The role of ryanodine receptor type 3 in a mouse model of Alzheimer disease

Dysregulated endoplasmic reticulum (ER) calcium (Ca²⁺) signaling is reported to play an important role in Alzheimer disease (AD) pathogenesis. The role of ER Ca²⁺ release channels, the ryanodine receptors (RyanRs), has been extensively studied in AD models and RyanR expression and activity are upregulated in the brains of various familial AD (FAD) models. The objective of this study was to utilize a genetic approach to evaluate the importance of RyanR type 3 (RyanR3) in the context of AD pathology.     

Stabilizing ER Ca2+ Channel Function as an Early Preventative Strategy for Alzheimer’s Disease

Alzheimer’s disease (AD) is a devastating neurodegenerative condition with no known cure. While current therapies target late-stage amyloid formation and cholinergic tone, to date, these strategies have proven ineffective at preventing disease progression. The reasons for this may be varied, and could reflect late intervention, or, that earlier pathogenic mechanisms have been overlooked and permitted to accelerate the disease process. One such example would include synaptic pathology, the disease component strongly associated with cognitive impairment. Dysregulated Ca²⁺ homeostasis may be one of the critical factors driving synaptic dysfunction. One of the earliest pathophysiological indicators in mutant presenilin (PS) AD mice is increased intracellular Ca²⁺ signaling, predominantly through the ER-localized inositol triphosphate (IP₃) and ryanodine receptors (RyR). In particular, the RyR-mediated Ca²⁺ upregulation within synaptic compartments is associated with altered synaptic homeostasis and network depression at early (presymptomatic) AD stages. Here, we offer an alternative approach to AD therapeutics by stabilizing early pathogenic mechanisms associated with synaptic abnormalities. We targeted the RyR as a means to prevent disease progression, and sub-chronically treated AD mouse models (4-weeks) with a novel formulation of the RyR inhibitor, dantrolene. Using 2-photon Ca²⁺ imaging and patch clamp recordings, we demonstrate that dantrolene treatment fully normalizes ER Ca²⁺ signaling within somatic and dendritic compartments in early and later-stage AD mice in hippocampal slices. Additionally, the elevated RyR2 levels in AD mice are restored to control levels with dantrolene treatment, as are synaptic transmission and synaptic plasticity. Aβ deposition within the cortex and hippocampus is also reduced in dantrolene-treated AD mice. In this study, we highlight the pivotal role of Ca²⁺ aberrations in AD, and propose a novel strategy to preserve synaptic function, and thereby cognitive function, in early AD patients.     

Presenilins as endoplasmic reticulum calcium leak channels and Alzheimer’s disease pathogenesis

Alzheimer disease (AD) is the most common neurodegenerative disorder worldwide and is at present, incurable. The accumulation of toxic amyloid-beta (Aβ) peptide aggregates in AD brain is thought to trigger the extensive synaptic loss and neurodegeneration linked to cognitive decline, an idea that underlies the ‘amyloid hypothesis’ of AD etiology in both the familal (FAD) and sporadic forms of the disease. Genetic mutations causing FAD also result in the dysregulation of neuronal calcium (Ca²⁺) handling and may contribute to AD pathogenesis, an idea termed the ‘calcium hypothesis’ of AD. Mutations in presenilin proteins account for majority of FAD cases. Presenilins function as catalytic subunit of γ-secretase involved in generation of Aβ peptide Recently, we discovered that presenilns function as low-conductance, passive ER Ca²⁺ leak channels, independent of γ-secretase activity. We further discovered that many FAD mutations in presenilins result in loss of ER Ca²⁺ leak function activity and Ca²⁺ overload in the ER. These results provided potential explanation for abnormal Ca²⁺ signaling observed in FAD cells with mutations in presenilns. Our latest work on studies of ER Ca²⁺ leak channel function of presenilins and implications of these findings for understanding AD pathogenesis are discussed in this article.     

