Endoplasmic Ca2 Research Articles

Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE4/5/6/11 negatively regulate hydrotropism via phosphorylation of MIZU-KUSSEI1.

Hydrotropism facilitates the orientation of plant roots towards regions of elevated water potential, enabling them to absorb adequate water. Although calcium signaling plays a crucial role in plant response to water tracking, the exact regulatory mechanisms remain a mystery. Here, we employed the Arabidopsis (Arabidopsis thaliana) hydrotropism-specific protein MIZU-KUSSEI1 (MIZ1) as bait and found that calcium-dependent protein kinases4/5/6/11 (CPK4/5/6/11) interacted with MIZ1 in vitro and in vivo. The cpk4/5/6/11 mutant exhibited increased sensitivity to water potential and enhanced root tip curvature. Furthermore, CPK4/5/6/11 primarily phosphorylated MIZ1 at Ser14/36 residues. Additionally, CPK-mediated phosphorylation of MIZ1 relieved its inhibitory effect on the activity of the endoplasmic reticulum-localized Ca2+ pump ECA1, altering the balance between cytoplasmic Ca2+ inflow and outflow, thereby negatively regulating the hydrotropic growth of plants. Overall, our findings unveil the molecular mechanisms by which the CPK4/5/6/11-MIZ1 module functions in regulating plant hydrotropism responses and provide a theoretical foundation for enhancing plant water use efficiency and promoting sustainable agriculture.

Biotoxicity of paraquat to lung cells mediated by endoplasmic reticulum-mitochondria interaction.

The high lethality caused by paraquat (PQ) poisoning has attracted much attention in public and human health due to its high toxicity and lethality. However, the understanding of the mechanism of PQ-induced apoptosis from the perspective of organelles, especially inter-organelle interactions, is still scarce. Exploring the linkage of multiple organelles during PQ poisoning and the molecular mechanisms of PQ poisoning under its mediation will help to gain insight into the mode of PQ poisoning at the organelle level. In this study, we observed that a certain dose of PQ gavage induced oxidative stress, mitochondrial dysfunction and endoplasmic reticulum stress in rat lung tissue cells. PQ toxicity led to the occurrence of Ca2+ overload in the endoplasmic reticulum, and the activated BIP and CHOP pathways directly/indirectly led to the expression of apoptogenic factors Caspase family factors. In addition, PQ promoted Ca2+ release from the endoplasmic reticulum and Ca2+ uptake by mitochondria, which induced the disruption of Bax/Bcl-2 channel proteins in response to the IP3R/RyR/VDAC1&2/MCU Ca2+ axis thereby leading to the release of CytoC, which ultimately induced endoplasmic reticulum stress and apoptotic cell death. In addition, 10 differential proteins were screened and validated by proteomics that may act as upstream and downstream active factors of mitochondria-endoplasmic reticulum interaction-mediated biotoxicity. Our findings provide new perspectives for researchers to explore the toxicity mechanisms of PQ to reduce their adverse effects.

Store-operated calcium entry is necessary for sustained K+-induced capillary-to-arteriole dilation in the brain

