Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 537
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Artif Cells Nanomed Biotechnol ; 47(1): 2948-2956, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31317779

RESUMO

Neurotoxicity of local anesthetics is often reported in the clinic, more and more people pay attention to them. CaMKIIß, a subtype of CaMKII, is detected in the central nervous system. Previous study found that CaMKIIß mRNA are up-regulated in DRG neurons treated with ropivacaine hydrochloride, as well as inhibition of Cav3.2 and Cav3.3 expression can improve the local anesthetics neurotoxicity. In this study, we observed the effect of CaMKIIß on neurotoxicity injury induced by ropivacaine hydrochloride with DRG cell in vitro. We first constructed the pAd-shRNA-CaMKIIß-DRG to inhibit CaMKIIß mRNA expression and detected the cell viability, cell apoptosis rate, CaMKIIß, Cav3.2 and Cav3.3 expression. The results showed that ropivacaine hydrochloride caused the DRG cell injury with cell viability decreased and cell apoptosis rate increased, CaMKIIß, Cav3.2 and Cav3.3 expression up-regulated. Interestingly, inhibition of CaMKIIß expression protected the DRG cell from the neurotoxicity injury induced by ropivacaine hydrochloride, increased the cell viability and decreased the apoptosis rate, as well as inhibition of CaMKIIß expression down-regulated Cav3.2 and Cav3.3 expression. In other words, CaMKIIß is involved with the DRG injury induced by ropivacaine hydrochloride. Inhibition CaMKIIß expression improved DRG injury, increased the cell viability and decreased cell apoptosis rate.


Assuntos
Anestésicos Locais/toxicidade , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Gânglios Espinais/citologia , Neurotoxinas/toxicidade , Ropivacaina/toxicidade , Animais , Apoptose/efeitos dos fármacos , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Sobrevivência Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley
2.
BMJ Case Rep ; 12(5)2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-31126930

RESUMO

The CACNA1H gene encodes the pore-forming α1 subunit of the T-type voltage-dependent calcium channel CaV3.2, expressed abundantly in the adrenal cortex. Mutations in CACNA1H are associated with various forms of primary aldosteronism (PA), including familial hyperaldosteronism type 4 (FH4). We describe a patient with refractory hypokalaemia and elevated aldosterone secretion independent of renin activity. Despite the absence of overt hypertension in this patient, the laboratory evaluation was consistent with a diagnosis of PA. Whole-exome sequencing revealed a de novo missense variant, R890H, in the voltage sensing domain of CACNA1H Expression of the variant channel in cells resulted in decreased whole-cell current, consistent with a loss-of-function. We hypothesise this variant is the genetic cause of pathological aldosterone secretion in this patient, and thereby expand the current understanding of the genetic basis of FH4.


Assuntos
Canais de Cálcio Tipo T/genética , Síndrome de Ehlers-Danlos , Predisposição Genética para Doença , Hiperaldosteronismo/diagnóstico , Hipopotassemia/etiologia , Adulto , Diagnóstico Diferencial , Feminino , Células Germinativas , Humanos , Hiperaldosteronismo/sangue , Hiperaldosteronismo/complicações , Hiperaldosteronismo/genética
3.
Neuroscience ; 409: 81-100, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31029730

RESUMO

Voltage-gated Ca2+ channels (VGCCs) play key roles in auditory perception and information processing within the inner ear and brainstem. Pharmacological inhibition of low voltage-activated (LVA) T-type Ca2+ channels is related to both age- and noise induced hearing loss in experimental animals and may represent a promising approach to the treatment of auditory impairment of various etiologies. Within the LVA Ca2+ channel subgroup, Cav3.2 is the most prominently expressed T-type channel entity in the cochlea and auditory brainstem. Thus, we performed a complete gender specific click and tone burst based auditory brainstem response (ABR) analysis of Cav3.2+/- and Cav3.2-/- mice, including i.a. temporal progression in hearing loss, amplitude growth function and wave latency analysis as well as a cochlear qPCR based evaluation of other VGCCs transcripts. Our results, based on a self-programmed automated wavelet approach, demonstrate that both heterozygous and Cav3.2 null mutant mice exhibit age-dependent increases in hearing thresholds at 5 months of age. In addition, complex alterations in WI-IV amplitudes and latencies were detected that were not attributable to alterations in the expression of other VGCCs in the auditory tract. Our results clearly demonstrate the important physiological role of Cav3.2 VGCCs in the spatiotemporal organization of auditory processing in young adult mice and suggest potential pharmacological targets for interventions in the future.


