Antiapoptotic B-cell lymphoma 2 (Bcl-2) targets the inositol 1,4,5-trisphosphate receptor (IP3R) via its BH4 domain, thereby suppressing IP3R Ca2+-flux properties and protecting against Ca2+-dependent apoptosis. Here, we directly compared IP3R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound the modulatory domain of IP3R nor inhibited IP3-induced Ca2+ release (IICR) in permeabilized and intact cells. We identified a critical residue in BH4-Bcl-2 (Lys17) not conserved in BH4-Bcl-Xl (Asp11). Changing Lys17 into Asp in BH4-Bcl-2 completely abolished its IP3R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP3R binding and inhibition. This difference in IP3R regulation between BH4-Bcl-2 and BH4-Bcl-Xl controls their antiapoptotic action. Although both BH4-Bcl-2 and BH4-Bcl-Xl had antiapoptotic activity, BH4-Bcl-2 was more potent than BH4-Bcl-Xl. The effect of BH4-Bcl-2, but not of BH4-Bcl-Xl, depended on its binding to IP3Rs. In agreement with the IP3R-binding properties, the antiapoptotic activity of BH4-Bcl-2 and BH4-Bcl-Xl was modulated by the Lys/Asp substitutions. Changing Lys17 into Asp in full-length Bcl-2 significantly decreased its binding to the IP3R, its ability to inhibit IICR and its protection against apoptotic stimuli. A single amino-acid difference between BH4-Bcl-2 and BH4-Bcl-Xl therefore underlies differential regulation of IP3Rs and Ca2+-driven apoptosis by these functional domains. Mutating this residue affects the function of Bcl-2 in Ca2+ signaling and apoptosis.

The amount of Ca(2+) 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 Ca(2+) ions mainly originate from the inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) stores of the endoplasmic reticulum (ER). The uptake of these Ca(2+) ions in the mitochondria depends on the functional properties and the subcellular localization of the IP(3) receptor (IP(3)R) in discrete domains near the mitochondria. To allow for an efficient transfer of the Ca(2+) ions from the ER to the mitochondria, structural interactions between IP(3)Rs 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.

IntroductionThe Bcl-2 protein contributes to the pathophysiology of cancer and the resistance of cancer to therapeutic agents by virtue of its ability to inhibit apoptosis. 1,2 The Bcl-2-positive lymphoid malignancies follicular lymphoma and chronic lymphocytic leukemia (CLL) are prime examples. They are associated with an elevation of Bcl-2 because of the t(14;18) chromosomal translocation in follicular lymphoma 3 and the loss of miR-15a and miR16-1 in CLL. 4,5 Cure of these malignancies is infrequently achieved with current therapeutic modalities, and thus a major challenge remains to develop new treatment modalities based on an understanding of the fundamental disease mechanisms. 6 Bcl-2 blocks apoptosis in part by binding its proapoptotic relatives, thus preserving mitochondrial integrity and preventing cytochrome c release. 7,8 Therefore, considerable investment has been made in the development of novel therapeutic agents, such as , that disrupt the inhibitory interaction of Bcl-2 with its proapoptotic relatives. 1,2,9-11 Several of these agents are currently undergoing clinical testing. However, Bcl-2 also inhibits apoptosis by regulating the release of Ca 2ϩ from the endoplasmic reticulum (ER). [12][13][14] This recently characterized mechanism involves a physical interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP 3 R) Ca 2ϩ -release channel on the ER. Through this interaction, Bcl-2 prevents cytoplasmic Ca 2ϩ elevation sufficient to trigger apoptosis. However, the potential contribution of the Bcl-2-IP 3 R interaction to the survival of CLL has not been investigated, so the opportunity for targeting this interaction therapeutically has not yet been realized.The IP 3 R is an IP 3 -gated Ca 2ϩ channel that is highly conserved, represented by 3 isoforms, and present in virtually all cell types. 15,16 IP 3 -dependent release of Ca 2ϩ from the ER into the cytoplasm produces Ca 2ϩ signals, generally in the form of Ca 2ϩ oscillations, which govern diverse cellular functions including cell proliferation and survival. 17,18 Ca 2ϩ oscillations support cell survival in part by positively regulating mitochondrial metabolism, but sustained high-amplitude elevations of Ca 2ϩ induce mitochondrial Ca 2ϩ overload and apoptosis. [19][20][21] Bcl-2 inhibits high-amplitude, proapoptotic Ca 2ϩ elevation but does not interfere with physiologic Ca 2ϩ oscillations. 22 In fact, under certain circumstances Bcl-2 and its homolog Bcl-xl enhance Ca 2ϩ oscillations, [22][23][24][25][26] and through this mechanism are predicted to promote efficient mitochondrial bioenergetics. 27 Thus, Bcl-2 supports cell survival both by enhancing physiologic Ca 2ϩ signals and by blocking proapoptotic Ca 2ϩ elevation.A major focus of our work has been to understand how Bcl-2 inhibits proapoptotic elevation of Ca 2ϩ based on evidence that Bcl-2 binds to the IP 3 R and thus inhibits ER Ca 2ϩ release. [28][29][30][31][32] Although this interaction has been mainly detected in cell extracts by coimmunoprecipitation or blue native gel ...

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-IDPS)) 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-IDPS 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-IDPS with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDPS 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-IDPS-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-IDPS-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.