Neurovascular coupling (NVC) is a physiological process that directs blood flow to the most active regions of the brain. The extensive network of capillaries in the brain acts as a sensory web that detects local neuronal activity and orchestrates the dilation of upstream arterioles to meet the brain’s metabolic demands. Activation of inwardly rectifying K+ (KIR) channels on brain capillary endothelial cells (cECs) by modest concentrations of extracellular K+ initiates retrograde propagating hyperpolarizing signals that induce vasodilation and increase blood flow. Brain capillaries exhibit diverse and dynamic patterns of Ca2+ signaling in vivo, but little is known about their origins or physiological function. Store-operated Ca2+ entry (SOCE) is fundamental to Ca2+homeostasis in non-excitable cells, but the contribution of SOCE to capillary-to-arteriole dilation has not been explored. Here, we report that brain cECs express the endoplasmic reticulum-resident Ca2+ sensing protein stromal interaction molecule 1 ( Stim1) and the Ca2+-selective Orai1 and Orai3 channels. Ca2+ imaging experiments using brain cECs from transgenic mice expressing GCaMP8 in the endothelium or loaded with Fluo-4 showed that these cells exhibited robust SOCE that was blocked by the Orai1 inhibitor Synta66. Using EC-specific knockout (ecKO) mice, we found that Stim1 and Orai1 but not Orai3 are required for SOCE. Ex vivo brain microvascular preparations consisting of cannulated and pressured parenchymal arterioles with intact capillary beds were used to examine the role of SOCE in capillary-to-arteriole dilation. Arteriole dilation in response to a train of K+ pulses applied to capillaries exhibited dramatic run-down when preparations were treated with Snyta66 or when tissues from Stim1-ecKO mice were used. Preparations from Orai3-ecKO mice did not demonstrate run-down. Moreover, inhibition of Ca2+-activated K+ (SK and IK) channels also induced run-down, suggesting that SOCE activates SK and IK channels to boost KIR-induced hyperpolarizing signals to dilate upstream arterioles during multiple stimulations. We conclude that brain cECs exhibit SOCE that requires Stim1 and Orai1. Our data further show that brain cEC SOCE is necessary for capillary-to-arteriole dilation during repeated stimulations, suggesting an essential role for SOCE in NVC. R35HL155008, R33NS115132, and P20GM130459 (to SE). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1132: Cellular Senescence Impairs The Maturity Of Cell-cell Junction In Vascular Endothelial Cells

Background and hypothesis: Vascular endothelial cells functionally differentiate upon formation of secure cell-cell junction. Disruption of cell-cell junction causes endothelial dysfunction and contributes to the early phase of pathogenesis of vascular diseases. Cellular senescence loosens cell-cell junction and causes endothelial dysfunction. We reported that the “maturity” of cell-cell junction determines how endothelial cells respond to the proinflammatory and proapoptotic insults. We hypothesize that cellular senescence affects the maturity of cell-cell junction. Methods and Results: Thrombin (1 U/mL) decreased a trans-endothelial electrical resistance (TEER) and transiently increased di-phosphorylation of myosin light chain (ppMLC) at 3 min in culture porcine aortic endothelial cells (PAEC) of lower passages (9-15 passages) at confluence. Fluorescence staining revealed that thrombin induced actin bundle formation, which was associated with ppMLC, but not pMLC (mono-phosphorylation), at cell periphery as initial events, followed by reorganization into actin stress fibers associated with both pMLC and ppMLC as later events. In contrast, PAEC of lower passages either at early culture day (3 days) or at confluence but in the absence of extracellular Ca 2+ exhibited instantaneous appearance of actin stress fibers associated with pMLC and ppMLC upon thrombin stimulation. Initial appearance of peripheral actin bundles in response to thrombin is one of the hallmarks of the mature cell-cell junction. When replicative senescence was induced by repeated passages (23-30 passages), senescent PAEC exhibited scattered actin stress fibers without any stimulation, and substantially increased actin stress fibers, with no peripheral actin bundles, after thrombin stimulation. Cellular senescence was confirmed by an increase in senescence-associated β-galactosidase activity. As a functional consequence, senescent PAEC exhibited augmented store-operated Ca 2+ entry induced by an inhibitor of endoplasmic Ca 2+ ATPase. Conclusions: Replicative cellular senescence impairs the maturity of cell-cell junction, thereby damaging the junctional integrity and physiological function of endothelial cells.