Assuntos
Limiar Auditivo/fisiologia , Canais de Cálcio Tipo T/metabolismo , Perda Auditiva/metabolismo , Audição/fisiologia , Animais , Canais de Cálcio Tipo T/genética , Cóclea/metabolismo , Perda Auditiva/genética , Camundongos , Camundongos Knockout
4.
Br J Anaesth ; 122(5): 643-651, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30916017

RESUMO

BACKGROUND: Hypnotics and general anaesthetics impair memory by altering hippocampal synaptic plasticity. We recently reported on a neurosteroid analogue with potent hypnotic activity [(3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile; 3ß-OH], which does not cause developmental neurotoxicity in rat pups. Here, we investigated the effects of 3ß-OH on neuronal excitability in the subiculum, the major output structure of the hippocampal formation, and synaptic plasticity at two key hippocampal synapses in juvenile rats. METHODS: Biophysical properties of isolated T-type calcium currents (T-currents) in the rat subiculum were investigated using acute slice preparations. Subicular T-type calcium channel (T-channel) subtype mRNA expression was compared using qRT-PCR. Using electrophysiological recordings, we examined the effects of 3ß-OH and an endogenous neuroactive steroid, allopregnanolone (Allo), on T-currents and burst firing properties of subicular neurones, and on the long-term potentiation (LTP) in CA3-CA1 and CA1-subiculum pathways. RESULTS: Biophysical and molecular studies confirmed that CaV3.1 channels represent the dominant T-channel isoform in the subiculum of juvenile rats. 3ß-OH and Allo inhibited rebound burst firing by decreasing the amplitude of T-currents in a voltage-dependent manner with similar potency, with 30-80% inhibition. Both neurosteroids suppressed LTP at the CA1-subiculum, but not at the CA3-CA1 Schaffer collateral synapse. CONCLUSIONS: Neurosteroid effects on T-channels modulate hippocampal output and provide possible molecular mechanisms for the amnestic action of the novel hypnotic 3ß-OH. Effects on T-channels in the subiculum provide a novel target for amnestic effects of hypnotics.


Assuntos
Androstanóis/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipnóticos e Sedativos/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Nitrilos/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/biossíntese , Canais de Cálcio Tipo T/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Masculino , RNA Mensageiro/genética , Ratos Sprague-Dawley
5.
BMC Res Notes ; 12(1): 157, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894204

RESUMO

OBJECTIVES: Voltage-gated Ca2+ channels (VGCCs) are of central relevance in regulating Ca2+ influx into living cells. The low-voltage activated (LVA) Cav3 T-type Ca2+ channels are widely distributed throughout the brain including the peripheral auditory system and ascending auditory tract. Their exact role in auditory information processing is still not fully understood. Within the LVA subgroup, Cav3.2 T-type Ca2+ channels seem to be of special importance as qPCR revealed a steady increase in Cav3.2 transcript levels over age, e.g. in the cochlea and spiral ganglion neurons (SGN). Furthermore, pharmacological studies suggested an association between Cav3.2 expression and both age-related and noise-induced hearing loss. Given the potential functional relevance of Cav3.2 VGGCs in sensorineural hearing loss, we recorded gender specific auditory evoked brainstem responses (ABRs) upon both click and tone burst presentation. Here we present auditory brainstem response (ABR) data from Cav3.2+/+, Cav3.2+/- and Cav3.2-/- mice from both genders which are of value for researchers who want to evaluate how Cav3.2 loss affects basic auditory parameters, e.g. click and tone burst based hearing thresholds, amplitude growth function and peak latencies. DATA DESCRIPTION: Information presented here includes ABR data from age-matched female and male Cav3.2+/+, Cav3.2+/- and Cav3.2-/- mice and technical aspects of the auditory recording protocol. Data were recorded using a commercially available ABR setup from Tucker Davis Technologies Inc. (TDT). Raw data files (arf.-file format) were exported as txt.-files with free access for analysis.


