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1.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34156466

RESUMO

Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER-PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER-PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER-PM junctions, subsequent binding to STIM1 and channel activation.


Assuntos
Canais de Cálcio , Cálcio , Acilação , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
2.
FASEB J ; 35(8): e21723, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34224609

RESUMO

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+ -dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.


Assuntos
Canais de Cálcio/metabolismo , Espermatozoides/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/deficiência , Canais de Cálcio/genética , Sinalização do Cálcio , AMP Cíclico/metabolismo , Feminino , Fertilização In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Transdução de Sinais , Capacitação Espermática/fisiologia , Motilidade Espermática/fisiologia , Cauda do Espermatozoide/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/ultraestrutura , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores
3.
Methods Mol Biol ; 2268: 193-205, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085270

RESUMO

Intracellular calcium mobilization can be measured using several methods varying in indicator dyes and devices used. In this chapter, we describe the fluorescence-based method (FLIPR Calcium 4 Assay) developed by Molecular Devices for a FlexStation and routinely used in our laboratory for detecting intracellular calcium changes. The assay is designed to study calcium mobilization induced by majority of GPCRs and calcium channels and allows for simultaneous concentration-dependent analysis of several receptor agonists and antagonists, useful in receptor characterization and drug discovery projects.


Assuntos
Bioensaio/métodos , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Descoberta de Drogas/métodos , Fluorometria/métodos , Ensaios de Triagem em Larga Escala/métodos , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Humanos , Transdução de Sinais
4.
Ecotoxicol Environ Saf ; 220: 112394, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34091186

RESUMO

Arsenic (As) and antimony (Sb) are known as an environmental contaminant with cardiotoxicity properties. The endoplasmic reticulum (ER) is the largest calcium reservoir in the cell, and its calcium homeostasis disorder plays a vital role in endoplasmic reticulum stress (ERS) and apoptosis. The objective of this study was to investigate whether As and Sb induced apoptosis via endoplasmic reticulum stress (ERS) linked to calcium homeostasis disturbance. In this study, thirty-two adult mice were gavage-fed daily with As2O3 (4 mg/kg), SbCl3 (15 mg/kg) and co-treat with SbCl3 (15 mg/kg) and As2O3 (4 mg/kg) daily for 60 days. It was observed that As or/and Sb caused histopathological lesions and ER expansion of the heart. Meanwhile, the gene expression of ER Ca2+ release channels (RyR2 and IP3R) and calmodulin-dependent protein kinase II (CaMKII) increased while the levels of mRNA and protein of ER Ca2+ uptake channel (SERCA2) downregulated significantly compared to the controls. Then, As or/and Sb induced ERS and triggered the ER apoptotic pathway by activating unfolded protein response (UPR)-associated genes ((PERK, ATF6, IRE1, XBP1, JNK, GRP78), and apoptosis-related genes (Caspase12, Caspase3, p53, CHOP). Above indicators in As + Sb group became more severe than that of As group and Sb group. Overall, our results proved that the cardiotoxicity caused by As or/and Sb might be concerning disturbing calcium homeostasis, which induced apoptosis through the ERS pathway.


Assuntos
Antimônio/toxicidade , Arsênio/toxicidade , Cálcio/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Coração/efeitos dos fármacos , Animais , Antimônio/metabolismo , Apoptose , Arsênio/metabolismo , Canais de Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiotoxicidade/metabolismo , Cardiotoxinas , Caspase 3/metabolismo , Morte Celular , Regulação para Baixo , Retículo Endoplasmático/metabolismo , Poluentes Ambientais/toxicidade , Homeostase/efeitos dos fármacos , Masculino , Metais Pesados/toxicidade , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Resposta a Proteínas não Dobradas
5.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068417

RESUMO

The CACNA1A gene encodes the pore-forming α1A subunit of the voltage-gated CaV2.1 Ca2+ channel, essential in neurotransmission, especially in Purkinje cells. Mutations in CACNA1A result in great clinical heterogeneity with progressive symptoms, paroxysmal events or both. During infancy, clinical and neuroimaging findings may be unspecific, and no dysmorphic features have been reported. We present the clinical, radiological and evolutionary features of three patients with congenital ataxia, one of them carrying a new variant. We report the structural localization of variants and their expected functional consequences. There was an improvement in cerebellar syndrome over time despite a cerebellar atrophy progression, inconsistent response to acetazolamide and positive response to methylphenidate. The patients shared distinctive facial gestalt: oval face, prominent forehead, hypertelorism, downslanting palpebral fissures and narrow nasal bridge. The two α1A affected residues are fully conserved throughout evolution and among the whole human CaV channel family. They contribute to the channel pore and the voltage sensor segment. According to structural data analysis and available functional characterization, they are expected to exert gain- (F1394L) and loss-of-function (R1664Q/R1669Q) effect, respectively. Among the CACNA1A-related phenotypes, our results suggest that non-progressive congenital ataxia is associated with developmental delay and dysmorphic features, constituting a recognizable syndromic neurodevelopmental disorder.