Tricyclic antidepressant amitriptyline attenuates Ca<sup>2+</sup> responses in rat peritoneal macrophages

Amitriptyline is a tricyclic antidepressant widely used in clinical practice for the treatment of anxiety, depression and chronic pain. These drugs have a multifaceted effect on cellular processes. One of their targets is sigma-1 receptors. Sigma-1 receptors are molecular chaperones located in endoplasmic reticulum membrane; they are characterized by a unique structure and pharmacological profile. Sigma-1 receptors regulate many cellular processes in health and disease, including Ca2+ signaling. Using Fura-2AM microfluorimetry, it was shown for the first time that sigma-1 receptor agonist, antidepressant amitriptyline, significantly suppresses both Ca2+ mobilization from intracellular Ca2+-stores and subsequent store-dependent Ca2+ entry into cells, induced by endoplasmic Ca2+-ATPase inhibitors thapsigargin and cyclopiazonic acid, as well as disulfide-containing immunomodulators glutoxim and molixan, in rat peritoneal macrophages. The results suggest the involvement of sigma-1 receptors in a complex signaling cascade induced by glutoxim or molixan, leading to an increase of intracellular Ca2+ concentration in macrophages. The results also indicate the participation of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

Proteomic analyses of urinary exosomes identify novel potential biomarkers for early diagnosis of sickle cell nephropathy, a sex-based study.

Sickle cell nephropathy (SCN) is a leading cause of morbidity and mortality in sickle cell disease (SCD). Early intervention is crucial for mitigating its effects. However, current diagnostic methods rely on generic tests and may not detect SCN until irreversible renal damage occurs. Therefore, specific biomarkers for early diagnosis of SCN are needed. Urinary exosomes, membrane-bound vesicles secreted by renal podocytes and epithelial cells, contain both common and cell type-specific membrane and cytosolic proteins, reflecting the physiologic and pathophysiologic states of the kidney. Using proteomics, we analyzed the proteomes of urinary exosomes from humanized SCD mice at 2months (without albuminuria) and 4months (with albuminuria) of age. Excretion of 164 proteins were significantly increased and 176 proteins was significantly decreased in the exosomes when mice developed albuminuria. Based on the relevance to SCD, chronic kidney disease and Western blot confirmation in mice, we analyzed protein abundance of heparanase, cathepsin C, α2-macroglobulin and sarcoplasmic endoplasmic Ca2+ ATPase-3 (SERCA3) in the urinary exosomes and urine of 18 SCD subjects without albuminuria and 12 subjects with albuminuria using Western blot analyses. Both male and female subjects increased or tended to increase the excretion of these proteins in their urinary exosomes upon developing albuminuria, but female subjects demonstrated stronger correlations between the excretion of these proteins and urine albumin creatinine ratio (UACR) compared to male subjects. In contrast, exosomal excretion of Tamm-Horsfall protein, β-actin and SHP-1 was independent of albuminuria. These findings provide a foundation for a time-course study to determine whether increases in the levels of these proteins precede the onset of albuminuria in patients, which will help determine the potential of these proteins as biomarkers for early detection of SCN.

Altered neuronal group 1 metabotropic glutamate receptor- and endoplasmic reticulum-mediated Ca2+ signaling in two rodent models of Alzheimer’s disease

Calcium mobilization from the endoplasmic reticulum (ER) induced by, for example, IP3 receptor (IP3R) stimulation, and its subsequent crosstalk with extracellular Ca2+ influx mediated through voltage-gated calcium channels (VGCCs) and neuronal store-operated calcium entry (nSOCE), is essential for normal neuronal signaling and cellular homeostasis. However, several studies suggest that chronic calcium dysregulation may play a key role in the onset and/or progression of neurodegenerative conditions, particularly Alzheimer’s disease (AD). Here, using early postnatal hippocampal tissue from two transgenic murine models of AD, we provide further evidence that not only are crucial calcium signaling pathways dysregulated, but also that such dysregulation occurs at very early stages of development.Utilizing epifluorescence calcium imaging, we investigated ER-, nSOCE- and VGCC-mediated calcium signaling in cultured primary hippocampal neurons from two transgenic rodent models of AD: 3xTg-AD mice (PS1M146V/APPSWE/TauP301L) and TgF344-AD rats (APPSWE/PS1ΔE9) between 2 and 9 days old.Our results reveal that, in comparison to control hippocampal neurons, those from 3xTg-AD mice possessed significantly greater basal ER calcium levels, as measured by larger responses to I-mGluR-mediated ER Ca2+ mobilization (amplitude; 4 (0–19) vs 21(12–36) a.u., non-Tg vs 3xTg-AD; median difference (95 % Cl) = 14 a.u. (11–18); p = 0.004)) but reduced nSOCE (15 (4–22) vs 8(5–11) a.u., non-Tg vs 3xTg-AD; median difference (95 % Cl) = -7 a.u. (-3– −10 a.u.); p < 0.0001). Furthermore, unlike non-Tg neurons, where depolarization enhanced the amplitude, duration and area under the curve (A.U.C.) of I-mGluR-evoked ER-mediated calcium signals when compared with basal conditions, this was not apparent in 3xTg-AD neurons. Whilst the amplitude of depolarization-enhanced I-mGluR-evoked ER-mediated calcium signals from both non-Tg F344 and TgF344-AD neurons was significantly enhanced relative to basal conditions, the A.U.C. and duration of responses were enhanced significantly upon depolarization in non-Tg F344, but not in TgF344-AD, neurons. Overall, the nature of basal I-mGluR-mediated calcium responses did not differ significantly between non-Tg F344 and TgF344-AD neurons.In summary, our results characterizing ER- and nSOCE-mediated calcium signaling in neurons demonstrate that ER Ca2+ dyshomeostasis is an early and potentially pathogenic event in familial AD.

Reconstitution of calcium channel protein Orai3 into liposomes for functional studies

Store-operated calcium entry (SOCE) is the main mechanism for the Ca2+ influx in non-excitable cells. The two major components of SOCE are stromal interaction molecule 1 (STIM1) in the endoplasmic reticulum and Ca2+ release-activated Ca2+ channel (CRAC) Orai on the plasma membrane. SOCE requires interaction between STIM1 and Orai. Mammals have three Orai homologs: Orai1, Orai2, and Orai3. Although Orai1 has been widely studied and proven to be essential for numerous cellular processes, Orai3 has also attracted a significant attention recently. The gating and activation mechanisms of Orai3 have yet to be fully elucidated. Here, we expressed, purified, and reconstituted Orai3 protein into liposomes and investigated its orientation and oligomeric state in the resulting proteoliposomes. STIM1 interacted with the Orai3-containing proteoliposomes and mediated calcium release from them, suggesting that the Orai3 channel was functional and that recombinant STIM1 could directly open the Orai3 channel in vitro. The developed in vitro calcium release system could be used to study the structure, function, and pharmacology of Orai3 channel.

Nuclear-cytoplasmic asynchrony in oocyte maturation caused by TUBB8 variants via impairing microtubule function: a novel pathogenic mechanism

BackgroundTUBB8, a crucial gene encoding microtubule protein, plays a pivotal role in cellular processes. Deleterious TUBB8 variants have been shown to significantly hinder oocyte maturation. In this study, we conducted an in vitro investigation using TUBB8 mutant mouse oocytes to elucidate the pathogenic mechanisms of TUBB8 variants in oocyte nuclear and cytoplasmic maturation.MethodsA mutant model was successfully established in mouse oocytes via microinjection to further investigate the effects of four novel discovered TUBB8 mutations on the nuclear and cytoplasmic maturation of mouse oocytes. Immunofluorescence and confocal microscopy were performed to observe the cortical polarity and spindle and of mutant oocytes. Active mitochondrial staining was performed to analyze mitochondrial distribution patterns. Endoplasmic reticulum and Ca2+ staining were conducted to assess ER distribution and cytoplasmic calcium ion concentration in oocytes.ResultsIn mouse oocytes, TUBB8 variants (p.A313V, p.C239W, p.R251Q, and p.G96R) resulted in a reduction of the first polar body extrusion rate, disruption of spindle assembly, and abnormal chromosome distribution. Additionally, these variants induced oocyte organelle abnormalities, including anomalies in mitochondrial redistribution and endoplasmic reticulum stress compared to the wild-type.ConclusionDeleterious TUBB8 variants could disrupt microtubule function, affecting critical processes such as spindle assembly, chromosome distribution, and organelle rearrangement during oocyte meiosis. These disruptions culminate in compromised nuclear-cytoplasmic maturation, consequently giving rise to oocyte maturation defects.

A multiple-oscillator mechanism underlies antigen-induced Ca2+ oscillations in Jurkat T-cells

T-cell receptor stimulation triggers cytosolic Ca2+ signaling by inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels gated by ER-located stromal-interacting molecules (STIM1/2). Physiologically, cytosolic Ca2+ signaling manifests as regenerative Ca2+ oscillations, which are critical for nuclear factor of activated T-cells-mediated transcription. In most cells, Ca2+ oscillations are thought to originate from IP3 receptor-mediated Ca2+ release, with CRAC channels indirectly sustaining them through ER refilling. Here, experimental and computational evidence support a multiple-oscillator mechanism in Jurkat T-cells whereby both IP3 receptor and CRAC channel activities oscillate and directly fuel antigen-evoked Ca2+ oscillations, with the CRAC channel being the major contributor. KO of either STIM1 or STIM2 significantly reduces CRAC channel activity. As such, STIM1 and STIM2 synergize for optimal Ca2+ oscillations and activation of nuclear factor of activated T-cells 1 and are essential for ER refilling. The loss of both STIM proteins abrogates CRAC channel activity, drastically reduces ER Ca2+ content, severely hampers cell proliferation and enhances cell death. These results clarify the mechanism and the contribution of STIM proteins to Ca2+ oscillations in T-cells.

Reconstitution of Calcium Channel Protein Orai3 into Liposomes for Functional Studies.

Store-operated calcium entry (SOCE) is the main mechanism for the Ca2+ influx in non-excitable cells. The two major components of SOCE are stromal interaction molecule 1 (STIM1) in the endoplasmic reticulum and Ca2+ release-activated Ca2+ channel (CRAC) Orai on the plasma membrane. SOCE requires interaction between STIM1 and Orai. Mammals have three Orai homologs: Orai1, Orai2, and Orai3. Although Orai1 has been widely studied and proven to essential for numerous cellular processes, Orai3 has also attracted a significant attention recently. The gating and activation mechanisms of Orai3 have yet to be fully elucidated. Here, we expressed, purified, and reconstituted Orai3 protein into liposomes and investigated its orientation and oligomeric state in the resulting proteoliposomes. STIM1 interacted with the Orai3-containing proteoliposomes and mediated calcium release from the them, suggesting that the Orai3 channel was functional and that recombinant STIM1 could directly open the Orai3 channel in vitro. The developed in vitro calcium release system could be used to study the structure, function, and pharmacology of Orai3 channel.

Nrh L11R single nucleotide polymorphism, a new prediction biomarker in breast cancer, impacts endoplasmic reticulum-dependent Ca2+ traffic and response to neoadjuvant chemotherapy

Overexpression of Bcl-2 proteins such as Bcl2L10, also referred to as Nrh, is associated with resistance to therapy and poor survival in various cancers, including breast cancer, lung cancer, and leukemia. The single nucleotide polymorphism (SNP) of BCL2L10 in its BH4 domain at position 11 (BCL2L10 Leu11Arg, rs2231292), corresponding to position 11 in the Nrh open reading frame, is reported to lower resistance towards chemotherapy, with patients showing better survival in the context of acute leukemia and colorectal cancer. Using cellular models and clinical data, we aimed to extend this knowledge to breast cancer. We report that the homozygous status of the Nrh Leu11Arg isoform (Nrh-R) is found in 9.7–11% percent of the clinical datasets studied. Furthermore, Nrh-R confers higher sensitivity towards Thapsigargin-induced cell death compared to the Nrh-L isoform, due to altered interactions with IP3R1 Ca2+ channels in the former case. Collectively, our data show that cells expressing the Nrh-R isoform are more prone to death triggered by Ca2+ stress inducers, compared to Nrh-L expressing cells. Analysis of breast cancer cohorts revealed that patients genotyped as Nrh-R/Nrh-R may have a better outcome. Overall, this study supports the notion that the rs2231292 Nrh SNP could be used as a predictive tool regarding chemoresistance, improving therapeutic decision-making processes. Moreover, it sheds new light on the contribution of the BH4 domain to the anti-apoptotic function of Nrh and identifies the IP3R1/Nrh complex as a potential therapeutic target in the context of breast cancer.

Neuroprotective properties of anti-apoptotic BCL-2 proteins in 5xFAD mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia. An early feature of the AD pathology is the dysregulation of intracellular Ca2+ signaling in neurons. In particular, increased Ca2+ release from endoplasmic reticulum-located Ca2+ channels, including inositol-1,4,5-trisphosphate type 1 receptors (IP3R1) and ryanodine receptors type 2 (RyR2), have been extensively reported. Known for its anti-apoptotic properties, Bcl-2 also has the ability to bind to and inhibit the Ca2+-flux properties of IP3Rs and RyRs. In this study, the hypothesis that the expression of Bcl-2 proteins can normalize dysregulated Ca2+ signaling in a mouse model of AD (5xFAD) and thereby prevent or slow the progression of AD was examined. Therefore, stereotactic injections of adeno-associated viral vectors expressing Bcl-2 proteins were performed in the CA1 region of the 5xFAD mouse hippocampus. In order to assess the importance of the association with IP3R1, the Bcl-2K17D mutant was also included in these experiments. This K17D mutation has been previously shown to decrease the association of Bcl-2 with IP3R1, thereby impairing its ability to inhibit IP3R1 while not affecting Bcl-2's ability to inhibit RyRs. Here, we demonstrate that Bcl-2 protein expression leads to synaptoprotective and amyloid-protective effects in the 5xFAD animal model. Several of these neuroprotective features are also observed by Bcl-2K17D protein expression, suggesting that these effects are not associated with Bcl-2-mediated inhibition of IP3R1. Potential mechanisms for this Bcl-2 synaptoprotective action may be related to its ability to inhibit RyR2 activity as Bcl-2 and Bcl-2K17D are equally potent in inhibiting RyR2-mediated Ca2+ fluxes. This work indicates that Bcl-2-based strategies hold neuroprotective potential in AD models, though the underlying mechanisms requires further investigation.

Role of Orai-family channels in the activation and regulation of transcriptional activity.

Store operated Ca2+ entry (SOCE) is a cornerstone for the maintenance of intracellular Ca2+ homeostasis and the regulation of a variety of cellular functions. SOCE is mediated by STIM and Orai proteins following the activation of inositol 1,4,5-trisphosphate receptors. Then, a reduction of the endoplasmic reticulumintraluminal Ca2+ concentration is sensed by STIM proteins, which undergo a conformational change and activate plasma membraneCa2+ channels comprised by Orai proteins. STIM1/Orai-mediated Ca2+ signals are finely regulated and modulate the activity of different transcription factors, including certain isoforms of the nuclear factor of activated T-cells, the cAMP-response element binding protein, the nuclear factor κ-light chain-enhancer of activated B cells, c-fos, and c-myc. These transcription factors associate SOCE with a plethora of signaling events and cellular functions. Here we provide an overview of the current knowledge about the role of Orai channels in the regulation of transcription factors through Ca2+ -dependent signaling pathways.

Long-term potentiation and spatial memory training stimulate the hippocampal expression of RyR2 calcium release channels.

Introduction: Neuronal Ca2+ signals generated through the activation of Ca2+-induced Ca2+ release in response to activity-generated Ca2+ influx play a significant role in hippocampal synaptic plasticity, spatial learning, and memory. We and others have previously reported that diverse stimulation protocols, or different memory-inducing procedures, enhance the expression of endoplasmic reticulum-resident Ca2+ release channels in rat primary hippocampal neuronal cells or hippocampal tissue. Methods and Results: Here, we report that induction of long-term potentiation (LTP) by Theta burst stimulation protocols of the CA3-CA1 hippocampal synapse increased the mRNA and protein levels of type-2 Ryanodine Receptor (RyR2) Ca2+ release channels in rat hippocampal slices. Suppression of RyR channel activity (1 h preincubation with 20 μM ryanodine) abolished both LTP induction and the enhanced expression of these channels; it also promoted an increase in the surface expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR1 and GluR2 and caused a moderate but significant reduction of dendritic spine density. In addition, training rats in the Morris water maze induced memory consolidation, which lasted for several days after the end of the training period, accompanied by an increase in the mRNA levels and the protein content of the RyR2 channel isoform. Discussion: We confirm in this work that LTP induction by TBS protocols requires functional RyR channels. We propose that the increments in the protein content of RyR2 Ca2+ release channels, induced by LTP or spatial memory training, play a significant role in hippocampal synaptic plasticity and spatial memory consolidation.

Arc ubiquitination regulates endoplasmic reticulum-mediated Ca2+ release and CaMKII signaling.

Synaptic plasticity relies on rapid, yet spatially precise signaling to alter synaptic strength. Arc is a brain enriched protein that is rapidly expressed during learning-related behaviors and is essential for regulating metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD). We previously showed that disrupting the ubiquitination capacity of Arc enhances mGluR-LTD; however, the consequences of Arc ubiquitination on other mGluR-mediated signaling events is poorly characterized. Here we find that pharmacological activation of Group I mGluRs with S-3,5-dihydroxyphenylglycine (DHPG) increases Ca2+ release from the endoplasmic reticulum (ER). Disrupting Arc ubiquitination on key amino acid residues enhances DHPG-induced ER-mediated Ca2+ release. These alterations were observed in all neuronal subregions except secondary branchpoints. Deficits in Arc ubiquitination altered Arc self-assembly and enhanced its interaction with calcium/calmodulin-dependent protein kinase IIb (CaMKIIb) and constitutively active forms of CaMKII in HEK293 cells. Colocalization of Arc and CaMKII was altered in cultured hippocampal neurons, with the notable exception of secondary branchpoints. Finally, disruptions in Arc ubiquitination were found to increase Arc interaction with the integral ER protein Calnexin. These results suggest a previously unknown role for Arc ubiquitination in the fine tuning of ER-mediated Ca2+ signaling that may support mGluR-LTD, which in turn, may regulate CaMKII and its interactions with Arc.

Xerostomia and Its Cellular Targets

Xerostomia, the subjective feeling of a dry mouth associated with dysfunction of the salivary glands, is mainly caused by radiation and chemotherapy, various systemic and autoimmune diseases, and drugs. As saliva plays numerous essential roles in oral and systemic health, xerostomia significantly reduces quality of life, but its prevalence is increasing. Salivation mainly depends on parasympathetic and sympathetic nerves, and the salivary glands responsible for this secretion move fluid unidirectionally through structural features such as the polarity of acinar cells. Saliva secretion is initiated by the binding of released neurotransmitters from nerves to specific G-protein-coupled receptors (GPCRs) on acinar cells. This signal induces two intracellular calcium (Ca2+) pathways (Ca2+ release from the endoplasmic reticulum and Ca2+ influx across the plasma membrane), and this increased intracellular Ca2+ concentration ([Ca2+]i) causes the translocation of the water channel aquaporin 5 (AQP5) to the apical membrane. Consequently, the GPCR-mediated increased [Ca2+]i in acinar cells promotes saliva secretion, and this saliva moves into the oral cavity through the ducts. In this review, we seek to elucidate the potential of GPCRs, the inositol 1,4,5-trisphosphate receptor (IP3R), store-operated Ca2+ entry (SOCE), and AQP5, which are essential for salivation, as cellular targets in the etiology of xerostomia.

Reticulocalbin3: A Ca2+ homeostasis regulator that promotes esophageal squamous cell carcinoma progression and cisplatin resistance.

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancers worldwide. There is a critical need to identify new mechanisms that contribute to ESCC progression. Reticulocalbin3 (RCN3) is mainly located in the endoplasmic reticulum and Ca2+‐binding protein containing EF‐hands. The function of RCN3 in tumor progression has not been clarified. We observed that the expression level of RCN3 was higher in ESCC tissues than in paired normal tissues. Overexpression of RCN3 was positively associated with tumor size, lymph node metastasis, TNM stage, lymphatic vessel infiltration, and poor outcome in patients with ESCC. Increased malignant phenotypes were observed in RCN3 overexpressing ESCC cells, whereas the opposite effects were achieved in RCN3‐silenced cells. Reticulocalbin3 promoted the expression of MMP‐2 and MMP‐9 by regulating the inositol 1,4,5‐trisphosphate receptor 1 (IP3R1)–Ca2+–calcium/calmodulin‐dependent protein kinase II–c‐Jun signaling pathway. Reticulocalbin3 induced cisplatin resistance by regulating IP3R1/Ca2+ to maintain intracellular Ca2+ homeostasis and reduced reactive oxygen species in ESCC cells. Finally, the expression of RCN3 was regulated by hypoxia inducible factor‐1α. Collectively, these data strongly support that RCN3 regulates Ca2+ homeostasis by targeting IP3R1 to promote the progression and platinum resistance of ESCC. Our studies suggest that RCN3 could serve as predictive factor of poor prognosis and potential therapeutic target for ESCC patients.

Sigma-1 Receptor Ligands Chlorpromazine and Trifluoperazine Attenuate Ca2+ Responses in Rat Peritoneal Macrophages.

Sigma-1 receptors are ubiquitous multifunctional ligand-regulated molecular chaperones in the endoplasmic reticulum membrane with a unique history, structure, and pharmacological profile. Sigma-1 receptors bind ligands of different chemical structure and pharmacological action and modulate a wide range of cellular processes in health and disease, including Ca2+ signaling. To elucidate the involvement of sigma-1 receptors in the processes of Ca2+ signaling in macrophages we studied the effect of sigma-1 receptor ligands, phenothiazine neuroleptics chlorpromazine and trifluoperazine, on Ca2+ responses induced by inhibitors of endoplasmic Ca2+–ATPases thapsigargin and cyclopiazonic acid, as well as by disulfide-containing immunomodulators Glutoxim and Molixan in rat peritoneal macrophages. Using Fura-2AM microfluorimetry we showed for the first time that chlorpromazine and trifluoperazine inhibit both phases of Ca2+ responses induced by Glutoxim, Molixan, thapsigargin, and cyclopiazonic acid in rat peritoneal macrophages. The data obtained indicate the participation of sigma-1 receptors in a complex signaling cascade caused by Glutoxim or Molixan and leading to an increase in intracellular Ca2+ concentration in macrophages. The results also indicate the involvement of sigma-1 receptors in the regulation of store-dependent Ca2+entry in macrophages.

Calreticulin and the Heart.

Calreticulin is an endoplasmic Ca2+ binding protein and molecular chaperone. As a cardiac embryonic gene, calreticulin is essential for heart development. The protein supports Ca2+-dependent signaling events that are critical to cardiomyocyte differentiation and cardiogenesis. The increased expression of calreticulin and endoplasmic reticulum/sarcoplasmic reticulum Ca2+ capacity produces cardiomyocytes with enhanced efficiency, and detrimental mechanical stretching of cardiac fibroblasts, leading to cardiac pathology. Deletion of the calreticulin gene in adult cardiomyocytes results in left ventricle dilation, an impaired electrocardiogram, and heart failure. These observations indicate that a well-adjusted endoplasmic reticulum and calreticulin-dependent Ca2+ pool in cardiomyocytes are critical for the maintenance of proper cardiac function.