Assuntos
Audiometria de Resposta Evocada/métodos , Canais de Cálcio Tipo T/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Perda Auditiva Neurossensorial/fisiopatologia , Animais , Canais de Cálcio Tipo T/deficiência , Canais de Cálcio Tipo T/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Fatores Sexuais
6.
Cancer Res ; 79(8): 1857-1868, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30755443

RESUMO

T-type Ca2+ channels (TTCC) have been identified as key regulators of cancer cell cycle and survival. In vivo studies in glioblastoma (GBM) murine xenografts have shown that drugs able to block TTCC in vitro (such as tetralol derivatives mibefradil/NNC-55-096, or different 3,4-dihydroquinazolines) slow tumor progression. However, currently available TTCC pharmacologic blockers have limited selectivity for TTCC and are unable to distinguish between TTCC isoforms. Here we analyzed the expression of TTCC transcripts in human GBM cells and show a prevalence of Cacna1g/Cav3.1 mRNAs. Infection of GBM cells with lentiviral particles carrying short hairpin RNA against Cav3.1 resulted in GBM cell death by apoptosis. We generated a murine GBM xenograft via subcutaneous injection of U87-MG GBM cells and found that tumor size was reduced when Cav3.1 expression was silenced. Furthermore, we developed an in vitro model of temozolomide-resistant GBM that showed increased expression of Cav3.1 accompanied by the activation of macroautophagy. We confirmed a positive correlation between Cav3.1 and autophagic markers in both GBM cultures and biopsies. Of note, Cav3.1 knockdown resulted in transcriptional downregulation of p62/SQSTM1 and deficient autophagy. Together, these data identify Cav3.1 channels as potential targets for slowing GBM progression and recurrence based on their role in regulating autophagy. SIGNIFICANCE: These findings identify Cav3.1 calcium channels as a molecular target to regulate autophagy and prevent progression and chemotherapeutic resistance in glioblastoma.


Assuntos
Neoplasias Encefálicas/patologia , Canais de Cálcio Tipo T/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/patologia , Temozolomida/farmacologia , Animais , Antineoplásicos Alquilantes/farmacologia , Apoptose , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Canais de Cálcio Tipo T/genética , Proliferação de Células , Progressão da Doença , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Mol Pain ; 15: 1744806919836569, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30803310

RESUMO

Cav3 channels play an important role in modulating chronic pain. However, less is known about the functional changes of Cav3 channels in superficial spinal dorsal horn in neuropathic pain states. Here, we examined the effect of partial sciatic nerve ligation (PSNL) on either expression or electrophysiological properties of Cav3 channels in superficial spinal dorsal horn. Our in vivo studies showed that the blockers of Cav3 channels robustly alleviated PSNL-induced mechanical allodynia and thermal hyperalgesia, which lasted at least 14 days following PSNL. Meanwhile, PSNL triggered an increase in both mRNA and protein levels of Cav3.2 but not Cav3.1 or Cav3.3 in rats. However, in Cav3.2 knockout mice, PSNL predominantly attenuated mechanical allodynia but not thermal hyperalgesia. In addition, the results of whole-cell patch-clamp recordings showed that both the overall proportion of Cav3 current-expressing neurons and the Cav3 current density in individual neurons were elevated in spinal lamina II neurons from PSNL rats, which could not be recapitulated in Cav3.2 knockout mice. Altogether, our findings reveal that the elevated functional Cav3.2 channels in superficial spinal dorsal horn may contribute to the mechanical allodynia in PSNL-induced neuropathic pain model.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Animais , Western Blotting , Canais de Cálcio Tipo T/genética , Eletrofisiologia , Hiperalgesia/genética , Hiperalgesia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Substância Gelatinosa/citologia
8.
Neuroscience ; 402: 78-89, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30677486

