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The endoplasmic reticulum: A multifunctional signaling organelle
Abstract
The endoplasmic reticulum (ER) is a multifunctional signaling organelle that controls a wide range of cellular processes such as the entry and release of Ca(2+), sterol biosynthesis, apoptosis and the release of arachidonic acid (AA). One of its primary functions is as a source of the Ca(2+) signals that are released through either inositol 1,4,5-trisphosphate (InsP(3)) or ryanodine receptors (RYRs). Since these receptors are Ca(2+)-sensitive, the ER functions as an excitable system capable of spreading signals throughout the cell through a process of Ca(2+)-induced Ca(2+) release (CICR). This regenerative capacity is particularly important in the control of muscle cells and neurons. Its role as an internal reservoir of Ca(2+) must be accommodated with its other major role in protein synthesis where a constant luminal level of Ca(2+) is essential for protein folding. The ER has a number of stress signaling pathways that activate various transcriptional cascades that regulate the luminal content of the Ca(2+)-dependent chaperones responsible for the folding and packaging of secretory proteins.Another emerging function of the ER is to regulate apoptosis by operating in tandem with mitochondria. Anti-apoptotic regulators of apoptosis such as Bcl-2 may act by reducing the ebb and flow of Ca(2+) through the ER/mitochondrial couple. Conversely, the presenilins that appear to increase the Ca(2+) content of the ER lumen make cells more susceptible to apoptosis.
... The endoplasmic reticulum is a reticular organelle that regulates the folding and posttranslational maturation of most membrane proteins and secretory proteins [53]. Proteins synthesized in the endoplasmic reticulum, often require a stable three-dimensional structure through the production of disulfide bonds, which are the basis of protein biological functions [54]. With the formation of disulfide bonds, H 2 O 2 , a byproduct, is produced in the endoplasmic reticulum, and ROS levels in the endoplasmic reticulum are subsequently upregulated [54]. ...
... Proteins synthesized in the endoplasmic reticulum, often require a stable three-dimensional structure through the production of disulfide bonds, which are the basis of protein biological functions [54]. With the formation of disulfide bonds, H 2 O 2 , a byproduct, is produced in the endoplasmic reticulum, and ROS levels in the endoplasmic reticulum are subsequently upregulated [54]. Under normal conditions, the reductive environment in the cytoplasm and endoplasmic reticulum prevents the production of disulfide bonds by cytoplasmic proteins [11], suggesting that ROS produced during protein folding and maturation in the endoplasmic reticulum contribute to disulfide bonds and disulfide production. ...
... The production of disulfide bonds mainly depends on the protein disulfide isomerase (PDI) family and other oxidoreductases [54]. In our enrichment analysis of SLC7A11 and its closely related genes, we found that these genes appear to regulate the activity of disulfide oxidoreductase, which regulates disulfide bonds and disulfide synthesis in ACC cells [55]. ...
Background
Disulfidptosis and the disulfidptosis-related gene SLC7A11 have recently attracted significant attention for their role in tumorigenesis and tumour management. However, its association with adrenocortical carcinoma (ACC) is rarely discussed.
Methods
Differential analysis, Cox regression analysis, and survival analysis were used to screen for the hub gene SLC7A11 in the TCGA and GTEx databases and disulfidptosis-related gene sets. Then, we performed an association analysis between SLC7A11 and clinically relevant factors in ACC patients. Univariate and multivariate Cox regression analyses were performed to evaluate the prognostic value of SLC7A11 and clinically relevant factors. Weighted gene coexpression analysis was used to find genes associated with SLC7A11. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and the LinkedOmics database were used to analyse the functions of SLC7A11-associated genes. The CIBERSORT and Xcell algorithms were used to analyse the relationship between SLC7A11 and immune cell infiltration in ACC. The TISIDB database was applied to search for the correlation between SLC7A11 expression and immune chemokines. In addition, we performed a correlation analysis for SLC7A11 expression and tumour mutational burden and immune checkpoint-related genes and assessed drug sensitivity based on SLC7A11 expression. Immunohistochemistry and RT‒qPCR were used to validate the upregulation of SLC7A11 in the ACC.
Results
SLC7A11 is highly expressed in multiple urological tumours, including ACC. SLC7A11 expression is strongly associated with clinically relevant factors (M-stage and MYL6 expression) in ACC. SLC7A11 and the constructed nomogram can accurately predict ACC patient outcomes. The functions of SLC7A11 and its closely related genes are tightly associated with the occurrence of disulfidptosis in ACC. SLC7A11 expression was tightly associated with various immune cell infiltration disorders in the ACC tumour microenvironment (TME). It was positively correlated with the expression of immune chemokines (CXCL8, CXCL3, and CCL20) and negatively correlated with the expression of immune chemokines (CXCL17 and CCL14). SLC7A11 expression was positively associated with the expression of immune checkpoint genes (NRP1, TNFSF4, TNFRSF9, and CD276) and tumour mutation burden. The expression level of SLC7A11 in ACC patients is closely associated withcthe drug sensitivity.
Conclusion
In ACC, high expression of SLC7A11 is associated with migration, invasion, drug sensitivity, immune infiltration disorders, and poor prognosis, and its induction of disulfidptosis is a promising target for the treatment of ACC.
... It performs a variety of cellular processes, including sterol and lipid biosynthesis, Ca 2+ storage, and folding of newly synthetized proteins. Disruption of these processes negatively impacts the ER homeostasis, leading to the accumulation of misfolded/unfolded proteins (proteotoxicity) in the ER lumen, a condition called ER stress [2][3][4]. Upon sensing ER stress, the cell activates signaling pathways known as the unfolded protein response (UPR) to restore ER homeostasis [5]. ...
... The ER serves as a reservoir for Ca 2+ and regulates Ca 2+ signaling through inositol 1,4,5-triphosphate (IP 3 ) receptors (IP 3 R) and ryanodine receptors. These receptors are located in regions enriched with signaling proteins that are in contact with mitochondria, called mitochondria-associated ER-membrane (MAM) [3,4,11,12]. A constant level of Ca 2+ in the ER lumen is essential for keeping Ca 2+ receptors in a sensitive state [4] and supporting protein folding through Ca 2+ -dependent chaperones, such as calnexin (CANX), calreticulin (CALR), and heat shock protein family A member 5 (HSPA5), also known as immunoglobulin heavy chain-binding protein (BiP/GRP-78) [3]. ...
... These receptors are located in regions enriched with signaling proteins that are in contact with mitochondria, called mitochondria-associated ER-membrane (MAM) [3,4,11,12]. A constant level of Ca 2+ in the ER lumen is essential for keeping Ca 2+ receptors in a sensitive state [4] and supporting protein folding through Ca 2+ -dependent chaperones, such as calnexin (CANX), calreticulin (CALR), and heat shock protein family A member 5 (HSPA5), also known as immunoglobulin heavy chain-binding protein (BiP/GRP-78) [3]. Hence, a decrease in ER luminal Ca 2+ may result in the accumulation of misfolded proteins, inducing ER stress. ...
The endoplasmic reticulum (ER) is a multifunctional organelle playing a vital role in maintaining cell homeostasis, and disruptions to its functions can have detrimental effects on cells. Dysregulated ER stress and the unfolded protein response (UPR) have been linked to various human diseases. For example, ER stress and the activation of the UPR signaling pathways in intestinal epithelial cells can either exacerbate or alleviate the severity of inflammatory bowel disease (IBD), contingent on the degree and conditions of activation. Our recent studies have shown that EPICERTIN, a recombinant variant of the cholera toxin B subunit containing an ER retention motif, can induce a protective UPR in colon epithelial cells, subsequently promoting epithelial restitution and mucosal healing in IBD models. These findings support the idea that compounds modulating UPR may be promising pharmaceutical candidates for the treatment of the disease. In this review, we summarize our current understanding of the ER stress and UPR in IBD, focusing on their roles in maintaining cell homeostasis, dysregulation, and disease pathogenesis. Additionally, we discuss therapeutic strategies that promote the cytoprotection of colon epithelial cells and reduce inflammation via pharmacological manipulation of the UPR.
... As the major internal Ca 2+ store, Ca 2+ release from SR/ER is mediated by two cation channels, ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs). [8][9][10] During the release, SR/ER membrane is charged upon the Ca 2+ efflux, which hinders the continued Ca 2+ release. Previously reported TRimeric Intracellular Cation channels (TRICs) act as counter-ion channels to balance the loss of positive charges from the SR/ER due to the release. ...
... Knockdown of CLCC1 by the two individual shRNAs markedly reduced internal Ca 2+ release induced by ATP (Fig. 3a), which triggers ER Ca 2+ release by generating IP3 that activates IP3Rs. 10 Compared to mock control and scrambled shRNA, knockdown of CLCC1 by the two individual shRNAs not only significantly reduced the amplitude, but also the rate (as reflected by the increase in time-to-peak), of ATP-induced Ca 2+ release (Fig. 3b, c). Although the two shRNAs had different CLCC1 knockdown efficiencies, they impaired the ATP-induced Ca 2+ amplitude and rate to a similar extent. ...
... Next, we asked whether CLCC1 regulates internal Ca 2+ release through RyRs, the predominant intracellular Ca 2+ channels expressed in cardiomyocytes. 10 To this end, we cultured cardiomyocytes from WT (+/+) and NM2453 mutant (NM/NM) mice; the latter carries a hypomorphic mutant allele, an intracisternal A-particle retrotransposon (IAP) insertion into Clcc1 that largely reduces CLCC1 protein expression 19 (Supplementary information, Fig. S1a). The cultured cardiomyocytes were stimulated with caffeine, an agonist for RyR-mediated Ca 2+ release. ...
Although anion channel activities have been demonstrated in sarcoplasmic reticulum/endoplasmic reticulum (SR/ER), their molecular identities and functions remain unclear. Here, we link rare variants of Chloride Channel CLIC Like 1 ( CLCC1 ) to amyotrophic lateral sclerosis (ALS)-like pathologies. We demonstrate that CLCC1 is a pore-forming component of an ER anion channel and that ALS-associated mutations impair channel conductance. CLCC1 forms homomultimers and its channel activity is inhibited by luminal Ca ²⁺ but facilitated by phosphatidylinositol 4,5-bisphosphate (PIP2). We identified conserved residues D25 and D181 in CLCC1 N-terminus responsible for Ca ²⁺ binding and luminal Ca ²⁺ -mediated inhibition on channel open probability and K298 in CLCC1 intraluminal loop as the critical PIP2-sensing residue. CLCC1 maintains steady-state [Cl – ] ER and [K ⁺ ] ER and ER morphology and regulates ER Ca ²⁺ homeostasis, including internal Ca ²⁺ release and steady-state [Ca ²⁺ ] ER . ALS-associated mutant forms of CLCC1 increase steady-state [Cl – ] ER and impair ER Ca ²⁺ homeostasis, and animals with the ALS-associated mutations are sensitized to stress challenge-induced protein misfolding. Phenotypic comparisons of multiple Clcc1 loss-of-function alleles, including ALS-associated mutations, reveal a CLCC1 dosage dependence in the severity of disease phenotypes in vivo. Similar to CLCC1 rare variations dominant in ALS, 10% of K298A heterozygous mice developed ALS-like symptoms, pointing to a mechanism of channelopathy dominant-negatively induced by a loss-of-function mutation. Conditional knockout of Clcc1 cell-autonomously causes motor neuron loss and ER stress, misfolded protein accumulation, and characteristic ALS pathologies in the spinal cord. Thus, our findings support that disruption of ER ion homeostasis maintained by CLCC1 contributes to ALS-like pathologies.
... Calcium ions enter into dendrite or synapse via voltage-gated calcium channels (VGCC) upon synaptic stimulation. The downstream of calcium wave propagation could produce functional differences in dendrites, where many mechanisms are responsive to the spatial-temporal dynamics of calcium (Adeoye et al. 2022;Berridge and Berridge 2002;Inoue et al. 2001;Johnston et al. 1996;Lee et al. 2016;Neymotin et al. 2015;Ross and Ross 2012;Schulte et al. 2022); for example,I h (Neymotin et al. 2013;Neymotin et al. 2015;Yuste 2013]and synaptic plasticity [Collin et al. 2005;Karagas et al. 2019;Khan and Khan 2022;LaFerla and LaFerla 2002). The information integration of dendrite is of great importance for neural communication, which is closely related with calcium dynamic (Johnston et al. 1996;Koch et al. 1983;LaFerla and LaFerla 2002;Neymotin et al. 2015;Stuart 2015;Stutzmann 2005). ...
... The calcium signal generated at the synapse could induce new gene expression at the soma, which is hundreds of microns away by the continuity between SER in the spines and SER of the dendrites (Berridge and Berridge 2002;Chanaday et al. 2022;De Schutter et al. 2008;Lopreore et al. 2008;McCormick and Huguenard 1992;Mori et al. 2011;Padamsey et al. 2019;Pozzo-Miller et al. 1997;Savić et al. 1998;Stuart 2015;Timofeeva 2003). Therefore, the questions as how far and how fast calcium can propagate are critically important, as well as its magnitude and sustaining time. ...
The source and dynamics of calcium is the key factor that regulates dendritic integration. Apart from the voltage-gated and ligand-gated calcium influx, an important source of calcium is from inner store of endoplasmic reticulum with a regenerative process of calcium-induced calcium release (CICR). To trigger this process, inositol 1,4,5-trisphosphate (IP3) and calcium are needed to satisfy certain requirements. The aim of our paper is to investigate how the CICR depends on the dynamics of membrane potential. We utilize one dimensional dendritic model to calculate membrane potential by Nernst–Planck Equation (NPE) and cable model and Pure Diffusion (PD) model, computational simulations are carried out to inject the calcium influx by synaptic stimulation and to predict subsequent CICR and calcium wave propagation. Our results demonstrate that CICR initiation and calcium wave propagation have much difference between electro-diffusion process of NPE and cable model. We find that cable model has lower threshold of IP3 stimulation to trigger CICR but is more difficult for calcium propagation than NPE, PD model requires even higher threshold of IP3 to initiate CICR process and calcium duration is shorter than NPE; the regenerative calcium wave propagates with faster speed in NPE than that in cable model and in PD model. Our work addresses the important role of electro-diffusion dynamics of charged ions in regulating CICR process in dendritic structure; and provides theoretical predictions for neurological process which requires sustaining calcium for downstream signaling processes.
... In this study we have sought to further characterize the pharmacology of SERCA-regulated Ca 2+ stores in T lymphocytes. There is a clear imperative to gain a better understanding of the roles played by Ca 2+ stores as essential regulators of the complex spatiotemporal Ca 2+ signal underlying critical early signaling events driving T cell activation [17][18][19]. A powerful approach to probing Ca 2+ store functions in T cell signaling networks is to modulate SERCA pump function using an array of small molecule pharmacological agents that can potentially provide a means for fine control of the various SERCA pump states as the primary regulators determining ER Ca 2+ store levels [9,11,12,16,19]. ...
The allosteric SERCA (Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase) activator CDN1163 has been recently added to the group of pharmacological tools for probing SERCA function. We chose to investigate the effects of the compound on T lymphocyte Ca2+ stores, using the well-described Jurkat T lymphocyte as a reliable cell system for Ca2+ signaling pathways. Our study identified the lowest concentrations of the SERCA inhibitors thapsigargin (TG) and 2,5-di-(tert butyl)-1,4-benzohydroquinone (tBHQ) capable of releasing Ca2+, permitting the differentiation of the TG-sensitive SERCA 2b Ca2+ store from the tBHQ-sensitive SERCA 3 Ca2+ store. We proceeded to test the effects of CDN1163 on Ca2+ stores, examining specific actions on the SERCA 2b and SERCA 3 Ca2+ pools using our low dose SERCA blocker regimen. In contrast to previous work, we find CDN1163 exerts complex time-sensitive and SERCA isoform-specific actions on Ca2+ stores. Surprisingly, short-term exposure (0-30 minutes) to CDN1163 perturbs T cell Ca2+ stores by suppressing Ca2+ uptake with diminished Ca2+ release from the SERCA 2b-controlled store. Concomitantly, we find evidence for a SERCA-activating effect of CDN1163 on the SERCA 3 regulated store, given the observation of increased Ca2+ release inducible by low dose tBHQ. Intriguingly, longer-term (>12 hours) CDN1163 exposure reversed this pattern, with increased Ca2+ release from SERCA 2b-regulated pools, yet decreased Ca2+ release responses from tBHQ-sensitive SERCA 3 pool. Our results reveal differential effects of CDN1163 on Ca2+ stores regulated by distinct SERCA isoforms, prompting the need for careful interpretation of the compound’s effects, particularly in cells expressing multiple SERCA isoforms.
... 27 This organelle also regulates numerous other molecular pathways and has a role in reduction-oxidation regulation, autophagy, and cell death. 28 Disturbance of ER internal balance causes the increase of unfolded or misfolded proteins in the cell, causing the cell to undergo apoptosis. It has been stated that ER homeostasis disorder plays a role in the pathophysiology of DM by causing β-cell dysfunction in type 1 and type 2 diabetes. ...
Background
Ischemia–reperfusion injury (IRI) poses a significant challenge for physicians, necessitating the management of cell damage and the preservation of organ functions. Various surgical procedures, such as vascular surgery on extremities, temporary cross-clamping of the abdominal aorta in aortic surgery, and the use of a tourniquet in extremity surgeries, may induce lower limb IRI. The susceptibility to IRI is heightened in individuals with diabetes. This study aimed to investigate the effects of fullerenol C60 and sevoflurane on mouse muscle tissue in a lower limb IRI model and to assess their potential in preventing complications arising from ischemia–reperfusion in mice with streptozocin-induced diabetes.
Methods
A total of 36 adult Swiss albino mice were randomly divided into six groups, each consisting of six mice: control group (group C), diabetes group (group D), diabetes–ischemia/reperfusion group (group DIR), diabetes–ischemia/reperfusion–fullerenol C60 group (group DIR-FC60), diabetes–ischemia/reperfusion–sevoflurane group (group DIR-S), and diabetes–ischemia/reperfusion–sevoflurane–fullerenol C60 group (DIR-S-FC60). Streptozocin (55 mg/kg) was intraperitoneally administered to induce diabetes in the relevant groups, with mice displaying blood glucose levels of 250 mg/dL or higher at 72 h were considered diabetic. After 4 weeks, all groups underwent laparotomy under anesthesia. In DIR-FC60 and DIR-S-FC60 groups, fullerenol C60 (100 mg/kg) was intraperitoneally administrated 30 min before the ischemia period. Sevoflurane, delivered in 100% oxygen at a rate of 2.3% and 4 L/min, was administered during the ischemia period in DIR-S and DIR-S-FC60 groups. In the IR groups, a microvascular clamp was placed on the infrarenal abdominal aorta for 120 min during the ischemia period, followed by the removal of the clamp and a 120-min reperfusion period. At the end of the reperfusion, gastrocnemius muscle tissues were removed for histopathological and biochemical parameter examinations.
Results
Histopathological examination revealed a significant reduction in the disorganization and degeneration of muscle cells in the DIR-S-FC60 group compared to the DIR group (p = 0.041). Inflammatory cell infiltration was notably lower in the DIR-S, DIR-FC60, and DIR-S-FC60 groups than in the DIR group (p = 0.031, p = 0.011, and p = 0.013, respectively). The total damage scores in the DIR-FC60 and DIR-S-FC60 groups were significantly lower than in the DIR group (p = 0.018 and p = 0.008, respectively). Furthermore, the levels of malondialdehyde (MDA) in the DIR-S, DIR-FC60, and DIR-S-FC60 groups were significantly lower than in the DIR group (p < 0.001, p < 0.001, and p < 0.001, respectively). Catalase (CAT) enzyme activity in the DIR-S, DIR-FC60, and DIR-S-FC60 groups was higher than in the DIR group (p = 0.001, p = 0.014, and p < 0.001, respectively). Superoxide dismutase (SOD) enzyme activity in the DIR-FC60 and DIR-S-FC60 groups was also higher than in the DIR group (p < 0.001 and p = 0.001, respectively).
Conclusion
Our findings indicate that administering fullerenol C60 30 min prior to ischemia in diabetic mice, in combination with sevoflurane, led to a reduction in oxidative stress and the correction of IR-related damage in muscle tissue histopathology. We believe that the administration of fullerenol C60 before IR, coupled with sevoflurane administration during IR, exerts a protective effect in mice.
... Calcium release by the ryanodine receptor and IP3 receptor from the intracellular sarcoplasmic reticulum/endoplasmic reticulum is required for cellular signaling. Countermovement of ions is also essential for balancing the transient negative membrane potential due to calcium release [64][65][66] , but the molecular identity of the counterion channel remains unknown. TMEM38 has been reported to play a physiological role in intracellular stores as a counterion channel for calcium handling 8 . ...
A transmembrane (TMEM) protein with an unknown function is a type of membrane-spanning protein expressed in the plasma membrane or the membranes of intracellular organelles. Recently, several TMEM proteins have been identified as functional ion channels. The structures and functions of these proteins have been extensively studied over the last two decades, starting with TMEM16A (ANO1). In this review, we provide a summary of the electrophysiological properties of known TMEM proteins that function as ion channels, such as TMEM175 (K EL ), TMEM206 (PAC), TMEM38 (TRIC), TMEM87A (GolpHCat), TMEM120A (TACAN), TMEM63 (OSCA), TMEM150C (Tentonin3), and TMEM43 (Gapjinc). Additionally, we examine the unique structural features of these channels compared to those of other well-known ion channels. Furthermore, we discuss the diverse physiological roles of these proteins in lysosomal/endosomal/Golgi pH regulation, intracellular Ca ²⁺ regulation, spatial memory, cell migration, adipocyte differentiation, and mechanical pain, as well as their pathophysiological roles in Parkinson’s disease, cancer, osteogenesis imperfecta, infantile hypomyelination, cardiomyopathy, and auditory neuropathy spectrum disorder. This review highlights the potential for the discovery of novel ion channels within the TMEM protein family and the development of new therapeutic targets for related channelopathies.
... 导产生，进而调控氧化代谢、线粒体呼吸和 ATP 合成从而影响细胞代谢 [11] 。而内质网作为 动物细胞中最大的一个细胞器，是蛋白质及脂类合成的关键场所，同时还与其它多种细胞 器存在广泛的互作。它的静息钙水平可以达到胞浆静息钙水平的几千倍，因而在细胞稳态 维持及钙信号转导中发挥着中心作用 [12] 。内质网膜上的钙泵，即肌/内质网 Ca 2+ ATP 酶 (sarco/endoplasmic reticulum Ca 2+ ATPase，SERCA) ；包括跨膜卷曲螺旋结构域蛋白 1 (transmembrane and coiled-coil domains，TMCO1)在内的漏钙通道 [13] ；钙释放通道 1,4,5-三磷酸肌醇受体(inositol-1,4,5-trsiphosphate receptors，IP 3 Rs)和兰尼碱受体(ryanodine receptors，RyRs)共同维持内质网钙水平的稳态。当 Ca 2+ 从内质网释放到胞浆或通过内质 网与细胞器之间的膜接触位点流入各个细胞器时，产生的钙信号会调控众多生理反应，如 内质网与线粒体之间的 Ca 2+ 传递参与调控脂类合成、细胞代谢等重要的细胞功能 [14] 。 高尔基体是一个高度动态的极性结构，由多个扁平膜囊紧密地堆叠在一起，分为靠近 细胞核一侧的顺式、圆盘状扁平的中部和面向质膜一侧的反式高尔基体三个主要的区室。 这三个区室中的 Ca 2+ 浓度和 pH 均有一定的差异。高尔基体主要参与细胞内翻译后修饰， 及脂质和蛋白质的分选，已有大量的证据证明高尔基体可以作为细胞内的钙库来发挥作用 [15,16] 。细胞中的各种囊泡是内膜系统的重要组成部分，承担着细胞内蛋白分选、定向运输、 物质储存及分泌等多种功能。细胞内的囊泡可大致分为：分泌囊泡、内体等。囊泡可响应 环境刺激，释放或回收 Ca 2+ ，进而调控下游反应 [17,18] 。 而溶酶体作为细胞中负责降解回收大分子物质的酸性细胞器，pH 低至 4.5，富含 Ca 2+ ， 静息游离钙水平维持在 500 μM 左右，是细胞中重要的-酸性钙库‖。溶酶体腔会发生碱化和 酸化，引发它向胞质的钙释放或溶酶体腔内钙水平的升高，以维持溶酶体生理功能正常进 行 [19] 。 线粒体及整个内体系统通过协调自身的钙转运蛋白质机器及细胞器互作来维持整个细 胞的钙稳态。当这些机制出现异常时，会损害细胞机能并导致各种疾病。内质网或线粒体 钙水平的失衡会引发细胞凋亡、代谢损伤等病理现象 [20] 。当参与钙信号调控的相关蛋白功 能异常，如 SERCA、Ca 2+ 单向转运体等的功能性突变，与细胞凋亡、免疫缺陷、心肌肥大、 神经退行性疾病等的发生密切相关 [21,22] 。高尔基体上的钙泵分泌通路 Ca 2+ -ATP 酶亚型 1 (secretory pathway Ca 2+ -ATPase 1, SPCA1)突变会导致一种名为 Hailey-Hailey(家族性良 性天疱疮)的遗传性皮肤病 [23,24] ，在人乳腺癌细胞系中也检测到 SPCA1 的升高 [25] 。并且， 中钙信号的监测 [38] 。而在腔内 pH 更低的溶酶体(pH=4.5)中，早期常通过联合使用钙指 示剂及 pH 指示剂，以校正 pH 对钙探针的影响。如有研究人员使用钙探针 Fura Dextran (FuraDx)及 pH 探针 Oregon Green Dextran (OGDx) 的混合物检测 HEK-293/HeLa 细胞中 pH 变化及内质网钙信号对于溶酶体钙的影响 [39][40][41] 。尽管上述化学钙探针能够一定程度上屏蔽 溶酶体腔内 pH 波动的影响，反应真实钙水平的变化，但由于很难控制每个溶酶体中两种 指示剂的量，因此，无法在单细胞器水平上实现信号的校正。为此，Nagarjun 等人开发了 一系列基于 DNA 的内体-溶酶体荧光钙指示剂--CalipHluor [42,43] 。该工具分别将 pH 探针 I-switch 及钙探针 Rhod-5F 与互补的两条 DNA 单链融合，并通过 DNA 杂交，控制 pH 探针 与钙探针的量始终保持 1:1 的比例。在 pH=4.6 时，CalipHluor Ly 的 K d 值约为 100 μM，适用 于溶酶体钙信号的检测。尽管如此，目前 CalipHluor 无法实现在特定的组织中定位，在注 射后需要经过各种生物屏障，限制了其在在体水平的应用 [44] 。关于其他化学类钙指示剂的 详细介绍，可参看 Pihán [ [47] ，GCaMP 的过表达会引发部分神经元死亡 [48] 。GCaMP2 [49] 转基因小鼠会发生心肌肥大 [50] ，GCaM5G 转基因小鼠的海马神经元的放电速率较高 [51] ， 多种表达 GCaMP6 的小鼠品系大脑神经元的活动异常 [52] , 等等。为了解决这类问题，2018 年刘晓冬团队在 GCaMP3 和 GCaMP6 的 N 末端引入一段 apoCaM 结合序列(apoCaM binding motif, CBM) ，来封闭低钙时 GCaMP 中游离的 CaM，避免过表达的 GECI 中 CaM 对神经元 L 型钙通道等介导的钙信号通路的影响，开发出了低细胞毒性的 GCaMP-X [53] ， 并进一步构建了反应更灵敏的 jGCaMP7b-X [ [77,80,82] [96] [46] 。基于 Cameleon 改造的 FRET 型 GECI 存在动态范围小(约两 倍)的问题 [99,100] ，而 GAP1 在内质网中测得其动态范围为 4 [46] ，相对来说有所提高。但与 其他细胞器钙探针对比仍有很大的提升空间，如熟知的 CEPIA1er 系列，其中 GEM-CEPIA1er 的体外测试动态范围能达到 20 [72] ...
... The endoplasmic reticulum (ER) is the primary storage site and modulator of intracellular Ca 2+ , in addition to its critical functions associated with the synthesis, modification, and trafficking of lipids and proteins (i.e., proteostasis) (Berridge, 2002;Kleizen & Braakman, 2004;Schwarz & Blower, 2016). Ca 2+ levels in the ER are estimated to be 5000-fold higher than calcium concentrations found in the cytosol (Brini & Carafoli, 2009), and this reservoir of intracellular Ca 2+ supports many cellular functions. ...
Dysregulation of synaptic glutamate levels can lead to excitotoxicity such as that observed in stroke, traumatic brain injury, and epilepsy. The role of increased intracellular calcium (Ca ²⁺ ) in the development of excitotoxicity is well established. However, less is known regarding the impact of glutamate on endoplasmic reticulum (ER)‐Ca ²⁺ ‐mediated processes such as proteostasis. To investigate this, we expressed a secreted ER Ca ²⁺ modulated protein (SERCaMP) in primary cortical neurons to monitor exodosis, a phenomenon whereby ER calcium depletion causes the secretion of ER‐resident proteins that perform essential functions to the ER and the cell. Activation of glutamatergic receptors (GluRs) led to an increase in SERCaMP secretion indicating that normally ER‐resident proteins are being secreted in a manner consistent with ER Ca ²⁺ depletion. Antagonism of ER Ca ²⁺ channels attenuated the effects of glutamate and GluR agonists on SERCaMP release. We also demonstrate that endogenous proteins containing an ER retention/retrieval sequence (ERS) are secreted in response to GluR activation supporting that neuronal activation by glutamate promotes ER exodosis. Ectopic expression of KDEL receptors attenuated the secretion of ERS‐containing proteins caused by GluR agonists. Taken together, our data indicate that excessive GluR activation causes disruption of neuronal proteostasis by triggering the secretion of ER‐resident proteins through ER Ca ²⁺ depletion and describes a new facet of excitotoxicity.
... In this study we have sought to further characterize the pharmacology of SERCA-regulated Ca 2+ stores in T lymphocytes. There is a clear imperative to gain a better understanding of the roles played by Ca 2+ stores as essential regulators of the complex spatiotemporal Ca 2+ signal underlying critical early signaling events driving T cell activation [17][18][19]. A powerful approach to probing Ca 2+ store functions in T cell signaling networks is to modulate SERCA pump function using an array of small molecule pharmacological agents that can potentially provide a means for fine control of the various SERCA pump states as the primary regulators determining ER Ca 2+ store levels [9,11,12,16,19]. ...
The allosteric SERCA (Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase) activator CDN1163 has been recently added to the group of pharmacological tools for probing SERCA function. We chose to investigate the effects of the compound on T lymphocyte Ca2+ stores, using the well-described Jurkat T lymphocyte as a reliable cell system for Ca2+ signaling pathways. Our study identified the lowest concentrations of the SERCA inhibitors thapsigargin (TG) and 2,5-di-(tert butyl)-1,4-benzohydroquinone (tBHQ) capable of releasing Ca2+, permitting the differentiation of the TG-sensitive SERCA 2b Ca2+ store from the tBHQ-sensitive SERCA 3 Ca2+ store. We proceeded to test the effects of CDN1163 on Ca2+ stores, examining specific actions on the SERCA 2b and SERCA 3 Ca2+ pools using our low dose SERCA blocker regimen. In contrast to previous work, we find CDN1163 exerts complex time-sensitive and SERCA isoform-specific actions on Ca2+ stores. Surprisingly, short-term exposure (0-30 minutes) to CDN1163 perturbs T cell Ca2+ stores by suppressing Ca2+ uptake with diminished Ca2+ release from the SERCA 2b-controlled store. Concomitantly, we find evidence for a SERCA-activating effect of CDN1163 on the SERCA 3 regulated store, given the observation of increased Ca2+ release inducible by low dose tBHQ. Intriguingly, longer-term (>12 hours) CDN1163 exposure reversed this pattern, with increased Ca2+ release from SERCA 2b-regulated pools, yet decreased Ca2+ release responses from tBHQ-sensitive SERCA 3 pool. Our results reveal differential effects of CDN1163 on Ca2+ stores regulated by distinct SERCA isoforms, prompting the need for careful interpretation of the compound’s effects, particularly in cells expressing multiple SERCA isoforms.
... Calcium can enter neurones via the NMDA, AMPA, kainite, and metabotropic receptors and leak channels and can be released through passive or active processes (leak channels, exclusion channels, diffusion channels). The calcium concentration is higher in the ER than in the cytosol (from the high micromolar to low millimolar range) (Berridge, 2002;Solovyova and Verkhratsky, 2002). This gradient is maintained by SERCA pumps, which fill the ER with cytosolic calcium. ...
... ER has several essential cellular functions including the synthesis and folding of secreted and transmembrane proteins, calcium storage, and lipid synthesis for membrane biogenesis or energy storage. ER also helps in maintenance of calcium homeostasis and regulation of intracellular signalling pathways [1] . Disruption of any of the ER functions can lead to ER stress. ...
Inflammation is a complex process which is associated with the initiation and progression of cancer. Prolonged Endoplasmic Reticulum (ER) stress triggers inflammation which is a key factor associated with cancer pathogenesis. ER stress also contributes to immune suppression in inflammatory and tumor microenvironment. It stimulates the production of pro-inflammatory cytokines by regulating the activation of various transcription factors and inflammatory signalling pathways. Targeting ER stress is an exciting possibility that can be used as a therapeutic strategy for cancer treatment. This mini review focuses on the emerging link between ER stress-induced inflammatory responses in cancer development.
... Other evidence shows that ROS content is significantly increased in the degenerative intervertebral disc tissue [48]. Importantly, ROS also induce ERS to synergistically damage cells [49,50]. Thus, ROS and ERS may be the key pathological processes during IDD. ...
The in-situ osmolarity is an important physicochemical factor that regulates cell fate of nucleus pulposus cells (NPCs). Our previous studies demonstrated that reduced N-cadherin (NCDH) expression in nucleus pulposus cells is associated with cellular damage under hyper-osmolarity microenvironment. This study was aimed at exploring the impacts of NCDH on senescence and apoptosis of NPCs, as well as the potential molecular mechanism. By comparing NPCs from patients with lumbar fractures and lumbar disc herniation, we identified a correlation between decreased NCDH expression and increased endoplasmic reticulum stress (ERS), resulting in undesirable cell fate (senescence and apoptosis). After blocking Reactive oxygen species (ROS) or ERS, it was indicated that hyper-osmolarity microenvironment induced ERS was ROS-dependent. Further results demonstrated the correlation in rat NPCs. Upregulation of NCDH expression reduced ROS-dependent ERS, thus limiting undesirable cell fates in vitro. This was further confirmed through the rat tail acupuncture injection model. NCDH overexpression successfully mitigated ERS, preserved extracellular matrix production and alleviating intervertebral disc degeneration in vivo. Together, NCDH can alleviate senescence and apoptosis of NPCs by suppressing ROS-dependent ERS via the ATF4-CHOP signaling axis in the hyper-osmolarity microenvironment, thus highlighting the therapeutic potential of NCDH in combating degenerative disc diseases.
... The protein aggregations seen in AD are indicative of an imbalance in proteostasis and an increase in endoplasmic reticulum (ER) stress 6,16 . ER stress occurs when proteins misfold and aggregate in the ER lumen [17][18][19][20] . ER stress in healthy organisms activates the unfolded protein response (UPR), a protein quality control and signal transduction pathway 21 . ...
... The endoplasmic reticulum also causes folded protein response and stress, which in turn leads to cell death. 9,10 In recent various studies, it has been suggested that agents that could pass the blood-brain barrier and have antioxidant, anti-inflammatory properties show preventive effects against ischemia and reperfusion injury of the cerebral tissue. [11][12][13] Agomelatine is a melatonin receptor (MT1/MT2) agonist and a serotonin receptor (5-HT2C) antagonist. ...
Cerebral ischemia and reperfusion are related to various situations like injuries after various traumas, oxidative stress, increased calcium ion, capillary hypoperfusion, microvascular hyperpermeability, leukocyte infiltration, and blood–brain barrier disruption. An antidepressant Agomelatine which is a melatonin receptor (MT1/MT2) agonist and serotonin receptor (5‐HT2C) antagonist has been reported by studies to have antioxidant and anti‐inflammatory effects. In our study, we aimed to detect the effects of citrate‐coated silver nanoparticle‐loaded agomelatine application on neurodegeneration, endoplasmic reticulum stress, autophagic and apoptotic cell death, inflammation, and P2X7R expression in the cerebral ischemia–reperfusion model to facilitate the passage of blood–brain barrier. Forty two Sprague–Dawley rats in total were divided into six equal groups (n:7) and applications were performed. Acute cerebral injury in the ischemia–reperfusion model was created 2 h after internal carotid artery ligation in rats and then at the 2nd hour of reperfusion citrate‐coated silver nanoparticles loaded with Agomelatine were applied. Twenty four hours later, neurologic analysis on animals in experimental groups was performed, animals were decapitated and GSH, GPx, SOD, CAT, MDA, IL‐1β, and TNF‐α parameters were examined after taking blood and the cerebral tissue samples. As a result, it was determined that ischemia–reperfusion caused endoplasmic reticulum stress in the cerebral tissues and thus caused cellular injury.
... Endoplasmic reticulum (ER) homeostasis is dependent on the proper folding and maturation of secretory pathway proteins, on lipid biosynthesis, and on cellular calcium homeostasis. Disruption of the ER homeostasis, however, leads to a stress response called the unfolded protein response (UPR), a multifunctional signaling pathway [1][2][3]. The UPR serves primarily as a cellular adaptive mechanism that counteracts the stress-related deregulation of ER function and promotes cellular survival from both intrinsic and extrinsic insults [4,5]. ...
The unfolded protein response is a survival signaling pathway that is induced during various types of ER stress. Here,
we determine IRE1’s role in miRNA regulation during ER stress. During induction of ER stress in human bronchial
epithelial cells, we utilized next generation sequencing to demonstrate that pre-miR-301a and pre-miR-106b were
significantly increased in the presence of an IRE1 inhibitor. Conversely, using nuclear-cytosolic fractionation on ER
stressed cells, we found that these pre-miRNAs were decreased in the nuclear fractions without the IRE1 inhibitor. We
also found that miR-301a-3p targets the proapoptotic UPR factor growth arrest and DNA-damage-inducible alpha
(GADD45A). Inhibiting miR-301a-3p levels or blocking its predicted miRNA binding site in GADD45A’s 3’ UTR with a tar-
get protector increased GADD45A mRNA expression. Furthermore, an elevation of XBP1s expression had no effect
on GADD45A mRNA expression. We also demonstrate that the introduction of a target protector for the miR-301a-3p
binding site in GADD45A mRNA during ER stress promoted cell death in the airway epithelial cells. In summary, these
results indicate that IRE1’s endonuclease activity is a two-edged sword that can splice XBP1 mRNA to stabilize survival
or degrade pre-miR-301a to elevate GADD45A mRNA expression to lead to apoptosis
... 12 Accumulating in myocardial tissue, semiquinone free radicals generate excessive reactive oxygen species (ROS), damaging the redox balance of cardiomyocytes, leading to oxidative stress in cardiomyocytes, thereby affecting the normal function of the myocardium. 13 Mechanistically, the DOX commonly destroys the permeability of the cardiomyocyte membrane by decreasing membrane potential, which promotes the release of cytochrome C to the interstitium through the ion channel in the cell membrane. This process results in overexpression of the pro-apoptotic protein caspase-3 and promotes apoptosis of cardiomyocytes. ...
The prevalence and mortality of heart disease have a persistent existence, and it is important to develop active substances with cardioprotective properties. It has been reported that peptides from animal heart hydrolysates possess cardioprotective activity, but those mechanisms and the sequence of peptides are still unrevealed. In the present study, the extracts of bovine myocardium were prepared by enzymatic hydrolysis (BHH-A) and water extraction (BHH-W). The cardioprotective function of peptides was verified in the DOX-induced H9c2 cells and myocardial injury mice. The mass spectrometry was used to contrast the differences of active ingredients between BHH-W and BHH-A. Results suggested that both BHH-A and BHH-W could increase the activity of antioxidant enzymes in cardiomyocytes and reduce the inflammatory level and apoptosis of myocardial cells. The improvement effects of BHH-A on myocardial injury in mice were better than those of BHH-W. The analysis of peptide composition demonstrated that the contents with N-segment hydrophobic amino acids were higher in the peptides identified in BHH-A. Hence, BHH-A could be used as a potential active substance to improve DOX-induced myocardial injury by reducing oxidative damage, inflammation, and cardiomyocyte apoptosis, and its activity may be related to the richness of small molecular peptides and hydrophobic amino acids.
... Overload of mitochondrial Ca 2+ The ER and mitochondria are major reservoirs of intracellular Ca 2+ [46,47]. Ca 2+ concentration changes increased mitochondrial permeation pressure. ...
Due to the sustained proliferative potential of cancer cells, inducing cell death is a potential strategy for cancer therapy. Paraptosis is a mode of cell death characterized by endoplasmic reticulum (ER) and/or mitochondrial swelling and cytoplasmic vacuolization, which is less investigated. Considerable evidence shows that paraptosis can be triggered by various chemical compounds, particularly in cancer cells, thus highlighting the potential application of this non-classical mode of cell death in cancer therapy. Despite these findings, there remain significant gaps in our understanding of the role of paraptosis in cancer. In this review, we summarize the current knowledge on chemical compound-induced paraptosis. The ER and mitochondria are the two major responding organelles in chemical compound-induced paraptosis, which can be triggered by the reduction of protein degradation, disruption of sulfhydryl homeostasis, overload of mitochondrial Ca2+, and increased generation of reactive oxygen species. We also discuss the stumbling blocks to the development of this field and the direction for further research. The rational use of paraptosis might help us develop a new paradigm for cancer therapy.
... 27 Then, IP 3 binds the type I IP 3 receptor on the endoplasmic reticulum, which stores calcium. 28,29 This binding leads to the release of calcium from the endoplasmic reticulum into the cytoplasm. Calcium then activates calmodulin-dependent protein kinase, which activates the anaphase-promoting complex (APC). ...
... The ER is a cellular organelle that functions as an adaptive response to cellular in ammation and oxidative stress. It plays a crucial role in processes such as autophagosome formation and the transmission of oxidative and in ammatory signals [40]. In models of kidney disease, when external stimuli exceed the ER's steady-state, there is a signi cant increase in ER stress (ERS), which disrupts cytoskeletal proteins in podocytes [41]. ...
Background
This study was conducted to investigate the expression patterns and biological roles of urinary long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in individuals diagnosed with lupus nephritis (LN).
Methods
The study cohort comprised six participants: three with systemic lupus erythematosus (SLE) involving LN, three with SLE without LN, and three healthy controls (CON). Microarray technology was employed to analyze urinary mRNAs and lncRNAs, thereby exploring alterations in overall RNA expression. Functional insights into dysregulated differentially expressed mRNAs (DEMs) associated with LN were derived through gene ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and gene set enrichment analysis (GSEA). Furthermore, the construction of a protein-protein interaction (PPI) network was accomplished using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). The identification of immune-related cell types was facilitated by Single-sample Gene Set Enrichment Analysis (ssGSEA). To predict potential drug candidates based on differentially expressed genes (DEGs), Connectivity Map (CMap) analysis was conducted.
Results
Within the urine samples of SLE patients, a total of 247 mRNAs and 602 lncRNAs exhibited differential expression relative to the control group. Among these, 83 down-regulated and 141 up-regulated DEMs were specifically discerned in patients with LN. GO analysis of the network highlighted enrichment in processes such as transcriptional regulation, intrinsic apoptotic signaling pathways in response to DNA damage, and the regulation of mitophagy. KEGG pathway analysis primarily revealed enrichment in protein processing within the endoplasmic reticulum, apoptosis, and the P53 signaling pathway. Co-expression and PPI network analysis suggested that nodes with higher degrees of connectivity were concentrated in pathways related to apoptosis and autophagy. An assessment of immune infiltration unveiled a correlation between activated B cells and CD56dim natural killer (NK) cells with LN pathogenesis. The prediction of drugs implicated inhibition of mechanistic Aurora kinase A (AURKA) as a primary targeted intervention. The molecular docking process confirmed the robust binding activity of hub genes' components.
Conclusion
This study has illuminated the distinct expression profiles of urinary long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in lupus nephritis (LN) patients. These profiles, particularly in the context of apoptosis, autophagy, and immune cell involvement, provide valuable insights into LN's underlying mechanisms. The identification of potential therapeutic targets, such as mechanistic AURKA, offers promising directions for future interventions in LN management.
... Vascular smooth muscle-dependent contraction is triggered by a rise in [Ca 2+ ] i due to Ca 2+ release from the intracellular stores and Ca 2+ influx from extracellular space via plasma membrane Ca 2+ channels [46,47]. Intracellular Ca 2+ release is essentially of SR origin, through activation of IP 3 Rs and ryanodine receptors [48,49]. Our results showed that TRPV4-dependent vasoconstriction in HSD-induced hypertension arose from IP 3 R activation (Fig. 3D). ...
Recent studies have focused on the contribution of vascular endothelial transient receptor potential vanilloid 4 (TRPV4) channels to hypertension. However, in hypertension, TRPV4 channels in vascular smooth muscle remain unexplored. In the present study, we performed wire myograph experiments in isolated aortas from endothelial cell specific TRPV4 channel knockout (TRPV4EC-/-) mice to demonstrate that GSK1016790A (a specific TRPV4 channel agonist) triggered aortic smooth muscle-dependent contractions from mice on a normal-salt diet, and the contractions were enhanced in high-salt diet (HSD) mice. Intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ imaging assays showed that TRPV4-induced [Ca2+]i was significantly higher in aortic smooth muscle cells (ASMCs) from HSD-induced hypertensive mice, and application of an inositol trisphosphate receptor (IP3R) inhibitor markedly attenuated TRPV4-induced [Ca2+]i. IP3R2 expression was enhanced in ASMCs from HSD-induced hypertensive mice and the contractile response induced by TRPV4 was inhibited by the IP3R inhibitor. Whole-transcriptome analysis by RNA-seq and western blot assays revealed the involvement of interferon regulatory factor 7 (IRF7) in TRPV4-IRF7-IP3R2 signaling in HSD-induced hypertension. These results suggested that TRPV4 channels regulate smooth muscle-dependent contractions in high salt-induced hypertension, and this contraction involves increased [Ca2+]i, IP3R2, and IRF7 activity. Our study revealed a considerable effect of TRPV4 channels in smooth muscle-dependent contraction in mice during high-salt induced hypertension.
... Many soluble and membrane proteins are located in the ER of cells for synthesis, folding, modification, and transport (26,27); unfolded/misfolded proteins can cause ERS, which impairs ER The degradation of CDV 851H protein depended on the ubiquitin-proteasome pathway. (A) CDV 851H protein was degraded in cells. ...
Canine distemper (CD) caused by canine distemper virus (CDV) is considered a highly contagious and acutely febrile disease in various animals around the world. Endoplasmic reticulum-associated protein degradation (ERAD) is an important biological effect induced by endoplasmic reticulum (ER) stress (ERS) for the degradation of unfolded/misfolded proteins in the ER of cells. CDV H glycoprotein is translocated into the ER for post-translational modifications. The effects of CDV H and ER on each other are unclear. In this study, we found that CDV H protein induced ERS through the PERK-mediated signaling pathway. The inhibition of ERS by 4-Phenylbutyric acid (4-PBA) increased the H protein amounts of an attenuated CDV, which was reduced by dithiothreitol (DTT)-induced ERS. Further, the H protein levels were increased when ERAD was inhibited by using Eeyarestatin I or interfering E3 ligase Hrd1 in ERAD, suggesting that the attenuated CDV H protein is degraded via ERAD. ERAD involved ubiquitin-dependent proteasome degradation (UPD) and/or autophagic-lysosome degradation (ALD). The attenuated CDV H protein was ubiquitinated and significantly increased after treatment with UPD inhibitor MG132 but not ALD inhibitor chloroquine (CQ), suggesting that ERAD degrading the attenuated CDV H protein selectively depends on UPD. Moreover, the inhibition of the degradation of CDV H protein with 4-PBA or MG132 treatment increased viral replication, whereas treatment with DTT promoting degradation of H protein was found to reduce viral replication. These findings suggest that the degradation of CDV H protein via ERAD negatively affects viral replication and provide a new idea for developing CDV prevention and control strategies.
... The endoplasmic reticulum performs crucial functions such as protein folding and lipid synthesis, acts as a Ca 2+ store, and effects its release [33]. Endoplasmic reticulummediated Ca 2+ uptake and release are mainly dependent on inositol 1,4,5-triphosphate receptors (IP3R), ryanodine receptors (RyR), and sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). ...
Diabetes mellitus is a chronic disease affecting over 500 million adults globally and is mainly categorized as type 1 diabetes mellitus (T1DM), where pancreatic beta cells are destroyed, and type 2 diabetes mellitus (T2DM), characterized by beta cell dysfunction. This review highlights the importance of the divalent cation calcium (Ca2+) and its associated signaling pathways in the proper functioning of beta cells and underlines the effects of Ca2+ dysfunction on beta cell function and its implications for the onset of diabetes. Great interest and promise are held by human pluripotent stem cell (hPSC) technology to generate functional pancreatic beta cells from diabetic patient-derived stem cells to replace the dysfunctional cells, thereby compensating for insulin deficiency and reducing the comorbidities of the disease and its associated financial and social burden on the patient and society. Beta-like cells generated by most current differentiation protocols have blunted functionality compared to their adult human counterparts. The Ca2+ dynamics in stem cell-derived beta-like cells and adult beta cells are summarized in this review, revealing the importance of proper Ca2+ homeostasis in beta-cell function. Consequently, the importance of targeting Ca2+ function in differentiation protocols is suggested to improve current strategies to use hPSCs to generate mature and functional beta-like cells with a comparable glucose-stimulated insulin secretion (GSIS) profile to adult beta cells.
... The protein aggregations seen in AD are indicative of an imbalance in proteostasis and an increase in endoplasmic reticulum (ER) stress (Hetz and Saxena, 2017;Hoozemans et al., 2012). ER stress occurs when proteins misfold and aggregate in the ER lumen (Berridge, 2002;Lee et al., 2002;Ron and Walter, 2007;Szegezdi et al., 2006). ER stress in healthy organisms activates the unfolded protein response (UPR), a protein quality control and signal transduction pathway (Hetz et al., 2020). ...
Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder that is pervasive among the aging population. Two distinct phenotypes of AD are deficits in cognition and proteostasis, including chronic activation of the unfolded protein response (UPR) and aberrant Aβ production. It is unknown if restoring proteostasis by reducing chronic and aberrant UPR activation in AD can improve pathology and cognition. Here, we present data using an APP knock-in mouse model of AD and several protein chaperone supplementation paradigms, including a late-stage intervention. We show that supplementing protein chaperones systemically and locally in the hippocampus reduces PERK signaling and increases XBP1s, which is associated with increased ADAM10 and decreased Aβ42. Importantly, chaperone treatment improves cognition which is correlated with increased CREB phosphorylation and BDNF. Together, this data suggests that chaperone treatment restores proteostasis in a mouse model of AD and that this restoration is associated with improved cognition and reduced pathology.
One-sentence summary
Chaperone therapy in a mouse model of Alzheimer’s disease improves cognition by reducing chronic UPR activity
... Intracellular Ca 2+ is one of the most crucial signaling ion in the cytosol, and it regulates various cellular functions, from fertilization to cell death (1). The cytosolic Ca 2+ concentration ([Ca 2+ ] i ) is strictly controlled in both a time-and space-dependent manner and is amplified in signal transduction pathways (2,3). Cytosolic Ca 2+ is internalized from the extracellular space or released from intracellular Ca 2+ stores, such as the endoplasmic reticulum (ER) (4). ...
Inositol 1,4,5-trisphosphate receptors (IP3Rs) are one of the two types of tetrameric ion channels that release calcium ion (Ca2+) from the endoplasmic reticulum (ER) into the cytosol. Ca2+ released via IP3Rs is a fundamental second messenger for numerous cell functions. Disturbances in the intracellular redox environment resulting from various diseases and aging interfere with proper calcium signaling, however, the details are unclear. Here, we elucidated the regulatory mechanisms of IP3Rs by protein disulfide isomerase family proteins localized in the ER by focusing on four cysteine residues residing in the ER lumen of IP3Rs. First, we revealed that two of the cysteine residues are essential for functional tetramer formation of IP3Rs. Two other cysteine residues, on the contrary, were revealed to be involved in the regulation of IP3Rs activity; its oxidation by ERp46 and the reduction by ERdj5 caused the activation and the inactivation of IP3Rs activity, respectively. We previously reported that ERdj5 can activate the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b) using its reducing activity [Ushioda et al., Proc. Natl. Acad. Sci. U.S.A. 113, E6055-E6063 (2016)]. Thus, we here established that ERdj5 exerts the reciprocal regulatory function for IP3Rs and SERCA2b by sensing the ER luminal Ca2+ concentration, which contributes to the calcium homeostasis in the ER.
... It has regulated numerous pivotal functions, including protein biosynthesis, folding, trafficking, and calcium storage, as well as lipogenesis. 6,7 Altering physiological conditions affect ER homeostasis for various reasons, such as genetic mutations, heat shock, oxidative stress, or multiple pathophysiologies. Furthermore, increased protein synthesis requirement, glucose deprivation, or imbalance in ER calcium stock levels can cause impaired functionality of ER, termed ER stress. ...
Objectives:
Drug-induced liver injury is a common adverse reaction that frequently occurs with chemotherapeutic agents, such as cisplatin (CIS). This study seeks to enhance our understanding of drug actions and their associated adverse effects by examining the toxicity of CIS on rat liver tissue. We aimed to investigate the potential hepatoprotective effects of irbesartan (IRB), an easily accessible angiotensin II receptor blocker, in mitigating CIS-induced hepatotoxicity.
Materials and methods:
Wistar albino rats were divided into four groups. These groups included a control group [saline, per oral (p.o.)] for seven days, and 1 mL saline intraperitoneal [(i.p.) on the fourth day]; a CIS group (1 mL saline for seven days and 7.5 mg/kg CIS i.p. on the fourth day); a CIS + IRB group (IRB: 50 mg/kg p.o. for seven days and 7.5 mg/kg CIS i.p. on the fourth day), and an IRB group (50 mg/kg IRB p.o. for seven days). The effect of IRB on interleukin-1 beta (IL-1β) and caspase 3 levels was evaluated by immunohistochemical analysis, and its effects on mRNA expression levels of CCAAT/enhancer-binding protein homologous protein (CHOP) and immunoglobulin-heavy-chain-binding protein (BiP) were tested by quantitative real-time polymerase chain reaction.
Results:
IRB administration mitigated CIS-induced liver toxicity by inhibiting endoplasmic reticulum (ER) stress. Specifically, this drug reduced the mRNA expression of ER stress markers, including CHOP and BiP. In addition, IRB treatment decreased oxidative stress, inflammatory responses, and apoptotic markers.
Conclusion:
These findings suggest that IRB is a promising therapeutic option for preventing CIS-induced liver injury, potentially by modulating ER stress-related pathways.
... The largest organelle in eukaryotes is the endoplasmic reticulum (ER). It is a major site of cellular translation, protein folding, and quality control [50,51], the major cellular calcium store and sink in most cells [7], and the primary site of lipid synthesis and regulation [66]. In addition to these crucial cellular functions, it also makes contact with and regulates the biology of essentially every other cellular compartment through specific tethering molecules [49]. ...
The endoplasmic reticulum (ER) is a structurally complex, membrane-enclosed compartment that stretches from the nuclear envelope to the extreme periphery of eukaryotic cells. The organelle is crucial for numerous distinct cellular processes, but how these processes are spatially regulated within the structure is unclear. Traditional imaging-based approaches to understanding protein dynamics within the organelle are limited by the convoluted structure and rapid movement of molecular components. Here, we introduce a combinatorial imaging and machine learning-assisted image analysis approach to track the motion of photoactivated proteins within the ER of live cells. We find that simultaneous knowledge of the underlying ER structure is required to accurately analyze fluorescently-tagged protein redistribution, and after appropriate structural calibration we see all proteins assayed show signatures of Brownian diffusion-dominated motion over micron spatial scales. Remarkably, we find that in some cells the ER structure can be explored in a highly asymmetric manner, likely as a result of uneven connectivity within the organelle. This remains true independently of the size, topology, or folding state of the fluorescently-tagged molecules, suggesting a potential role for ER connectivity in driving spatially regulated biology in eukaryotes.
... The endoplasmic reticulum (ER) is one of the two main reservoirs for releasable Ca 2+ in the cell and usually maintains free Ca 2+ concentrations of 100-800 μM, which amounts to at least three orders of magnitude higher than in the cytosol (Berridge et al., 2000;Berridge, 2002) ( Figure 1A). Therefore, it is remarkable that the ER membrane is not tight to ions; it has indeed a distinct permeability to ions and even small molecules. ...
... In the cytoplasm, a majority of the M29 + CaM dimers were found to be co-localizing with the ER. In eukaryotes, ER plays a vital role in the synthesis, folding, and sorting of proteins as well as in acting as a dynamic reservoir of intracellular Ca 2+ essential for cellular calcium signalling [48]. The co-localization of M29 + CaM dimers with ER and their intensified sequestration under cellular Ca 2+ depleted conditions further indicate that a yet unknown ER protein (probably, membrane-bound) could be involved in the interaction with either CaM or M29 + CaM dimers in a Ca 2+ -dependent manner (Fig. 10). ...
OsMADS29 (M29) is a crucial regulator of seed development in rice. The expression of M29 is strictly regulated at transcriptional as well as post‐transcriptional levels. The MADS‐box proteins are known to bind to DNA as dimers. However, in the case of M29, the dimerization also plays a vital role in its localization into the nucleus. The factor(s) that affect oligomerization and nuclear transport of MADS proteins have not yet been characterized. By using BiFC in transgenic BY‐2 cell lines and Yeast‐2‐hybrid assay (Y2H), we show that calmodulin (CaM) interacts with M29 in a Ca²⁺‐dependent manner. This interaction specifically takes place in the cytoplasm, probably in association with the endoplasmic reticulum. By generating domain‐specific deletions, we show that both sites in M29 are involved in this interaction. Further, by using BiFC‐FRET‐FLIM, we demonstrate that CaM may also help in the dimerization of two M29 monomers. Since most MADS proteins have CaM binding domains, the interaction between these proteins could be a general regulatory mechanism for oligomerization and nuclear transport.
... Most secreted proteins, transmembrane proteins, and lipids are produced in the ER. In addition, the ER maintains cellular homeostasis by tightly regulating calcium dynamics, phospholipid biogenesis, and various intracellular signals by releasing Ca 2+ (Chevet et al. 2001;Berridge 2002;Park et al. 2021). Alterations in calcium levels and metabolism, redox imbalances, nutrient deprivation, and hypoxia can disrupt ER homeostasis (Schonthal 2012). ...
Cells activate protective mechanisms to overcome stressful conditions that threaten cellular homeostasis, including imbalances in calcium, redox, and nutrient levels. Endoplasmic reticulum (ER) stress activates an intracellular signaling pathway, known as the unfolded protein response (UPR), to mitigate such circumstances and protect cells. Although ER stress is sometimes a negative regulator of autophagy, UPR induced by ER stress typically activates autophagy, a self-degradative pathway that further supports its cytoprotective role. Sustained activation of ER stress and autophagy is known to trigger cell death and is considered a therapeutic target for certain diseases. However, ER stress-induced autophagy can also lead to treatment resistance in cancer and exacerbation of certain diseases. Since the ER stress response and autophagy affect each other, and the degree of their activation is closely related to various diseases, understanding their relationship is very important. In this review, we summarize the current understanding of two fundamental cellular stress responses, the ER stress response and autophagy, and their crosstalk under pathological conditions to help develop therapies for inflammatory diseases, neurodegenerative disorders, and cancer.
... Therefore, research in this area should be focused on the study and application of new approaches for the treatment of this type of cancer. MEL, a hormone produced by the pineal gland, has the structure of an indolamine, and its precursor is tryptophan [41,42]. MEL has been shown to affect various types of cancer, in particular, breast cancer, colorectal cancer, hematopoietic tissue tumors, etc. [43][44][45]. ...
Melatonin (N-acetyl-5-methoxytryptamine, MEL), secreted by the pineal gland, plays an important role in regulation of various functions in the human body. There is evidence that MEL exhibits antitumor effect in various types of cancer. We studied the combined effect of MEL and drugs from different pharmacological groups, such as cytarabine (CYT) and navitoclax (ABT-737), on the state of the pool of acute myeloid leukemia (AML) tumor cell using the MV4-11 cell line as model. The combined action of MEL with CYT or ABT-737 contributed to the decrease in proliferative activity of leukemic cells, decrease in the membrane potential of mitochondria, and increase in the production of reactive oxygen species (ROS) and cytosolic Ca2+. We have shown that introduction of MEL together with CYT or ABT-737 increases expression of the C/EBP homologous protein (CHOP) and the autophagy marker LC3A/B and decreases expression of the protein disulfide isomerase (PDI) and binding immunoglobulin protein (BIP), and, therefore, could modulate endoplasmic reticulum (ER) stress and initiate autophagy. The findings support an early suggestion that MEL is able to provide benefits for cancer treatment and be considered as an adjunct to the drugs used in cancer therapy.
... The ER is an intracellular organelle found in eukaryotic cells. The main functions of the ER are the folding of secreted proteins, participating in calcium signaling as one of the largest calcium stores, and posttranslational modification (Gething and Sambrook, 1992;Helenius et al., 1992;Berridge, 2002). Ca 2+ is responsible for the transmission of neuronal depolarization and synaptic activity, and Ca 2+ homeostasis and signaling are responsible for proper synaptic plasticity and survival. ...
The endoplasmic reticulum (ER) is a major organelle involved in protein quality control and cellular homeostasis. ER stress results from structural and functional dysfunction of the organelle, along with the accumulation of misfolded proteins and changes in calcium homeostasis, it leads to ER stress response pathway such as unfolded protein response (UPR). Neurons are particularly sensitive to the accumulation of misfolded proteins. Thus, the ER stress is involved in neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, prion disease and motor neuron disease (MND). Recently, the complex involvement of ER stress pathways has been demonstrated in experimental models of amyotrophic lateral sclerosis (ALS)/MND using pharmacological and genetic manipulation of the unfolded protein response (UPR), an adaptive response to ER stress. Here, we aim to provide recent evidence demonstrating that the ER stress pathway is an essential pathological mechanism of ALS. In addition, we also provide therapeutic strategies that can help treat diseases by targeting the ER stress pathway.
Melatonin (N-acetyl-5-methoxytryptamine, MEL), secreted by the pineal gland, plays an important role in the regulation of the different functions in human. However, there are the facts that MEL has an antitumor effect in various types of cancer. We have studied the combined effect of MEL and targeted drugs such as cytarabine (CYT) and navitoclax (ABT-737) on the development of acute myeloid leukemia in the MV4-11 cell model. The combined action of MEL with CYT or ABT-737 contributed to a decrease in the proliferative activity of leukemia cells, a drop in the membrane potential of mitochondria, and an increase in the production of reactive oxygen species and cytosolic Ca2+. We have shown that MEL, together with CYT or ABT-737, increases expression of homologous C/EBP protein and autophagy marker LC3A/B and decreases protein disulfide isomerase and immunoglobulin-binding protein levels, and consequently modulate endoplasmic stress. reticulum and initiate autophagy. The obtained data support the earlier suggestion that MEL may have potential benefits in cancer treatment and may be considered as an additive to drugs used in therapy.
Endoplasmic/sarcoplasmic reticulum (ER/SR) sits at the heart of the calcium (Ca ²⁺ ) signaling machinery, yet current genetically encoded Ca ²⁺ indicators (GECIs) lack the ability to detect elementary Ca ²⁺ release events from ER/SR, particularly in muscle cells. Here we report a set of organellar GECIs, termed NEMOer, to efficiently capture ER Ca ²⁺ dynamics with increased sensitivity and responsiveness. Compared to G-CEPIA1er, NEMOer indicators exhibit dynamic ranges that are an order of magnitude larger, which enables up to 5-fold more sensitive detection of Ca ²⁺ oscillation in both non-excitable and excitable cells. The ratiometric version further allows super-resolution monitoring of local ER Ca ²⁺ homeostasis and dynamics. Notably, the NEMOer-f variant enabled the inaugural detection of Ca ²⁺ blinks, elementary Ca ²⁺ releasing signals from the SR of cardiomyocytes, as well as in vivo spontaneous SR Ca ²⁺ releases in zebrafish. In summary, the highly dynamic NEMOer sensors expand the repertoire of organellar Ca ²⁺ sensors that allow real-time monitoring of intricate Ca ²⁺ dynamics and homeostasis in live cells with high spatiotemporal resolution.
Dysregulation of synaptic glutamate levels can lead to excitotoxicity such as that observed in stroke, traumatic brain injury, and epilepsy. The role of increased intracellular calcium (Ca ²⁺ ) in the development of excitotoxicity is well established. However, less is known regarding the impact of glutamate on endoplasmic reticulum (ER)-Ca ²⁺ -mediated processes such as proteostasis. To investigate this, we expressed a secreted ER Ca ²⁺ modulated protein (SERCaMP) in primary cortical neurons to monitor exodosis, a phenomenon whereby ER calcium depletion causes the secretion of ER resident proteins that perform essential functions to the ER and the cell. Activation of glutamatergic receptors (GluRs) led to an increase in SERCaMP secretion indicating that normally ER resident proteins are being secreted in a manner consistent with ER Ca ²⁺ depletion. Antagonism of ER Ca ²⁺ channels attenuated the effects of glutamate and GluR agonists on SERCaMP release. We also demonstrate that endogenous proteins containing an ER retention sequence (ERS) are secreted in response to GluR activation supporting that neuronal activation by glutamate promotes ER exodosis. Ectopic expression of KDEL receptors attenuated the secretion of ERS-containing proteins caused by GluR agonists. Taken together, our data indicate that excessive GluR activation causes disruption of neuronal proteostasis by triggering the secretion of ER resident proteins through ER Ca ²⁺ depletion and describes a new facet of excitotoxicity.
Significance
During excitotoxicity, the excessive activation of glutamate receptors causes elevated intracellular calcium (Ca ²⁺ ) that promotes cellular dysfunction and death. While the role of cytosolic Ca ²⁺ in excitotoxicity has been well-studied, the consequences of changes in endoplasmic reticulum (ER) Ca ²⁺ during excitotoxicity remains unclear. The relatively high concentration of calcium in the ER is necessary for ER resident proteins to function prop out essential functions and maintain cellular proteostasis. We show here that excitotoxic conditions destabilize the ER proteome by triggering ER resident protein secretion. Stabilizing ER Ca ²⁺ or overexpressing receptors that interact with ER resident proteins can prevent disruption of proteostasis associated with excitotoxicity. The present study provides a new link between excitotoxicity, ER Ca ²⁺ homeostasis, and the ER proteome.
Calcium ions (Ca2+) play a critical role in triggering neurotransmitter release. The rate of release is directly related to the concentration of Ca2+ at the presynaptic site, with a supralinear relationship. There are two main sources of Ca2+ that trigger synaptic vesicle fusion: influx through voltage-gated Ca2+ channels in the plasma membrane and release from the endoplasmic reticulum via ryanodine receptors. This chapter will cover the sources of Ca2+ at the presynaptic nerve terminal, the relationship between neurotransmitter release rate and Ca2+ concentration, and the mechanisms that achieve the necessary Ca2+ concentrations for triggering synaptic exocytosis at the presynaptic site.
Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels is not well understood. To determine the effects of tunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13 cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA, and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis, and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrate silencing RyR1 suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis, although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these results show changes in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.
The impact of microplastic particles on organisms is currently intensely researched. Although it is well established that macrophages ingest polystyrene (PS) microparticles, little is known about the subsequent fate of the particles, such as entrapment in organelles, distribution during cell division, as well as possible mechanisms of excretion. Here, submicrometer (0.2 and 0.5 µm) and micron-sized (3 µm) particles were used to analyze particle fate upon ingestion of murine macrophages (J774A.1 and ImKC). Distribution and excretion of PS particles was investigated over cycles of cellular division. The distribution during cell division seems cell-specific upon comparing two different macrophage cell lines, and no apparent active excretion of microplastic particles could be observed. Using polarized cells, M1 polarized macrophages show higher phagocytic activity and particle uptake than M2 polarized ones or M0 cells. While particles with all tested diameters were found in the cytoplasm, submicron particles were additionally co-localized with the endoplasmic reticulum. Further, 0.5 µm particles were occasionally found in endosomes. Our results indicate that a possible reason for the previously described low cytotoxicity upon uptake of pristine PS microparticles by macrophages may be due to the preferential localization in the cytoplasm.
Endoplasmic reticulum-plasma membrane contact sites (ER-PM MCS) are a specialised domain involved in the control of Ca2+ dynamics and various Ca2+-dependent cellular processes. Intracellular Ca2+ signals are broadly supported by Ca2+ release from intracellular Ca2+ channels such as inositol 1,4,5-trisphosphate receptors (IP3Rs) and subsequent store-operated Ca2+ entry (SOCE) across the PM to replenish store content. IP3Rs sit in close proximity to the PM where they can easily access newly synthesised IP3, interact with binding partners such as actin, and localise adjacent to ER-PM MCS populated by the SOCE machinery, STIM1-2 and Orai1-3, to possibly form a locally regulated unit of Ca2+ influx. PtdIns(4,5)P2 is a multiplex regulator of Ca2+ signalling at the ER-PM MCS interacting with multiple proteins at these junctions such as actin and STIM1, whilst also being consumed as a substrate for phospholipase C to produce IP3 in response to extracellular stimuli. In this review, we consider the mechanisms regulating the synthesis and turnover of PtdIns(4,5)P2 via the phosphoinositide cycle and its significance for sustained signalling at the ER-PM MCS. Furthermore, we highlight recent insights into the role of PtdIns(4,5)P2 in the spatiotemporal organization of signalling at ER-PM junctions and raise outstanding questions on how this multi-faceted regulation occurs.
Background:
Cardiac contractile function requires high energy from mitochondria, and Ca2+ from the sarcoplasmic reticulum (SR). Via local Ca2+ transfer at close mitochondria-SR contacts, cardiac excitation feedforward regulates mitochondrial ATP production to match surges in demand (excitation-bioenergetics coupling). However, pathological stresses may cause mitochondrial Ca2+ overload, excessive reactive oxygen species production and permeability transition, risking homeostatic collapse and myocyte loss. Excitation-bioenergetics coupling involves mitochondria-SR tethers but the role of tethering in cardiac physiology/pathology is debated. Endogenous tether proteins are multifunctional; therefore, nonselective targets to scrutinize interorganelle linkage. Here, we assessed the physiological/pathological relevance of selective chronic enhancement of cardiac mitochondria-SR tethering.
Methods:
We introduced to mice a cardiac muscle-specific engineered tether (linker) transgene with a fluorescent protein core and deployed 2D/3D electron microscopy, biochemical approaches, fluorescence imaging, in vivo and ex vivo cardiac performance monitoring and stress challenges to characterize the linker phenotype.
Results:
Expressed in the mature cardiomyocytes, the linker expanded and tightened individual mitochondria-junctional SR contacts; but also evoked a marked remodeling with large dense mitochondrial clusters that excluded dyads. Yet, excitation-bioenergetics coupling remained well-preserved, likely due to more longitudinal mitochondria-dyad contacts and nanotunnelling between mitochondria exposed to junctional SR and those sealed away from junctional SR. Remarkably, the linker decreased female vulnerability to acute massive β-adrenergic stress. It also reduced myocyte death and mitochondrial calcium-overload-associated myocardial impairment in ex vivo ischemia/reperfusion injury.
Conclusions:
We propose that mitochondria-SR/endoplasmic reticulum contacts operate at a structural optimum. Although acute changes in tethering may cause dysfunction, upon chronic enhancement of contacts from early life, adaptive remodeling of the organelles shifts the system to a new, stable structural optimum. This remodeling balances the individually enhanced mitochondrion-junctional SR crosstalk and excitation-bioenergetics coupling, by increasing the connected mitochondrial pool and, presumably, Ca2+/reactive oxygen species capacity, which then improves the resilience to stresses associated with dysregulated hyperactive Ca2+ signaling.
Eukaryotes depend upon the proper localization, accumulation, and release of intracellular Ca2+. This is regulated through specialized cellular compartments, signaling pathways, and Ca2+-binding proteins and channels. Cytosolic and extracellular signaling governing intracellular Ca2+ stores are well explored. However, regulatory signals within Ca2+ storage organelles like the endoplasmic/sarcoplasmic reticulum are not well understood. This is due to a lack of identified signaling molecules - like protein kinases - within these compartments, limited information on their regulation, and incomplete understanding of mechanisms involving modified substrates. Here we review recent advances in intralumenal signaling focusing on the secretory pathway protein kinase FAM20C and its regulation, Ca2+-binding protein substrates, and potential mechanisms through which FAM20C may regulate Ca2+ storage.
Cardiac fibrosis is defined as excessive deposition of extracellular matrix (ECM) in pathological conditions. Cardiac fibroblasts (CFs) activated by injury or inflammation differentiate into myofibroblasts (MFs) with secretory and contractile functions. In the fibrotic heart, MFs produce ECM which is composed mainly of collagen and is initially involved in maintaining tissue integrity. However, persistent fibrosis disrupts the coordination of excitatory contractile coupling, leading to systolic and diastolic dysfunction, and ultimately heart failure. Numerous studies have demonstrated that both voltage- and non-voltage-gated ion channels alter intracellular ion levels and cellular activity, contributing to myofibroblast proliferation, contraction, and secretory function. However, an effective treatment strategy for myocardial fibrosis has not been established. Therefore, this review describes the progress made in research related to transient receptor potential (TRP) channels, Piezo1, Ca2+ release-activated Ca2+ (CRAC) channels, voltage-gated Ca2+ channels (VGCCs), sodium channels, and potassium channels in myocardial fibroblasts with the aim of providing new ideas for treating myocardial fibrosis.
Oxygen is a critical factor for most organisms and this is especially true for aquatic animals. Unfortunately, high-density aquaculture farming practices and environmental degradation will inevitably lead to hypoxic stress in fishes such as largemouth bass (Micropterus salmoides). Thus, characterizing the physiological responses during acute hypoxia exposure is extremely important for understanding the adaptation mechanisms of largemouth bass to hypoxia. The present study aimed to investigate mitochondrial function and Ca2+ exchange in largemouth bass under hypoxic conditions. Largemouth bass were subjected to hypoxia (1.2 ± 0.2 mg/L) for 24 h Liver mitochondria and endoplasmic reticulum (ER) parameters were analyzed. We used Liquid chromatography-mass spectrometry (LC-MS) to further elucidate the pattern of energy metabolism. Changes of Ca2+ concentrations were observed in primary hepatocytes of largemouth bass under hypoxic conditions. Our results indicate that the morphology and function of the mitochondria and ER were altered under hypoxia. First, the occurrence of autophagy was accompanied by reactive oxygen species (ROS) generation and electron transport chain (ETC) activity modulation under hypoxia. Second, hypoxia enhanced mitochondrial fusion and fission, mitochondrial biosynthesis, and ER quality control in the early stages of hypoxic stress (before 8 h). Third, hypoxia modulated tricarboxylic acid (TCA) cycle flux and caused the accumulation of TCA intermediate metabolites (citric acid and oxoglutaric acid). Additionally, Ca2+ efflux in the ER was observed., and the genes for Ca2+ transporters presented high expression levels in cellular and mitochondrial membranes. Collectively, the above physiological responses of the mitochondria and ER contributed to maintaining energy production to withstand the hypoxic stress in largemouth bass. These results provide novel insights into the physiological and metabolic changes in largemouth bass under hypoxic conditions.
Release of Ca2+ ions from intracellular stores can occur via two classes of Ca2+-release channel (CRC) protein, the inositol 1,4,5-trisphosphate receptors (InsP3Rs) and the ryanodine receptors (RyRs). Multiple isoforms and subtypes of each CRC class display distinct but overlapping distributions within mammalian tissues. InsP3Rs and RyRs interact with a plethora of accessory proteins which modulate the activity of their intrinsic channels. Although many aspects of CRC structure and function have been reviewed in recent years, the properties of proteins with which they interact has not been comprehensively surveyed, despite extensive current research on the roles of these modulators. The aim of this article is to review the regulation of CRC activity by accessory proteins and, wherever possible, to outline the structural details of such interactions. The CRCs are large transmembrane proteins, with the bulk of their structure located cytoplasmically. Intra- and inter-complex protein–protein interactions between these cytoplasmic domains also regulate CRC function. Some accessory proteins modulate channel activity of all CRC subtypes characterized, whereas other have class- or even isoform-specific effects. Certain accessory proteins exert both direct and indirect forms of regulation on CRCs, occasionally with opposing effects. Others are themselves modulated by changes in Ca2+ concentration, thereby participating in feedback mechanisms acting on InsP3R and RyR activity. CRCs are therefore capable of integrating numerous signalling events within a cell by virtue of such protein–protein interactions. Consequently, the functional properties of InsP3Rs and RyRs within particular cells and subcellular domains are ‘customized ’ by the accessory proteins present.
Calpains and caspases are two cysteine protease families that play important roles in regulating pathological cell death. Here, we report that m-calpain may be responsible for cleaving procaspase-12, a caspase localized in the ER, to generate active caspase-12. In addition, calpain may be responsible for cleaving the loop region in Bcl-xL and, therefore, turning an antiapoptotic molecule into a proapoptotic molecule. We propose that disturbance to intracellular calcium storage as a result of ischemic injury or amyloid β peptide cytotoxicity may induce apoptosis through calpain- mediated caspase-12 activation and Bcl-xL inactivation. These data suggest a novel apoptotic pathway involving calcium-mediated calpain activation and cross-talks between calpain and caspase families.
Calreticulin (CRT) and calnexin (CLNX) are lectin chaperones that participate in protein folding in the endoplasmic reticulum (ER). CRT is a soluble ER lumenal protein, whereas CLNX is a transmembrane protein with a cytosolic domain that contains two consensus motifs for protein kinase (PK) C/proline- directed kinase (PDK) phosphorylation. Using confocal Ca²⁺ imaging in Xenopus oocytes, we report here that coexpression of CLNX with sarco endoplasmic reticulum calcium ATPase (SERCA) 2b results in inhibition of intracellular Ca²⁺ oscillations, suggesting a functional inhibition of the pump. By site-directed mutagenesis, we demonstrate that this interaction is regulated by a COOH-terminal serine residue (S562) in CLNX. Furthermore, inositol 1,4,5-trisphosphate– mediated Ca²⁺ release results in a dephosphorylation of this residue. We also demonstrate by coimmunoprecipitation that CLNX physically interacts with the COOH terminus of SERCA2b and that after dephosphorylation treatment, this interaction is significantly reduced. Together, our results suggest that CRT is uniquely regulated by ER lumenal conditions, whereas CLNX is, in addition, regulated by the phosphorylation status of its cytosolic domain. The S562 residue in CLNX acts as a molecular switch that regulates the interaction of the chaperone with SERCA2b, thereby affecting Ca²⁺ signaling and controlling Ca²⁺-sensitive chaperone functions in the ER.
Nuclear DNA damage and ligation of plasma-membrane death receptors have long been recognized as initial triggers of apoptosis that induce mitochondrial membrane permeabilization (MMP) and/or the direct activation of caspases. Accumulating evidence suggests that other organelles, including the endoplasmic reticulum (ER), lysosomes and the Golgi apparatus, are also major points of integration of pro-apoptotic signalling or damage sensing. Each organelle possesses sensors that detect specific alterations, locally activates signal transduction pathways and emits signals that ensure inter-organellar cross-talk. The ER senses local stress through chaperones, Ca2+-binding proteins and Ca2+ release channels, which might transmit ER Ca2+ responses to mitochondria. The ER also contains several Bcl-2-binding proteins, and Bcl-2 has been reported to exert part of its cytoprotective effect within the ER. Upon membrane destabilization, lysosomes release cathepsins that are endowed with the capacity of triggering MMP. The Golgi apparatus constitutes a privileged site for the generation of the pro-apoptotic mediator ganglioside GD3, facilitates local caspase-2 activation and might serve as a storage organelle for latent death receptors. Intriguingly, most organelle-specific death responses finally lead to either MMP or caspase activation, both of which might function as central integrators of the death pathway, thereby streamlining lysosome-, Golgi- or ER-elicited responses into a common pathway.
• The cellular mechanisms governing cardiac atrial pacemaker activity are not clear. In the present study we used perforated patch voltage clamp and confocal fluorescence microscopy to study the contribution of intracellular Ca2+ release to automaticity of pacemaker cells isolated from cat right atrium. • In spontaneously beating pacemaker cells, an increase in subsarcolemmal intracellular Ca2+ concentration occurred concomitantly with the last third of diastolic depolarization due to local release of Ca2+ from the sarcoplasmic reticulum (SR), i.e. Ca2+ sparks. Nickel (Ni2+; 25–50 M), a blocker of low voltage-activated T-type Ca2+ current ((ICa,T), decreased diastolic depolarization, prolonged pacemaker cycle length and suppressed diastolic Ca2+ release. • Voltage clamp analysis indicated that the diastolic Ca2+ release was voltage dependent and triggered at about -60 mV. Ni2+ suppressed low voltage-activated Ca2+ release. Moreover, low voltage-activated Ca2+ release was paralleled by a slow inward current presumably due to stimulation of Na+-Ca2+ exchange (INa-Ca). Low voltage-activated Ca2+ release was found in both sino-atrial node and latent atrial pacemaker cells but not in working atrial myocytes. • These findings suggest that low voltage-activated ICa,T triggers subsarcolemmal Ca2+ sparks, which in turn stimulate INa-Ca to depolarize the pacemaker potential to threshold. This novel mechanism indicates a pivotal role for ICa,T and subsarcolemmal intracellular Ca2+ release in normal atrial pacemaker activity and may contribute to the development of ectopic atrial arrhythmias.
By resolving immunoprecipitates on nonreducing sodium dodecyl sulfate gels, we have detected several disulfide-bonded intermediates in folding within the endoplasmic reticulum of newly made H1 subunits of the asialoglycoprotein receptor. H1 in the endoplasmic reticulum (ER) can be partially unfolded by treatment of cells with dithiothreitol, but H1 in Golgi or post-Golgi organelles is resistant to such unfolding. This defines a late step in H1 folding that occurs just prior to exit from the ER. Depletion of calcium from the endoplasmic reticulum, either by treatment with A23187 or thapsigargin, has no effect on folding or secretion of newly made albumin, but totally blocks H1 maturation from the ER. No ER intermediates in H1 folding are formed in cells treated with A23187 or thapsigargin, indicating that at least an early step in H1 folding requires a high Ca2+ concentration in the ER lumen. As judged by cross-linking experiments, formation of H1 dimers and trimers occurs immediately after biosynthesis of the peptide chain, before monomer folding, and occurs normally in cells in which ER Ca2+ is reduced and where the monomer never folds properly. Calcium is essential for the asialoglycoprotein receptor to bind galactose, and our results suggest that Ca2+ is also essential for the receptor polypeptides to fold in the ER.
HepG2 cells were employed as model system to investigate potential relationships between early protein processing and Ca2+ storage by the endoplasmic reticulum. Ca2+ was required for glycoprotein processing and export by intact cells. The processing and export of alpha 1-antitrypsin and the secretion of complement factor 3, which are glycosylated proteins, were inhibited by the Ca2+ ionophore ionomycin whereas the export of albumin, a non-glycoprotein, was little affected. Ionomycin blocked processing of alpha 1-antitrypsin at the conversion from the high mannose to the complex glycosylated form without affecting ATP or GTP contents. Pre-existing inhibition of intracellular processing of alpha 1-antitrypsin by ionomycin was fully reversible upon removal of the ionophore with fatty acid-free bovine serum albumin. This reversal required Ca2+. After reversal the arrested form of alpha 1-antitrypsin was fully converted to the mature form and exported to the medium. Inhibitions of alpha 1-antitrypsin processing and complement factor 3 secretion by the metalloendoprotease antagonist Cbz-Gly-Phe-NH2 (where Cbz is benzyloxycarbonyl) were strongest at low extracellular Ca2+ but were reduced or prevented by high extracellular Ca2+. Processing and secretion of alpha 1-antitrypsin were reduced upon incubation in low Ca2+ medium. Exposure to dithiothreitol reduced albumin export while affecting alpha 1-antitrypsin export minimally. Suppression of amino acid incorporation into total cellular proteins of HepG2 cells accompanied inhibitions of protein processing by agents depleting sequestered Ca2+ stores or by dithiothreitol. Putative control of rates of translational initiation by the endoplasmic reticulum through linkage to rates of early protein processing is discussed.
In the cultured human hepatoma HepG2, Ca2+ ionophores block secretion of different secretary proteins to different extents, alpha 1-antitrypsin secretion being more sensitive to A23187 and ionomycin than is alpha 1-antichymotrypsin, and albumin secretion the least of the three proteins studied. As judged by subcellular fractionation experiments and by treatment of pulse chase labeled protein with endoglycosidase H, A23187 and ionomycin cause newly made secretory proteins to remain within the rough endoplasmic reticulum (ER). Experiments in which A23187 is added at different times during a pulse or chase show that secretion of newly made alpha 1-antitrypsin becomes resistant to the ionophore, on average, 15 min after synthesis; this is about 20 min before it reaches the trans-Golgi, and while it is still within the rough ER. We speculate that a high concentration of Ca2+ within the ER may be essential for certain secretory proteins to fold properly, that folding is inhibited when ER Ca2+ levels are lowered by ionophore treatment, and that unfolded proteins, particularly alpha 1-antitrypsin, cannot exit the rough ER. Treatment of murine 3T3 fibroblasts or human hepatoma HepG2 cells with the Ca2+ ionophores A23187 or ionomycin also induces a severalfold accumulation of the ER lumenal protein Bip (Grp78). These findings disagree with a recent report that Ca2+ ionophores cause secretion of Bip and other resident ER proteins, but is consistent with other reports that A23187 causes accumulation of mRNAs for Bip and other ER lumenal proteins.
The axon initial segment of cortical principal neurones contains an organelle consisting of two to four stacks of flat, membrane-delineated cisternae alternating with electron-dense, fibrillar material. These cisternal organelles are situated predominantly close to the synaptic junctions of GABAergic axo-axonic cell terminals. To examine the possibility that the cisternal organelle is involved in Ca2+ sequestration, we tested for the presence of Ca(2+)-ATPase in the cisternal organelles of pyramidal cell axons in the CA1 and CA3 regions of the hippocampus. Electron microscopic immunocytochemistry using antibodies to muscle sarcoplasmic reticulum ATPase revealed immunoreactivity associated with cisternal organelle membranes. The localisation of Ca(2+)-ATPase in cisternal organelles was also confirmed by enzyme cytochemistry, which produced reaction product in the lumen of the cisternae. These experiments provide evidence for the presence of a Ca2+ pump in the cisternal organelle membrane, which may play a role in the sequestration and release of Ca2+. Cisternal organelles are very closely aligned to the axolemma and the outermost cisternal membrane is connected to the plasma membrane by periodic electron-dense bridges as detected in electron micrographs. It is suggested that the interface acts as a voltage sensor, releasing Ca2+ from cisternal organelles upon depolarisation of the axon initial segment, in a manner similar to the sarcoplasmic reticulum of skeletal muscle. The increase in intra-axonal Ca2+ may regulate the GABAA receptors associated with the axo-axonic cell synapses, and could affect the excitability of pyramidal cells.
Expression studies with skeletal and cardiac muscle cDNAs have suggested that the putative cytoplasmic loop region of the dihydropyridine receptor (DHPR) alpha 1 subunit between transmembrane repeats II and III (DCL) is a major determinant of the type of excitation-contraction coupling (skeletal or cardiac) in rescued dysgenic muscle cells (Tanabe, T., Beam, K. G., Adams, B. A., Niidome, T., and Numa, S. (1990) Nature 346, 567-569). In this study, the possibility of a direct functional interaction with the sarcoplasmic reticulum ryanodine receptor/Ca2+ release channel has been tested by expressing the DCLs of the mammalian skeletal and cardiac muscle DHPR alpha 1 subunit in Escherichia coli. The purified peptides activated the skeletal muscle ryanodine receptor/Ca2+ release channel in single channel and [3H]ryanodine binding measurements, by increasing channel open probability and the affinity of [3H]ryanodine binding, respectively. The two peptides did not activate the cardiac muscle Ca2+ release channel. Other proteins (polylysine, serum albumin) also increased [3H]ryanodine binding and Ca2+ release channel activity, but their activation mechanisms were distinguishable from DCLs. These results show that the II-III cytoplasmic loop of the skeletal and cardiac DHPR alpha 1 subunit functionally interacts with the skeletal, but not cardiac, muscle Ca2+ release channel. Furthermore, our studies suggest that in addition to the DHPR, the sarcoplasmic reticulum Ca2+ release channel may determine the type of E-C coupling that exists in muscle.
The major proteins in the lumen of the endoplasmic reticulum (ER) are thought to function in Ca2+ sequestration or as "molecular chaperones" in the folding and assembly of membrane or secreted proteins. Based on the ability of many chaperones to bind selectively to unfolded proteins and to dissociate from them upon ATP hydrolysis, we developed an affinity chromatography method to isolate proteins with these characteristics from pancreatic or liver ER. Seven ER proteins bound selectively to denatured protein columns and were specifically eluted by ATP (10(-6) M) but not by a nonhydrolyzable ATP analog. These proteins were identified with antibodies and microsequencing as the ER chaperone BiP (grp78), grp94, calreticulin, a novel 46-kDa protein that binds azido-ATP, as well as three members of the thioredoxin superfamily: protein-disulfide isomerase, ERp72, and a previously reported 50-kDa protein (p50). This set of seven proteins bound to and was eluted with ATP from a variety of denatured proteins, including histone, gelatin, alpha fetoprotein, thyroglobulin, lysozyme, casein, and IgG. The release of grp94, protein-disulfide isomerase, ERp72, calreticulin, and p50 was stimulated by Ca2+ in the presence of ATP. These proteins thus appear to function as Ca(2+)-dependent chaperones, which may account for the Ca2+ and ATP requirement for protein folding in the ER.
Mobilization of Ca2+ from the endoplasmic reticulum (ER) suppresses translational initiation and inhibits post-translational processing and secretion of glycoproteins. This study explores the mechanism whereby ionomycin, a Ca2+ ionophore, and thapsigargin, an ER Ca(2+)-ATPase inhibitor, promote retention of alpha 1-antitrypsin (alpha 1-AT) bearing high mannose, endoglycosidase H (Endo H)-sensitive oligosaccharide side chains within the ER of HepG2 cells. Arrest occurred at the removal of mannose residues such that intermediates with Man7-9GlcNAc2 side chains accumulated with the Man8-9GlcNAc2 structures predominating. Maturation of alpha 1-AT bearing Man5-6GlcNAc2 side chains was unaffected. Inhibition of alpha 1-AT processing by ionomycin occurred independently of translational suppression. Forms of alpha 1-AT identical to those retained with ionomycin or thapsigargin were observed upon treatment with the alpha-1,2-mannosidase inhibitor 1-deoxymannojirimycin whereas castanospermine, an inhibitor of ER alpha-glucosidase I, produced different forms of the glycoprotein. Neither inhibitor impaired transport or secretion of alpha 1-AT. With brefeldin A, which causes redistribution of Golgi enzymes to the ER, alpha 1-AT was retained intracellularly but acquired resistance to Endo H. With ionomycin, thapsigargin, or 1-deoxymannojirimycin-treated cells, however, brefeldin A failed to promote further processing of the glycoprotein. Possible mechanisms for the suppression of alpha 1-AT processing at the alpha-1,2-mannosidase step by Ca(2+)-mobilizing agents are discussed. Excepting tunicamycin, traditional inhibitors of protein processing did not affect amino acid incorporation.
During the first cell cycle of the ascidian egg, two phases of ooplasmic segregation create distinct cytoplasmic domains that are crucial for later development. We recently defined a domain enriched in ER in the vegetal region of Phallusia mammillata eggs. To explore the possible physiological and developmental function of this ER domain, we here investigate its organization and fate by labeling the ER network in vivo with DiIC16(3), and observing its distribution before and after fertilization in the living egg. In unfertilized eggs, the ER-rich vegetal cortex is overlaid by the ER-poor but mitochondria-rich subcortical myoplasm. Fertilization results in striking rearrangements of the ER network. First, ER accumulates at the vegetal-contraction pole as a thick layer between the plasma membrane and the myoplasm. This accompanies the relocation of the myoplasm toward that region during the first phase of ooplasmic segregation. In other parts of the cytoplasm, ER becomes progressively redistributed into ER-rich and ER-poor microdomains. As the sperm aster grows, ER accumulates in its centrosomal area and along its astral rays. During the second phase of ooplasmic segregation, which takes place once meiosis is completed, the concentrated ER domain at the vegetal-contraction pole moves with the sperm aster and the bulk of the myoplasm toward the future posterior side of the embryo. These results show that after fertilization, ER first accumulates in the vegetal area from which repetitive calcium waves are known to originate (Speksnijder, J. E. 1992. Dev. Biol. 153:259-271). This ER domain subsequently colocalizes with the myoplasm to the presumptive primary muscle cell region.
Calnexin (CNX) and calreticulin (CRT) are molecular chaperones that bind preferentially to monoglucosylated trimming intermediates of glycoproteins in the endoplasmic reticulum. To determine their role in the maturation of newly synthesized glycoproteins, we analyzed the folding and trimerization of in vitro translated influenza hemagglutinin (HA) in canine pancreas microsomes under conditions in which HA's interactions with CNX and CRT could be manipulated. While CNX bound to all folding intermediates (IT1, IT2 and NT), CRT was found to associate preferentially with the earliest oxidative form (IT1). If HA's binding to CNX and CRT was inhibited using a glucosidase inhibitor, castanospermine (CST), the rate of disulfide formation and oligomerization was doubled but the overall efficiency of maturation of HA decreased due to aggregation and degradation. If, on the other hand, HA was arrested in CNX-CRT complexes, folding and trimerization were inhibited. This suggested that the action of CNX and CRT, like that of other chaperones, depended on an 'on-and-off' cycle. Taken together, these results indicated that CNX and CRT promote correct folding by inhibiting aggregation, preventing premature oxidation and oligomerization, and by suppressing degradation of incompletely folded glycopolypeptides.
Synaptic activity-dependent changes in the spatio-temporal distribution of calcium ions regulate important neuronal functions such as dendritic integration and synaptic plasticity, but the processes that terminate the free Ca2+ transients associated with these changes remain unclear. We have characterized at the electron microscopic level the intracellular compartments involved in buffering free Ca2+ transients in dendritic cytoplasm of CA3 neurons by measuring the larger changes in the concentrations of total Ca that persist for several minutes after neuronal activity. Quantitative energy-dispersive x-ray microanalysis of cryosections from hippocampal slice cultures rapidly frozen 3 min after afferent synaptic activity identified a subset of dendritic endoplasmic reticulum (ER) as a high-capacity Ca2+ buffer. Calcium sequestration by cisterns of this subset of ER was graded, reversible, and dependent on a thapsigargin-sensitive Ca2+-ATPase. Sequestration was so robust that after repetitive high-frequency stimulation the Ca content of responsive ER cisterns increased as much as 20-fold. These results demonstrate that a subpopulation of ER is the major dendritic Ca sequestration compartment in the minutes after neuronal activity.
Oxidants are important human toxicants. Increased intracellular free Ca2+ may be critical for oxidant toxicity, but this mechanism remains controversial. Furthermore, oxidants damage the endoplasmic reticulum (ER) and release ER Ca2+, but the role of the ER in oxidant toxicity and Ca2+ regulation during toxicity is also unclear. tert-Butylhydroperoxide (TBHP), a prototypical organic oxidant, causes oxidative stress and an increase in intracellular free Ca2+. Therefore, we addressed the mechanism of oxidant-induced cell death and investigated the role of ER stress proteins in Ca2+ regulation and cytoprotection after treating renal epithelial cells with TBHP. Prior ER stress induces expression of the ER stress proteins Grp78, Grp94, and calreticulin and rendered cells resistant to cell death caused by a subsequent TBHP challenge. Expressing antisense RNA targeted to grp78 prevents grp78 induction sensitized cells to TBHP and disrupted their ability to develop cellular tolerance. In addition, overexpressing calreticulin, another ER chaperone and Ca2+-binding protein, also protected cells against TBHP. Interestingly, neither prior ER stress nor calreticulin expression prevented lipid peroxidation, but both blocked the rise in intracellular free Ca2+ after TBHP treatment. Loading cells with EGTA, even after peroxidation had already occurred, also prevented TBHP-induced cell death, indicating that buffering intracellular Ca2+ prevents cell killing. Thus, Ca2+ plays an important role in TBHP-induced cell death in these cells, and the ER is an important regulator of cellular Ca2+ homeostasis during oxidative stress. Given the importance of oxidants in human disease, it would appear that the role of ER stress proteins in protection from oxidant damage warrants further consideration.
Networks of interstitial cells of Cajal embedded in the musculature of the gastrointestinal tract are involved in the generation of electrical pacemaker activity for gastrointestinal motility. This pacemaker activity manifests itself as rhythmic slow waves in membrane potential, and controls the frequency and propagation characteristics of gut contractile activity. Mice that lack a functional Kit receptor fail to develop the network of interstitial cells of Cajal associated with Auerbach's plexus in the mouse small intestine and do not generate slow wave activity. These cells could provide an essential component of slow wave activity (for example, a biochemical trigger that would be transferred to smooth muscle cells), or provide an actual pacemaker current that could initiate slow waves. Here we provide direct evidence that a single cell, identified as an interstitial cell of Cajal by light microscopy, electron microscopy and expression of Kit mRNA, generates spontaneous contractions and a rhythmic inward current that is insensitive to L-type calcium channel blockers. Identification of the pacemaker of gut motility will aid in the elucidation of the pathophysiology of intestinal motor disorders, and provide a target cell for pharmacological treatment.
The kinetic relationship between depletion of endoplasmic reticulum calcium stores and the activation of a calcium release-activated calcium current (Icrac) was investigated in the RBL-1 mast cell line. The inositol trisphosphate receptor activator, inositol 2,4, 5-trisphosphate ((2,4,5)IP3), the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, and the calcium ionophore, ionomycin, were used to deplete stored calcium. For (2,4,5)IP3 and thapsigargin, a significant delay was observed between the initiation of calcium store depletion and the activation of Icrac. However, for ionomycin, little or no delay was observed. This may indicate that a specialized subcompartment of the endoplasmic reticulum functions as a regulator of calcium entry and that this compartment is relatively resistant to depletion by (2,4,5)IP3 and thapsigargin but not to depletion by ionomycin. For all three calcium-depleting agents, the rate of development of Icrac, once initiated, was relatively constant, suggesting an all-or-none mechanism. However, there were also clear experimental situations in which submaximal, graded depletion of stored calcium resulted in submaximal activation of Icrac. This complex behavior could also result from the existence of a specific subcompartment of endoplasmic reticulum regulating Icrac. The kinetic behavior of this compartment may not be accurately reflected by the kinetics of calcium changes in the bulk of endoplasmic reticulum. These findings add to the growing body of evidence suggesting specialization of the endoplasmic reticulum calcium stores with regard to the control of capacitative calcium entry.
In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca2+ oscillations in a manner consistent with inhibition of Ca2+ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)2 gene display differential Ca2+ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.
The heterogeneity of the endoplasmic reticulum (ER) is well established in cell biology. To a large extent, however, the differences among ER domains (such as the nuclear envelope and the rough- and smooth-surfaced cisternae) are believed to reflect their different involvement in the synthesis,
One of the most versatile and universal signalling agents in the human body is the calcium ion, Ca2+. How does this simple ion act during cell birth, life and death, and how does it regulate so many different cellular processes?
Excitotoxicity, a form of neuronal injury in which excessive activation of glutamate receptors results in cellular calcium overload, has been implicated in the pathogenesis of Alzheimer disease (AD), although direct evidence is lacking. Mutations in the presenilin-1 (PS1) gene on chromosome 14 are causally linked to many cases of early-onset inherited AD (refs. 5,6). We generated PS1 mutant mice (PS1M146VKI) that express the PS1 M146V targeted allele at normal physiological levels. Although PS1M146VKI mice have no overt mutant phenotype, they are hypersensitive to seizure-induced synaptic degeneration and necrotic neuronal death in the hippocampus. Cultured hippocampal neurons from PS1M146VKI mice have increased vulnerability to death induced by glutamate, which is correlated with perturbed calcium homeostasis, increased oxidative stress and mitochondrial dysfunction. Agents that suppress calcium influx or release and antioxidants protect neurons against the excitotoxic action of the PS1 mutation. These findings establish a direct link between a genetic defect that causes AD and excitotoxic neuronal degeneration, and indicate new avenues for therapeutic intervention in AD patients.
Release of Ca2+ ions from intracellular stores can occur via two classes of Ca2+-release channel (CRC) protein, the inositol 1,4, 5-trisphosphate receptors (InsP3Rs) and the ryanodine receptors (RyRs). Multiple isoforms and subtypes of each CRC class display distinct but overlapping distributions within mammalian tissues. InsP3Rs and RyRs interact with a plethora of accessory proteins which modulate the activity of their intrinsic channels. Although many aspects of CRC structure and function have been reviewed in recent years, the properties of proteins with which they interact has not been comprehensively surveyed, despite extensive current research on the roles of these modulators. The aim of this article is to review the regulation of CRC activity by accessory proteins and, wherever possible, to outline the structural details of such interactions. The CRCs are large transmembrane proteins, with the bulk of their structure located cytoplasmically. Intra- and inter-complex protein-protein interactions between these cytoplasmic domains also regulate CRC function. Some accessory proteins modulate channel activity of all CRC subtypes characterized, whereas other have class- or even isoform-specific effects. Certain accessory proteins exert both direct and indirect forms of regulation on CRCs, occasionally with opposing effects. Others are themselves modulated by changes in Ca2+ concentration, thereby participating in feedback mechanisms acting on InsP3R and RyR activity. CRCs are therefore capable of integrating numerous signalling events within a cell by virtue of such protein-protein interactions. Consequently, the functional properties of InsP3Rs and RyRs within particular cells and subcellular domains are 'customized' by the accessory proteins present.
Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer's disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer's disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer's disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid beta-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.
Eicosanoids mediate complement-dependent glomerular epithelial injury in experimental membranous nephropathy. The release of arachidonic acid from phospholipids by cytosolic phospholipase A2 (cPLA2) is the rate-limiting step in eicosanoid synthesis. The present study examines the association of cPLA2 with membranes of organelles. Glomerular epithelial cells were disrupted by homogenization in Ca²⁺-free buffer; organelles were separated by gradient centrifugation. The distribution of cPLA2 and organelles was analysed by immunoblotting with antibodies against cPLA2 and organelle markers, or by enzyme assay. In cells incubated with or without the Ca²⁺ ionophore ionomycin plus PMA, cPLA2 co-localized with plasma membrane, endoplasmic reticulum and nuclei, but not with mitochondria or Golgi. A greater amount of cPLA2 was associated with membranes in stimulated cells, but membrane-associated cPLA2 was readily detectable under resting conditions. The pattern of association of cPLA2 with membrane in cells treated with antibody and complement was similar to that in cells stimulated with ionomycin plus PMA; however, complement did not enhance the membrane association of cPLA2 protein. To determine the functional role of membrane association of cPLA2, phospholipids were labelled with [³H]arachidonic acid. Cells were then incubated with or without antibody and complement and were fractionated. Complement induced a loss of radio-activity from the plasma membrane, endoplasmic reticulum and nuclei, but not from the mitochondrial fraction. Thus the release of arachidonic acid by cPLA2 is due to the hydrolysis of phospholipids at multiple subcellular membrane sites, including the endoplasmic reticulum, plasma membrane and nucleus.
The recognition that apoptosis is regulated by an evolutionarily conserved set of polypeptides from the nematode Caenorhabditis elegant to humans suggests that a conserved set of biochemical mechanism(s) may also be involved in the response. Work from a number of independent laboratories suggests that alterations in cytosolic Ca2+ homeostasis represent one such candidate mechanism, and molecular targets for Ca2+ are now being identified. This review will summarize what is known about the role of Ca2+ in the regulation of apoptosis and discuss how Ca2+ might interact with some of the other biochemical signals implicated in cell death.
Persistence of capacitative Ca²⁺ influx in inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-deficient DT40 cells (DT40IP3R-/−) raises the question of whether gating of Ca²⁺-release activated Ca²⁺ current (Icrac) by conformational coupling to Ca²⁺-release channels is a general mechanism of gating of these channels. In the present work we examined the properties and mechanism of activation of Icrac Ca²⁺ current in wild-type and DT40IP3R-/− cells. In both cell types passive depletion of internal Ca²⁺ stores by infusion of EGTA activated a Ca²⁺ current with similar characteristics and time course. The current was highly Ca²⁺-selective and showed strong inward rectification, all typical of Icrac. The activator of ryanodine receptor (RyR), cADP-ribose (cADPR), facilitated activation of Icrac, and the inhibitors of the RyRs, 8-N-cADPR, ryanodine and Ruthenium Red, all inhibited Icrac activation in DT40IP3R-/− cells, even after complete depletion of intracellular Ca²⁺ stores by ionomycin. Wild-type and DT40IP3R-/− cells express RyR isoforms 1 and 3. RyR levels were adapted in DT40IP3R-/− cells to a lower RyR3/RyR1 ratio than in wild-type cells. These results suggest that IP3Rs and RyRs can efficiently gate Icrac in DT40 cells and explain the persistence of Icrac gating by internal stores in the absence of IP3Rs.
During the first cell cycle of the ascidian egg, two phases of ooplasmic segregation create distinct cytoplasmic domains that are crucial for later development. We recently defined a domain enriched in ER in the vegetal region of Phallusia mammillata eggs. To explore the possible physiological and developmental function of this ER domain, we here investigate its organization and fate by labeling the ER network in vivo with DiIC16(3), and observing its distribution before and after fertilization in the living egg. In unfertilized eggs, the ER-rich vegetal cortex is overlaid by the ER-poor but mitochondria-rich subcortical myoplasm. Fertilization results in striking rearrangements of the ER network. First, ER accumulates at the vegetal-contraction pole as a thick layer between the plasma membrane and the myoplasm. This accompanies the relocation of the myoplasm toward that region during the first phase of ooplasmic segregation. In other parts of the cytoplasm, ER becomes progressively redistributed into ER-rich and ER-poor microdomains. As the sperm aster grows, ER accumulates in its centrosomal area and along its astral rays. During the second phase of ooplasmic segregation, which takes place once meiosis is completed, the concentrated ER domain at the vegetal-contraction pole moves with the sperm aster and the bulk of the myoplasm toward the future posterior side of the embryo. These results show that after fertilization, ER first accumulates in the vegetal area from which repetitive calcium waves are known to originate (Speksnijder, J. E. 1992. Dev. Biol. 153:259-271). This ER domain subsequently colocalizes with the myoplasm to the presumptive primary muscle cell region.
