between both genotypes. Thus, Cav 2·3 does not only contribute to the cardiac autonomous nervous system but also to intrinsic rhythm propagation.
Assuntos
Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Frequência Cardíaca/efeitos dos fármacos , Coração/efeitos dos fármacos , Isoproterenol/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Propranolol/farmacologia , Processamento Alternativo , Animais , Antiarrítmicos/farmacologia , Cálcio/metabolismo , Canais de Cálcio Tipo R/deficiência , Cardiotônicos/farmacologia , Proteínas de Transporte de Cátions/deficiência , Células Cultivadas , Coração/fisiologia , Frequência Cardíaca/fisiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , TelemetriaRESUMO
Mutations in distal S6 were shown to significantly alter the stability of the open state of Ca(V)2.3 (Raybaud, A., Baspinar, E. E., Dionne, F., Dodier, Y., Sauvé, R., and Parent, L. (2007) J. Biol. Chem. 282, 27944-27952). By analogy with K(V) channels, we tested the hypothesis that channel activation involves electromechanical coupling between S6 and the S4S5 linker in Ca(V)2.3. Among the 11 positions tested in the S4S5 linker of domain II, mutations of the leucine residue at position 596 were found to destabilize significantly the closed state with a -50 mV shift in the activation potential and a -20 mV shift in its charge-voltage relationship as compared with Ca(V)2.3 wt. A double mutant cycle analysis was performed by introducing pairs of glycine residues between S4S5 and S6 of Domain II. Strong coupling energies (ΔΔG(interact) > 2 kcal mol(-1)) were measured for the activation gating of 12 of 39 pairs of mutants. Leu-596 (IIS4S5) was strongly coupled with distal residues in IIS6 from Leu-699 to Asp-704. In particular, the double mutant L596G/I701G showed strong cooperativity with a ΔΔG(interact) ≈6 kcal mol(-1) suggesting that both positions contribute to the activation gating of the channel. Altogether, our results highlight the role of a leucine residue in S4S5 and provide the first series of evidence that the IIS4S5 and IIS6 regions are energetically coupled during the activation of a voltage-gated Ca(V) channel.