RESUMO

Here we report that the low-voltage-dependent T-type calcium (Ca2+) channel Cav3.2, encoded by the CACNA1H gene, regulates neuronal differentiation during early embryonic brain development through activating caspase-3. At the onset of neuronal differentiation, neural progenitor cells exhibited spontaneous Ca2+ activity. This activity strongly correlated with the upregulation of CACNA1H mRNA. Cells exhibiting robust spontaneous Ca2+ signaling had increased caspase-3 activity unrelated to apoptosis. Inhibition of Cav3.2 by drugs or viral CACNA1H knock down resulted in decreased caspase-3 activity followed by suppressed neurogenesis. In contrast, when CACNA1H was overexpressed, increased neurogenesis was detected. Cortical slices from Cacna1h knockout mice showed decreased spontaneous Ca2+ activity, a significantly lower protein level of cleaved caspase-3, and microanatomical abnormalities in the subventricular/ventricular and cortical plate zones when compared to their respective embryonic controls. In summary, we demonstrate a novel relationship between Cav3.2 and caspase-3 signaling that affects neurogenesis in the developing brain.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Caspase 3/metabolismo , Diferenciação Celular , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Canais de Cálcio Tipo T/genética , Sinalização do Cálcio , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos Laterais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Neuroepiteliais/metabolismo
9.
Nat Commun ; 10(1): 314, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30659191

RESUMO

Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp-dependent, Cre-mediated Cav3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research.


Assuntos
Encéfalo/efeitos da radiação , DNA Nucleotidiltransferases/genética , Animais , Comportamento Animal/fisiologia , Encéfalo/enzimologia , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , DNA Nucleotidiltransferases/metabolismo , DNA Nucleotidiltransferases/efeitos da radiação , Regulação da Expressão Gênica , Inativação Gênica , Células HEK293 , Humanos , Luz , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Optogenética , Recombinação Genética
11.
Toxicology ; 413: 33-39, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30552955

RESUMO

Bortezomib, a first-line agent for treatment of multiple myeloma, exhibits anticancer activity through proteasome inhibition. However, bortezomib-induced peripheral neuropathy (BIPN) is one of the most serious side effects. Since decreased proteasomal degradation of Cav3.2 T-type calcium channels in the primary afferents is involved in persistent pain, we investigated whether BIPN involves increased protein levels of Cav3.2 in mice. Six repeated i.p. administrations of bortezomib for 12 days developed persistent mechanical allodynia. Systemic administration of novel T-type calcium channel blockers, (2R/S)-6-prenylnaringenin and KTt-45, and of TTA-A2, the well-known blocker, reversed the BIPN. Ascorbic acid, known to block Cav3.2, but not Cav3.1 or 3.3, and silencing of Cav3.2 gene also suppressed BIPN. Protein levels of Cav3.2 in the dorsal root ganglion (DRG) at L4-L6 levels increased throughout days 1-21 after the onset of bortezomib treatment. Protein levels of USP5, a deubiquitinating enzyme that specifically inhibits proteasomal degradation of Cav3.2, increased in DRG on days 3-21, but not day 1, in bortezomib-treated mice. In DRG-derived ND7/23 cells, bortezomib increased protein levels of Cav3.2 and T-channel-dependent currents, as assessed by a patch-clamp method, but did not upregulate expression of Cav3.2 mRNA or USP5 protein. MG-132, another proteasome inhibitor, also increased Cav3.2 protein levels in the cultured cells. Given the previous evidence for USP5 induction following nociceptor excitation, our data suggest that BIPN involves the increased protein levels of Cav3.2 in nociceptors through inhibition of proteasomal degradation of Cav3.2 by bortezomib itself and then by USP5 that is upregulated probably in an activity-dependent manner.


Assuntos
Antineoplásicos/toxicidade , Bortezomib/toxicidade , Canais de Cálcio Tipo T/biossíntese , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/metabolismo , Inibidores de Proteassoma/toxicidade , Animais , Canais de Cálcio Tipo T/deficiência , Canais de Cálcio Tipo T/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes/métodos , Masculino , Camundongos , Doenças do Sistema Nervoso Periférico/genética , Ratos
12.
ACS Nano ; 13(1): 274-283, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30566319

RESUMO

Multidrug resistance (MDR) is the key cause that accounts for the failure of clinical cancer chemotherapy. To address the problem, herein, we presented an alternative strategy to conquer drug-resistant breast cancer through the combinatorial delivery of Ca2+ channel siRNA with cytotoxic drugs. Mesoporous silica nanocapsules (MSNCs) with mesoporous and hollow structure were fabricated for co-delivery of T-type Ca2+ channel siRNA and doxorubicin (DOX) with high drug loading efficiency. The DOX/siRNA co-loaded MSNCs showed a synergistic therapeutic effect on drug-resistant breast cancer cells MCF-7/ADR, while had only an additive effect on the drug-sensitive MCF-7 counterpart. It was found that the combination of T-type Ca2+ channel siRNA and DOX had a similar effect on MCF-7 and MCF-7/ADR in the knockdown of overexpressed T-type Ca2+ channels and decrease in cytosolic Ca2+ concentration ([Ca2+]i), but it specifically induced G0/G1 phase cell-cycle arrest and intracellular drug accumulation enhancement in MCF-7/ADR. The in vitro and in vivo results demonstrated that the MSNCs with good biocompatibility had a high efficiency for conquering the drug-resistant breast cancer with the DOX/calcium channel siRNA cocktail co-delivery. It provides a biological target for drug/gene delivery enhanced cancer therapy with nanoformulations.


Assuntos
Antineoplásicos/administração & dosagem , Sinalização do Cálcio , Doxorrubicina/administração & dosagem , Neoplasias Mamárias Experimentais/tratamento farmacológico , Nanocápsulas/química , Terapêutica com RNAi/métodos , Animais , Antineoplásicos/uso terapêutico , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Células MCF-7 , Masculino , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Nanocápsulas/efeitos adversos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Dióxido de Silício/química
13.
PLoS One ; 13(12): e0206986, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30571684

RESUMO

T-type calcium channels in the brain mediate the pathophysiology of epilepsy, pain, and sleep. Recently, we developed a novel therapeutic candidate, SAK3 (ethyl 8'-methyl-2',4-dioxo-2-(piperidin-1-yl)-2'H-spiro[cyclopentane-1,3'-imidazo[1,2-a] pyridine]-2-ene-3-carboxylate), for Alzheimer's disease (AD). The cognitive improvement by SAK3 is closely associated with enhanced acetylcholine (ACh) release in the hippocampus. Since monoamines such as dopamine (DA), noradrenaline (NA), and serotonin (5-HT) are also involved in hippocampus-dependent learning and psychomotor behaviors in mice, we investigated the effects of SAK3 on these monoamine releases in the mouse brain. Oral administration of SAK3 (0.5 mg/kg, p.o.) significantly promoted DA and 5-HT releases in the naive mouse hippocampal CA1 region but not in the medial prefrontal cortex (mPFC), while SAK3 did not affect NA release in either brain region. The T-type calcium channel-specific inhibitor, NNC 55-0396 (1 µM) significantly antagonized SAK3-enhanced DA and 5-HT releases in the hippocampus. Interestingly, the α7 nicotinic ACh receptor (nAChR) antagonist, methyllycaconitine (1 nM) significantly inhibited DA release, and the α4 nAChR antagonist, dihydro-ß-erythroidine (100 µM) significantly blocked both DA and 5-HT releases following SAK3 (0.5 mg/kg, p.o.) administration in the hippocampus. SAK3 did not alter basal monoamine contents both in the mPFC and hippocampus. SAK3 (0.5 mg/kg, p.o.) administration also significantly elevated DA and 5-HT releases in the hippocampal CA1 region of amyloid-precursor protein (APP)NL-GF knock-in (KI) mice. Moreover, hippocampal DA and 5-HT contents were significantly decreased in APPNL-GF KI mice. Taken together, our data suggest that SAK3 promotes monoamine DA and 5-HT releases by enhancing the T-type calcium channel and nAChR in the mouse hippocampus.


Assuntos
Precursor de Proteína beta-Amiloide/genética , Região CA1 Hipocampal/efeitos dos fármacos , Canais de Cálcio Tipo T/metabolismo , Dopamina/metabolismo , Técnicas de Introdução de Genes , Imidazóis/farmacologia , Serotonina/metabolismo , Compostos de Espiro/farmacologia , Animais , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/fisiologia , Canais de Cálcio Tipo T/deficiência , Canais de Cálcio Tipo T/genética , Cognição/efeitos dos fármacos , Técnicas de Inativação de Genes , Masculino , Camundongos
14.
Channels (Austin) ; 12(1): 378-387, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30403912

RESUMO

Contributions of voltage sensing S4 segments in domains I - IV of CaV3.1 channel to channel activation were analyzed. Neutralization of the uppermost charge in individual S4 segments by exchange of arginine for cysteine was employed. Mutant channels with single exchange in domains I - IV, in two adjacent domains, and in all four domains were constructed and expressed in HEK 293 cells. Changes in maximal gating charge Qmax and the relation between Qmax and maximal conductance Gmax were evaluated. Qmax was the most affected by single mutation in domain I and by double mutations in domains I + II and I + IV. The ratio Gmax/Qmax proportional to opening probability of the channel was significantly decreased by the mutation in domain III and increased by mutations in domains I and II. In channels containing double mutations Gmax/Qmax ratio increased significantly when the mutation in domain I was included. Mutations in domains II and III zeroed each other. Mutation in domain IV prevented the decrease caused by the mutation in domain III. Neither ion current nor gating current was observed when channels with quadruple mutations were expressed. Immunocytochemistry analysis did not reveal the presence of channel protein in the cell membrane. Likely, quadruple mutation results in a structural change that affects the channel's trafficking mechanism. Altogether, S4 segments in domains I-IV of the CaV3.1 channel unequally contribute to channel gating by voltage. We suggest the most important role of the voltage sensor in the domain I and lesser roles of voltage sensors in domains II and III.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Animais , Canais de Cálcio Tipo T/análise , Canais de Cálcio Tipo T/genética , Membrana Celular/química , Membrana Celular/metabolismo , Células HEK293 , Humanos , Camundongos , Mutação
15.
Elife ; 72018 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-30465523

RESUMO

Learning the spatial organization of the environment is essential for most animals' survival. This requires the animal to derive allocentric spatial information from egocentric sensory and motor experience. The neural mechanisms underlying this transformation are mostly unknown. We addressed this problem in electric fish, which can precisely navigate in complete darkness and whose brain circuitry is relatively simple. We conducted the first neural recordings in the preglomerular complex, the thalamic region exclusively connecting the optic tectum with the spatial learning circuits in the dorsolateral pallium. While tectal topographic information was mostly eliminated in preglomerular neurons, the time-intervals between object encounters were precisely encoded. We show that this reliable temporal information, combined with a speed signal, can permit accurate estimation of the distance between encounters, a necessary component of path-integration that enables computing allocentric spatial relations. Our results suggest that similar mechanisms are involved in sequential spatial learning in all vertebrates.


Assuntos
Egocentrismo , Peixe Elétrico/fisiologia , Percepção Espacial/fisiologia , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Modelos Biológicos , Movimento (Física) , Neurônios/fisiologia , Tálamo/fisiologia , Fatores de Tempo , Vias Visuais/fisiologia
16.
Arterioscler Thromb Vasc Biol ; 38(10): 2371-2381, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30354206