Akt kinase reducing endoplasmic reticulum Ca2+ release protects cells from Ca2+-dependent apoptotic stimuli

The proto-oncogene Akt is a potent inhibitor of apoptosis, and it is activated in many human cancers. A number of recent studies have highlighted the importance of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) in mediating calcium (Ca²⁺) transfer from the endoplasmic reticulum (ER) to the mitochondria in several models of apoptosis. Akt is a serine-threonine kinase and recent data indicate the IP3R as a target of its phosphorylation activity.Here we show that HeLa cells, overexpressing the constitutively active myristoylated/palmitylated AKT1 (m/p-AKT1), were found to have a reduced Ca²⁺ release from ER after stimulation with agonist coupled to the generation of IP3. In turn, this affected cytosolic and mitochondria Ca²⁺ response after Ca²⁺ release from the ER induced either by agonist stimulation or by apoptotic stimuli releasing Ca²⁺ from intracellular stores.Most importantly, this alteration of ER Ca²⁺ content and release, reduces significantly cellular sensitivity to Ca²⁺ mediated proapoptotic stimulation. These results reveal a primary role of Akt in shaping intracellular Ca²⁺ homeostasis, that may underlie its protective role against some proapoptotic stimuli.     

A Highlights from MBoC Selection: Dendritic and axonal mechanisms of Ca2+ elevation impair BDNF transport in Aβ oligomer–treated hippocampal neurons

Disruption of fast axonal transport (FAT) and intracellular Ca²⁺ dysregulation are early pathological events in Alzheimer''s disease (AD). Amyloid-β oligomers (AβOs), a causative agent of AD, impair transport of BDNF independent of tau by nonexcitotoxic activation of calcineurin (CaN). Ca²⁺-dependent mechanisms that regulate the onset, severity, and spatiotemporal progression of BDNF transport defects from dendritic and axonal AβO binding sites are unknown. Here we show that BDNF transport defects in dendrites and axons are induced simultaneously but exhibit different rates of decline. The spatiotemporal progression of FAT impairment correlates with Ca²⁺ elevation and CaN activation first in dendrites and subsequently in axons. Although many axonal pathologies have been described in AD, studies have primarily focused only on the dendritic effects of AβOs despite compelling reports of presynaptic AβOs in AD models and patients. Indeed, we observe that dendritic CaN activation converges on Ca²⁺ influx through axonal voltage-gated Ca²⁺ channels to impair FAT. Finally, FAT defects are prevented by dantrolene, a clinical compound that reduces Ca²⁺ release from the ER. This work establishes a novel role for Ca²⁺ dysregulation in BDNF transport disruption and tau-independent Aβ toxicity in early AD.     

A new target for Alzheimer’s disease: A small molecule SERCA activator is neuroprotective in vitro and improves memory and cognition in APP/PS1 mice

Amongst the cellular cacophony of altered signals in Alzheimer’s disease (AD), disrupted Ca²⁺ homeostasis and consequential endoplasmic reticulum (ER) stress signals have been recognized as key determinants of neuron fate. This altered Ca²⁺ state is accompanied by a failing sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump, which has been recognized as a causal feature of the underlying disease state. Repair of the Ca²⁺ dyshomeostasis represents a putative drug target via alleviation of ER stress and rescue of injured neurons, effectively modifying the AD state. Herein, we report a small molecule SERCA activator that rescues brain cells and raises ER Ca²⁺ in vitro, and shows efficacy in the APP/PS1 double transgenic mouse model of Alzheimer’s disease. These results support SERCA activation as a therapeutic target for AD.     

