The BRCA1 Tumor Suppressor Binds to Inositol 1,4,5-Trisphosphate Receptors to Stimulate Apoptotic Calcium Release

The inositol 1,4,5-trisphosphate receptor (IP₃R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP₃Rs influences many signaling pathways, including those regulating apoptosis. IP₃R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP₃R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP₃R to its ligand, IP₃. BRCA1 is recruited to the ER during apoptosis in an IP₃R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.     

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.     

Haloperidol Affects Plasticity of Differentiated NG-108 Cells Through σ1R/IP<Subscript>3</Subscript>R1 Complex

Haloperidol is an antipsychotic agent that primarily acts as an antagonist of D2 dopamine receptors. Besides other receptor systems, it targets sigma 1 receptors (σ1Rs) and inositol 1,4,5-trisphosphate receptors (IP₃Rs). Aim of this work was to investigate possible changes in IP₃Rs and σ1Rs resulting from haloperidol treatment and to propose physiological consequences in differentiated NG-108 cells, i.e., effect on cellular plasticity. Haloperidol treatment resulted in up-regulation of both type 1 IP₃Rs (IP₃R1s) and σ1Rs at mRNA and protein levels. Haloperidol treatment did not alter expression of other types of IP₃Rs. Calcium release from endoplasmic reticulum (ER) mediated by increased amount of IP₃R1s elevated cytosolic calcium and generated ER stress. IP₃R1s were bound to σ1Rs, and translocation of this complex from ER to nucleus occurred in the group of cells treated with haloperidol, which was followed by increased nuclear calcium levels. Haloperidol-induced changes in cytosolic, reticular, and nuclear calcium levels were similar when specific σ1 blocker -BD 1047- was used. Changes in calcium levels in nucleus, ER, and cytoplasm might be responsible for alterations in cellular plasticity, because length of neurites increased and number of neurites decreased in haloperidol-treated differentiated NG-108 cells.     

Functional Inositol 1,4,5-Trisphosphate Receptors Assembled from Concatenated Homo- and Heteromeric Subunits

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R1, -2, and -3). Individual IP₃R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca²⁺ release from intracellular stores. IP₃R subtypes are regulated differentially by IP₃, Ca²⁺, ATP, and various other cellular factors and events. IP₃R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP₃R subtypes. When multiple subtypes of IP₃R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP₃R channels contributes to shaping the spatio-temporal properties of IP₃-mediated Ca²⁺ signals has been difficult to evaluate. To address this question, we created concatenated IP₃R linked by short flexible linkers. Dimeric constructs were expressed in DT40–3KO cells, an IP₃R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP₃R. Importantly, IP₃R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca²⁺ release. Using single channel “on-nucleus” patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP₃R1 and IP₃R2 heterodimers was dominated by IP₃R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP₃R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP₃R monomers.     

Re-evaluation of the Role of Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) in Cellular Calcium Signaling

Changes in cytoplasmic Ca²⁺ concentration, resulting from activation of intracellular Ca²⁺ channels within the endoplasmic reticulum, regulate several aspects of cellular growth and differentiation. Ca²⁺ homeostasis endoplasmic reticulum protein (CHERP) is a ubiquitously expressed protein that has been proposed as a regulator of both major families of endoplasmic reticulum Ca²⁺ channels, inositol 1,4,5-trisphosphate receptors (IP₃Rs) and ryanodine receptors (RyRs), with resulting effects on mitotic cycling. However, the manner by which CHERP regulates intracellular Ca²⁺ channels to impact cellular growth is unknown. Here, we challenge previous findings that CHERP acts as a direct cytoplasmic regulator of IP₃Rs and RyRs and propose that CHERP acts in the nucleus to impact cellular proliferation by regulating the function of the U2 snRNA spliceosomal complex. The previously reported effects of CHERP on cellular growth therefore are likely indirect effects of altered spliceosomal function, consistent with prior data showing that loss of function of U2 snRNP components can interfere with cell growth and induce cell cycle arrest.     