Background: VDAC1 mediates the transfer of pro-apoptotic Ca 2ϩ signals into mitochondria. Results: The BH4 domain of Bcl-XL, but not that of Bcl-2, targets VDAC1 and suppresses its pro-apoptotic Ca 2ϩ -flux properties. N-terminal VDAC1 peptide alleviates this effect of BH4-Bcl-XL. Conclusion: Bcl-XL via its BH4 domain inhibits VDAC1 activity. Significance: Bcl-2 and Bcl-XL differ in their BH4 domain biology by regulating ER and mitochondrial Ca 2ϩ -transport systems, respectively.

The tight interplay between endoplasmic-reticulum-(ER-) and mitochondria-mediated Ca2+ signaling is a key determinant of cellular health and cellular fate through the control of apoptosis and autophagy. Proteins that prevent or promote apoptosis and autophagy can affect intracellular Ca2+ dynamics and homeostasis through binding and modulation of the intracellular Ca2+-release and Ca2+-uptake mechanisms. During aging, oxidative stress becomes an additional factor that affects ER and mitochondrial function and thus their role in Ca2+ signaling. Importantly, mitochondrial dysfunction and sustained mitochondrial damage are likely to underlie part of the aging process. In this paper, we will discuss the different mechanisms that control intracellular Ca2+ signaling with respect to apoptosis and autophagy and review how these processes are affected during aging through accumulation of reactive oxygen species.

The anti-apoptotic Bcl-2 protein is emerging as an efficient inhibitor of IP3R function, contributing to its oncogenic properties. Yet, the underlying molecular mechanisms remain not fully understood. Using mutations or pharmacological inhibition to antagonize Bcl-2's hydrophobic cleft, we excluded this functional domain as responsible for Bcl-2-mediated IP3Rs inhibition. In contrast, the deletion of the C-terminus, containing the trans-membrane domain, which is only present in Bcl-2α, but not in Bcl-2β, led to impaired inhibition of IP3R-mediated Ca2+ release and staurosporine-induced apoptosis. Strikingly, the trans-membrane domain was sufficient for IP3R binding and inhibition. We therefore propose a novel model, in which the Bcl-2's C-terminus serves as a functional anchor, which beyond mere ER-membrane targeting, underlies efficient IP3R inhibition by (i) positioning the BH4 domain in the close proximity of its binding site on IP3R, thus facilitating their interaction; (ii) inhibiting IP3R-channel openings through a direct interaction with the C-terminal region of the channel downstream of the channel-pore. Finally, since the hydrophobic cleft of Bcl-2 was not involved in IP3R suppression, our findings indicate that ABT-199 does not interfere with IP3R regulation by Bcl-2 and its mechanism of action as a cell-death therapeutic in cancer cells likely does not involve Ca2+ signaling.

A distinctive feature in the topographic organization of the olfactory system in mammals is the dual function of the odorant receptor (OR): it detects odors in the nasal epithelium and plays an instructive role in the axonal convergence of olfactory sensory neurons (OSN) into the olfactory bulb (OB). The latter function is supported by genetic experiments and by the expression of the OR not only on the cilia, but also on the axon termini of the OSN. The signaling pathway coupled to the OR on the cilia is well known and is recognized to involve cAMP and Ca 2؉ , whereas, until now, nothing was known on the functional characteristics of the OR on the axon termini-growth cone. Here, by analyzing the spatiotemporal dynamics of cAMP and Ca 2؉ in living OSN in vitro and in situ, we found that the OR at the growth cone is capable of binding odors and is coupled to cAMP synthesis and Ca 2؉ influx through cyclic nucleotide gated (CNG) channels. Furthermore we found that selective odor activation of the OR on the growth cone is followed by nuclear translocation of protein kinase A catalytic subunit. These results define the functional properties of the OR on the growth cone and suggest a potential role of OR activation in axonal convergence and sensory map formation.olfactory sensory neurons ͉ real-time imaging ͉ second messengers I n sensory systems, neurons in the peripheral sheet send their axons in precise loci of the CNS to create an internal representation of the external world. The spatial segregation of afferent inputs from primary sensory neurons provides a topographic map that defines the quality and the location of the stimulus. In the olfactory system, each OSN expresses only one OR gene out of a repertoire of approximately 1000 (1). OSNs expressing different ORs are randomly dispersed in the nasal epithelium. However, spatial order is achieved in the olfactory bulb (OB), where OSNs expressing the same odorant receptor converge with exquisite precision to form glomeruli both on the lateral and the medial side of each OB. Each odor is thus encoded by a specific spatial pattern of activated glomeruli. The OR, a G-protein-coupled receptor, upon binding of odorant ligands at the cilia, activates a specific G protein, Golf, that stimulates adenylyl cyclase, AC, to synthesize cAMP. The cAMP then directly activates cyclic nucleotide gated (CNG) channels, leading to action potential generation (2, 3).A unique feature in the topographic organization of the OB is the ''dual role'' of the OR. Although it is well established that the OR is involved in the transduction of chemical signals (odors) at the cilia level, a number of evidence suggests that this receptor plays also an instructive role in glomerular convergence of OSN axons to the bulb (4-6). The latter property is supported by genetic observations demonstrating that alterations of OR sequence perturb the sensory map (7-9) and by the demonstration that the OR is expressed not only at the cilia but also on the OSN axon termini (10, 11). The OR is not the only determ...