Presenilins function in ER calcium leak and Alzheimer's disease pathogenesis

Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide and is at present, incurable. The accumulation of toxic amyloid-beta (Aβ) peptide aggregates in AD brain is thought to trigger the extensive synaptic loss and neurodegeneration linked to cognitive decline, an idea that underlies the ‘amyloid hypothesis’ of AD etiology in both the familal (FAD) and sporadic forms of the disease. Genetic mutations causing FAD also result in the dysregulation of neuronal calcium (Ca²⁺) handling and may contribute to AD pathogenesis, an idea termed the ‘calcium hypothesis’ of AD. Mutations in presenilin proteins account for the majority of FAD cases. Presenilins function as catalytic subunits of γ-secretase involved in the generation of Aβ peptide. Recently, we discovered that presenilns function as low-conductance, passive ER Ca²⁺ leak channels, independent of γ-secretase activity. We further discovered that many FAD mutations in presenilins results in the loss of ER Ca²⁺ leak function activity and Ca²⁺ overload in the ER. These results provided potential explanation for abnormal Ca²⁺ signaling observed in FAD cells with mutations in presenilns. The implications of these findings for understanding AD pathogenesis are discussed in this article.     

IP3 receptors in cell survival and apoptosis: Ca2+ release and beyond

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) serve to discharge Ca²⁺ from ER stores in response to agonist stimulation. The present review summarizes the role of these receptors in models of Ca²⁺-dependent apoptosis. In particular we focus on the regulation of IP₃Rs by caspase-3 cleavage, cytochrome c, anti-apoptotic proteins and Akt kinase. We also address the evidence that some of the effects of IP₃Rs in apoptosis may be independent of their ion-channel function. The role of IP₃Rs in delivering Ca²⁺ to the mitochondria is discussed from the perspective of the factors determining inter-organellar dynamics and the spatial proximity of mitochondria and ER membranes.     

Role of SOCE architects STIM and Orai proteins in Cell Death

Calcium (Ca²⁺) signaling plays a critical role in regulating plethora of cellular functions including cell survival, proliferation and migration. The perturbations in cellular Ca²⁺ homeostasis can lead to cell death either by activating autophagic pathways or through induction of apoptosis. Endoplasmic reticulum (ER) is the major storehouse of Ca²⁺ within cells and a number of physiological agonists mediate ER Ca²⁺ release by activating IP₃ receptors (IP₃R). This decrease in ER Ca²⁺ levels is sensed by STIM, which physically interacts and activates plasma membrane Ca²⁺ selective Orai channels. Emerging literature implicates a key role for STIM1, STIM2, Orai1 and Orai3 in regulating both cell survival and death pathways. In this review, we will retrospect the work highlighting the role of STIM and Orai homologs in regulating cell death signaling. We will further discuss the rationales that could explain the dual role of STIM and Orai proteins in regulating cell fate decisions.     

Bcl-2 family in inter-organelle modulation of calcium signaling; roles in bioenergetics and cell survival

Bcl-2 family proteins, known for their apoptosis functioning at the mitochondria, have been shown to localize to other cellular compartments to mediate calcium (Ca²⁺) signals. Since the proper supply of Ca²⁺ in cells serves as an important mechanism for cellular survival and bioenergetics, we propose an integrating role for Bcl-2 family proteins in modulating Ca²⁺ signaling. The endoplasmic reticulum (ER) is the main Ca²⁺ storage for the cell and Bcl-2 family proteins competitively regulate its Ca²⁺ concentration. Bcl-2 family proteins also regulate the flux of Ca²⁺ from the ER by physically interacting with inositol 1,4,5-trisphosphate receptors (IP₃Rs) to mediate their opening. Type 1 IP₃Rs reside at the bulk ER to coordinate cytosolic Ca²⁺ signals, while type 3 IP₃Rs reside at mitochondria-associated ER membrane (MAM) to facilitate mitochondrial Ca²⁺ uptake. In healthy cells, mitochondrial Ca²⁺ drives pyruvate into the citric acid (TCA) cycle to facilitate ATP production, while a continuous accumulation of Ca²⁺ can trigger the release of cytochrome c, thus initiating apoptosis. Since multiple organelles and Bcl-2 family proteins are involved in Ca²⁺ signaling, we aim to clarify the role that Bcl-2 family proteins play in facilitating Ca²⁺ signaling and how mitochondrial Ca²⁺ is relevant in both bioenergetics and apoptosis. We also explore how these insights could be useful in controlling bioenergetics in apoptosis-resistant cell lines.     

Mitochondria adjust Ca signaling regime to a pattern of stimulation in salivary acinar cells

The salivary acinar cells have unique Ca²⁺ signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca²⁺ signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca²⁺ entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca²⁺ machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca²⁺ imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca²⁺ signaling regime and ER refilling according to a pattern of agonist stimulation. Under the sustained stimulation, SOCE is increased proportionally to the degree of ER depletion. Cell adapts its Ca²⁺ handling system directing more Ca²⁺ into mitochondria via microdomains of high [Ca²⁺] providing positive feedback on SOCE while intra-mitochondrial tunneling provides adequate ER refilling. In the absence of an agonist, the bulk of ER refilling occurs through Ca²⁺-ATPase-mediated Ca²⁺ uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca²⁺ fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion.     

Ca2+‐dependent endoplasmic reticulum stress correlates with astrogliosis in oligomeric amyloid β‐treated astrocytes and in a model of Alzheimer's disease

Neurotoxic effects of amyloid β peptides are mediated through deregulation of intracellular Ca²⁺ homeostasis and signaling, but relatively little is known about amyloid β modulation of Ca²⁺ homeostasis and its pathological influence on glia. Here, we found that amyloid β oligomers caused a cytoplasmic Ca²⁺ increase in cultured astrocytes, which was reduced by inhibitors of PLC and ER Ca²⁺ release. Furthermore, amyloid β peptides triggered increased expression of glial fibrillary acidic protein (GFAP), as well as oxidative and ER stress, as indicated by eIF2α phosphorylation and overexpression of chaperone GRP78. These effects were decreased by ryanodine and 2APB, inhibitors of ryanodine receptors and InsP₃ receptors, respectively, in both primary cultured astrocytes and organotypic cultures of hippocampus and entorhinal cortex. Importantly, intracerebroventricular injection of amyloid β oligomers triggered overexpression of GFAP and GRP78 in astrocytes of the hippocampal dentate gyrus. These data were validated in a triple‐transgenic mouse model of Alzheimer's disease (AD). Overexpression of GFAP and GRP78 in the hippocampal astrocytes correlated with the amyloid β oligomer load in 12‐month‐old mice, suggesting that this parameter drives astrocytic ER stress and astrogliosis in vivo. Together, these results provide evidence that amyloid β oligomers disrupt ER Ca²⁺ homeostasis, which induces ER stress that leads to astrogliosis; this mechanism may be relevant to AD pathophysiology.     

4TM-TRPM8 channels are new gatekeepers of the ER-mitochondria Ca2+ transfer

Calcium (Ca²⁺) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca²⁺ release was associated with the ER Ca²⁺ release channels, inositol 1,4,5?triphosphate receptor (IP₃R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca²⁺ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca²⁺ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca²⁺ concentration ([Ca²⁺]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca²⁺ release mechanism distinct from classical Ca²⁺ release channels.     

Modulation of Endoplasmic Reticulum Ca2+ Store Filling by Cyclic ADP-ribose Promotes Inositol Trisphosphate (IP3)-evoked Ca2+ Signals

In addition to its well established function in activating Ca²⁺ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca²⁺ into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca²⁺ signals via changes in ER Ca²⁺ store content, by imaging Ca²⁺ liberation through inositol trisphosphate receptors (IP₃R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca²⁺] was transiently elevated by applying voltage-clamp pulses to induce Ca²⁺ influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca²⁺ signals evoked by strong photorelease of IP₃, and increased numbers of local Ca²⁺ puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca²⁺ elevation alone, indicating that they did not arise through direct actions of cADPR or Ca²⁺ on the IP₃R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca²⁺ may modulate IP₃R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.     

In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates

Cerebral ischemia is a key pathophysiological feature of various brain insults. Inadequate oxygen supply can manifest regionally in stroke or as a result of traumatic brain injury or globally following cardiac arrest, all leading to irreversible brain damage. Mitochondrial function is essential for neuronal survival, since neurons critically depend on ATP synthesis generated by mitochondrial oxidative phosphorylation. Mitochondrial activity depends on Ca²⁺ and is fueled either by Ca²⁺ from the extracellular space when triggered by neuronal activity or by Ca²⁺ released from the endoplasmic reticulum (ER) and taken up through specialized contact sites between the ER and mitochondria known as mitochondrial-associated ER membranes. The coordination of these Ca²⁺ pools is required to synchronize mitochondrial respiration rates and ATP synthesis to physiological demands. In this review, we discuss the role of the proteins involved in mitochondrial Ca²⁺ homeostasis in models of ischemia. The proteins include those important for the Ca²⁺-dependent motility of mitochondria and for Ca²⁺ transfer from the ER to mitochondria, the tethering proteins that bring the two organelles together, inositol 1,4,5-triphosphate receptors that enable Ca²⁺ release from the ER, voltage-dependent anion channels that allow Ca²⁺ entry through the highly permeable outer mitochondrial membrane and the mitochondrial Ca²⁺ uniporter together with its regulatory proteins that permit Ca²⁺ entry into the mitochondrial matrix. Finally, we address those proteins important for the extrusion of Ca²⁺ from the mitochondria such as the mitochondrial Na⁺/Ca²⁺ exchanger or, if the mitochondrial Ca²⁺ concentration exceeds a certain threshold, the mitochondrial permeability transition pore.     

Role of calcium in adult onset polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in genes encoding the polycystin (PC) 1 and 2 proteins. The goal of this study was to determine the role of calcium in regulating cyst growth. Stromal interaction molecule 1 (STIM1) protein expression was 15-fold higher in PC1-null proximal tubule cells (PN) than in heterozygote (PH) controls and 2-fold higher in an inducible, PC1 knockout, mouse model of ADPKD compared to a non-cystic match control. IP3 receptor protein expression was also higher in the cystic mice. Knocking down STIM1 with siRNA reduced cyst growth and lowered cAMP levels in PN cells. Fura2 measurements of intracellular Ca²⁺ showed higher levels of intracellular Ca²⁺, SOCE and thaspigargin-stimulated ER Ca²⁺ release in PN vs. PH cells. There was a dramatic reduction in thapsigargin-stimulated release of ER Ca²⁺ following STIM1 silencing or application of 2-APB, consistent with altered ER Ca²⁺ movement; the protein expression of the Ca²⁺-dependent adenylyl cyclases (AC) AC3 and AC6 was up- and down-regulated, respectively. Like STIM1 knockdown, application of the calmodulin inhibitor W7 lowered cAMP levels, further indicating that STIM1 regulates AC3 via Ca²⁺ We conclude that the high levels of STIM1 in ADPKD cells play a role in supporting cyst growth and promoting high cAMP levels and an increased release of Ca²⁺ from the ER. Thus, our results provide novel therapeutic targets for treating ADPKD.     

Familial Alzheimer’s disease-linked presenilin mutants and intracellular Ca2+ handling: A single-organelle,FRET-based analysis