Identification of Functionally Critical Residues in the Channel Domain of Inositol Trisphosphate Receptors

We have combined alanine mutagenesis and functional assays to identify amino acid residues in the channel domain that are critical for inositol 1,4,5-trisphosphate receptor (IP₃R) channel function. The residues selected were highly conserved in all three IP₃R isoforms and were located in the cytosolic end of the S6 pore-lining helix and proximal portion of the C-tail. Two adjacent hydrophobic amino acids (Ile-2588 and Ile-2589) at the putative cytosolic interface of the S6 helix inactivated channel function and could be candidates for the channel gate. Of five negatively charged residues mutated, none completely eliminated channel function. Of five positively charged residues mutated, only one inactivated the channel (Arg-2596). In addition to the previously identified role of a pair of cysteines in the C-tail (Cys-2610 and Cys-2613), a pair of highly conserved histidines (His-2630 and His-2635) were also essential for channel function. Expression of the H2630A and H2635A mutants (but not R2596A) produced receptors with destabilized interactions between the N-terminal fragment and the channel domain. A previously unrecognized association between the cytosolic C-tail and the TM 4,5-loop was demonstrated using GST pulldown assays. However, none of the mutations in the C-tail interfered with this interaction or altered the ability of the C-tail to assemble into dimers. Our present findings and recent information on IP₃R structure from electron microscopy and crystallography are incorporated into a revised model of channel gating.     

Modulation of Inositol 1,4,5-Trisphosphate Receptor Type 2 Channel Activity by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Phosphorylation

InsP₃-mediated calcium release through the type 2 inositol 1,4,5-trisphosphate receptor (InsP₃R2) in cardiac myocytes results in the activation of associated CaMKII, thus enabling the kinase to act on downstream targets, such as histone deacetylases 4 and 5 (HDAC4 and HDAC5). The CaMKII activity also feedback modulates InsP₃R2 function by direct phosphorylation and results in a dramatic decrease in the receptor-channel open probability (P_o). We have identified S150 in the InsP₃R2 core suppressor domain (amino acids 1–225) as the specific residue that is phosphorylated by CaMKII. Site-directed mutagenesis reveals that S150 is the CaMKII phosphorylation site responsible for modulation of channel activity. Nonphosphorylatable (S150A) and phosphomimetic (S150E) mutations were studied in planar lipid bilayers. The InsP₃R2 S150A channel showed no decrease in activity when treated with CaMKII. Conversely, the phosphomimetic (S150E) channel displayed a very low P_o under normal recording conditions in the absence of CaMKII (2 μm InsP₃ and 250 nm [Ca²⁺]_FREE) and mimicked a WT channel that has been phosphorylated by CaMKII. Phopho-specific antibodies demonstrate that InsP₃R2 Ser-150 is phosphorylated in vivo by CaMKIIδ. The results of this study show that serine 150 of the InsP₃R2 is phosphorylated by CaMKII and results in a decrease in the channel open probability.     

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca²⁺ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP₃R), an intracellular Ca²⁺ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP₃R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2^−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca²⁺ release in intact cells and IP₃-induced Ca²⁺ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca²⁺ signal by a local Ca²⁺-induced Ca²⁺-release mechanism, which only occurred in the presence of the TRPP2-IP₃R interaction, and not via altered IP₃R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca²⁺ microdomains necessary for initiating Ca²⁺-induced Ca²⁺ release. The data strongly suggest that defects in this mechanism may account for the altered Ca²⁺ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.     

Termination of calcium puffs and coupled closings of inositol trisphosphate receptor channels

Calcium puffs are localized Ca²⁺ signals mediated by Ca²⁺ release from the endoplasmic reticulum (ER) through clusters of inositol trisphosphate receptor (IP₃R) channels. The recruitment of IP₃R channels during puffs depends on Ca²⁺-induced Ca²⁺ release, a regenerative process that must be terminated to maintain control of cell signaling and prevent Ca²⁺ cytotoxicity. Here, we studied puff termination using total internal reflection microscopy to resolve the gating of individual IP₃R channels during puffs in intact SH-SY5Y neuroblastoma cells. We find that the kinetics of IP₃R channel closing differ from that expected for independent, stochastic gating, in that multiple channels tend to remain open together longer than predicted from their individual open lifetimes and then close in near-synchrony. This behavior cannot readily be explained by previously proposed termination mechanisms, including Ca²⁺-inhibition of IP₃Rs and local depletion of Ca²⁺ in the ER lumen. Instead, we postulate that the gating of closely adjacent IP₃Rs is coupled, possibly via allosteric interactions, suggesting an important mechanism to ensure robust puff termination in addition to Ca²⁺-inactivation.     

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.     

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.     

