Characterizing Modulators of Protease-activated Receptors with a Calcium Mobilization Assay Using a Plate Reader.

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of Gαq signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism.

One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.

A Model for the Coexistence of Competing Mechanisms for Ca 2 + Oscillations in T-lymphocytes.

Ca 2 + is a ubiquitous signaling mechanism across different cell types. In T-cells, it is associated with cytokine production and immune function. Benson et al. have shown the coexistence of competing Ca 2 + oscillations during antigen stimulation of T-cell receptors, depending on the presence of extracellular Ca 2 + influx through the Ca 2 + release-activated Ca 2 + channel (Benson in J Biol Chem 29:105310, 2023). In this paper, we construct a mathematical model consisting of five ordinary differential equations and analyze the relationship between the competing oscillatory mechanisms.. We perform bifurcation analysis on two versions of our model, corresponding to the two oscillatory types, to find the defining characteristics of these two families.

Protein carbonylation causes sarcoplasmic reticulum Ca2+ overload by increasing intracellular Na+ level in ventricular myocytes.

Diabetes is commonly associated with an elevated level of reactive carbonyl species due to alteration of glucose and fatty acid metabolism. These metabolic changes cause an abnormality in cardiac Ca2+ regulation that can lead to cardiomyopathies. In this study, we explored how the reactive α-dicarbonyl methylglyoxal (MGO) affects Ca2+ regulation in mouse ventricular myocytes. Analysis of intracellular Ca2+ dynamics revealed that MGO (200 µM) increases action potential (AP)-induced Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load, with a limited effect on L-type Ca2+ channel-mediated Ca2+ transients and SERCA-mediated Ca2+ uptake. At the same time, MGO significantly slowed down cytosolic Ca2+ extrusion by Na+/Ca2+ exchanger (NCX). MGO also increased the frequency of Ca2+ waves during rest and these Ca2+ release events were abolished by an external solution with zero [Na+] and [Ca2+]. Adrenergic receptor activation with isoproterenol (10 nM) increased Ca2+ transients and SR Ca2+ load, but it also triggered spontaneous Ca2+ waves in 27% of studied cells. Pretreatment of myocytes with MGO increased the fraction of cells with Ca2+ waves during adrenergic receptor stimulation by 163%. Measurements of intracellular [Na+] revealed that MGO increases cytosolic [Na+] by 57% from the maximal effect produced by the Na+-K+ ATPase inhibitor ouabain (20 µM). This increase in cytosolic [Na+] was a result of activation of a tetrodotoxin-sensitive Na+ influx, but not an inhibition of Na+-K+ ATPase. An increase in cytosolic [Na+] after treating cells with ouabain produced similar effects on Ca2+ regulation as MGO. These results suggest that protein carbonylation can affect cardiac Ca2+ regulation by increasing cytosolic [Na+] via a tetrodotoxin-sensitive pathway. This, in turn, reduces Ca2+ extrusion by NCX, causing SR Ca2+ overload and spontaneous Ca2+ waves.

Exploring Ca2+ Dynamics in Myelinating Oligodendrocytes through rAAV-Mediated jGCaMP8s Expression in Developing Spinal Cord Organ Cultures.

Oligodendrocytes, the myelin-producing glial cells of the central nervous system (CNS), crucially contribute to myelination and circuit function. An increasing amount of evidence suggests that intracellular calcium (Ca2+) dynamics in oligodendrocytes mediates activity-dependent and activity-independent myelination. Unraveling how myelinating oligodendrocytes orchestrate and integrate Ca2+ signals, particularly in relation to axonal firing, is crucial for gaining insights into their role in the CNS development and function, both in health and disease. In this framework, we used the recombinant adeno-associated virus/Olig001 capsid variant to express the genetically encoded Ca2+ indicator jGCaMP8s, under the control of the myelin basic protein promoter. In our study, this tool exhibits excellent tropism and selectivity for myelinating and mature oligodendrocytes, and it allows monitoring Ca2+ activity in myelin-forming cells, both in isolated primary cultures and organotypic spinal cord explants. By live imaging of myelin Ca2+ events in oligodendrocytes within organ cultures, we observed a rapid decline in the amplitude and duration of Ca2+ events across different in vitro developmental stages. Active myelin sheath remodeling and growth are modulated at the level of myelin-axon interface through Ca2+ signaling, and, during early myelination in organ cultures, this phase is finely tuned by the firing of axon action potentials. In the later stages of myelination, Ca2+ events in mature oligodendrocytes no longer display such a modulation, underscoring the involvement of complex Ca2+ signaling in CNS myelination.

FGF13 deficiency ameliorates calcium signaling abnormality in heart failure by regulating microtubule stability.

Calcium signaling abnormality in cardiomyocytes, as a key mechanism, is closely associated with developing heart failure. Fibroblast growth factor 13 (FGF13) demonstrates important regulatory roles in the heart, but its association with cardiac calcium signaling in heart failure remains unknown. This study aimed to investigate the role and mechanism of FGF13 on calcium mishandling in heart failure. Mice underwent transaortic constriction to establish a heart failure model, which showed decreased ejection fraction, fractional shortening, and contractility. FGF13 deficiency alleviated cardiac dysfunction. Heart failure reduces calcium transients in cardiomyocytes, which were alleviated by FGF13 deficiency. Meanwhile, FGF13 deficiency restored decreased Cav1.2 and Serca2α expression and activity in heart failure. Furthermore, FGF13 interacted with microtubules in the heart, and FGF13 deficiency inhibited the increase of microtubule stability during heart failure. Finally, in isoproterenol-stimulated FGF13 knockdown neonatal rat ventricular myocytes (NRVMs), wildtype FGF13 overexpression, but not FGF13 mutant, which lost the binding site of microtubules, promoted calcium transient abnormality aggravation and Cav1.2 downregulation compared with FGF13 knockdown group. Generally, FGF13 deficiency improves abnormal calcium signaling by inhibiting the increased microtubule stability in heart failure, indicating the important role of FGF13 in cardiac calcium homeostasis and providing new avenues for heart failure prevention and treatment.

Berberine inhibits intracellular Ca2+ signals in mouse pancreatic acinar cells through M3 muscarinic receptors: Novel target, mechanism, and implication.

Berberine, a natural isoquinoline alkaloid, exhibits a variety of pharmacological effects, but the pharmacological targets and mechanisms remain elusive. Here, we report a novel finding that berberine inhibits acetylcholine (ACh)-induced intracellular Ca2+ oscillations, mediated through an inhibition of the muscarinic subtype 3 (M3) receptor. Patch-clamp recordings and confocal Ca2+ imaging were applied to acute dissociated pancreatic acinar cells prepared from CD1 mice to examine the effects of berberine on ACh-induced Ca2+ oscillations. Whole-cell patch-clamp recordings showed that berberine (from 0.1 to 10 µM) reduced ACh-induced Ca2+ oscillations in a concentration-dependent manner, and this inhibition also depended on ACh concentrations. The inhibitory effect of berberine neither occurred in intracellular targets nor extracellular cholecystokinin (CCK) receptors, chloride (Cl-) channels, and store-operated Ca2+ channels. Together, the results demonstrate that berberine directly inhibits the muscarinic M3 receptors, further confirmed by evidence of the interaction between berberine and M3 receptors in pancreatic acinar cells.

Calcium signaling crosstalk between the endoplasmic reticulum and mitochondria, a new drug development strategies of kidney diseases.

Calcium (Ca2+) acts as a second messenger and constitutes a complex and large information exchange system between the endoplasmic reticulum (ER) and mitochondria; this process is involved in various life activities, such as energy metabolism, cell proliferation and apoptosis. Increasing evidence has suggested that alterations in Ca2+ crosstalk between the ER and mitochondria, including alterations in ER and mitochondrial Ca2+ channels and related Ca2+ regulatory proteins, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), inositol 1,4,5-trisphosphate receptor (IP3R), and calnexin (CNX), are closely associated with the development of kidney disease. Therapies targeting intracellular Ca2+ signaling have emerged as an emerging field in the treatment of renal diseases. In this review, we focused on recent advances in Ca2+ signaling, ER and mitochondrial Ca2+ monitoring methods and Ca2+ homeostasis in the development of renal diseases and sought to identify new targets and insights for the treatment of renal diseases by targeting Ca2+ channels or related Ca2+ regulatory proteins.

Temporal multiscale modeling of biochemical regulatory networks: Calcium-regulated hepatocyte lipid and glucose metabolism.

Hepatocyte lipid and glucose metabolism is regulated not only by major hormones like insulin and glucagon but also by many other factors, including calcium ions. Recently, mitochondria-associated membrane (MAM) dysfunction combined with incorrect IP3-receptor regulation has been shown to result in abnormal calcium signaling in hepatocytes. This dysfunction could further lead to hepatic metabolism pathology. However, the exact contribution of MAM dysfunction, incorrect IP3-receptor regulation and insulin resistance to the calcium-insulin-glucagon interplay is not understood yet. In this work, we analyze the role of abnormal calcium signaling and insulin dysfunction in hepatocytes by proposing a model of hepatocyte metabolic regulatory network with a detailed focus on the model construction details besides the biological aspect. In this work, we analyze the role of abnormal calcium signaling and insulin dysfunction in hepatocytes by proposing a model of hepatocyte metabolic regulatory network. We focus on the model construction details, model validation, and predictions. We describe the dynamic regulation of signaling processes by sigmoid Hill function. In particular, we study the effect of both the Hill function slope and the distance between Hill function extremes on metabolic processes in hepatocytes as a model of nonspecific insulin dysfunction. We also address the significant time difference between characteristic time of glucose hepatic processing and a typical calcium oscillation period in hepatocytes. Our modeling results show that calcium signaling dysfunction results in an abnormal increase in postprandial glucose levels, an abnormal glucose decrease in fasting, and a decreased amount of stored glycogen. An insulin dysfunction of glucose phosphorylation, glucose dephosphorylation, and glycogen breakdown also cause a noticeable effect. We also get some insight into the so-called hepatic insulin resistance paradox, confirming the hypothesis regarding indirect insulin action on hepatocytes via dysfunctional adipocyte lipolysis.

A PACAP-activated network for secretion requires coordination of Ca2+ influx and Ca2+ mobilization.

Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca2+ which are disrupted when Ca2+ influx through L-type channels is blocked or internal Ca2+ stores are depleted. PACAP liberates stored Ca2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca2+ mobilization to Ca2+ influx and supporting Ca2+-induced Ca2+-release. These Ca2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ, is regulated by a signaling network that promotes sustained elevations in intracellular Ca2+ through multiple pathways.

Network representation of multicellular activity in pancreatic islets: Technical considerations for functional connectivity analysis.

Within the islets of Langerhans, beta cells orchestrate synchronized insulin secretion, a pivotal aspect of metabolic homeostasis. Despite the inherent heterogeneity and multimodal activity of individual cells, intercellular coupling acts as a homogenizing force, enabling coordinated responses through the propagation of intercellular waves. Disruptions in this coordination are implicated in irregular insulin secretion, a hallmark of diabetes. Recently, innovative approaches, such as integrating multicellular calcium imaging with network analysis, have emerged for a quantitative assessment of the cellular activity in islets. However, different groups use distinct experimental preparations, microscopic techniques, apply different methods to process the measured signals and use various methods to derive functional connectivity patterns. This makes comparisons between findings and their integration into a bigger picture difficult and has led to disputes in functional connectivity interpretations. To address these issues, we present here a systematic analysis of how different approaches influence the network representation of islet activity. Our findings show that the choice of methods used to construct networks is not crucial, although care is needed when combining data from different islets. Conversely, the conclusions drawn from network analysis can be heavily affected by the pre-processing of the time series, the type of the oscillatory component in the signals, and by the experimental preparation. Our tutorial-like investigation aims to resolve interpretational issues, reconcile conflicting views, advance functional implications, and encourage researchers to adopt connectivity analysis. As we conclude, we outline challenges for future research, emphasizing the broader applicability of our conclusions to other tissues exhibiting complex multicellular dynamics.

Centripetal integration of past events in hippocampal astrocytes regulated by locus coeruleus.

An essential feature of neurons is their ability to centrally integrate information from their dendrites. The activity of astrocytes, in contrast, has been described as mostly uncoordinated across cellular compartments without clear central integration. Here we report conditional integration of calcium signals in astrocytic distal processes at their soma. In the hippocampus of adult mice of both sexes, we found that global astrocytic activity, as recorded with population calcium imaging, reflected past neuronal and behavioral events on a timescale of seconds. Salient past events, indicated by pupil dilations, facilitated the propagation of calcium signals from distal processes to the soma. Centripetal propagation to the soma was reproduced by optogenetic activation of the locus coeruleus, a key regulator of arousal, and reduced by pharmacological inhibition of α1-adrenergic receptors. Together, our results suggest that astrocytes are computational units of the brain that slowly and conditionally integrate calcium signals upon behaviorally relevant events.

Voltage-induced calcium release in Caenorhabditis elegans body muscles.

Type 1 voltage-activated calcium channels (CaV1) in the plasma membrane trigger calcium release from the sarcoplasmic reticulum (SR) by two mechanisms. In voltage-induced calcium release (VICR), CaV1 voltage sensing domains are directly coupled to ryanodine receptors (RYRs), an SR calcium channel. In calcium-induced calcium release (CICR), calcium ions flowing through activated CaV1 channels bind and activate RYR channels. VICR is thought to occur exclusively in vertebrate skeletal muscle while CICR occurs in all other muscles (including all invertebrate muscles). Here, we use calcium-activated SLO-2 potassium channels to analyze CaV1-SR coupling in Caenorhabditis elegans body muscles. SLO-2 channels were activated by both VICR and external calcium. VICR-mediated SLO-2 activation requires two SR calcium channels (RYRs and IP3 Receptors), JPH-1/Junctophilin, a PDZ (PSD95, Dlg1, ZO-1 domain) binding domain (PBD) at EGL-19/CaV1's carboxy-terminus, and SHN-1/Shank (a scaffolding protein that binds EGL-19's PBD). Thus, VICR occurs in invertebrate muscles.

Nonlinear super-resolution signal processing allows intracellular tracking of calcium dynamics.

Objective.Traditional quantification of fluorescence signals, such asΔF/F, relies on ratiometric measures that necessitate a baseline for comparison, limiting their applicability in dynamic analyses. Our goal here is to develop a baseline-independent method for analyzing fluorescence data that fully exploits temporal dynamics to introduce a novel approach for dynamical super-resolution analysis, including in subcellular resolution.Approach.We introduce ARES (Autoregressive RESiduals), a novel method that leverages the temporal aspect of fluorescence signals. By focusing on the quantification of residuals following linear autoregression, ARES obviates the need for a predefined baseline, enabling a more nuanced analysis of signal dynamics.Main result.We delineate the foundational attributes of ARES, illustrating its capability to enhance both spatial and temporal resolution of calcium fluorescence activity beyond the conventional ratiometric measure (ΔF/F). Additionally, we demonstrate ARES's utility in elucidating intracellular calcium dynamics through the detailed observation of calcium wave propagation within a dendrite.Significance.ARES stands out as a robust and precise tool for the quantification of fluorescence signals, adept at analyzing both spontaneous and evoked calcium dynamics. Its ability to facilitate the subcellular localization of calcium signals and the spatiotemporal tracking of calcium dynamics-where traditional ratiometric measures falter-underscores its potential to revolutionize baseline-independent analyses in the field.

Transcriptional Regulation of SugarCane Response to Sporisorium scitamineum: Insights from Time-Course Gene Coexpression and Ca2+ Signaling.

Sugarcane response to Sporisorium scitamineum is determined by multiple major genes and numerous microeffector genes. Here, time-ordered gene coexpression networks were applied to explore the interaction between sugarcane and S. scitamineum. Totally, 2459 differentially expressed genes were identified and divided into 10 levels, and several stress-related subnetworks were established. Interestingly, the Ca2+ signaling pathway was activated to establish the response to sugarcane smut disease. Accordingly, two CAX genes (ScCAX2 and ScCAX3) were cloned and characterized from sugarcane. They were significantly upregulated under ABA stress but inhibited by MeJA treatment. Furthermore, overexpression of ScCAX2 and ScCAX3 enhanced the susceptibility of transgenic plants to the pathogen infection, suggesting its negative role in disease resistance. A regulatory model for ScCAX genes in disease response was thus depicted. This work helps to clarify the transcriptional regulation of sugarcane response to S. scitamineum stress and the function of the CAX gene in disease response.

Mitochondrial calcium signaling in non-neuronal cells: Implications for Alzheimer's disease pathogenesis.

Mitochondrial dysregulation is pivotal in Alzheimer's disease (AD) pathogenesis. Calcium governs vital mitochondrial processes impacting energy conversion, oxidative stress, and cell death signaling. Disruptions in mitochondrial calcium (mCa2+) handling induce calcium overload and trigger the opening of mitochondrial permeability transition pore, ensuing energy deprivation and resulting in AD-related neuronal cell death. However, the role of mCa2+ in non-neuronal cells (microglia, astrocytes, oligodendrocytes, endothelial cells, and pericytes) remains elusive. This review provides a comprehensive exploration of mitochondrial heterogeneity and calcium signaling, offering insights into specific differences among various brain cell types in AD.

Polycystin-2 Mediated Calcium Signalling in the Dictyostelium Model for Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) occurs when the proteins Polycystin-1 (PC1, PKD1) and Polycystin-2 (PC2, PKD2) contain mutations. PC1 is a large membrane receptor that can interact and form a complex with the calcium-permeable cation channel PC2. This complex localizes to the plasma membrane, primary cilia and ER. Dysregulated calcium signalling and consequential alterations in downstream signalling pathways in ADPKD are linked to cyst formation and expansion; however, it is not completely understood how PC1 and PC2 regulate calcium signalling. We have studied Polycystin-2 mediated calcium signalling in the model organism Dictyostelium discoideum by overexpressing and knocking down the expression of the endogenous Polycystin-2 homologue, Polycystin-2. Chemoattractant-stimulated cytosolic calcium response magnitudes increased and decreased in overexpression and knockdown strains, respectively, and analysis of the response kinetics indicates that Polycystin-2 is a significant contributor to the control of Ca2+ responses. Furthermore, basal cytosolic calcium levels were reduced in Polycystin-2 knockdown transformants. These alterations in Ca2+ signalling also impacted other downstream Ca2+-sensitive processes including growth rates, endocytosis, stalk cell differentiation and spore viability, indicating that Dictyostelium is a useful model to study Polycystin-2 mediated calcium signalling.

Multicore fiber optic imaging reveals that astrocyte calcium activity in the mouse cerebral cortex is modulated by internal motivational state.

Astrocytes are a direct target of neuromodulators and can influence neuronal activity on broad spatial and temporal scales in response to a rise in cytosolic calcium. However, our knowledge about how astrocytes are recruited during different animal behaviors remains limited. To measure astrocyte activity calcium in vivo during normative behaviors, we utilize a high-resolution, long working distance multicore fiber optic imaging system that allows visualization of individual astrocyte calcium transients in the cerebral cortex of freely moving mice. We define the spatiotemporal dynamics of astrocyte calcium changes during diverse behaviors, ranging from sleep-wake cycles to the exploration of novel objects, showing that their activity is more variable and less synchronous than apparent in head-immobilized imaging conditions. In accordance with their molecular diversity, individual astrocytes often exhibit distinct thresholds and activity patterns during explorative behaviors, allowing temporal encoding across the astrocyte network. Astrocyte calcium events were induced by noradrenergic and cholinergic systems and modulated by internal state. The distinct activity patterns exhibited by astrocytes provides a means to vary their neuromodulatory influence in different behavioral contexts and internal states.

Non-ionotropic voltage-gated calcium channel signaling.

Voltage-gated calcium channels (VGCCs) are the major conduits for calcium ions (Ca2+) within excitable cells. Recent studies have highlighted the non-ionotropic functionality of VGCCs, revealing their capacity to activate intracellular pathways independently of ion flow. This non-ionotropic signaling mode plays a pivotal role in excitation-coupling processes, including gene transcription through excitation-transcription (ET), synaptic transmission via excitation-secretion (ES), and cardiac contraction through excitation-contraction (EC). However, it is noteworthy that these excitation-coupling processes require extracellular calcium (Ca2+) and Ca2+ occupancy of the channel ion pore. Analogous to the "non-canonical" characterization of the non-ionotropic signaling exhibited by the N-methyl-D-aspartate receptor (NMDA), which requires extracellular Ca2+ without the influx of ions, VGCC activation requires depolarization-triggered conformational change(s) concomitant with Ca2+ binding to the open channel. Here, we discuss the contributions of VGCCs to ES, ET, and EC coupling as Ca2+ binding macromolecules that transduces external stimuli to intracellular input prior to elevating intracellular Ca2+. We emphasize the recognition of calcium ion occupancy within the open ion-pore and its contribution to the excitation coupling processes that precede the influx of calcium. The non-ionotropic activation of VGCCs, triggered by the upstroke of an action potential, provides a conceptual framework to elucidate the mechanistic aspects underlying the microseconds nature of synaptic transmission, cardiac contractility, and the rapid induction of first-wave genes.

Exploring the intricacies of calcium dysregulation in ischemic stroke: Insights into neuronal cell death and therapeutic strategies.

Calcium ion (Ca2+) dysregulation is one of the main causes of neuronal cell death and brain damage after cerebral ischemia. During ischemic stroke, the ability of neurons to maintain Ca2+ homeostasis is compromised. Ca2+ regulates various functions of the nervous system, including neuronal activity and adenosine triphosphate (ATP) production. Disruptions in Ca2+ homeostasis can trigger a cascade of events, including activation of the unfolded protein response (UPR) pathway, which is associated with endoplasmic reticulum (ER) stress and mitochondrial dysfunction. This response occurs when the cell is unable to manage protein folding within the ER due to various stressors, such as a high influx of Ca2+. Consequently, the UPR is initiated to restore ER function and alleviate stress, but prolonged activation can lead to mitochondrial dysfunction and, ultimately, cell death. Hence, precise regulation of Ca2+ within the cell is mandatory. The ER and mitochondria are two such organelles that maintain intracellular Ca2+ homeostasis through various calcium-operating channels, including ryanodine receptors (RyRs), inositol trisphosphate receptors (IP3Rs), sarco/endoplasmic reticulum calcium ATPases (SERCAs), the mitochondrial Na+/Ca2+ exchanger (NCLX), the mitochondrial calcium uniporter (MCU) and voltage-dependent anion channels (VDACs). These channels utilize Ca2+ sequestering and release mechanisms to maintain intracellular Ca2+ homeostasis and ensure proper cellular function and survival. The present review critically evaluates the significance of Ca2+ and its physiological role in cerebral ischemia. We have compiled recent findings on calcium's role and emerging treatment strategies, particularly targeting mitochondria and the endoplasmic reticulum, to address Ca2+ overload in cerebral ischemia.