An imbalance in Ca²⁺ homeostasis represents an early event in the pathogenesis of Alzheimer’s disease (AD). Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial AD (FAD), have been extensively associated with alterations in different Ca²⁺ signaling pathways, in particular those handled by storage compartments. However, FAD-PSs effect on organelles Ca²⁺ content is still debated and the mechanism of action of mutant proteins is unclear.To fulfil the need of a direct investigation of intracellular stores Ca²⁺ dynamics, we here present a detailed and quantitative single-cell analysis of FAD-PSs effects on organelle Ca²⁺ handling using specifically targeted, FRET (Fluorescence/Förster Resonance Energy Transfer)-based Ca²⁺ indicators. In SH-SY5Y human neuroblastoma cells and in patient-derived fibroblasts expressing different FAD-PSs mutations, we directly measured Ca²⁺ concentration within the main intracellular Ca²⁺ stores, e.g., Endoplasmic Reticulum (ER) and Golgi Apparatus (GA) medial- and trans-compartment. We unambiguously demonstrate that the expression of FAD-PS2 mutants, but not FAD-PS1, in either SH-SY5Y cells or FAD patient-derived fibroblasts, is able to alter Ca²⁺ handling of ER and medial-GA, but not trans-GA, reducing, compared to control cells, the Ca²⁺ content within these organelles by partially blocking SERCA (Sarco/Endoplasmic Reticulum Ca²⁺-ATPase) activity. Moreover, by using a cytosolic Ca²⁺ probe, we show that the expression of both FAD-PS1 and -PS2 reduces the Ca²⁺ influx activated by stores depletion (Store-Operated Ca²⁺ Entry; SOCE), by decreasing the expression levels of one of the key molecules, STIM1 (STromal Interaction Molecule 1), controlling this pathway.Our data indicate that FAD-linked PSs mutants differentially modulate the Ca²⁺ content of intracellular stores yet leading to a complex dysregulation of Ca²⁺ homeostasis, which represents a common disease phenotype of AD.     

Activation of galanin receptor 2 stimulates large conductance Ca-dependent K (BK) channels through the IP3 pathway in human embryonic kidney (HEK293) cells

The large conductance Ca²⁺-activated K⁺ (BK) channels are widely distributed in the brain, and act as intracellular calcium sensors in neurons. They play an important feedback role in controlling Ca²⁺ flux and Ca²⁺-dependent processes, including neurotransmitter release and cellular excitability. In this study, the effects of the neuropeptide galanin on BK channels were examined by determining the whole-cell currents and single-channel activities in human embryonic kidney (HEK293) cells co-expressing GalR2 and the BK alpha subunit. Galanin enhanced the currents of BK channels, in a concentration-dependent and PTX-independent manner, with an ED50 value of 71.8 ± 16.9 nM. This activation was mediated by GalR2, since its agonist AR-M1896 mimicked the effect of galanin, and since galanin did not facilitate BK currents in cells co-expressing cDNAs of BK and GalR1 or GalR3. The galanin-induced BK current persisted after replacement with Ca²⁺-free solution, suggesting that extracellular Ca²⁺ is not essential. Chelating intracellular Ca²⁺ by either the slow Ca²⁺ buffer EGTA or the fast Ca²⁺ buffer BAPTA abolished galanin-mediated activation of BK channels, indicating the important role of intracellular Ca²⁺. The role of Ca²⁺ efflux from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) was confirmed by application of thapsigargin, an irreversible inhibitor that depletes Ca²⁺ from SR/ER. Moreover, the inositol-1,4,5-triphosphate receptor (IP3R) was identified as the mediator responsible for increased intracellular Ca²⁺ activating BK channels. Taken together, activation of GalR2 leads to elevation of intracellular Ca²⁺ is due to Ca²⁺ efflux from ER through IP3R sequentially opening BK channels.     

Orai3 channel is the 2-APB-induced endoplasmic reticulum calcium leak

We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca²⁺ leak using synthetic Ca²⁺ indicators that report changes in both the cytoplasmic ([Ca²⁺]_i) and the luminal ER ([Ca²⁺]_ER) Ca²⁺ concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca²⁺ leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca²⁺]_ER but did not decrease the ER Ca²⁺ leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP₃R and only Orai3 channel supported the 2-APB-induced ER Ca²⁺ leak, while Orai1 and Orai2 inhibited this type of ER Ca²⁺ leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca²⁺ leak but did not modify the ER Ca²⁺ leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca²⁺]_i response after TG but only when the ER store had been overloaded with Ca²⁺ by eliminating the acidic internal Ca²⁺ store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca²⁺ leak but forms an ER Ca²⁺ leak channel that is limiting the overloading with Ca²⁺ of the ER store.