RESUMO

Objective- This study examined whether caveolae position CaV3.2 (T-type Ca2+ channel encoded by the α-3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca2+ influx to trigger Ca2+ sparks and large-conductance Ca2+-activated K+ channel feedback. Approach and Results- Using smooth muscle cells from mouse mesenteric arteries, the proximity ligation assay confirmed that CaV3.2 reside within 40 nm of caveolin 1, a key caveolae protein. Methyl-ß-cyclodextrin, a cholesterol depleting agent that disrupts caveolae, suppressed CaV3.2 activity along with large-conductance Ca2+-activated K+-mediated spontaneous transient outward currents in cells from C57BL/6 but not CaV3.2-/- mice. Genetic deletion of caveolin 1, a perturbation that prevents caveolae formation, also impaired spontaneous transient outward current production but did so without impairing Ca2+ channel activity, including CaV3.2. These observations indicate a mistargeting of CaV3.2 in caveolin 1-/- mice, a view supported by a loss of Ni2+-sensitive Ca2+ spark generation and colocalization signal (CaV3.2-RyR) from the proximity ligation assay. Vasomotor and membrane potential measurements confirmed that cellular disruption of the CaV3.2-RyR axis functionally impaired the ability of large-conductance Ca2+-activated K+ to set tone in pressurized caveolin 1-/- arteries. Conclusions- Caveolae play a critical role in protein targeting and preserving the close structural relationship between CaV3.2 and RyR needed to drive negative feedback control in resistance arteries.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio , Cavéolas/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Canais de Cálcio Tipo T/deficiência , Canais de Cálcio Tipo T/genética , Caveolina 1/genética , Caveolina 1/metabolismo , Retroalimentação Fisiológica , Masculino , Potenciais da Membrana , Artérias Mesentéricas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Vasoconstrição , Vasodilatação
17.
Mar Drugs ; 16(11)2018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-30366389

RESUMO

Memory retrieval dysfunction is a symptom of schizophrenia, autism spectrum disorder (ASD), and absence epilepsy (AE), as well as an early sign of Alzheimer's disease. To date, few drugs have been reported to enhance memory retrieval. Here, we found that a coral-derived natural product, excavatolide-B (Exc-B), enhances contextual memory retrieval in both wild-type and Cav3.2-/- mice via repressing the delayed rectifier potassium current, thus lowering the threshold for action potential initiation and enhancing induction of long-term potentiation (LTP). The human CACNA1H gene encodes a T-type calcium channel (Cav3.2), and its mutation is associated with schizophrenia, ASD, and AE, which are all characterized by abnormal memory function. Our previous publication demonstrated that Cav3.2-/- mice exhibit impaired contextual-associated memory retrieval, whilst their retrieval of spatial memory and auditory cued memory remain intact. The effect of Exc-B on enhancing the retrieval of context-associated memory provides a hope for novel drug development.


Assuntos
Canais de Potássio de Retificação Tardia/antagonistas & inibidores , Diterpenos/farmacologia , Hipocampo/efeitos dos fármacos , Memória/efeitos dos fármacos , Animais , Comportamento Animal , Canais de Cálcio Tipo T/genética , Canais de Potássio de Retificação Tardia/metabolismo , Espinhas Dendríticas/efeitos dos fármacos , Medo/psicologia , Hipocampo/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células
18.
Int J Mol Sci ; 19(10)2018 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-30262720

RESUMO

Activation of the mineralocorticoid receptor (MR) in the heart is considered to be a cardiovascular risk factor. MR activation leads to heart hypertrophy and arrhythmia. In ventricular cardiomyocytes, aldosterone induces a profound remodeling of ion channel expression, in particular, an increase in the expression and activity of T-type voltage-gated calcium channels (T-channels). The molecular mechanisms immediately downstream from MR activation, which lead to the increased expression of T-channels and, consecutively, to an acceleration of spontaneous cell contractions in vitro, remain poorly investigated. Here, we investigated the putative role of a specific microRNA in linking MR activation to the regulation of T-channel expression and cardiomyocyte beating frequency. A screening assay identified microRNA 204 (miR-204) as one of the major upregulated microRNAs after aldosterone stimulation of isolated neonatal rat cardiomyocytes. Aldosterone significantly increased the level of miR-204, an effect blocked by the MR antagonist spironolactone. When miR-204 was overexpressed in isolated cardiomyocytes, their spontaneous beating frequency was significantly increased after 24 h, like upon aldosterone stimulation, and messenger RNAs coding T-channels (CaV3.1 and CaV3.2) were increased. Concomitantly, T-type calcium currents were significantly increased upon miR-204 overexpression. Specifically repressing the expression of miR-204 abolished the aldosterone-induced increase of CaV3.1 and CaV3.2 mRNAs, as well as T-type calcium currents. Finally, aldosterone and miR-204 overexpression were found to reduce REST-NRSF, a known transcriptional repressor of CaV3.2 T-type calcium channels. Our study thus strongly suggests that miR-204 expression stimulated by aldosterone promotes the expression of T-channels in isolated rat ventricular cardiomyocytes, and therefore, increases the frequency of the cell spontaneous contractions, presumably through the inhibition of REST-NRSF protein.


