RESUMO
Patients with recurrent acute pancreatitis (RAP) are at significant risk of developing early chronic pancreatitis (CP), which progresses into irreversible, end-stage CP with severe symptoms. There is no specific therapy in RAP or in early CP that may hinder disease progression. The pathogenesis of CP is complex and involves interactions among multiple cell types, including pancreatic acinar, ductal, and stellate cells (PSC). Therefore, it is pivotal to identify common pathogenic pathways in these cells that could be targeted pharmacologically. The Orai1-mediated store-operated Ca2+ entry (SOCE) is a ubiquitous signaling mechanism that may become overactivated in pathological states resulting in intracellular Ca2+ overload. In this study, we used ex vivo and in vivo preclinical disease models to demonstrate that Orai1 inhibition prevents progression of RAP and early CP. The selective Orai1 inhibitor CM5480 restored the expression of SOCE-associated regulatory factor in acinar cells, prevented uncontrolled Ca2+ elevation, protected acinar and ductal functions, mitigated immune cell infiltration, and diminished PSC activation, proliferation, and migration. We suggest that the overactivation of Orai1 is a crucial pathogenetic event in the progression of early CP and that inhibition of Orai1 could prevent the development of end-stage CP.
Assuntos
Cálcio , Pancreatite Crônica , Humanos , Cálcio/metabolismo , Doença Aguda , Canais de Cálcio/metabolismo , Proteína ORAI1/metabolismoRESUMO
Microglia are important mediators of neuroinflammation, which underlies neuropathic pain. However, the molecular checkpoints controlling microglial reactivity are not well-understood. Here, we investigated the role of Orai1 channels for microglia-mediated neuroinflammation following nerve injury and find that deletion of Orai1 in microglia attenuates Ca2+ signaling and the production of inflammatory cytokines by proalgesic agonists. Conditional deletion of Orai1 attenuated microglial proliferation in the dorsal horn, spinal cytokine levels, and potentiation of excitatory neurotransmission following peripheral nerve injury. These cellular effects were accompanied by mitigation of pain hyperalgesia in microglial Orai1 knockout mice. A small-molecule Orai1 inhibitor, CM4620, similarly mitigated allodynia in male mice. Unexpectedly, these protective effects were not seen in female mice, revealing sexual dimorphism in Orai1 regulation of microglial reactivity and hyperalgesia. Together, these findings indicate that Orai1 channels are key regulators of the sexually dimorphic role of microglia for the neuroinflammation that underlies neuropathic pain.
Assuntos
Microglia , Neuralgia , Camundongos , Masculino , Feminino , Animais , Microglia/metabolismo , Hiperalgesia/genética , Doenças Neuroinflamatórias , Neuralgia/genética , Camundongos Knockout , Citocinas/metabolismo , Medula Espinal , Proteína ORAI1/genéticaRESUMO
T cell activation and function depend on Ca2+ signals mediated by store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels formed by ORAI1 proteins. We here investigated how SOCE controls T cell function in pulmonary inflammation during a T helper 1 (TH1) cell-mediated response to influenza A virus (IAV) infection and TH2 cell-mediated allergic airway inflammation. T cell-specific deletion of Orai1 did not exacerbate pulmonary inflammation and viral burdens following IAV infection but protected mice from house dust mite-induced allergic airway inflammation. ORAI1 controlled the expression of genes including p53 and E2F transcription factors that regulate the cell cycle in TH2 cells in response to allergen stimulation and the expression of transcription factors and cytokines that regulate TH2 cell function. Systemic application of a CRAC channel blocker suppressed allergic airway inflammation without compromising immunity to IAV infection, suggesting that inhibition of SOCE is a potential treatment for allergic airway disease.
Assuntos
Canais de Cálcio , Vírus da Influenza A , Alérgenos , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Citocinas/metabolismo , Fatores de Transcrição E2F , Inflamação , Camundongos , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Inflammatory bowel disease (IBD) is characterized by dysregulated intestinal immune responses. Using mass cytometry (CyTOF) to analyze the immune cell composition in the lamina propria (LP) of patients with ulcerative colitis (UC) and Crohn's disease (CD), we observed an enrichment of CD4+ effector T cells producing IL-17A and TNF, CD8+ T cells producing IFNγ, T regulatory (Treg) cells, and innate lymphoid cells (ILC). The function of these immune cells is regulated by store-operated Ca2+ entry (SOCE), which results from the opening of Ca2+ release-activated Ca2+ (CRAC) channels formed by ORAI and STIM proteins. We observed that the pharmacologic inhibition of SOCE attenuated the production of proinflammatory cytokines including IL-2, IL-4, IL-6, IL-17A, TNF, and IFNγ by human colonic T cells and ILCs, reduced the production of IL-6 by B cells and the production of IFNγ by myeloid cells, but had no effect on the viability, differentiation, and function of intestinal epithelial cells. T cell-specific deletion of CRAC channel genes in mice showed that Orai1, Stim1, and Stim2-deficient T cells have quantitatively distinct defects in SOCE, which correlate with gradually more pronounced impairment of cytokine production by Th1 and Th17 cells and the severity of IBD. Moreover, the pharmacologic inhibition of SOCE with a selective CRAC channel inhibitor attenuated IBD severity and colitogenic T cell function in mice. Our data indicate that SOCE inhibition may be a suitable new approach for the treatment of IBD.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio , Doenças Inflamatórias Intestinais , Animais , Linfócitos T CD8-Positivos/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Humanos , Imunidade Inata , Interleucina-17/metabolismo , Interleucina-6/metabolismo , Camundongos , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/genética , Células Th17/metabolismoRESUMO
BACKGROUND: Calcium release-activated calcium (CRAC) channel inhibitors block proinflammatory cytokine release, preserve endothelial integrity and may effectively treat patients with severe COVID-19 pneumonia. METHODS: CARDEA was a phase 2, randomized, double-blind, placebo-controlled trial evaluating the addition of Auxora, a CRAC channel inhibitor, to corticosteroids and standard of care in adults with severe COVID-19 pneumonia. Eligible patients were adults with ≥ 1 symptom consistent with COVID-19 infection, a diagnosis of COVID-19 confirmed by laboratory testing using polymerase chain reaction or other assay, and pneumonia documented by chest imaging. Patients were also required to be receiving oxygen therapy using either a high flow or low flow nasal cannula at the time of enrolment and have at the time of enrollment a baseline imputed PaO2/FiO2 ratio > 75 and ≤ 300. The PaO2/FiO2 was imputed from a SpO2/FiO2 determine by pulse oximetry using a non-linear equation. Patients could not be receiving either non-invasive or invasive mechanical ventilation at the time of enrolment. The primary endpoint was time to recovery through Day 60, with secondary endpoints of all-cause mortality at Day 60 and Day 30. Due to declining rates of COVID-19 hospitalizations and utilization of standard of care medications prohibited by regulatory guidance, the trial was stopped early. RESULTS: The pre-specified efficacy set consisted of the 261 patients with a baseline imputed PaO2/FiO2≤ 200 with 130 and 131 in the Auxora and placebo groups, respectively. Time to recovery was 7 vs. 10 days (P = 0.0979) for patients who received Auxora vs. placebo, respectively. The all-cause mortality rate at Day 60 was 13.8% with Auxora vs. 20.6% with placebo (P = 0.1449); Day 30 all-cause mortality was 7.7% and 17.6%, respectively (P = 0.0165). Similar trends were noted in all randomized patients, patients on high flow nasal cannula at baseline or those with a baseline imputed PaO2/FiO2 ≤ 100. Serious adverse events (SAEs) were less frequent in patients treated with Auxora vs. placebo and occurred in 34 patients (24.1%) receiving Auxora and 49 (35.0%) receiving placebo (P = 0.0616). The most common SAEs were respiratory failure, acute respiratory distress syndrome, and pneumonia. CONCLUSIONS: Auxora was safe and well tolerated with strong signals in both time to recovery and all-cause mortality through Day 60 in patients with severe COVID-19 pneumonia. Further studies of Auxora in patients with severe COVID-19 pneumonia are warranted. Trial registration NCT04345614.
Assuntos
Benzamidas , Tratamento Farmacológico da COVID-19 , Canais de Cálcio Ativados pela Liberação de Cálcio , Pirazinas , Síndrome do Desconforto Respiratório , Adulto , Benzamidas/uso terapêutico , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Humanos , Pirazinas/uso terapêutico , Respiração Artificial , SARS-CoV-2 , Resultado do TratamentoRESUMO
The airway epithelial cells (AECs) lining the conducting passageways of the lung secrete a variety of immunomodulatory factors. Among these, PGE2 limits lung inflammation and promotes bronchodilation. By contrast, IL-6 drives intense airway inflammation, remodeling, and fibrosis. The signaling that differentiates the production of these opposing mediators is not understood. In this study, we find that the production of PGE2 and IL-6 following stimulation of human AECs by the damage-associated molecular pattern extracellular ATP shares a common requirement for Ca2+ release-activated Ca2+ (CRAC) channels. ATP-mediated synthesis of PGE2 required activation of metabotropic P2Y2 receptors and CRAC channel-mediated cytosolic phospholipase A2 signaling. By contrast, ATP-evoked synthesis of IL-6 occurred via activation of ionotropic P2X receptors and CRAC channel-mediated calcineurin/NFAT signaling. In contrast to ATP, which elicited the production of both PGE2 and IL-6, the uridine nucleotide, UTP, stimulated PGE2 but not IL-6 production. These results reveal that human AECs employ unique receptor-specific signaling mechanisms with CRAC channels as a signaling nexus to regulate release of opposing immunomodulatory mediators. Collectively, our results identify P2Y2 receptors, CRAC channels, and P2X receptors as potential intervention targets for airway diseases.
Assuntos
Dinoprostona/metabolismo , Inflamação/imunologia , Interleucina-6/metabolismo , Mucosa Respiratória/metabolismo , Trifosfato de Adenosina/farmacocinética , Alarminas/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Células Cultivadas , Humanos , Imunomodulação , Interleucina-6/genética , Fatores de Transcrição NFATC/metabolismo , Fosfolipases A2/metabolismo , Receptores Purinérgicos P2X/metabolismo , Mucosa Respiratória/patologia , Transdução de Sinais , Nucleotídeos de Uracila/metabolismoRESUMO
OBJECTIVES: To assess the safety of Auxora in patients with acute pancreatitis (AP), systemic inflammatory response syndrome (SIRS), and hypoxemia, and identify efficacy endpoints to prospectively test in future studies. METHODS: This phase 2, open-label, dose-response study randomized patients with AP, accompanying SIRS, and hypoxemia (n = 21) to receive low-dose or high-dose Auxora plus standard of care (SOC) or SOC alone. All patients received pancreatic contrast-enhanced computed tomography scans at screenings, day 5/discharge, and as clinically required 90 days postrandomization; scans were blinded and centrally read to determine AP severity using computed tomography severity index. Solid food tolerance was assessed at every meal and SIRS every 12 hours. RESULTS: The number of patients experiencing serious adverse events was not increased with Auxora versus SOC alone. Three (36.5%) patients with moderate AP receiving low-dose Auxora improved to mild AP; no computed tomography severity index improvements were observed with SOC. By study end, patients receiving Auxora better tolerated solid foods, had less persistent SIRS, and had reduced hospitalization versus SOC. CONCLUSIONS: The favorable safety profile and patient outcomes suggest Auxora may be an appropriate early treatment for patients with AP and SIRS. Clinical development will continue in a randomized, controlled, blinded, dose-ranging study.
Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Pancreatite/tratamento farmacológico , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Doença Aguda , Adulto , Idoso , Bloqueadores dos Canais de Cálcio/administração & dosagem , Bloqueadores dos Canais de Cálcio/efeitos adversos , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Estudos de Coortes , Relação Dose-Resposta a Droga , Feminino , Humanos , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Pancreatite/complicações , Insuficiência Respiratória/induzido quimicamente , Síndrome de Resposta Inflamatória Sistêmica/complicações , Resultado do TratamentoRESUMO
BACKGROUND AND PURPOSE: Ischemic injury triggers multiple pathological responses in the brain tissue, including spreading depolarizations across the cerebral cortex (cortical spreading depolarizations [CSD]). Microglia have been recently shown to play a significant role in the propagation of CSD. However, the intracellular responses of myeloid cells during ischemic stroke have not been investigated. METHODS: We have studied intracellular calcium activity in cortical microglia in the stroke model of the middle cerebral artery occlusion, using the murine Polr2a-based and Cre-dependent GCaMP5 and tdTomato reporter (PC::G5-tdT). High-speed 2-photon microscopy through cranial windows was employed to record signals from genetically encoded indicators of calcium. Inflammatory stimuli and pharmacological inhibition were used to modulate microglial calcium responses in the somatosensory cortex. RESULTS: In vivo imaging revealed periodical calcium activity in microglia during the hyperacute phase of ischemic stroke. This activity was more frequent during the first 6 hours after occlusion, but the amplitudes of calcium transients became larger at later time points. Consistent with CSD nature of these events, we reproducibly triggered comparable calcium transients with microinjections of potassium chloride (KCl) into adjacent cortical areas. Furthermore, lipopolysaccharide-induced peripheral inflammation, mimicking sterile inflammation during ischemic stroke, produced significantly greater microglial calcium transients during CSD. Finally, in vivo pharmacological analysis with CRAC (calcium release-activated channel) inhibitor CM-EX-137 demonstrated that CSD-associated microglial calcium transients after KCl microinjections are mediated at least in part by the CRAC mechanism. CONCLUSIONS: Our findings demonstrate that microglia participate in ischemic brain injury via previously undetected mechanisms, which may provide new avenues for therapeutic interventions.
Assuntos
Sinalização do Cálcio , AVC Isquêmico/fisiopatologia , Microglia , Doença Aguda , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Encefalite/induzido quimicamente , Encefalite/fisiopatologia , Processamento de Imagem Assistida por Computador , Infarto da Artéria Cerebral Média/fisiopatologia , Lipopolissacarídeos , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Células Mieloides , Cloreto de Potássio/farmacologia , Córtex Somatossensorial/fisiopatologiaRESUMO
BACKGROUND: Calcium release-activated calcium (CRAC) channel inhibitors stabilize the pulmonary endothelium and block proinflammatory cytokine release, potentially mitigating respiratory complications observed in patients with COVID-19. This study aimed to investigate the safety and efficacy of Auxora, a novel, intravenously administered CRAC channel inhibitor, in adults with severe or critical COVID-19 pneumonia. METHODS: A randomized, controlled, open-label study of Auxora was conducted in adults with severe or critical COVID-19 pneumonia. Patients were randomized 2:1 to receive three doses of once-daily Auxora versus standard of care (SOC) alone. The primary objective was to assess the safety and tolerability of Auxora. Following FDA guidance, study enrollment was halted early to allow for transition to a randomized, blinded, placebo-controlled study. RESULTS: In total, 17 patients with severe and three with critical COVID-19 pneumonia were randomized to Auxora and nine with severe and one with critical COVID-19 pneumonia to SOC. Similar proportions of patients receiving Auxora and SOC experienced ≥ 1 adverse event (75% versus 80%, respectively). Fewer patients receiving Auxora experienced serious adverse events versus SOC (30% versus 50%, respectively). Two patients (10%) receiving Auxora and two (20%) receiving SOC died during the 30 days after randomization. Among patients with severe COVID-19 pneumonia, the median time to recovery with Auxora was 5 days versus 12 days with SOC; the recovery rate ratio was 1.87 (95% CI, 0.72, 4.89). Invasive mechanical ventilation was needed in 18% of patients with severe COVID-19 pneumonia receiving Auxora versus 50% receiving SOC (absolute risk reduction = 32%; 95% CI, - 0.07, 0.71). Outcomes measured by an 8-point ordinal scale were significantly improved for patients receiving Auxora, especially for patients with a baseline PaO2/FiO2 = 101-200. CONCLUSIONS: Auxora demonstrated a favorable safety profile in patients with severe or critical COVID-19 pneumonia and improved outcomes in patients with severe COVID-19 pneumonia. These results, however, are limited by the open-label study design and small patient population resulting from the early cessation of enrollment in response to regulatory guidance. The impact of Auxora on respiratory complications in patients with severe COVID-19 pneumonia will be further assessed in a planned randomized, blinded, placebo-controlled study. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04345614 . Submitted on 7 April 2020.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Infecções por Coronavirus/terapia , Cuidados Críticos/métodos , Pneumonia Viral/terapia , Idoso , COVID-19 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Índice de Gravidade de Doença , Padrão de Cuidado , Resultado do TratamentoRESUMO
KEY POINTS: This work confirms previous reports that CM4620, a small molecule inhibitor of Ca2+ entry via store operated Ca2+ entry (SOCE) channels formed by stromal interaction molecule 1 (STIM1)/Orai complexes, attenuates acinar cell pathology and acute pancreatitis in mouse experimental models. Here we report that intravenous administration of CM4620 reduces the severity of acute pancreatitis in the rat, a hitherto untested species. Using CM4620, we probe further the mechanisms whereby SOCE via STIM1/Orai complexes contributes to the disease in pancreatic acinar cells, supporting a role for endoplasmic reticulum stress/cell death pathways in these cells. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes neutrophil oxidative burst and inflammatory gene expression during acute pancreatitis, including in immune cells which may be either circulating or invading the pancreas. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes activation and fibroinflammatory gene expression within pancreatic stellate cells. ABSTRACT: Key features of acute pancreatitis include excess cellular Ca2+ entry driven by Ca2+ depletion from the endoplasmic reticulum (ER) and subsequent activation of store-operated Ca2+ entry (SOCE) channels in the plasma membrane. In several cell types, including pancreatic acinar, stellate cells (PaSCs) and immune cells, SOCE is mediated via channels composed primarily of Orai1 and stromal interaction molecule 1 (STIM1). CM4620, a selective Orai1 inhibitor, prevents Ca2+ entry in acinar cells. This study investigates the effects of CM4620 in preventing or reducing acute pancreatitis features and severity. We tested the effects of CM4620 on SOCE, trypsinogen activation, acinar cell death, activation of NFAT and NF-κB, and inflammatory responses in ex vivo and in vivo rodent models of acute pancreatitis and human pancreatic acini. We also examined whether CM4620 inhibited cytokine release in immune cells, fibro-inflammatory responses in PaSCs, and oxidative burst in neutrophils, all cell types participating in pancreatitis. CM4620 administration to rats by i.v. infusion starting 30 min after induction of pancreatitis significantly diminished pancreatitis features including pancreatic oedema, acinar cell vacuolization, intrapancreatic trypsin activity, cell death signalling and acinar cell death. CM4620 also decreased myeloperoxidase activity and inflammatory cytokine expression in pancreas and lung tissues, fMLF peptide-induced oxidative burst in human neutrophils, and cytokine production in human peripheral blood mononuclear cells (PBMCs) and rodent PaSCs, indicating that Orai1/STIM1 channels participate in the inflammatory responses of these cell types during acute pancreatitis. These findings support pathological Ca2+ entry-mediated cell death and proinflammatory signalling as central mechanisms in acute pancreatitis pathobiology.
Assuntos
Amidinas/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Bloqueadores dos Canais de Cálcio/uso terapêutico , Proteína ORAI1/antagonistas & inibidores , Pancreatite/tratamento farmacológico , Prolina/análogos & derivados , Células Acinares/metabolismo , Amidinas/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Ceruletídeo , Citocinas/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Células Estreladas do Pâncreas/metabolismo , Pancreatite/induzido quimicamente , Pancreatite/imunologia , Pancreatite/metabolismo , Peroxidase/metabolismo , Prolina/farmacologia , Prolina/uso terapêutico , Ratos , Superóxidos/metabolismoRESUMO
Calcium release-activated calcium (CRAC) channels have been the target of drug discovery for many years. The identification of STIM and Orai proteins as key components of CRAC channels greatly facilitated this process because their co-expression in cell lines produced electrophysiological currents (ICRAC) much larger than those in native cells, making it easier to confirm and characterize the effects of modulatory compounds. A driving force in the quest for CRAC channel drugs has been the immunocompromised phenotype displayed by humans and mice with null or loss-of-function mutations in STIM1 or Orai1, suggesting that CRAC channel inhibitors could be useful therapeutics for autoimmune or inflammatory conditions. Emerging data also suggests that other therapeutic conditions may benefit from CRAC channel inhibition. However, only recently have CRAC channel inhibitors reached clinical trials. This review discusses the challenges associated with drug discovery and development on CRAC channels and the approaches employed to date, as well as the results, starting from initial high-throughput screens for CRAC channel modulators and progressing through target selection and justification, descriptions of pharmacological, safety and toxicological profiles of compounds, and finally the entry of CRAC channel inhibitors into clinical trials.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Desenvolvimento de Medicamentos/métodos , Descoberta de Drogas/métodos , Animais , Bloqueadores dos Canais de Cálcio/administração & dosagem , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Ensaios Clínicos como Assunto/métodos , Sistemas de Liberação de Medicamentos/tendências , Desenvolvimento de Medicamentos/tendências , Descoberta de Drogas/tendências , HumanosRESUMO
Alzheimer's disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Abeta(42) levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Abeta(40) levels while concomitantly elevating levels of Abeta(38) and Abeta(37). Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Abeta(42); chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g., intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs).
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/imunologia , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Anticorpos/farmacologia , Butiratos/farmacologia , Caderinas/metabolismo , Células Cultivadas , Cricetinae , Cricetulus , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Transferência Ressonante de Energia de Fluorescência/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrocarbonetos Halogenados/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Ratos , Receptores Notch/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Transfecção/métodosRESUMO
For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai in Drosophila cells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca2+ concentration near the interface, indicating Ca2+ influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca2+ signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca2+ influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca2+ signaling during subsequent antigen encounter.
Assuntos
Canais de Cálcio/fisiologia , Ativação Linfocitária , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Linfócitos T/imunologia , Regulação para Cima , Cálcio/metabolismo , Linhagem Celular , Humanos , Transporte de Íons , Proteína ORAI1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Molécula 1 de Interação EstromalRESUMO
Ca(2+) release-activated Ca(2+) (CRAC) channels, located in the plasma membrane, are opened upon release of Ca(2+) from intracellular stores, permitting Ca(2+) entry and sustained [Ca(2+)](i) signaling that replenishes the store in numerous cell types. This mechanism is particularly important in T lymphocytes of the immune system, providing the missing link in the signal transduction cascade that is initiated by T cell receptor engagement and leads to altered expression of genes that results ultimately in the production of cytokines and cell proliferation. In the past three years, RNA interference screens together with over-expression and site-directed mutagenesis have identified the triggering molecule (Stim) that links store depletion to CRAC channel-mediated Ca(2+) influx and the pore subunit (Orai) of the CRAC channel that allows highly selective entry of Ca(2+) ions into cells.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Drosophila/fisiologia , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteína ORAI1 , Interferência de RNA , Molécula 1 de Interação Estromal , Linfócitos T/imunologiaRESUMO
Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Drosophila melanogaster/genética , Genoma de Inseto , Interferência de RNA , Animais , Canais de Cálcio/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Inibidores Enzimáticos/metabolismo , Humanos , Técnicas de Patch-Clamp , RNA de Cadeia Dupla/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Tapsigargina/metabolismoRESUMO
As the sole Ca2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca2+ signalling and many other cellular processes. A calcium-release-activated calcium (CRAC) channel in T lymphocytes is the best-characterized SOC influx channel and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously we identified Stim and the mammalian homologue STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes. Here we show that the expression of EF-hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca2+ store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulum-like sites to the plasma membrane upon depletion of the Ca2+ store. We propose that STIM1 functions as the missing link between Ca2+ store depletion and SOC influx, serving as a Ca2+ sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Animais , Biotinilação , Sinalização do Cálcio , Linhagem Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Motivos EF Hand/genética , Retículo Endoplasmático/metabolismo , Imunofluorescência , Humanos , Transporte de Íons , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia Imunoeletrônica , Modelos Biológicos , Mutação/genética , Transporte Proteico , Ratos , Molécula 1 de Interação EstromalRESUMO
Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Sequência Conservada/fisiologia , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Evolução Molecular , Humanos , Células Jurkat , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Neoplasias/genética , Técnicas de Patch-Clamp , Interferência de RNA , Molécula 1 de Interação Estromal , Tapsigargina/farmacologiaRESUMO
The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels and their modulation by the auxiliary beta1b, beta2e, beta3a, and beta4a subunits were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing human CaV2.1 channels. These calcium channels showed a complex modal gating, which is described in this and the following paper (Fellin, T., S. Luvisetto, M. Spagnolo, and D. Pietrobon. 2004. J. Gen. Physiol. 124:463-474). Here, we report the characterization of two modes of gating of human CaV2.1 channels, the slow mode and the fast mode. A channel in the two gating modes differs in mean closed times and latency to first opening (both longer in the slow mode), in voltage dependence of the open probability (larger depolarizations are necessary to open the channel in the slow mode), in kinetics of inactivation (slower in the slow mode), and voltage dependence of steady-state inactivation (occurring at less negative voltages in the slow mode). CaV2.1 channels containing any of the four beta subtypes can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. In both modes, CaV2.1 channels display different rates of inactivation and different steady-state inactivation depending on the beta subtype. The type of beta subunit also modulates the relative occurrence of the slow and the fast gating mode of CaV2.1 channels; beta3a promotes the fast mode, whereas beta4a promotes the slow mode. The prevailing mode of gating of CaV2.1 channels lacking a beta subunit is a gating mode in which the channel shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages than in either the fast or slow mode.
Assuntos
Canais de Cálcio Tipo N/fisiologia , Ativação do Canal Iônico/fisiologia , Rim/fisiologia , Potenciais da Membrana/fisiologia , Células Cultivadas , Condutividade Elétrica , Humanos , Técnicas de Patch-Clamp/métodos , Subunidades Proteicas/fisiologia , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20-50 pA at -110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing approximately 300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/fisiologia , Proteínas de Drosophila/fisiologia , Ácido Egtázico/análogos & derivados , Animais , Soluções Tampão , Canais de Cálcio/metabolismo , Linhagem Celular , Diálise , Drosophila , Proteínas de Drosophila/metabolismo , Ácido Egtázico/farmacologia , Técnicas de Patch-Clamp , Tapsigargina/farmacologiaRESUMO
Regardless of the voltage-gated ion channel that is targeted in a drug discovery effort for the treatment of epilepsy, two routes have been followed historically: 1). a compound initially, and often surreptitiously, discovered due to activity in animal seizure models is further optimized by medicinal chemistry, or 2). a molecular target is identified based on the phenotype of transgenic animals, or linkage studies from humans with the disease, and compounds are then investigated within a mechanistic framework. Antagonists of voltage-gated sodium channels have been pursued utilizing primarily the first approach; many of these compounds also have significant activity at other ion channels. Both approaches have been utilized to discover voltage-gated calcium channel antagonists, although most efforts to date have used the first approach. Several spontaneous mutant mice and transgenic animals have been utilized to probe the role of the numerous voltage-gated calcium channel subunits and their isoforms as potential molecular targets. Compounds that open or prolong the opening of voltage-gated potassium channels have been discovered using the first approach, with a detailed understanding of the molecular target and mechanism of action coming to light several years later. Genetic evidence from humans is limited to relatively rare forms of epilepsy, and transgenic animals with interesting phenotypes do not always translate into good molecular targets in humans. No clinically-useful antiepileptic drug (AED) has been developed to date that specifically interacts with one, or even one class, of ion channels to produce a therapeutic effect. The tools now exist to search for potent, selective, and safe ion channel modulators for the treatment of epilepsy. This review seeks to summarize the most recent pre-clinical and clinical efforts focused on voltage-gated ion-channels for the development of AEDs.