Assuntos
Ataxia/patologia , Canais de Cálcio/genética , Mutação , Adulto , Sequência de Aminoácidos , Ataxia/congênito , Ataxia/etiologia , Ataxia/metabolismo , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Criança , Feminino , Humanos , Masculino , Neuroimagem , Fenótipo , Conformação Proteica , Homologia de Sequência , Relação Estrutura-Atividade , Adulto Jovem
6.
Methods Mol Biol ; 2277: 69-89, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080145

RESUMO

The mitochondrial calcium uniporter (MCU ) is an essential protein of the inner mitochondrial membrane that mediates the uptake of calcium into mitochondria of virtually all mammalian tissues, regulating cell metabolism, signaling, and death. MCU-mediated calcium uptake has been shown to play a pathophysiological role in diverse human disease contexts, which qualifies this channel as a druggable target for therapeutic intervention.Here, we present a protocol to perform drug screens to identify effective and specific MCU-targeting inhibitors. The methodology is based on the use of cryopreserved mitochondria that are isolated from a yeast strain engineered to express the human MCU and its essential regulator EMRE together with the luminescence calcium sensor aequorin. Yeast mitochondria with a functionally reconstituted MCU-mediated calcium uptake are then employed as a ready-to-use screening reagent. False discovery rate is further minimized by energizing mitochondria with D-lactate in a mannitol/sucrose-based medium, which provides a mean to discriminate between direct and secondary effects of drugs on mitochondrial calcium uptake. This screening assay is sensitive and robust and can be easily implemented in any laboratory.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Mitocôndrias/efeitos dos fármacos , Equorina/farmacologia , Cálcio/metabolismo , Canais de Cálcio/genética , Descoberta de Drogas/métodos , Humanos , Ácido Láctico/farmacologia , Mitocôndrias/metabolismo , Mitoxantrona/farmacologia , Saccharomyces cerevisiae/citologia
7.
Int J Mol Sci ; 22(10)2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-34063554

RESUMO

Acute lung injury (ALI) afflicts approximately 200,000 patients annually and has a 40% mortality rate. The COVID-19 pandemic has massively increased the rate of ALI incidence. The pathogenesis of ALI involves tissue damage from invading microbes and, in severe cases, the overexpression of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). This study aimed to develop a therapy to normalize the excess production of inflammatory cytokines and promote tissue repair in the lipopolysaccharide (LPS)-induced ALI. Based on our previous studies, we tested the insulin-like growth factor I (IGF-I) and BTP-2 therapies. IGF-I was selected, because we and others have shown that elevated inflammatory cytokines suppress the expression of growth hormone receptors in the liver, leading to a decrease in the circulating IGF-I. IGF-I is a growth factor that increases vascular protection, enhances tissue repair, and decreases pro-inflammatory cytokines. It is also required to produce anti-inflammatory 1,25-dihydroxyvitamin D. BTP-2, an inhibitor of cytosolic calcium, was used to suppress the LPS-induced increase in cytosolic calcium, which otherwise leads to an increase in proinflammatory cytokines. We showed that LPS increased the expression of the primary inflammatory mediators such as toll like receptor-4 (TLR-4), IL-1ß, interleukin-17 (IL-17), TNF-α, and interferon-γ (IFN-γ), which were normalized by the IGF-I + BTP-2 dual therapy in the lungs, along with improved vascular gene expression markers. The histologic lung injury score was markedly elevated by LPS and reduced to normal by the combination therapy. In conclusion, the LPS-induced increases in inflammatory cytokines, vascular injuries, and lung injuries were all improved by IGF-I + BTP-2 combination therapy.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Anilidas/farmacologia , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Tiadiazóis/farmacologia , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/virologia , Anilidas/uso terapêutico , Animais , COVID-19/complicações , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Citocinas/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/genética , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/uso terapêutico , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tiadiazóis/uso terapêutico , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
8.
Cells ; 10(5)2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067054

RESUMO

The flavonoid naringenin (Nar), present in citrus fruits and tomatoes, has been identified as a blocker of an emerging class of human intracellular channels, namely the two-pore channel (TPC) family, whose role has been established in several diseases. Indeed, Nar was shown to be effective against neoangiogenesis, a process essential for solid tumor progression, by specifically impairing TPC activity. The goal of the present review is to illustrate the rationale that links TPC channels to the mechanism of coronavirus infection, and how their inhibition by Nar could be an efficient pharmacological strategy to fight the current pandemic plague COVID-19.


Assuntos
COVID-19/tratamento farmacológico , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Flavanonas/farmacologia , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Arabidopsis/metabolismo , COVID-19/epidemiologia , COVID-19/patologia , COVID-19/virologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/virologia , Flavanonas/uso terapêutico , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/virologia , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Pandemias/prevenção & controle , SARS-CoV-2/patogenicidade , Vacúolos/metabolismo , Internalização do Vírus/efeitos dos fármacos
9.
Cell Mol Life Sci ; 78(11): 4993-5014, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33884443

RESUMO

Transient receptor potential (TRP) channels play prominent roles in ion homeostasis by their ability to control cation influx. Mouse placentation is governed by the processes of trophoblast proliferation, invasion, differentiation, and fusion, all of which require calcium signaling. Although certain TRP channels have been shown to contribute to maternal-fetal transport of magnesium and calcium, a role for TRP channels in specific trophoblast functions has been disregarded. Using qRT-PCR and in situ hybridisation, the spatio-temporal expression pattern of TRP channels in the mouse placenta across gestation (E10.5-E18.5) was assessed. Prominent expression was observed for Trpv2, Trpm6, and Trpm7. Calcium microfluorimetry in primary trophoblast cells isolated at E14.5 of gestation further revealed the functional activity of TRPV2 and TRPM7. Finally, comparing TRP channels expression in mouse trophoblast stem cells (mTSCs) and mouse embryonic stem cells (mESC) confirmed the specific expression of TRPV2 during placental development. Moreover, TRP channel expression was similar in mTSCs compared to primary trophoblasts and validate mTSC as a model to study TRP channels in placental development. Collectivity, our results identify a specific spatio-temporal TRP channel expression pattern in trophoblasts, suggesting a possible involvement in regulating the process of placentation.


Assuntos
Placenta/metabolismo , Placentação/genética , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Diferenciação Celular , Proliferação de Células , Feminino , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Gravidez , Células-Tronco/citologia , Células-Tronco/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Canais de Potencial de Receptor Transitório/genética , Trofoblastos/citologia , Trofoblastos/metabolismo
10.
Toxins (Basel) ; 13(4)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33808507

RESUMO

The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Genes Reporter , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Neuroblastoma/metabolismo , Neurotoxinas/farmacologia , Vesículas Secretórias/efeitos dos fármacos , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Luciferases/genética , Luciferases/metabolismo , Neuroblastoma/genética , Neuroblastoma/patologia , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , Vesículas Secretórias/genética , Vesículas Secretórias/metabolismo , Venenos de Aranha/farmacologia
11.
Yakugaku Zasshi ; 141(4): 491-499, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-33790116

RESUMO

Mitochondria play a role as intracellular calcium stores as well as energy conversion functions. Excessive calcium accumulation in mitochondria induces cell death and induces diseases such as ischemia-reperfusion injury. Mitochondrial calcium uptake is considered to be mediated by calcium uniporters, which have attracted much attention as potential drug targets. Although calcium uniporter was shown to function as an ion channel, the molecular mechanisms have long been unclear. In this decade, the molecular composition of the calcium uniporter complex was discovered; the calcium uniporter consists of the 7 subunits. Each subunit has no structural similarity to other Ca ion channels; thus, the novel molecular mechanism of the Ca2+ uptake by calcium uniporter is of interest. Although calcium uniporter is conserved in human to warm, yeast lack mitochondrial calcium uptake activity. In the previous study, various subunits of mammalian calcium uniporter were expressed in the yeast mitochondria. As a result, although the expression of each subunit alone did not affect on the mitochondrial calcium uptake activity, the co-expression of mitochondrial calcium uniporter (MCU) and essential MCU regulator (EMRE) enabled to reconstitute calcium uptake activity in yeast mitochondria. This indicated that MCU and EMRE are key factors of the calcium uptake activity in mitochondria. This yeast reconstitution technique has also enabled us to perform detailed structure-function analysis of the MCU and EMRE. In this paper, we will discuss the molecular mechanism of Ca2+ uptake and the prospects for drug discovery.


Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Canais de Cálcio/fisiologia , Descoberta de Drogas , Saccharomyces cerevisiae/metabolismo
12.
Int J Mol Sci ; 22(5)2021 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-33799975

RESUMO

Migraine is a common neurological disease that affects about 11% of the adult population. The disease is divided into two main clinical subtypes: migraine with aura and migraine without aura. According to the neurovascular theory of migraine, the activation of the trigeminovascular system (TGVS) and the release of numerous neuropeptides, including calcitonin gene-related peptide (CGRP) are involved in headache pathogenesis. TGVS can be activated by cortical spreading depression (CSD), a phenomenon responsible for the aura. The mechanism of CSD, stemming in part from aberrant interactions between neurons and glia have been studied in models of familial hemiplegic migraine (FHM), a rare monogenic form of migraine with aura. The present review focuses on those interactions, especially as seen in FHM type 1, a variant of the disease caused by a mutation in CACNA1A, which encodes the α1A subunit of the P/Q-type voltage-gated calcium channel.


Assuntos
Canais de Cálcio/metabolismo , Transtornos de Enxaqueca/etiologia , Neuroglia/patologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Humanos , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/fisiopatologia , Mutação , Neuroglia/metabolismo
13.
Int J Mol Sci ; 22(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806007

RESUMO

The Transient Receptor Potential Ankyrin 1 cation channel (TRPA1) is a broadly-tuned chemosensor expressed in nociceptive neurons. Multiple TRPA1 agonists are chemically unrelated non-electrophilic compounds, for which the mechanisms of channel activation remain unknown. Here, we assess the hypothesis that such chemicals activate TRPA1 by inducing mechanical perturbations in the plasma membrane. We characterized the activation of mouse TRPA1 by non-electrophilic alkylphenols (APs) of different carbon chain lengths in the para position of the aromatic ring. Having discarded oxidative stress and the action of electrophilic mediators as activation mechanisms, we determined whether APs induce mechanical perturbations in the plasma membrane using dyes whose fluorescence properties change upon alteration of the lipid environment. APs activated TRPA1, with potency increasing with their lipophilicity. APs increased the generalized polarization of Laurdan fluorescence and the anisotropy of the fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH), also according to their lipophilicity. Thus, the potency of APs for TRPA1 activation is an increasing function of their ability to induce lipid order and membrane rigidity. These results support the hypothesis that TRPA1 senses non-electrophilic compounds by detecting the mechanical alterations they produce in the plasma membrane. This may explain how structurally unrelated non-reactive compounds induce TRPA1 activation and support the role of TRPA1 as an unspecific sensor of potentially noxious compounds.


Assuntos
Membrana Celular/metabolismo , Fenóis/farmacologia , Canal de Cátion TRPA1/agonistas , Animais , Anisotropia , Células CHO , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Carbono/química , Cricetulus , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligantes , Lipídeos de Membrana , Camundongos , Nociceptores/metabolismo , Estresse Oxidativo
14.
Int J Mol Sci ; 22(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807167

RESUMO

Atractylodin (ATR) is a bioactive component found in dried rhizomes of Atractylodes lancea (AL) De Candolle. Although AL has accumulated empirical evidence for the treatment of pain, the molecular mechanism underlying the anti-pain effect of ATR remains unclear. In this study, we found that ATR increases transient receptor potential ankyrin-1 (TRPA1) single-channel activity in hTRPA1 expressing HEK293 cells. A bath application of ATR produced a long-lasting calcium response, and the response was completely diminished in the dorsal root ganglion neurons of TRPA1 knockout mice. Intraplantar injection of ATR evoked moderate and prolonged nociceptive behavior compared to the injection of allyl isothiocyanate (AITC). Systemic application of ATR inhibited AITC-induced nociceptive responses in a dose-dependent manner. Co-application of ATR and QX-314 increased the noxious heat threshold compared with AITC in vivo. Collectively, we concluded that ATR is a unique agonist of TRPA1 channels, which produces long-lasting channel activation. Our results indicated ATR-mediated anti-nociceptive effect through the desensitization of TRPA1-expressing nociceptors.


Assuntos
Furanos/metabolismo , Furanos/farmacologia , Canal de Cátion TRPA1/metabolismo , Analgésicos/metabolismo , Analgésicos/farmacologia , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Isotiocianatos/farmacologia , Lidocaína/análogos & derivados , Lidocaína/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Nociceptividade/efeitos dos fármacos , Nociceptores/metabolismo , Dor/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Canal de Cátion TRPA1/agonistas , Canal de Cátion TRPA1/efeitos dos fármacos , Canais de Cátion TRPC/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo
15.
Int J Mol Sci ; 22(6)2021 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-33806823

RESUMO

Sperm motility is linked to the activation of signaling pathways that trigger movement. These pathways are mainly dependent on Ca2+, which acts as a secondary messenger. The maintenance of adequate Ca2+ concentrations is possible thanks to proper concentrations of other ions, such as K+ and Na+, among others, that modulate plasma membrane potential and the intracellular pH. Like in every cell, ion homeostasis in spermatozoa is ensured by a vast spectrum of ion channels supported by the work of ion pumps and transporters. To achieve success in fertilization, sperm ion channels have to be sensitive to various external and internal factors. This sensitivity is provided by specific channel structures. In addition, novel sperm-specific channels or isoforms have been found with compositions that increase the chance of fertilization. Notably, the most significant sperm ion channel is the cation channel of sperm (CatSper), which is a sperm-specific Ca2+ channel required for the hyperactivation of sperm motility. The role of other ion channels in the spermatozoa, such as voltage-gated Ca2+ channels (VGCCs), Ca2+-activated Cl-channels (CaCCs), SLO K+ channels or voltage-gated H+ channels (VGHCs), is to ensure the activation and modulation of CatSper. As the activation of sperm motility differs among metazoa, different ion channels may participate; however, knowledge regarding these channels is still scarce. In the present review, the roles and structures of the most important known ion channels are described in regard to regulation of sperm motility in animals.


Assuntos
Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/metabolismo , Motilidade Espermática , Espermatozoides/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/química , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Canais de Cloreto/química , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Humanos , Canais Iônicos/genética , Masculino , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Sódio/química , Canais de Sódio/genética , Canais de Sódio/metabolismo , Relação Estrutura-Atividade
16.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806419

RESUMO

Arterial smooth muscle exhibits rhythmic oscillatory contractions called vasomotion and believed to be a protective mechanism against tissue hypoperfusion or hypoxia. Oscillations of vascular tone depend on voltage and follow oscillations of the membrane potential. Voltage-gated sodium channels (Nav), responsible for the initiation and propagation of action potentials in excitable cells, have also been evidenced both in animal and human vascular smooth muscle cells (SMCs). For example, they contribute to arterial contraction in rats, but their physiopathological relevance has not been established in human vessels. In the present study, we investigated the functional role of Nav in the human artery. Experiments were performed on human uterine arteries obtained after hysterectomy and on SMCs dissociated from these arteries. In SMCs, we recorded a tetrodotoxin (TTX)-sensitive and fast inactivating voltage-dependent INa current. Various Nav genes, encoding α-subunit isoforms sensitive (Nav 1.2; 1.3; 1.7) and resistant (Nav 1.5) to TTX, were detected both in arterial tissue and in SMCs. Nav channels immunostaining showed uniform distribution in SMCs and endothelial cells. On arterial tissue, we recorded variations of isometric tension, ex vivo, in response to various agonists and antagonists. In arterial rings placed under hypoxic conditions, the depolarizing agent KCl and veratridine, a specific Nav channels agonist, both induced a sustained contraction overlaid with rhythmic oscillations of tension. After suppression of sympathetic control either by blocking the release of catecholamine or by antagonizing the target adrenergic response, rhythmic activity persisted while the sustained contraction was abolished. This rhythmic activity of the arteries was suppressed by TTX but, in contrast, only attenuated by antagonists of calcium channels, Na+/Ca2+ exchanger, Na+/K+-ATPase and the cardiac Nav channel. These results highlight the role of Nav as a novel key element in the vasomotion of human arteries. Hypoxia promotes activation of Nav channels involved in the initiation of rhythmic oscillatory contractile activity.


Assuntos
Artérias/metabolismo , Hipóxia/metabolismo , Contração Muscular/fisiologia , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Adulto , Animais , Artérias/efeitos dos fármacos , Canais de Cálcio/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Tetrodotoxina/farmacologia
17.
Neuron ; 109(11): 1836-1847.e5, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-33915110

RESUMO

Mature behaviors emerge from neural circuits sculpted by genetic programs and spontaneous and evoked neural activity. However, how neural activity is refined to drive maturation of learned behavior remains poorly understood. Here, we explore how transient hormonal signaling coordinates a neural activity state transition and maturation of associative learning. We identify spontaneous, asynchronous activity in a Drosophila learning and memory brain region, the mushroom body. This activity declines significantly over the first week of adulthood. Moreover, this activity is generated cell-autonomously via Cacophony voltage-gated calcium channels in a single cell type, α'/ß' Kenyon cells. Juvenile hormone, a crucial developmental regulator, acts transiently in α'/ß' Kenyon cells during a young adult sensitive period to downregulate spontaneous activity and enable subsequent enhanced learning. Hormone signaling in young animals therefore controls a neural activity state transition and is required for improved associative learning, providing insight into the maturation of circuits and behavior.


Assuntos
Hormônios Juvenis/metabolismo , Aprendizagem , Corpos Pedunculados/metabolismo , Neurogênese , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Corpos Pedunculados/citologia , Corpos Pedunculados/crescimento & desenvolvimento , Corpos Pedunculados/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transmissão Sináptica
18.
Sci Signal ; 14(675)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758061

RESUMO

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.


Assuntos
COVID-19/metabolismo , COVID-19/virologia , Sinalização do Cálcio/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , NADP/análogos & derivados , SARS-CoV-2/fisiologia , Marcadores de Afinidade , Animais , Canais de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Química Click/métodos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , NADP/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transcriptoma , Internalização do Vírus
19.
FASEB J ; 35(4): e21528, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33742713

RESUMO

We have recently reported two different methodologies that improve sperm functionality. The first method involved transient exposure to the Ca2+ ionophore A23187 , and the second required sperm incubation in the absence of energy nutrients (starvation). Both methods were associated with an initial loss of motility followed by a rescue step involving ionophore removal or addition of energy metabolites, respectively. In this work, we show that starvation is accompanied by an increase in intracellular Ca2+ ([Ca2+ ]i ). Additionally, the starved cells acquire a significantly enhanced capacity to undergo a progesterone-induced acrosome reaction. Electrophysiological measurements show that CatSper channel remains active in starvation conditions. However, the increase in [Ca2+ ]i was also observed in sperm from CatSper null mice. Upon starvation, addition of energy nutrients reversed the effects on [Ca2+ ]i and decreased the effect of progesterone on the acrosome reaction to control levels. These data indicate that both methods have common molecular features.


Assuntos
Cálcio/metabolismo , Progesterona/farmacologia , Capacitação Espermática/efeitos dos fármacos , Inanição/metabolismo , Reação Acrossômica/efeitos dos fármacos , Animais , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Feminino , Masculino , Camundongos , Progesterona/metabolismo , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo
20.
Am J Physiol Cell Physiol ; 320(6): C966-C973, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33788632

RESUMO

Two types of voltage-dependent inward currents were evoked by depolarization in murine antral smooth muscle cells (SMCs) bathed in Ca2+-containing physiological solution: high-voltage-activated (HVA) and low-voltage-activated (LVA) inward currents. We examined whether the LVA current was due to: 1) T-type Ca2+ channels, 2) Ca2+-activated Cl-channels, 3) nonselective cation channels (NSCC), or 4) voltage-dependent K+ channels. Replacement of external Ca2+ (2 mM) with equimolar Ba2+ increased the amplitude of the HVA current but blocked the LVA current. Nicardipine blocked the HVA current, and in the presence of nicardipine, T-type Ca2+ blockers failed to block LVA current. A Cl- channel antagonist had little effect on LVA current. Cation-free external solution completely abolished both HVA and LVA currents. Addition of Ca2+ to the solution restored only HVA currents. Addition of K+ (5 mM) to otherwise cation-free solution induced LVA current that reversed at -20 mV. These data suggest that LVA current is not due to T-type Ca2+ channels, Ca2+-activated Cl- channels, or NSCC. A-type K+ (KA) currents and delayed rectifying K+ (KDR) currents can be resolved in antral SMCs dialyzed with a solution containing 140 mM K+. When cells were exposed to high K+ external solution and dialyzed with Cs+-rich solution in the presence of nicardipine, LVA current was evoked and reversed at positive potentials. LVA currents were blocked by K+ channel blockers, 4-aminopyridine, and tetraethylammonium. In conclusion, LVA inward currents can be generated by K+ influx via KA channels in murine antral SMCs when cells were dialyzed with Cs+-rich solution.


Assuntos
Potenciais da Membrana/fisiologia , Miócitos de Músculo Liso/metabolismo , Animais , Artefatos , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Células Cultivadas , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Canais de Potássio/metabolismo
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