The BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as an evolutionary conserved inhibitor of IP3 receptor channels

Ca²⁺ signalling plays an important role in various physiological processes in vertebrates. In mammals, the highly conserved anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein is an important modulator of the inositol 1,4,5-trisphosphate receptor (IP₃R), i.e. the main intracellular Ca²⁺ - release channel located at the endoplasmic reticulum (ER). The Bcl-2 Homology (BH) 4 domain of Bcl-2 (BH4-Bcl-2) is a critical determinant for inhibiting IP₃Rs, by directly targeting a region in the modulatory domain of the receptor (domain 3). In this paper, we aimed to track the evolutionary history of IP₃R regulation by the BH4 domain of Bcl-2 orthologues from different classes of vertebrates, including Osteichthyes, Amphibia, Reptilia, Aves and Mammalia. The high degree of conservation of the BH4 sequences correlated with the ability of all tested peptides to bind to the domain 3 of mouse IP₃R1 in GST-pull downs and their overall ability to inhibit IP₃-induced Ca²⁺ release (IICR) in permeabilized cells. Nevertheless, the BH4 domains differed in their potency to suppress IICR. The peptide derived from X. laevis was the least potent inhibitor. We identified a critical residue in BH4-Bcl-2 from H. sapiens, Thr7, which is replaced by Gly7 in X. laevis. Compared to the wild type X. laevis BH4-Bcl-2, a “humanized” version of the peptide (BH4-Bcl-2 Gly7Thr), displayed increased IP₃R-inhibitory properties. Despite the differences in the inhibitory efficiency, our data indicate that the BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as a binding partner and inhibitor of IP₃R channels.     

Targeting Bcl-2-IP3 receptor interaction to treat cancer: A novel approach inspired by nearly a century treating cancer with adrenal corticosteroid hormones

Bcl-2 inhibits cell death by at least two different mechanisms. On the one hand, its BH3 domain binds to pro-apoptotic proteins such as Bim and prevents apoptosis induction. On the other hand, the BH4 domain of Bcl-2 binds to the inositol 1,4,5-trisphosphate receptor (IP₃R), preventing Ca²⁺ signals that mediate cell death. In normal T-cells, Bcl-2 levels increase during the immune response, protecting against cell death, and then decline as apoptosis ensues and the immune response dissipates. But in many cancers Bcl-2 is aberrantly expressed and exploited to prevent cell death by inhibiting IP₃R-mediated Ca²⁺ elevation. This review summarizes what is known about the mechanism of Bcl-2's control over IP₃R-mediated Ca²⁺ release and cell death induction. Early insights into the role of Ca²⁺ elevation in corticosteroid-mediated cell death serves as a model for how targeting IP₃R-mediated Ca²⁺ elevation can be a highly effective therapeutic approach for different types of cancer. Moreover, the successful development of ABT-199 (Venetoclax), a small molecule targeting the BH3 domain of Bcl-2 but without effects on Ca²⁺, serves as proof of principle that targeting Bcl-2 can be an effective therapeutic approach. BIRD-2, a synthetic peptide that inhibits Bcl-2-IP₃R interaction, induces cell death induction in ABT-199 (Venetoclax)-resistant cancer models, attesting to the value of developing therapeutic agents that selectively target Bcl-2-IP₃R interaction, inducing Ca²⁺-mediated cell death.     

Tracing the evolutionary history of Ca2+-signaling modulation by human Bcl-2: Insights from the Capsaspora owczarzaki IP3 receptor ortholog

Recently, a functional IP₃R ortholog (CO.IP₃R-A) capable of IP₃-induced Ca²⁺ release has been discovered in Capsaspora owczarzaki, a close unicellular relative to Metazoa. In contrast to mammalian IP₃Rs, CO.IP₃R-A is not modulated by Ca²⁺, ATP or PKA. Protein-sequence analysis revealed that CO.IP₃R-A contained a putative binding site for anti-apoptotic Bcl-2, although Bcl-2 was not detected in Capsaspora owczarzaki and only appeared in Metazoa. Here, we examined whether human Bcl-2 could form a complex with CO.IP₃R-A channels and modulate their Ca²⁺-flux properties using ectopic expression approaches in a HEK293 cell model in which all three IP₃R isoforms were knocked out. We demonstrate that human Bcl-2 via its BH4 domain could functionally interact with CO.IP₃R-A, thereby suppressing Ca²⁺ flux through CO.IP₃R-A channels. The BH4 domain of Bcl-2 was sufficient for interaction with CO.IP₃R-A channels. Moreover, mutating the Lys17 of Bcl-2's BH4 domain, the residue critical for Bcl-2-dependent modulation of mammalian IP₃Rs, abrogated Bcl-2's ability to bind and inhibit CO.IP₃R-A channels. Hence, this raises the possibility that a unicellular ancestor of animals already had an IP₃R that harbored a Bcl-2-binding site. Bcl-2 proteins may have evolved as controllers of IP₃R function by exploiting this pre-existing site, thereby counteracting Ca²⁺-dependent apoptosis.     

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.     

An Interaction between Bcl-xL and the Voltage-dependent Anion Channel (VDAC) Promotes Mitochondrial Ca2+ Uptake

The role of the antiapoptotic protein Bcl-x_L in regulating mitochondrial Ca²⁺ ([Ca²⁺]_mito) handling was examined in wild-type (WT) and Bcl-x_L knock-out (Bcl-x_L-KO) mouse embryonic fibroblast cells. Inositol 1,4,5-trisphosphate-generating agonist evoked cytosolic Ca²⁺ transients that produced a larger [Ca²⁺]_mito uptake in WT cells compared with Bcl-x_L-KO. In permeabilized cells, stepping external [Ca²⁺] from 0 to 3 μm also produced a larger [Ca²⁺]_mito uptake in WT; moreover, the [Ca²⁺]_mito uptake capacity of Bcl-x_L-KO cells was restored by re-expression of mitochondrially targeted Bcl-x_L. Bcl-x_L enhancement of [Ca²⁺]_mito uptake persisted after dissipation of the mitochondrial membrane potential but was absent in mitoplasts lacking an outer mitochondrial membrane. The outer membrane-localized voltage-dependent anion channel (VDAC) is a known Ca²⁺ permeability pathway that directly interacts with Bcl-x_L. Bcl-x_L interacted with VDAC1 and -3 isoforms, and peptides based on the VDAC sequence disrupted Bcl-x_L binding. Peptides reduced [Ca²⁺]_mito uptake in WT but were without effect in Bcl-x_L-KO cells. In addition, peptides reduced [Ca²⁺]_mito uptake in VDAC1 and VDAC3 knock-out but not VDAC1 and -3 double knock-out mouse embryonic fibroblast cells, confirming that Bcl-x_L interacts functionally with VDAC1 and -3 but not VDAC2. Thus, an interaction between Bcl-x_L and VDAC promotes matrix Ca²⁺ accumulation by increasing Ca²⁺ transfer across the outer mitochondrial membrane.     

TRPC3 Regulates Agonist-stimulated Ca2+ Mobilization by Mediating the Interaction between Type I Inositol 1,4,5-Trisphosphate Receptor, RACK1, and Orai1

There is a body of evidence suggesting that Ca²⁺ handling proteins assemble into signaling complexes required for a fine regulation of Ca²⁺ signals, events that regulate a variety of critical cellular processes. Canonical transient receptor potential (TRPC) and Orai proteins have both been proposed to form Ca²⁺-permeable channels mediating Ca²⁺ entry upon agonist stimulation. A number of studies have demonstrated that inositol 1,4,5-trisphosphate receptors (IP₃Rs) interact with plasma membrane TRPC channels; however, at present there is no evidence supporting the interaction between Orai proteins and IP₃Rs. Here we report that treatment with thapsigargin or cellular agonists results in association of Orai1 with types I and II IP₃Rs. In addition, we have found that TRPC3, RACK1 (receptor for activated protein kinase C-1), and STIM1 (stromal interaction molecule 1) interact with Orai1 upon stimulation with agonists. TRPC3 expression silencing prevented both the interaction of Orai1 with TRPC3 and, more interestingly, the association of Orai1 with the type I IP₃R, but not with the type II IP₃R, thus suggesting that TRPC3 selectively mediates interaction between Orai1 and type I IP₃R. In addition, TRPC3 expression silencing attenuated ATP- and CCh-stimulated interaction between RACK1 and the type I IP₃R, as well as Ca²⁺ release and entry. In conclusion, our results indicate that agonist stimulation results in the formation of an Orai1-STIM1-TRPC3-RACK1-type I IP₃R complex, where TRPC3 plays a central role. This Ca²⁺ signaling complex might be important for both agonist-induced Ca²⁺ release and entry.     

Bcl-2 interaction with the inositol 1,4,5-trisphosphate receptor: role in Ca(2+) signaling and disease

The Bcl-2 protein, best known for its ability to inhibit apoptosis, interacts with the inositol 1,4,5-trisphosphate receptor (IP₃R) Ca²⁺ channel to regulate IP₃-mediated Ca²⁺ release from the endoplasmic reticulum. This review summarizes the current state of knowledge regarding the interaction of Bcl-2, and also its homologue Bcl-xl, with the IP₃R and how these interactions regulate Ca²⁺ signaling. The dual role of these interactions in promoting prosurvival Ca²⁺ signals, while at the same time inhibiting proapoptotic Ca²⁺ signals, is discussed. Moreover, this review will elucidate the recently recognized importance of the Bcl-2-IP₃R interaction in human disease.     

IP3R2 levels dictate the apoptotic sensitivity of diffuse large B-cell lymphoma cells to an IP3R-derived peptide targeting the BH4 domain of Bcl-2

Disrupting inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP₃R-derived peptide (TAT-IDP^S)) that selectively targets the BH4 domain of Bcl-2 but not that of B-cell lymphoma 2-extra large (Bcl-Xl) potentiated pro-apoptotic Ca²⁺ signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP₃R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDP^S in a more heterogeneous Bcl-2-dependent cancer model using a set of ‘primed to death'' diffuse large B-cell lymphoma (DL-BCL) cell lines containing elevated Bcl-2 levels. We discovered a large heterogeneity in the apoptotic responses of these cells to TAT-IDP^S with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDP^S to promote IP₃R-mediated Ca²⁺ release. Although total IP₃R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP₃R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDP^S-induced Ca²⁺ rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP₃R2-protein level, but not with IP₃R1-, IP₃R3- or total IP₃R-expression levels. Inhibiting or knocking down IP₃R2 activity in SU-DHL-4-reduced TAT-IDP^S-induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP₃R2 and to promote IP₃-induced pro-apoptotic Ca²⁺ signaling. Thus, certain chronically activated B-cell lymphoma cells are addicted to high Bcl-2 levels for their survival not only to neutralize pro-apoptotic Bcl-2-family members but also to suppress IP₃R hyperactivity. In particular, cancer cells expressing high levels of IP₃R2 are addicted to IP₃R/Bcl-2 complex formation and disruption of these complexes using peptide tools results in pro-apoptotic Ca²⁺ signaling and cell death.     

Modulation of Ca2+ signaling by antiapoptotic Bcl-2 versus Bcl-xL: From molecular mechanisms to relevance for cancer cell survival

Members of the Bcl-2-protein family are key controllers of apoptotic cell death. The family is divided into antiapoptotic (including Bcl-2 itself, Bcl-xL, Mcl-1, etc.) and proapoptotic members (Bax, Bak, Bim, Bim, Puma, Noxa, Bad, etc.). These proteins are well known for their canonical role in the mitochondria, where they control mitochondrial outer membrane permeabilization and subsequent apoptosis. However, several proteins are recognized as modulators of intracellular Ca²⁺ signals that originate from the endoplasmic reticulum (ER), the major intracellular Ca²⁺-storage organelle. More than 25 years ago, Bcl-2, the founding member of the family, was reported to control apoptosis through Ca²⁺ signaling. Further work elucidated that Bcl-2 directly targets and inhibits inositol 1,4,5-trisphosphate receptors (IP₃Rs), thereby suppressing proapoptotic Ca²⁺ signaling. In addition to Bcl-2, Bcl-xL was also shown to impact cell survival by sensitizing IP₃R function, thereby promoting prosurvival oscillatory Ca²⁺ release. However, new work challenges this model and demonstrates that Bcl-2 and Bcl-xL can both function as inhibitors of IP₃Rs. This suggests that, depending on the cell context, Bcl-xL could support very distinct Ca²⁺ patterns. This not only raises several questions but also opens new possibilities for the treatment of Bcl-xL-dependent cancers. In this review, we will discuss the similarities and divergences between Bcl-2 and Bcl-xL regarding Ca²⁺ homeostasis and IP₃R modulation from both a molecular and a functional point of view, with particular emphasis on cancer cell death resistance mechanisms.     

The Bcl-2 Protein Family Member Bok Binds to the Coupling Domain of Inositol 1,4,5-Trisphosphate Receptors and Protects Them from Proteolytic Cleavage

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP₃Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP₃R1 and IP₃R2, and barely to IP₃R3, and essentially all cellular Bok is IP₃R bound in cells that express substantial amounts of IP₃Rs. Binding to IP₃Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP₃Rs (amino acids 1895–1903 of IP₃R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP₃R binding, the following was observed: (i) Bok does not appear to control the ability of IP₃Rs to release ER calcium stores, (ii) Bok regulates IP₃R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP₃R degradation, and (iv) Bok protects IP₃Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP₃Rs and that the most significant consequence of this binding appears to be protection of IP₃Rs from proteolysis. Thus, Bok may govern IP₃R cleavage and activity during apoptosis.     

Bcl-xL acts as an inhibitor of IP3R channels,thereby antagonizing Ca2+-driven apoptosis

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca²⁺ dynamics by controlling IP₃ receptor (IP₃R) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IP₃Rs and preventing pro-apoptotic Ca²⁺ release and Bcl-xL sensitizing IP₃Rs to low [IP₃] and promoting pro-survival Ca²⁺ oscillations. We here demonstrate that Bcl-xL too inhibits IP₃R-mediated Ca²⁺ release by interacting with the same IP₃R regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2’s IP₃R-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IP₃R and abrogated Bcl-xL’s inhibitory effect on IP₃Rs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xL^K87D, suppressed IP₃R single-channel openings stimulated by sub-maximal and threshold [IP₃]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IP₃Rs contributes to its anti-apoptotic properties against Ca²⁺-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca²⁺ elevations in wild-type but not in IP₃R-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca²⁺ signals and cell death, while Bcl-xL^K87D was much less effective in doing so. In the absence of IP₃Rs, Bcl-xL and Bcl-xL^K87D were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IP₃R activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IP₃R-mediated Ca²⁺ release and increased the sensitivity towards STS, without altering the ER Ca²⁺ content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca²⁺-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IP₃R-mediated Ca²⁺ release and IP₃R-driven cell death. Our work further underpins that IP₃R inhibition is an integral part of Bcl-xL’s anti-apoptotic function.Subject terms: Cancer, Cell biology, Molecular biology     

The Voltage-dependent Anion Channel in Endoplasmic/Sarcoplasmic Reticulum: Characterization,Modulation and Possible Function

In recent years, it has been recognized that there is a metabolic coupling between the cytosol, ER/SR and mitochondria. In this cross-talk, mitochondrial Ca²⁺ homeostasis and ATP production and supply play a major role. The primary transporter of adenine nucleotides, Ca²⁺and other metabolites into and out of mitochondria is the voltage-dependent anion channel (VDAC) located at the outer mitochondrial membrane, at a crucial position in the cell. VDAC has been established as a key player in mitochondrial metabolite and ion signaling and it has also been proposed that VDAC is present in extramitochondrial membranes. Thus, regulation of VDAC, as the main interface between mitochondrial and cellular metabolism, by other molecules is of utmost importance. This article reviews localization and function of VDAC, and focuses on VDAC as a skeletal muscle sarcoplasmic reticulum channel. The regulation of VDAC activity by associated proteins and by inhibitors is also presented. Several aspects of the physiological relevance of VDAC to Ca²⁺ homeostasis and mitochondria-mediated apoptosis will be discussed.     

Alpha-Helical Destabilization of the Bcl-2-BH4-Domain Peptide Abolishes Its Ability to Inhibit the IP3 Receptor

The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP₃R), the primary Ca²⁺-release channel in the endoplasmic reticulum (ER). Bcl-2 can thereby reduce pro-apoptotic IP₃R-mediated Ca²⁺ release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4) has been identified as essential and sufficient for this IP₃R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP ₃R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine “hinges” replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG). By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP ₃R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP₃R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP₃R with significant pharmacological implications.     

High Bcl-2/Bax ratio in Walker tumor cells protects mitochondria but does not prevent H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced apoptosis via calcineurin pathways

It has been previously shown that Walker 256 tumor cells express a high content of the anti-apoptotic protein Bcl-2 which protects mitochondria against the damaging effects of Ca²⁺. In the present study, we analyze H₂O₂-induced apoptotic death in two different types of tumor cells: Walker 256 and SCC-25. Treatment with H₂O₂ (4mM) increased reactive oxygen species generation and the concentration of cytosolic free Ca²⁺. These alterations preceded apoptosis in both cell lines. In Walker cells, which show a high Bcl-2/Bax ratio, apoptosis was dependent on calcineurin activation and independent of changes in mitochondrial membrane potential (Δ < eqid1 > _m), as well as cytochrome c release. In contrast, in SCC-25 cells, which show a lower Bcl-2/Bax ratio, apoptosis was preceded by a decrease in Δ < eqid2 > _m, mitochondrial permeability transition, and cytochrome c release. Caspase-3 activation occurred in both cell lines. The data suggest that although the high Bcl-2/Bax ratio protected the mitochondria of Walker cells from oxidative stress, it was not sufficient to prevent apoptosis through calcineurin pathways.     

GT-repeat polymorphism in the heme oxygenase-1 gene promoter and the risk of carotid atherosclerosis related to arsenic exposure

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca²⁺ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP₃Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca²⁺ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP₃Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP₃Rs were located in the SR. [Ca²⁺]i, [Ca²⁺]_m and [Ca²⁺]_SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_i and [Ca²⁺]_m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca²⁺ release from the SR into the mitochondria through IP₃Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.     

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.     

Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca²⁺ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP₃Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca²⁺ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP₃Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP₃Rs were located in the SR. [Ca²⁺]i, [Ca²⁺]_m and [Ca²⁺]_SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_i and [Ca²⁺]_m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca²⁺ release from the SR into the mitochondria through IP₃Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.     

Disruption of the VDAC2–Bak interaction by Bcl-xS mediates efficient induction of apoptosis in melanoma cells

The proapoptotic B-cell lymphoma (Bcl)-2 protein Bcl-x_S encloses the Bcl-2 homology (BH) domains BH3 and BH4 and triggers apoptosis via the multidomain protein Bak, however, the mechanism remained elusive. For investigating Bcl-x_S efficacy and pathways, an adenoviral vector was constructed with its cDNA under tetracycline-off control. Bcl-x_S overexpression resulted in efficient apoptosis induction and caspase activation in melanoma cells. Indicative of mitochondrial apoptosis pathways, Bcl-x_S translocated to the mitochondria, disrupted the mitochondrial membrane potential and induced release of cytochrome c, apoptosis-inducing factor and second mitochondria-derived activator of caspases. In melanoma cells, Bcl-x_S resulted in significant Bak activation, and Bak knockdown as well as Bcl-x_L overexpression abrogated Bcl-x_S-induced apoptosis, whereas Mcl-1 (myeloid cell leukemia-1) knockdown resulted in a sensitization. With regard to the particular role of voltage-dependent anion channel 2 (VDAC2) for inhibition of Bak, we identified here a notable interaction between Bcl-x_S and VDAC2 in melanoma cells, which was proven in reciprocal coimmunoprecipitation analyses. On the other hand, Bcl-x_S showed no direct interaction with Bak, and its binding to VDAC2 appeared as also independent of Bak expression. Suggesting a new proapoptotic mechanism, Bcl-x_S overexpression resulted in disruption of the VDAC2–Bak interaction leading to release of Bak. Further supporting this pathway, overexpression of VDAC2 strongly decreased apoptosis by Bcl-x_S. New proapoptotic pathways are of principle interest for overcoming apoptosis deficiency of melanoma cells.     

Isoform- and Species-specific Control of Inositol 1,4,5-Trisphosphate (IP3) Receptors by Reactive Oxygen Species

Reactive oxygen species (ROS) stimulate cytoplasmic [Ca²⁺] ([Ca²⁺]_c) signaling, but the exact role of the IP₃ receptors (IP₃R) in this process remains unclear. IP₃Rs serve as a potential target of ROS produced by both ER and mitochondrial enzymes, which might locally expose IP₃Rs at the ER-mitochondrial associations. Also, IP₃Rs contain multiple reactive thiols, common molecular targets of ROS. Therefore, we have examined the effect of superoxide anion (O₂^⨪) on IP₃R-mediated Ca²⁺ signaling. In human HepG2, rat RBL-2H3, and chicken DT40 cells, we observed [Ca²⁺]_c spikes and frequency-modulated oscillations evoked by a O₂^⨪ donor, xanthine (X) + xanthine oxidase (XO), dose-dependently. The [Ca²⁺]_c signal was mediated by ER Ca²⁺ mobilization. X+XO added to permeabilized cells promoted the [Ca²⁺]_c rise evoked by submaximal doses of IP₃, indicating that O₂^⨪ directly sensitizes IP₃R-mediated Ca²⁺ release. In response to X+XO, DT40 cells lacking two of three IP₃R isoforms (DKO) expressing either type 1 (DKO1) or type 2 IP₃Rs (DKO2) showed a [Ca²⁺]_c signal, whereas DKO expressing type 3 IP₃R (DKO3) did not. By contrast, IgM that stimulates IP₃ formation, elicited a [Ca²⁺]_c signal in every DKO. X+XO also facilitated the Ca²⁺ release evoked by submaximal IP₃ in permeabilized DKO1 and DKO2 but was ineffective in DKO3 or in DT40 lacking every IP₃R (TKO). However, X+XO could also facilitate the effect of suboptimal IP₃ in TKO transfected with rat IP₃R3. Although in silico studies failed to identify a thiol missing in the chicken IP₃R3, an X+XO-induced redox change was documented only in the rat IP₃R3. Thus, ROS seem to specifically sensitize IP₃Rs through a thiol group(s) within the IP₃R, which is probably inaccessible in the chicken IP₃R3.