Initiation and propagation of a neuronal intracellular calcium wave

The ability to image calcium movement within individual neurons inspires questions of functionality including whether calcium entry into the nucleus is related to genetic regulation for phenomena such as long term potentiation. Calcium waves have been initiated in hippocampal pyramidal cells with glutmatergic signals both in the presence and absence of back propagating action potentials (BPAPs). The dendritic sites of initiation of these calcium waves within about 100 μm of the soma are thought to be localized near oblique junctions. Stimulation of synapses on oblique dendrites leads to production of inositol 1,4,5-trisphosphate (IP₃) which diffuses to the apical dendrite igniting awaiting IP₃ receptors (IP₃Rs) and initiating and propagating catalytic calcium release from the endoplasmic reticulum. We construct a reduced mathematical system which accounts for calcium wave initiation and propagation due to elevated IP₃. Inhomogeneity in IP₃ distribution is responsible for calcium wave initiation versus subthreshold or spatially uniform suprathreshold activation. However, the likelihood that a calcium wave is initiated does not necessarily increase with more calcium entering from BPAPs. For low transient synaptic stimuli, timing between IP₃ generation and BPAPs is critical for calcium wave initiation. We also show that inhomogeneity in IP₃R density can account for calcium wave directionality. Simulating somatic muscarinic receptor production of IP₃, we can account for the critical difference between calcium wave entry into the soma and failure to do so.     

Selective modulation of subtype III IP3R by Akt regulates ER Ca2+ release and apoptosis

Ca²⁺ transfer from endoplasmic reticulum (ER) to mitochondria can trigger apoptotic pathways by inducing release of mitochondrial pro-apoptotic factors. Three different types of inositol 1,4,5-trisphosphate receptor (IP₃R) serve to discharge Ca²⁺ from ER, but possess some peculiarities, especially in apoptosis induction. The anti-apoptotic protein Akt can phosphorylate all IP₃R isoforms and protect cells from apoptosis, reducing ER Ca²⁺ release. However, it has not been elucidated which IP₃R subtypes mediate these effects. Here, we show that Akt activation in COS7 cells, which lack of IP₃R I, strongly suppresses IP₃-mediated Ca²⁺ release and apoptosis. Conversely, in SH-SY 5Y cells, which are type III-deficient, Akt is unable to modulate ER Ca²⁺ flux, losing its anti-apoptotic activity. In SH-SY 5Y-expressing subtype III, Akt recovers its protective function on cell death, by reduction of Ca²⁺ release. Moreover, regulating Ca²⁺ flux to mitochondria, Akt maintains the mitochondrial integrity and delays the trigger of apoptosis, in a type III-dependent mechanism. These results demonstrate a specific activity of Akt on IP₃R III, leading to diminished Ca²⁺ transfer to mitochondria and protection from apoptosis, suggesting an additional level of cell death regulation mediated by Akt.     

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.     

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.     

The IP3 receptor-mitochondria connection in apoptosis and autophagy

The amount of Ca²⁺ taken up in the mitochondrial matrix is a crucial determinant of cell fate; it plays a decisive role in the choice of the cell between life and death. The Ca²⁺ ions mainly originate from the inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores of the endoplasmic reticulum (ER). The uptake of these Ca²⁺ ions in the mitochondria depends on the functional properties and the subcellular localization of the IP₃ receptor (IP₃R) in discrete domains near the mitochondria. To allow for an efficient transfer of the Ca²⁺ ions from the ER to the mitochondria, structural interactions between IP₃Rs and mitochondria are needed. This review will focus on the key proteins involved in these interactions, how they are regulated, and what are their physiological roles in apoptosis, necrosis and autophagy. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Regulation of transient receptor potential melastatin 4 channel by sarcoplasmic reticulum inositol trisphosphate receptors: Role in human detrusor smooth muscle function

We recently reported key physiologic roles for Ca²⁺-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca²⁺-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca²⁺, inositol 1,4,5-trisphosphate receptor (IP₃R)-mediated Ca²⁺ release from the sarcoplasmic reticulum represents a potential Ca²⁺ source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP₃Rs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IP₃Rs and are activated by IP₃R-mediated Ca²⁺ release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IP₃Rs in human DSM cells. As the TRPM4 channels and IP₃Rs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca²⁺-mediated TRPM4-IP₃R regulatory mechanism. To investigate IP₃R regulation of TRPM4 channel activity, we sought to determine the consequences of IP₃R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IP₃Rs with the selective IP₃R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP₃Rs have a key role in mediating the Ca²⁺-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IP₃Rs are spatially and functionally coupled in human DSM.     

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.