Assuntos
Canais de Cálcio Tipo T/genética , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Potenciais de Ação , Aldosterona/farmacologia , Animais , Canais de Cálcio Tipo T/metabolismo , Células Cultivadas , MicroRNAs/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Ratos , Ratos Wistar
19.
Cell ; 175(1): 239-253.e17, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30197081

RESUMO

Many disease-causing missense mutations affect intrinsically disordered regions (IDRs) of proteins, but the molecular mechanism of their pathogenicity is enigmatic. Here, we employ a peptide-based proteomic screen to investigate the impact of mutations in IDRs on protein-protein interactions. We find that mutations in disordered cytosolic regions of three transmembrane proteins (GLUT1, ITPR1, and CACNA1H) lead to an increased clathrin binding. All three mutations create dileucine motifs known to mediate clathrin-dependent trafficking. Follow-up experiments on GLUT1 (SLC2A1), the glucose transporter causative of GLUT1 deficiency syndrome, revealed that the mutated protein mislocalizes to intracellular compartments. Mutant GLUT1 interacts with adaptor proteins (APs) in vitro, and knocking down AP-2 reverts the cellular mislocalization and restores glucose transport. A systematic analysis of other known disease-causing variants revealed a significant and specific overrepresentation of gained dileucine motifs in structurally disordered cytosolic domains of transmembrane proteins. Thus, several mutations in disordered regions appear to cause "dileucineopathies."


Assuntos
Transportador de Glucose Tipo 1/fisiologia , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/fisiologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/fisiologia , Erros Inatos do Metabolismo dos Carboidratos , Clatrina/metabolismo , Citoplasma/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Proteínas Intrinsicamente Desordenadas/metabolismo , Leucina/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Transporte de Monossacarídeos/deficiência , Mutação/genética , Peptídeos , Ligação Proteica , Proteômica/métodos
20.
Medicine (Baltimore) ; 97(36): e12148, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30200108

RESUMO

RATIONALE: Spinocerebellar ataxia (SCA), a genetically inherited heterogeneous disorder, is characterized by gait ataxia, dysarthria, parkinsonism, choreic movements, dystonia, epilepsy, cognitive and psychiatric symptoms. Spinocerebellar ataxia-42 (SCA42), caused by heterozygous mutation in the calcium channel 1G (CACNA1G) gene, is a rare SCA subtype and the transmission pattern is autosomal dominant inheritance. PATIENT CONCERNS: We presented a novel mutation (c.4721T>A; p.Met1574Lys) in 3 patients from a Chinese family using whole-exome sequencing. All patients exhibited cerebellar ataxia and the clinical manifestations were similar to those that were previously reported in the French and Japanese families. In addition, cerebral magnetic resonance imaging (MRI) showed cerebellar atrophy, and the hot cross bun sign of brainstem was found in the proband and her sister. DIAGNOSES: The clinical features and MRI findings indicated the diagnosis of SCA. Taken together, the symptoms, MRI findings, as well as whole-exome sequencing made the diagnosis of SCA42 most likely candidate. INTERVENTIONS AND OUTCOMES: The patient was treated with cobamamide (1.5 mg once daily) for nerve nutrition and further physical therapy. At the 4-month follow-up visit, the patient's condition did not improve obviously. LESSONS: Recently, a missense mutation in CACNA1G gene (c.5144G4A; p.Arg1715His) was identified in French and Japanese families with SCA42. However, there has been no report of SCA42 or its mutant loci in Chinese patients. Our finding showed a novel mutation in CACNA1G gene and provided important insights into the pathogenesis of SCA42.


Assuntos
Canais de Cálcio Tipo T/genética , Mutação , Ataxias Espinocerebelares/genética , Família , Feminino , Predisposição Genética para Doença , Humanos , Pessoa de Meia-Idade , Ataxias Espinocerebelares/diagnóstico por imagem , Ataxias Espinocerebelares/terapia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA