RESUMEN
T-cell receptor stimulation triggers cytosolic Ca2+ signaling by inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels gated by ER-located stromal-interacting molecules (STIM1/2). Physiologically, cytosolic Ca2+ signaling manifests as regenerative Ca2+ oscillations, which are critical for nuclear factor of activated T-cells-mediated transcription. In most cells, Ca2+ oscillations are thought to originate from IP3 receptor-mediated Ca2+ release, with CRAC channels indirectly sustaining them through ER refilling. Here, experimental and computational evidence support a multiple-oscillator mechanism in Jurkat T-cells whereby both IP3 receptor and CRAC channel activities oscillate and directly fuel antigen-evoked Ca2+ oscillations, with the CRAC channel being the major contributor. KO of either STIM1 or STIM2 significantly reduces CRAC channel activity. As such, STIM1 and STIM2 synergize for optimal Ca2+ oscillations and activation of nuclear factor of activated T-cells 1 and are essential for ER refilling. The loss of both STIM proteins abrogates CRAC channel activity, drastically reduces ER Ca2+ content, severely hampers cell proliferation and enhances cell death. These results clarify the mechanism and the contribution of STIM proteins to Ca2+ oscillations in T-cells.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio , Señalización del Calcio , Humanos , Calcio/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio/genética , Células Jurkat , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/genética , Molécula de Interacción Estromal 2/metabolismo , Técnicas de Inactivación de Genes , Modelos Biológicos , Isoformas de Proteínas , Transporte de Proteínas/genética , Proliferación Celular/genética , Supervivencia Celular/genéticaRESUMEN
The essential role of store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels in T cells is well established. In contrast, the contribution of individual Orai isoforms to SOCE and their downstream signaling functions in B cells are poorly understood. Here, we demonstrate changes in the expression of Orai isoforms in response to B cell activation. We show that both Orai3 and Orai1 mediate native CRAC channels in B cells. The combined loss of Orai1 and Orai3, but not Orai3 alone, impairs SOCE, proliferation and survival, nuclear factor of activated T cells (NFAT) activation, mitochondrial respiration, glycolysis, and the metabolic reprogramming of primary B cells in response to antigenic stimulation. Nevertheless, the combined deletion of Orai1 and Orai3 in B cells did not compromise humoral immunity to influenza A virus infection in mice, suggesting that other in vivo co-stimulatory signals can overcome the requirement of BCR-mediated CRAC channel function in B cells. Our results shed important new light on the physiological roles of Orai1 and Orai3 proteins in SOCE and the effector functions of B lymphocytes.
Asunto(s)
Linfocitos B , Canales de Calcio , Proteína ORAI1 , Animales , Ratones , Linfocitos B/metabolismo , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismoRESUMEN
Antigen receptor stimulation triggers cytosolic Ca2+ signals, which activate transcriptional and metabolic programs critical for immune function. B-cell receptor (BCR) engagement causes rapid cytosolic Ca2+ rise through the ubiquitous store-operated calcium entry (SOCE) pathway. Slc8b1, which encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), extrudes Ca2+ out of the mitochondria and maintains optimal SOCE activity. Inhibition of NCLX in DT40 and A20 B lymphocyte lines was recently shown to impair cytosolic Ca2+ transients in response to antigen-receptor stimulation, however the downstream functional consequences of this impairment remain unclear. Here, we generated Slc8b1 knockout A20 B-cell lines using CRISPR/Cas9 technology and B-cell specific Slc8b1 knockout mice. Surprisingly, while loss of Slc8b1 in B lymphocytes led to reduction in SOCE, it had a marginal effect on mitochondrial Ca2+ extrusion, suggesting that NCLX is not the major mitochondrial Ca2+ extrusion mechanism in B cells. Furthermore, endoplasmic reticulum (ER) Ca2+ content and rates of ER depletion and refilling remained unaltered in Slc8b1 knockout B cells. Slc8b1 deficiency increased mitochondrial production of oxidants, reduced mitochondrial bioenergetics and altered mitochondrial ultrastructure. B-cell specific Slc8b1 knockout mice showed reduced germinal center B cell responses following foreign antigen and pathogen driven immune responses. Our studies provide novel insights into the function of Slc8b1 in germinal center B cells and its contribution to B-cell signaling and effector function.
Asunto(s)
Calcio , Intercambiador de Sodio-Calcio , Animales , Ratones , Linfocitos B/metabolismo , Calcio/metabolismo , Señalización del Calcio/fisiología , Ratones Noqueados , Mitocondrias/metabolismo , Sodio/metabolismo , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
The structural changes of airway smooth muscle (ASM) that characterize airway remodeling (AR) are crucial to the pathogenesis of asthma. During AR, ASM cells dedifferentiate from a quiescent to a proliferative, migratory, and secretory phenotype. Calcium (Ca2+) is a ubiquitous second messenger that regulates many cellular processes, including proliferation, migration, contraction, and metabolism. Furthermore, mitochondria have emerged as major Ca2+ signaling organelles that buffer Ca2+ through uptake by the mitochondrial Ca2+ uniporter and extrude it through the Na+/Ca2+ exchanger (NCLX/Slc8b1). Here, we show using mitochondrial Ca2+-sensitive dyes that NCLX only partially contributes to mitochondrial Ca2+ extrusion in ASM cells. Yet, NCLX is necessary for ASM cell proliferation and migration. Through cellular imaging, RNA-Seq, and biochemical assays, we demonstrate that NCLX regulates these processes by preventing mitochondrial Ca2+ overload and supporting store-operated Ca2+ entry, activation of Ca2+/calmodulin-dependent kinase II, and transcriptional and metabolic reprogramming. Using small animal respiratory mechanic measurements and immunohistochemistry, we show that smooth muscle-specific NCLX KO mice are protected against AR, fibrosis, and hyperresponsiveness in an experimental model of asthma. Our findings support NCLX as a potential therapeutic target in the treatment of asthma.
Asunto(s)
Asma , Intercambiador de Sodio-Calcio , Remodelación de las Vías Aéreas (Respiratorias) , Animales , Asma/genética , Calcio/metabolismo , Ratones , Músculo Liso/metabolismo , Sodio/metabolismo , Intercambiador de Sodio-Calcio/genética , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
Airway remodeling and airway hyperresponsiveness are central drivers of asthma severity. Airway remodeling is a structural change involving the dedifferentiation of airway smooth muscle (ASM) cells from a quiescent to a proliferative and secretory phenotype. Here, we show up-regulation of the endoplasmic reticulum Ca2+ sensor stromal-interacting molecule 1 (STIM1) in ASM of asthmatic mice. STIM1 is required for metabolic and transcriptional reprogramming that supports airway remodeling, including ASM proliferation, migration, secretion of cytokines and extracellular matrix, enhanced mitochondrial mass, and increased oxidative phosphorylation and glycolytic flux. Mechanistically, STIM1-mediated Ca2+ influx is critical for the activation of nuclear factor of activated T cells 4 and subsequent interleukin-6 secretion and transcription of pro-remodeling transcription factors, growth factors, surface receptors, and asthma-associated proteins. STIM1 drives airway hyperresponsiveness in asthmatic mice through enhanced frequency and amplitude of ASM cytosolic Ca2+ oscillations. Our data advocates for ASM STIM1 as a target for asthma therapy.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Asma/fisiopatología , Músculo Liso/fisiopatología , Hipersensibilidad Respiratoria , Molécula de Interacción Estromal 1/fisiología , Animales , Asma/patología , Calcio/metabolismo , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Reprogramación Celular/fisiología , Enfermedad Crónica , Transporte Iónico , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Músculo Liso/patología , Molécula de Interacción Estromal 1/genética , Transcripción Genética/fisiologíaRESUMEN
Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling pathway that is evolutionarily conserved across eukaryotes. SOCE is triggered physiologically when the endoplasmic reticulum (ER) Ca2+ stores are emptied through activation of inositol 1,4,5-trisphosphate receptors. SOCE is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which are highly Ca2+ selective. Upon store depletion, the ER Ca2+-sensing STIM proteins aggregate and gain extended conformations spanning the ER-plasma membrane junctional space to bind and activate Orai, the pore-forming proteins of hexameric CRAC channels. In recent years, studies on STIM and Orai tissue-specific knockout mice and gain- and loss-of-function mutations in humans have shed light on the physiological functions of SOCE in various tissues. Here, we describe recent findings on the composition of native CRAC channels and their physiological functions in immune, muscle, secretory, and neuronal systems to draw lessons from transgenic mice and human diseases caused by altered CRAC channel activity.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/fisiología , Señalización del Calcio/fisiología , Humanos , Proteínas de la Membrana/fisiología , Ratones , Molécula de Interacción Estromal 1/metabolismoRESUMEN
Mitochondrial Ca2+ uptake tailors the strength of stimulation of plasma membrane phospholipase C-coupled receptors to that of cellular bioenergetics. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, subcellular Ca2+ imaging, and mathematical modeling to show that MCU is a universal regulator of intracellular Ca2+ signaling across mammalian cell types. MCU activity sustains cytosolic Ca2+ signaling by preventing Ca2+-dependent inactivation of store-operated Ca2+ release-activated Ca2+ channels and by inhibiting Ca2+ extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca2+ responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, nuclear translocation of nuclear factor for activated T cells transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol-mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca2+ clearance and buffering capacity of mitochondria reciprocally regulate interorganellar Ca2+ transfer and nuclear factor for activated T cells nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca2+ buffering by mitochondria but also in shaping endoplasmic reticulum and cytosolic Ca2+ signals that regulate cellular transcription and function.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Citosol/metabolismo , Mitocondrias/metabolismo , Factores de Transcripción NFATC/metabolismo , Sistemas CRISPR-Cas , Canales de Calcio/genética , Retículo Endoplásmico , Técnicas de Inactivación de Genes , Células HCT116 , Células HEK293 , Humanos , Células Jurkat , Activación de Linfocitos , Factores de Transcripción NFATC/genética , Linfocitos T/metabolismoRESUMEN
This protocol outlines steps to visualize and detect Ca2+ puffs following photo-liberation of caged inositol-1,4,5-trisphosphate (IP3) from HEK-293 cells expressing only the native IP3R type 1 receptor using total internal reflection fluorescence (TIRF) microscopy. TIRF microscopy offers high axial resolution and allows imaging at high speed, with a higher signal-to-background ratio. Additionally, we shed light on commonly encountered pitfalls, which should be considered while recording Ca2+ puffs using TIRF microscopy. For complete details on the use and execution of this protocol, please refer to Emrich et al. (2021) and Lock et al. (2015a).
Asunto(s)
Señalización del Calcio , Microscopía Fluorescente/métodos , Animales , Células HEK293 , HumanosRESUMEN
BACKGROUND: Inflammation of diverticula, which are outpouchings of the colonic bowl wall, causes diverticulitis. Severe cases of diverticulitis require surgical intervention. Through RNA-seq analysis of intestinal tissues, we previously found that the innate immune response was deregulated in surgical diverticulitis patients. In that study, pro-inflammatory and macrophage markers were differentially expressed in the colons of diverticulitis versus control patients. Here we investigate CD163L1+ macrophages and the pro-inflammatory chemokine, CXCL10, in diverticulitis. MATERIALS AND METHODS: We assessed tissue from an uninvolved area adjacent to a region of the sigmoid colon chronically affected by diverticulitis and performed Spearman's correlation on transcripts associated with macrophage signaling. We identified altered CD163L1 and CXCL10 gene expression levels that we confirmed by RT-qPCR analysis on an independent cohort of diverticulitis patients and controls. We used immunofluorescence microscopy to localize CD163L1+ macrophages and CXCL10 levels in intestinal tissue and ELISA to measure CXCL10 levels in patient serum. RESULTS: We found a positive correlation between intestinal CD163L1 and CXCL10 gene expression and an increased number of CD163L1+ macrophages in the sigmoid colons of diverticulitis patients relative to controls (P = 0.036). Macrophages at the apices of colonic crypts expressed the chemokine CXCL10. Correspondingly, these diverticulitis patients also displayed heightened CXCL10 levels in their serum (P = 0.007). CONCLUSIONS: We identified a novel population of CD163L1+CXCL10+ macrophages in the colonic crypts of diverticulitis patients and demonstrated increased expression of serum CXCL10 in these patients. CXCL10 may serve as a prognostic biomarker to aid in clinical decision making for diverticulitis patients.
Asunto(s)
Quimiocina CXCL10 , Diverticulitis , Macrófagos , Glicoproteínas de Membrana , Receptores Depuradores , Quimiocina CXCL10/sangre , Quimiocina CXCL10/inmunología , Colon/inmunología , Colon/patología , Colon Sigmoide/patología , Colon Sigmoide/cirugía , Diverticulitis/sangre , Diverticulitis/inmunología , Diverticulitis/patología , Diverticulitis/cirugía , Humanos , Mucosa Intestinal/inmunología , Macrófagos/inmunología , Macrófagos/patología , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/inmunología , Receptores Depuradores/sangre , Receptores Depuradores/inmunologíaRESUMEN
Stromal-interaction molecules (STIM1/2) sense endoplasmic reticulum (ER) Ca2+ depletion and activate Orai channels. However, the choreography of interactions between native STIM/Orai proteins under physiological agonist stimulation is unknown. We show that the five STIM1/2 and Orai1/2/3 proteins are non-redundant and function together to ensure the graded diversity of mammalian Ca2+ signaling. Physiological Ca2+ signaling requires functional interactions between STIM1/2, Orai1/2/3, and IP3Rs, ensuring that receptor-mediated Ca2+ release is tailored to Ca2+ entry and nuclear factor of activated T cells (NFAT) activation. The N-terminal Ca2+-binding ER-luminal domains of unactivated STIM1/2 inhibit IP3R-evoked Ca2+ release. A gradual increase in agonist intensity and STIM1/2 activation relieves IP3R inhibition. Concomitantly, activated STIM1/2 C termini differentially interact with Orai1/2/3 as agonist intensity increases. Thus, coordinated and omnitemporal functions of all five STIM/Orai and IP3Rs translate the strength of agonist stimulation to precise levels of Ca2+ signaling and NFAT induction, ensuring the fidelity of complex mammalian Ca2+ signaling.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Proteína ORAI2/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Canales de Calcio/genética , Señalización del Calcio/efectos de los fármacos , Carbacol/farmacología , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Potenciales de la Membrana , Modelos Biológicos , Agonistas Muscarínicos/farmacología , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Proteína ORAI2/genética , Unión Proteica , Receptor Cross-Talk , Molécula de Interacción Estromal 1/agonistas , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 2/agonistas , Molécula de Interacción Estromal 2/genética , Factores de TiempoRESUMEN
The ubiquitous store-operated Ca2+ entry pathway mediated by plasma membrane Ca2+ release-activated Ca2+ (CRAC) channels regulates a wide variety of physiological functions. While it is clearly established that the ORAI1 protein is essential for native mammalian CRAC channels, the contribution of ORAI2 and ORAI3 have remained nebulous. The crystal structure of the sole Orai isoform in drosophila (dOrai) revealed a hexameric assembly, suggesting that mammalian CRAC channels are hexamers of ORAI. Nevertheless, the relative contribution of each isoform of the mammalian ORAI trio to the stoichiometry of native CRAC channels remains elusive. The recent generation of ORAI isoform single, double and triple knockout cell lines and tissue-specific knockout mice has shed light on how native ORAI isoform heteromerization fine tunes CRAC-mediated Ca2+ signaling.
RESUMEN
Despite the established role of mitochondria in cancer, the mechanisms by which mitochondrial Ca2+ (mtCa2+) regulates tumorigenesis remain incompletely understood. The crucial role of mtCa2+ in tumorigenesis is highlighted by altered expression of proteins mediating mtCa2+ uptake and extrusion in cancer. Here, we demonstrate decreased expression of the mitochondrial Na+/Ca2+/Li+ exchanger NCLX (SLC8B1) in human colorectal tumors and its association with advanced-stage disease in patients. Downregulation of NCLX causes mtCa2+ overload, mitochondrial depolarization, decreased expression of cell-cycle genes and reduced tumor size in xenograft and spontaneous colorectal cancer mouse models. Concomitantly, NCLX downregulation drives metastatic spread, chemoresistance, and expression of epithelial-to-mesenchymal, hypoxia, and stem cell pathways. Mechanistically, mtCa2+ overload leads to increased mitochondrial reactive oxygen species, which activate HIF1α signaling supporting metastasis of NCLX-null tumor cells. Thus, loss of NCLX is a novel driver of metastasis, indicating that regulation of mtCa2+ is a novel therapeutic approach in metastatic colorectal cancer.
Colorectal cancer is the second largest cause of cancer deaths worldwide. Even in cases where the cancer is diagnosed and treated early, cells can sometimes survive treatment and spread to other organs. Once the cancer has spread, the survival rate is less than 15%. Mitochondria are compartments in the cell that produce energy, and they play an important role in supporting the rapid growth of cancer cells. The levels of calcium ions in mitochondria control how they produce energy, a process that is altered in cancer cells. To better understand how calcium ions influence colorectal cancer growth, Pathak, Gueguinou et al. studied a protein called NCLX, which controls calcium levels by pumping them out of the mitochondria. Two mouse strains that were used to study what happens if NCLX is missing. The first strain was genetically modified to disable the gene for NCLX and then exposed to carcinogens. The second strain was injected with colorectal cancer cells from a human tumor that were lacking NCLX. In both strains, the tumors that formed were smaller than in mice with NCLX. However, the human cancer cells in the second model were more likely to spread to other organs. This is likely because the build-up of calcium ions in the mitochondria of mice lacking NCLX led to an increase in the production of hypoxia-inducible factor-1a, a protein that is a common driver of cancer spread. Pathak, Gueguinou et al. demonstrated how NCLX can affect colorectal cancer progression. It suggests that it may have opposing effects during early and late-stage colorectal cancer, encouraging tumor growth but also decreasing the spread to other organs. Further research could help refine treatments at different stages of the disease.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteínas Mitocondriales/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Calcio/metabolismo , Colon/patología , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Metástasis de la NeoplasiaRESUMEN
The ubiquitous Ca2+ release-activated Ca2+ (CRAC) channel is crucial to many physiological functions. Both gain and loss of CRAC function is linked to disease. While ORAI1 is a crucial subunit of CRAC channels, recent evidence suggests that ORAI2 and ORAI3 heteromerize with ORAI1 to form native CRAC channels. Furthermore, ORAI2 and ORAI3 can form CRAC channels independently of ORAI1, suggesting diverse native CRAC stoichiometries. Yet, most available CRAC modifiers are presumed to target ORAI1 with little knowledge of their effects on ORAI2/3 or heteromers of ORAIs. Here, we used ORAI1/2/3 triple-null cells to express individual ORAI1, ORAI2, ORAI3 or ORAI1/2/3 concatemers. We reveal that GSK-7975A and BTP2 essentially abrogate ORAI1 and ORAI2 activity while causing only a partial inhibition of ORAI3. Interestingly, Synta66 abrogated ORAI1 channel function, while potentiating ORAI2 with no effect on ORAI3. CRAC channel activities mediated by concatenated ORAI1-1, ORAI1-2 and ORAI1-3 dimers were inhibited by Synta66, while ORAI2-3 dimers were unaffected. The CRAC enhancer IA65 significantly potentiated ORAI1 and ORAI1-1 activity with marginal effects on other ORAIs. Further, we characterized the profiles of individual ORAI isoforms in the presence of Gd3+ (5µM), 2-APB (5⯵M and 50⯵M), as well as changes in intracellular and extracellular pH. Our data reveal unique pharmacological features of ORAI isoforms expressed in an ORAI-null background and provide new insights into ORAI isoform selectivity of widely used CRAC pharmacological compounds.
Asunto(s)
Canales de Calcio/metabolismo , Proteína ORAI1/metabolismo , Proteína ORAI2/metabolismo , Anilidas/farmacología , Benzamidas/farmacología , Células HEK293 , Humanos , Isoformas de Proteínas/metabolismo , Multimerización de Proteína/efectos de los fármacos , Pirazoles/farmacología , Tiadiazoles/farmacologíaRESUMEN
Voltage-gated L-type Ca2+ channel (Cav1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca2+ signaling machinery, including down-regulation of Cav1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca2+ sensor proteins and the plasma membrane ORAI Ca2+ channels. STIM/ORAI proteins mediate store-operated Ca2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca2+, causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Cav1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Hipertensión/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Moléculas de Interacción Estromal/metabolismo , Remodelación Vascular/fisiología , Animales , Antihipertensivos/farmacología , Calcio/metabolismo , Canales de Calcio Tipo L/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Insuficiencia Cardíaca , Humanos , Proteínas de la Membrana/genética , Miocitos del Músculo Liso , Proteínas de Neoplasias , Proteína ORAI1/genética , Ratas , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 2/genéticaRESUMEN
The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells. The roles of ORAI2 and ORAI3 have remained obscure. We show that ORAI2 and ORAI3 channels play a critical role in mediating the regenerative Ca2+ oscillations induced by physiological receptor activation, yet ORAI1 is dispensable in generation of oscillations. We reveal that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation. This broadened bandwidth of Ca2+ influx is translated by cells into differential activation of NFAT1 and NFAT4 isoforms. Our results uncover a long-sought role for ORAI2 and ORAI3, revealing an intricate control mechanism whereby heteromerization of ORAI channels mediates graded Ca2+ signals that extend the agonist-sensitivity to fine-tune transcriptional control.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Carbacol/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Factores de Transcripción NFATC/metabolismo , Proteína ORAI1/metabolismo , Unión Proteica/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Multimerización de Proteína/efectos de los fármacos , Molécula de Interacción Estromal 1/metabolismo , Imagen de Lapso de TiempoRESUMEN
Store-operated Ca2+ entry (SOCE) is a ubiquitous pathway for Ca2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca2+-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1-/-, STIM2-/-, and STIM1/2-/- knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.