We have studied the Ca2+ leak pathways in the endoplasmic reticulum of pancreatic acinar cells by directly measuring Ca2+ in the endoplasmic reticulum ([Ca2+]ER). Cytosolic Ca2+ ([Ca2+]C) was clamped to the resting level by a BAPTA-Ca2+ mixture. Administration of cholecystokinin within the physiological concentration range caused a graded decrease of [Ca2+]ER, and the rate of Ca2+ release generated by 10 pm cholecystokinin is at least 3× as fast as the basal Ca2+ leak revealed by inhibition of the endoplasmic reticulum Ca2+-ATPase. Acetylcholine also evokes a dose-dependent decrease of [Ca2+]ER, with an EC50 of 0.98 ± 0.06 μm. Inhibition of receptors for inositol 1,4,5-trisphosphate (IP3) by heparin or flunarizine blocks the effect of acetylcholine but only partly blocks the effect of cholecystokinin. 8-NH2 cyclic ADP-ribose (20 μm) inhibits the action of cholecystokinin, but not of acetylcholine. The basal Ca2+ leak from the endoplasmic reticulum is not blocked by antagonists of the IP3 receptor, the ryanodine receptor, or the receptor for nicotinic acid adenine dinucleotide phosphate. However, treatment with
puromycin (0.1–1 mm) to remove nascent polypeptides from ribosomes increases Ca2+leak from the endoplasmic reticulum by a mechanism independent of the endoplasmic reticulum Ca2+ pumps and of the receptors for IP3 or ryanodine.
Various conditions that perturb the function of the endoplasmic reticulum (ER) were recently shown to activate the transcription factor NF-κB. Activation of NF-κB is caused by the accumulation of proteins in the ER membrane, a condition we have called ER overload. Both the release of Ca2+ from the ER and the subsequent production of reactive oxygen intermediates are required for ER-overload-mediated NF-κB activation. This novel intracellular signal transduction pathway might be important in antiviral defense and play a role in various diseases as well as in B-cell development.
Apoptosis (programmed cell death) has gained widespread attention due to its roles in a variety of physiological and pathological processes, yet precisely how apoptosis is regulated by external and internal cues remains unclear. Work from our laboratories and others has implicated alterations in intracellular Ca2+in apoptosis, and more recent work has defined particular biochemical processes that are targeted by Ca2+in apoptotic cells. This review will summarize the role of Ca2+in apoptosis within the context of what is known about the core components of the effector machinery for apoptosis.
Oocytes of a large fraction of Xenopus females exhibit a complex response to acetylcholine (ACh) consisting of rapid, transient and prolonged, slow chloride currents. Frequent consecutive challenges or a single prolonged challenge with ACh result in a marked decrease in response amplitudes, i.e. refractoriness. In ACh-refractory oocytes, the response to injected inositol 1,4,5-trisphosphate (InsP3), the intracellular mediator of the ACh response, is not affected. Similarly, InsP3-evoked responses were obtained in oocytes that lacked muscarinic response or that lost their responsiveness as a result of progesterone-induced maturation. To investigate the mechanism of this phenomenon, we have depleted intracellular calcium stores by repeated challenges with ACh in calcium-free medium. Disappearance of the ACh response through depletion of the ACh-coupled calcium store did not prevent a subsequent response to InsP3. These results imply that InsP3 can mobilize calcium from other stores, not depleted by previous exposure to ACh. This finding is further reinforced by our results that demonstrate that ACh causes 45Ca efflux in responsive oocytes, while InsP3 in supramaximal concentrations does not induce 45Ca efflux. Indeed, InsP3 can induce 45Ca efflux only when more than 2 pmol/oocyte is injected. This is also the concentration of InsP3 that desensitizes the InsP3 response. These data suggest that InsP3 also releases cellular calcium from stores different from those mobilized by ACh.
The release of Ca2+ from intracellular stores by sub-optimal doses of inositol trisphosphate has been shown to be dose-related ('quantal'), and a simple model is proposed here to account for this phenomenon. It is suggested that there is a regulatory Ca2(+)-binding site on, or associated with, the luminal domain of the InsP3 receptor, which allosterically controls Ca2+ efflux, and the affinity for Ca2+ of that site is modulated by InsP3 binding to the cytoplasmic domain of the receptor; a similar mechanism applied to the ryanodine receptor might also explain some aspects of Ca2(+)-induced Ca2+ release. The stimulated entry of Ca2+ into a cell which occurs upon activation of inositide-linked receptors has been variously and confusingly proposed to be regulated by InsP3, InsP4, and/or a 'capacitative' Ca2+ pool; the mechanism of InsP3 receptor action suggested here is shown to lead to a potential reconciliation of all these conflicting proposals.
It is suggested that a link in excitation-contraction coupling involves
the movement of a fixed amount of charge free to move between different
locations across the membrane.
1. Ryanodine-sensitive intracellular Ca2+ release activated by Ca2+ entry was studied with fura-2 fluorescence and intracellular voltage recording techniques in rabbit otic ganglion cells. 2. The removal of extracellular Ca2+ reduced sustained, transient or oscillatory rises in intracellular Ca2+ ([Ca2+]i) induced at high extracellular K+ and abolished the [Ca2+]i oscillation in cultured neurones. 3. Ryanodine (10 microM) transiently increased [Ca2+]i and reduced the amplitude and rate of rise of the high-K(+)-induced rise in [Ca2+]i, while caffeine (5 mM) produced a few transient rises in [Ca2+]i in most cultured cells and [Ca2+]i oscillation only in one cell. 4. The two components of the slow after-hyperpolarization (AHP) of an action potential in neurones of freshly isolated ganglia were dependent on extracellular Ca2+ and abolished by Ca2+ channel blockers, Cd2+ or Co2+. 5. The late component of AHP (LAHP), but not the initial component, in 'fresh' neurones increased in area with an increase in the preceding interval, was abolished by ryanodine (10 microM) and intracellularly injected EGTA, and mimicked by intracellular injection of Ca2+. 6. A ryanodine-sensitive Ca(2+)-induced Ca2+ release thus exists, operates in response to an action potential-induced Ca2+ entry and underlies LAHP in rabbit otic ganglion cells.
The orderly sequence of events that constitutes the cell cycle is carefully regulated. A part of this regulation depends upon the ubiquitous calcium signalling system. Many growth factors utilize the messenger inositol trisphosphate (InsP3) to set up prolonged calcium signals, often organized in an oscillatory pattern. These repetitive calcium spikes require both the entry of external calcium and its release from internal stores. One function of this calcium signal is to activate the immediate early genes responsible for inducing resting cells (G0) to re-enter the cell cycle. It may also promote the initiation of DNA synthesis at the G1/S transition. Finally, calcium contributes to the completion of the cell cycle by stimulating events at mitosis. The role of calcium in cell proliferation is highlighted by the increasing number of anticancer therapies and immunosuppressant drugs directed towards this calcium signalling pathway.
To study the distribution of a major Ca(2+)-sequestering site in PtK2 cells, a rat kangaroo kidney epithelial cell line, during interphase and mitosis, we prepared an affinity-purified polyclonal antibody against bovine liver calreticulin (CRT), a major Ca(2+)-binding protein of the endoplasmic reticulum (ER). Immunofluorescence microscopy and immunoperoxidase electron microscopy showed that the anti-CRT antibody labeled a continuous reticular network of the ER and the nuclear envelope in interphase PtK2 cells. The same PtK2 cells double-stained with DiOC6 (3) and the anti-CRT antibody revealed labeling of identical reticular membranes. In contrast to the localization in the ER localization, the mitochondria and the Golgi apparatus were not labeled. These results confirm the exclusive localization of CRT in the ER and that this organelle is a major site for Ca2+ storage in non-muscle cells. In mitotic cells, marked changes of the labeled structure began at prophase-prometaphase and persisted throughout all phases of mitosis. The cytoplasm of the mitotic cells showed diffuse fluorescence, this being more intense around, but not inside, the mitotic spindle. Confocal microscopy and immunoelectron microscopy demonstrated that the CRT-containing membranes changed to segmented tubuloreticular structures, which were concentrated around the mitotic spindle. The ER containing CRT could be responsible for the sequestration of Ca2+ and for the regulation of the concentration of this cation during mitosis, as well as during interphase.
Subcellular gradients of cytosolic free Ca2+ concentration, [Ca2+]i, are thought to be critical for the localization of functional responses within a cell. A potential but previously unexplored mechanism for the generation of gradients of [Ca2+]i is the accumulation of Ca2+ stores at the site of Ca2+ action. The distribution of the Ca2+ store markers Ca(2+)-dependent adenosine triphosphatase and calreticulin was investigated in resting and phagocytosing human neutrophils. Both proteins showed an evenly distributed fine granular pattern in nonphagocytosing cells, but became markedly concentrated in the filamentous actin-rich cytoplasmic area around the ingested particle during phagocytosis. This redistribution began at early stages of phagocytosis and did not depend on an increase in [Ca2+]i. Thus, accumulation of Ca2+ stores in a restricted area of the cell may contribute to the generation of localized increases in [Ca2+]i.
Release of Ca2+ stored in endoplasmic reticulum is a ubiquitous mechanism involved in cellular signal transduction, proliferation, and apoptosis. Recently, sphingolipid metabolites have been recognized as mediators of intracellular Ca2+ release, through their action at a previously undescribed intracellular Ca2+ channel. Here we describe the molecular cloning and characterization of a protein that causes the expression of sphingosyl-phosphocholine-mediated Ca2+ release when its complementary RNA is injected into Xenopus oocytes. SCaMPER (for sphingolipid Ca2+ release-mediating protein of endoplasmic reticulum) is an 181 amino acid protein with two putative membrane-spanning domains. SCaMPER is incorporated into microsomes upon expression in SO cells or after translation in vitro. It mediates Ca2+ release at 4 degrees C as well as 22 degrees C, consistent with having ion channel function. The EC50 for Ca2+ release from Xenopus oocytes is 40 microM, similar to sphingosyl-phosphocholine-mediated Ca2+ release from permeabilized mammalian cells. Because Ca2+ release is not blocked by ryanodine or La3+, the activity described here is distinct from the Ca2+ release activity of the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor. The properties of SCaMPER are identical to those of the sphingolipid-gated Ca2+ channel that we have previously described. These findings suggest that SCaMPER is a sphingolipid-gated Ca2+-permeable channel and support its role as a mediator of this pathway for intracellular Ca2+ signal transduction.
Green fluorescent protein (GFP) was targeted to the lumen of the endoplasmic reticulum (ER) of starfish eggs by injecting mRNA coding for a chimeric protein containing a signal sequence and the KDEL ER retention sequence. By confocal microscopy, the GFP chimeric protein was localized in intracellular cisternae (membrane sheets) and the nuclear envelope, showing that it had been successfully targeted to the ER. The labeling pattern closely resembled that produced by the fluorescent dicarbocyanine DiI, which has been used previously to label the ER (Jaffe and Terasaki, Dev. Biol. 164, 579-587, 1994). Eggs expressing the GFP chimera were used to examine whether there is a loss of ER continuity at fertilization. The time required for recovery of fluorescence after photobleaching for both the GFP chimera and DiI was much longer in eggs at 1 min postfertilization than in unfertilized eggs or in 20-min-postfertilized eggs. This result provides strong evidence for a transient loss of continuity of the ER associated with Ca release at fertilization.
Calcium is a ubiquitous second messenger used to regulate a wide range of cellular processes. This role in signalling has to be conducted against the rigid homeostatic mechanisms that ensure that the resting level of Ca2+ is kept low (i.e. between 20 and 100 nmol l-1) in order to avoid the cytotoxic effects of a prolonged elevation of [Ca2+]. Cells have evolved a sophisticated signalling system based on the generation of brief pulses of Ca2+ which enables this ion to be used as a messenger, thus avoiding its toxic effects. Such Ca2+ spikes usually result from the coordinated release of Ca2+ from internal stores using either inositol 1,4,5-trisphosphate or ryanodine receptors. Using Ca2+ imaging techniques, the opening of individual channels has now been visualized and models have been proposed to explain how these elementary events are coordinated to generate the global Ca2+ signals that regulate cellular activity.
The spatial organization of endoplasmic reticulum (ER) and nuclear envelope (NE) calcium stores is important for the regulation of localized calcium signals and sustained calcium gradients. Here, we have used a lumenal GFP fusion protein and shown that, in resting cells, large molecules can rapidly diffuse across the cell within the lumenal storage space defined by the ER and NE membranes. Increases in cytosolic calcium concentration reversibly fragmented ER tubules and prevented lumenal diffusion. However, the integrity of the NE was maintained, and a significant fraction of NE lumenal protein accumulated in an NE-associated vesicle. These dynamic properties of ER-NE calcium stores provide insights into the spatiotemporal control of calcium signaling.
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Group I metabotropic glutamate receptors (mGluRs) activate PI turnover and thereby trigger intracellular calcium release. Previously, we demonstrated that mGluRs form natural complexes with members of a family of Homer-related synaptic proteins. Here, we present evidence that Homer proteins form a physical tether linking mGluRs with the inositol trisphosphate receptors (IP3R). A novel proline-rich "Homer ligand" (PPXXFr) is identified in group 1 mGluRs and IP3R, and these receptors coimmunoprecipitate as a complex with Homer from brain. Expression of the IEG form of Homer, which lacks the ability to cross-link, modulates mGluR-induced intracellular calcium release. These studies identify a novel mechanism in calcium signaling and provide evidence that an IEG, whose expression is driven by synaptic activity, can directly modify a specific synaptic function.
The recognition that apoptosis is regulated by an evolutionarily conserved set of polypeptides from the nematode Caenorhabditis elegans to humans suggests that a conserved set of biochemical mechanism(s) may also be involved in the response. Early evidence suggested that the endogenous endonuclease implicated in apoptosis in most model systems is Ca(2+)-dependent, and subsequent work from a number of independent laboratories suggests that alterations in cytosolic Ca2+ homeostasis are one of the conserved biochemical pathways regulating the response. Molecular targets for Ca2+ are now being identified and include signal transduction intermediates, endonuclease(s) and proteases, and the enzymes involved in the maintenance of phospholipid asymmetry in the plasma membrane. Furthermore, interesting preliminary work suggests that BCL-2 suppresses apoptosis via a mechanism that is linked to intracellular Ca2+ compartmentalization, and it appears that Ca2+ alterations in some examples of apoptosis occur as the result of changes within the mitochondria. This review will summarize what is known about the role of Ca2+ in the regulation of apoptosis and discuss how Ca2+ might interact with some of the other biochemical signals implicated in cell death.
Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current (I(crac)). A critical question in cell signalling is how SOC and I(crac) sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion; in an alternative model, InsP3 receptors in the stores are coupled to SOC and I(crac). The mammalian Htrp3 protein forms a well defined store-operated channel and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and I(crac). We show here that Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. Our results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac).