Bcl-2 and IP3 compete for the ligand-binding domain of IP3Rs modulating Ca2+ signaling output.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca2+ dynamics by directly targeting and inhibiting the IP3 receptor (IP3R), a major intracellular Ca2+-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IP3R activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IP3R function provoked by low concentration of agonist or IP3, while fails to attenuate against high concentration of agonist or IP3. Surprisingly, Bcl-2 remained capable of inhibiting IP3R1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380-1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IP3R1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP3 and adenophostin A, ligands of the IP3R. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP3 to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IP3R activity at the level of the LBD. This allows for exquisite modulation of Bcl-2's inhibitory properties on IP3Rs that is tunable to the level of IP3 signaling in cells.

Fundamental insights into autosomal dominant polycystic kidney disease from human-based cell models.

Several animal- and human-derived models are used in autosomal dominant polycystic kidney disease (ADPKD) research to gain insight in the disease mechanism. However, a consistent correlation between animal and human ADPKD models is lacking. Therefore, established human-derived models are relevant to affirm research results and translate findings into a clinical set-up. In this review, we give an extensive overview of the existing human-based cell models. We discuss their source (urine, nephrectomy and stem cell), immortalisation procedures, genetic engineering, kidney segmental origin and characterisation with nephron segment markers. We summarise the most studied pathways and lessons learned from these different ADPKD models. Finally, we issue recommendations for the derivation of human-derived cell lines and for experimental set-ups with these cell lines.

Polycystin-2-dependent control of cardiomyocyte autophagy.

AIMS: Considerable evidence points to critical roles of intracellular Ca2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca2+ homeostasis and autophagy. METHODS AND RESULTS: Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca2+ chelation using BAPTA-AM, whereas removal of extracellular Ca2+ had no effect, pointing to a role of intracellular Ca2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca2+-channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca2+. Furthermore, PC2 ablation was associated with impaired Ca2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca2+ stores. Finally, we provide evidence that Ca2+-mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types. CONCLUSION: Together, this study unveils PC2 as a novel regulator of autophagy acting through control of intracellular Ca2+ homeostasis.

The BH4 domain of anti-apoptotic Bcl-XL, but not that of the related Bcl-2, limits the voltage-dependent anion channel 1 (VDAC1)-mediated transfer of pro-apoptotic Ca2+ signals to mitochondria.

Excessive Ca(2+) fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca(2+)-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP3Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca(2+)-dependent apoptosis by binding and inhibiting IP3Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP3Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca(2+) uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1(-/-) cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca(2+) uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca(2+) release in the cytosol or to prevent apoptosis, as done instead by an IP3R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca(2+) uptake into the mitochondria.

Bcl-2 binds to and inhibits ryanodine receptors.

The anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein not only counteracts apoptosis at the mitochondria by scaffolding pro-apoptotic Bcl-2-family members, but also acts at the endoplasmic reticulum, thereby controlling intracellular Ca(2+) dynamics. Bcl-2 inhibits Ca(2+) release by targeting the inositol 1,4,5-trisphosphate receptor (IP3R). Sequence analysis has revealed that the Bcl-2-binding site on the IP3R displays strong similarity with a conserved sequence present in all three ryanodine receptor (RyR) isoforms. We now report that Bcl-2 co-immunoprecipitated with RyRs in ectopic expression systems and in native rat hippocampi, indicating that endogenous RyR-Bcl-2 complexes exist. Purified RyR domains containing the putative Bcl-2-binding site bound full-length Bcl-2 in pulldown experiments and interacted with the BH4 domain of Bcl-2 in surface plasmon resonance (SPR) experiments, suggesting a direct interaction. Exogenous expression of full-length Bcl-2 or electroporation loading of the BH4 domain of Bcl-2 dampened RyR-mediated Ca(2+) release in HEK293 cell models. Finally, introducing the BH4-domain peptide into hippocampal neurons through a patch pipette decreased RyR-mediated Ca(2+) release. In conclusion, this study identifies Bcl-2 as a new inhibitor of RyR-based intracellular Ca(2+)-release channels.

Altered mTOR signalling in nephropathic cystinosis.

Lysosomes play a central role in regulating autophagy via activation of mammalian target of rapamycin complex 1 (mTORC1). We examined mTORC1 signalling in the lysosomal storage disease nephropathic cystinosis (MIM 219800), in which accumulation of autophagy markers has been previously demonstrated. Cystinosis is caused by mutations in the lysosomal cystine transporter cystinosin and initially affects kidney proximal tubules causing renal Fanconi syndrome, followed by a gradual development of end-stage renal disease and extrarenal complications. Using proximal tubular kidney cells obtained from healthy donors and from cystinotic patients, we demonstrate that cystinosin deficiency is associated with a perturbed mTORC1 signalling, delayed reactivation of mTORC1 after starvation and abnormal lysosomal retention of mTOR during starvation. These effects could not be reversed by treatment with cystine-depleting drug cysteamine. Altered mTORC1 signalling can contribute to the development of proximal tubular dysfunction in cystinosis and points to new possibilities in therapeutic intervention through modulation of mTORC-dependent signalling cascades.

Autophagy in renal diseases.

Autophagy is the cell biology process in which cytoplasmic components are degraded in lysosomes to maintain cellular homeostasis and energy production. In the healthy kidney, autophagy plays an important role in the homeostasis and viability of renal cells such as podocytes and tubular epithelial cells and of immune cells. Recently, evidence is mounting that (dys)regulation of autophagy is implicated in the pathogenesis of various renal diseases, and might be an attractive target for new renoprotective therapies. In this review, we provide an overview of the role of autophagy in kidney physiology and kidney diseases.

Targeting Bcl-2-IP3 receptor interaction to reverse Bcl-2's inhibition of apoptotic calcium signals.

The antiapoptotic protein Bcl-2 inhibits Ca2+ release from the endoplasmic reticulum (ER). One proposed mechanism involves an interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel localized with Bcl-2 on the ER. Here we document Bcl-2-IP3R interaction within cells by FRET and identify a Bcl-2 interacting region in the regulatory and coupling domain of the IP3R. A peptide based on this IP3R sequence displaced Bcl-2 from the IP3R and reversed Bcl-2-mediated inhibition of IP3R channel activity in vitro, IP3-induced ER Ca2+ release in permeabilized cells, and cell-permeable IP3 ester-induced Ca2+ elevation in intact cells. This peptide also reversed Bcl-2's inhibition of T cell receptor-induced Ca2+ elevation and apoptosis. Thus, the interaction of Bcl-2 with IP3Rs contributes to the regulation of proapoptotic Ca2+ signals by Bcl-2, suggesting the Bcl-2-IP3R interaction as a potential therapeutic target in diseases associated with Bcl-2's inhibition of cell death.

Inositol 1,4,5-trisphosphate receptor-isoform diversity in cell death and survival.

Cell-death and -survival decisions are critically controlled by intracellular Ca(2+) homeostasis and dynamics at the level of the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) play a pivotal role in these processes by mediating Ca(2+) flux from the ER into the cytosol and mitochondria. Hence, it is clear that many pro-survival and pro-death signaling pathways and proteins affect Ca(2+) signaling by directly targeting IP3R channels, which can happen in an IP3R-isoform-dependent manner. In this review, we will focus on how the different IP3R isoforms (IP3R1, IP3R2 and IP3R3) control cell death and survival. First, we will present an overview of the isoform-specific regulation of IP3Rs by cellular factors like IP3, Ca(2+), Ca(2+)-binding proteins, adenosine triphosphate (ATP), thiol modification, phosphorylation and interacting proteins, and of IP3R-isoform specific expression patterns. Second, we will discuss the role of the ER as a Ca(2+) store in cell death and survival and how IP3Rs and pro-survival/pro-death proteins can modulate the basal ER Ca(2+) leak. Third, we will review the regulation of the Ca(2+)-flux properties of the IP3R isoforms by the ER-resident and by the cytoplasmic proteins involved in cell death and survival as well as by redox regulation. Hence, we aim to highlight the specific roles of the various IP3R isoforms in cell-death and -survival signaling. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

A dual role for the anti-apoptotic Bcl-2 protein in cancer: mitochondria versus endoplasmic reticulum.

Anti-apoptotic Bcl-2 contributes to cancer formation and progression by promoting the survival of altered cells. Hence, it is a prime target for novel specific anti-cancer therapeutics. In addition to its canonical anti-apoptotic role, Bcl-2 has an inhibitory effect on cell-cycle progression. Bcl-2 acts at two different intracellular compartments, the mitochondria and the endoplasmic reticulum (ER). At the mitochondria, Bcl-2 via its hydrophobic cleft scaffolds the Bcl-2-homology (BH) domain 3 (BH3) of pro-apoptotic Bcl-2-family members. Small molecules (like BH3 mimetics) can disrupt this interaction, resulting in apoptotic cell death in cancer cells. At the ER, Bcl-2 modulates Ca(2+) signaling, thereby promoting proliferation while increasing resistance to apoptosis. Bcl-2 at the ER acts via its N-terminal BH4 domain, which directly binds and inhibits the inositol 1,4,5-trisphosphate receptor (IP3R), the main intracellular Ca(2+)-release channel. Tools targeting the BH4 domain of Bcl-2 reverse Bcl-2's inhibitory action on IP3Rs and trigger pro-apoptotic Ca(2+) signaling in cancer B-cells, including chronic lymphocytic leukemia (CLL) cells and diffuse large B-cell lymphoma (DLBCL) cells. The sensitivity of DLBCL cells to BH4-domain targeting tools strongly correlated with the expression levels of the IP3R2 channel, the IP3R isoform with the highest affinity for IP3. Interestingly, bio-informatic analysis of a database of primary CLL patient cells also revealed a transcriptional upregulation of IP3R2. Finally, this review proposes a model, in which cancer cell survival depends on Bcl-2 at the mitochondria and/or the ER. This dependence likely will have an impact on their responses to BH3-mimetic drugs and BH4-domain targeting tools. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Regulation of inositol 1,4,5-trisphosphate receptors during endoplasmic reticulum stress.

The endoplasmic reticulum (ER) performs multiple functions in the cell: it is the major site of protein and lipid synthesis as well as the most important intracellular Ca(2+) reservoir. Adverse conditions, including a decrease in the ER Ca(2+) level or an increase in oxidative stress, impair the formation of new proteins, resulting in ER stress. The subsequent unfolded protein response (UPR) is a cellular attempt to lower the burden on the ER and to restore ER homeostasis by imposing a general arrest in protein synthesis, upregulating chaperone proteins and degrading misfolded proteins. This response can also lead to autophagy and, if the stress can not be alleviated, to apoptosis. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and IP3-induced Ca(2+) signaling are important players in these processes. Not only is the IP3R activity modulated in a dual way during ER stress, but also other key proteins involved in Ca(2+) signaling are modulated. Changes also occur at the structural level with a strengthening of the contacts between the ER and the mitochondria, which are important determinants of mitochondrial Ca(2+) uptake. The resulting cytoplasmic and mitochondrial Ca(2+) signals will control cellular decisions that either promote cell survival or cause their elimination via apoptosis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Polycystin-1 but not polycystin-2 deficiency causes upregulation of the mTOR pathway and can be synergistically targeted with rapamycin and metformin.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss-of-function mutations in either PKD1 or PKD2 genes, which encode polycystin-1 (TRPP1) and polycystin-2 (TRPP2), respectively. Increased activity of the mammalian target of rapamycin (mTOR) pathway has been shown in PKD1 mutants but is less documented for PKD2 mutants. Clinical trials using mTOR inhibitors were disappointing, while the AMP-activated kinase (AMPK) activator, metformin is not yet tested in patients. Here, we studied the mTOR activity and its upstream pathways in several human and mouse renal cell models with either siRNA or stable knockdown and with overexpression of TRPP2. Our data reveal for the first time differences between TRPP1 and TRPP2 deficiency. In contrast to TRPP1 deficiency, TRPP2-deficient cells did neither display excessive activation of the mTOR-kinase complex nor inhibition of AMPK activity, while ERK1/2 and Akt activity were similarly affected among TRPP1- and TRPP2-deficient cells. Furthermore, cell proliferation was more pronounced in TRPP1 than in TRPP2-deficient cells. Interestingly, combining low concentrations of rapamycin and metformin was more effective for inhibiting mTOR complex 1 activity in TRPP1-deficient cells than either drug alone. Our results demonstrate a synergistic effect of a combination of low concentrations of drugs suppressing the increased mTOR activity in TRPP1-deficient cells. This novel insight can be exploited in future clinical trials to optimize the efficiency and avoiding side effects of drugs in the treatment of ADPKD patients with PKD1 mutations. Furthermore, as TRPP2 deficiency by itself did not affect mTOR signaling, this may underlie the differences in phenotype, and genetic testing has to be considered for selecting patients for the ongoing trials.

The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore.

Bax inhibitor-1 (BI-1) is a multitransmembrane domain-spanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca(2+) homeostasis by acting as a Ca(2+)-leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca(2+) pore responsible for its Ca(2+)-leak properties. We utilized a set of C-terminal peptides to screen for Ca(2+) leak activity in unidirectional (45)Ca(2+)-flux experiments and identified an α-helical 20-amino acid peptide causing Ca(2+) leak from the ER. The Ca(2+) leak was independent of endogenous ER Ca(2+)-release channels or other Ca(2+)-leak mechanisms, namely translocons and presenilins. The Ca(2+)-permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca(2+)-leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca(2+)-leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca(2+)-channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca(2+) leak. Thus, we elucidated residues critically important for BI-1-mediated Ca(2+) leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca(2+)-leak properties of BI-1 are an added function during evolution.

Induction of Ca²+-driven apoptosis in chronic lymphocytic leukemia cells by peptide-mediated disruption of Bcl-2-IP3 receptor interaction.

Bcl-2 contributes to the pathophysiology and therapeutic resistance of chronic lymphocytic leukemia (CLL). Therefore, developing inhibitors of this protein based on a thorough understanding of its mechanism of action is an active and promising area of inquiry. One approach centers on agents (eg, ABT-737) that compete with proapoptotic members of the Bcl-2 protein family for binding in the hydrophobic groove formed by the BH1-BH3 domains of Bcl-2. Another region of Bcl-2, the BH4 domain, also contributes to the antiapoptotic activity of Bcl-2 by binding to the inositol 1,4,5-trisphosphate receptor (IP3R) Ca²(+) channel, inhibiting IP(3)-dependent Ca²(+) release from the endoplasmic reticulum. We report that a novel synthetic peptide, modeled after the Bcl-2-interacting site on the IP3R, binds to the BH4 domain of Bcl-2 and functions as a competitive inhibitor of the Bcl-2-IP3R interaction. By disrupting the Bcl-2-IP3R interaction, this peptide induces an IP3R-dependent Ca²(+) elevation in lymphoma and leukemia cell lines and in primary CLL cells. The Ca²(+) elevation evoked by this peptide induces apoptosis in CLL cells, but not in normal peripheral blood lymphocytes, suggesting the involvement of the Bcl-2-IP3R interaction in the molecular mechanism of CLL and indicating the potential merit of targeting this interaction therapeutically.

The IP(3) receptor-mitochondria connection in apoptosis and autophagy.

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.

F508del-CFTR increases intracellular Ca(2+) signaling that causes enhanced calcium-dependent Cl(-) conductance in cystic fibrosis.

In many cells, increase in intracellular calcium ([Ca(2+)](i)) activates a Ca(2+)-dependent chloride (Cl(-)) conductance (CaCC). CaCC is enhanced in cystic fibrosis (CF) epithelial cells lacking Cl(-) transport by the CF transmembrane conductance regulator (CFTR). Here, we show that in freshly isolated nasal epithelial cells of F508del-homozygous CF patients, expression of TMEM16A and bestrophin 1 was unchanged. However, calcium signaling was strongly enhanced after induction of expression of F508del-CFTR, which is unable to exit the endoplasmic reticulum (ER). Since receptor-mediated [Ca(2+)](i) increase is Cl(-) dependent, we suggested that F508del-CFTR may function as an ER chloride counter-ion channel for Ca(2+). This was confirmed by expression of the double mutant F508del/G551D-CFTR, which remained in the ER but had no effects on [Ca(2+)](i). Moreover, F508del-CFTR could serve as a scavenger for inositol-1,4,5-trisphosphate [IP3] receptor binding protein released with IP(3) (IRBIT). Our data may explain how ER-localized F508del-CFTR controls intracellular Ca(2+) signaling.

Profiling of the Bcl-2/Bcl-X(L)-binding sites on type 1 IP(3) receptor.

Several members of the anti-apoptotic Bcl-2-protein family, including Bcl-2, Bcl-X(L) and Mcl-1, directly bind and regulate the inositol 1,4,5-trisphosphate receptor (IP(3)R), one of the two main intracellular Ca(2+)-release channel types present in the endoplasmic reticulum. However, the molecular determinants underlying their binding to the IP(3)R remained a matter of debate. One interaction site for Bcl-2 was proposed in the central part of the modulatory domain [Y.P. Rong, A.S. Aromolaran, G. Bultynck, F. Zhong, X. Li, K. McColl, S. Matsuyama, S. Herlitze, H.L. Roderick, M.D. Bootman, G.A. Mignery, J.B. Parys, H. De Smedt, C.W. Distelhorst, Targeting Bcl-2-IP3 receptor interaction to reverse Bcl-2's inhibition of apoptotic calcium signals, Mol. Cell 31 (2008) 255-265] and another site in the C-terminal domain of the IP(3)R encompassing the sixth transmembrane domain, to which Bcl-2, Bcl-X(L) and Mcl-1 can bind [E.F. Eckenrode, J. Yang, G.V. Velmurugan, J.K. Foskett, C. White, Apoptosis protection by Mcl-1 and Bcl-2 modulation of inositol 1,4,5-trisphosphate receptor-dependent Ca(2+) signaling, J. Biol. Chem. 285 (2010) 13678-13684]. Here, we investigated and compared the binding of Bcl-2 and Bcl-X(L) to both sites. Two different IP(3)R domains were used for the C-terminal site: one lacking and one containing the sixth transmembrane domain. Our results show that elements preceding the C-terminal cytosolic tail located at the sixth transmembrane domain of IP(3)R1 were critical for recruiting both Bcl-2 and Bcl-X(L) to the C-terminal part of the IP(3)R. Furthermore, consistent with our previous observations, Bcl-X(L) bound with higher efficiency to the C-terminal part of the IP(3)R and to a much lesser extent to the central, modulatory domain, while Bcl-2 targeted both sites with similar efficiencies. In conclusion, IP(3)R harbors two different binding sites for anti-apoptotic Bcl-2 proteins, one in the central, modulatory domain and one in the C-terminal domain near the Ca(2+)-channel pore.

Inositol 1,4,5-trisphosphate and its receptors.

Activation of cells by many extracellular agonists leads to the production of inositol 1,4,5-trisphosphate (IP3). IP3 is a global messenger that easily diffuses in the cytosol. Its receptor (IP3R) is a Ca(2+)-release channel located on intracellular membranes, especially the endoplasmic reticulum (ER). The IP3R has an affinity for IP(3) in the low nanomolar range. A prime regulator of the IP3R is the Ca(2+) ion itself. Cytosolic Ca(2+) is considered as a co-agonist of the IP3R, as it strongly increases IP(3)R activity at concentrations up to about 300 nM. In contrast, at higher concentrations, cytosolic Ca(2+) inhibits the IP3R. Also the luminal Ca(2+) sensitizes the IP3R. In higher organisms three genes encode for an IP3R and additional diversity exists as a result of alternative splicing mechanisms and the formation of homo- and heterotetramers. The various IP3R isoforms have a similar structure and a similar function, but due to differences in their affinity for IP3, their variable sensitivity to regulatory parameters, their differential interaction with associated proteins, and the variation in their subcellular localization, they participate differently in the formation of intracellular Ca(2+) signals and this affects therefore the physiological consequences of these signals.

The BH4 domain of Bcl-2 inhibits ER calcium release and apoptosis by binding the regulatory and coupling domain of the IP3 receptor.

Although the presence of a BH4 domain distinguishes the antiapoptotic protein Bcl-2 from its proapoptotic relatives, little is known about its function. BH4 deletion converts Bcl-2 into a proapoptotic protein, whereas a TAT-BH4 fusion peptide inhibits apoptosis and improves survival in models of disease due to accelerated apoptosis. Thus, the BH4 domain has antiapoptotic activity independent of full-length Bcl-2. Here we report that the BH4 domain mediates interaction of Bcl-2 with the inositol 1,4,5-trisphosphate (IP3) receptor, an IP3-gated Ca(2+) channel on the endoplasmic reticulum (ER). BH4 peptide binds to the regulatory and coupling domain of the IP3 receptor and inhibits IP3-dependent channel opening, Ca(2+) release from the ER, and Ca(2+)-mediated apoptosis. A peptide inhibitor of Bcl-2-IP3 receptor interaction prevents these BH4-mediated effects. By inhibiting proapoptotic Ca(2+) signals at their point of origin, the Bcl-2 BH4 domain has the facility to block diverse pathways through which Ca(2+) induces apoptosis.

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 Ca(2+) signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP(3)R), an intracellular Ca(2+) 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 IP(3)R 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 Ca(2+) release in intact cells and IP(3)-induced Ca(2+) release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca(2+) signal by a local Ca(2+)-induced Ca(2+)-release mechanism, which only occurred in the presence of the TRPP2-IP(3)R interaction, and not via altered IP(3)R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca(2+) microdomains necessary for initiating Ca(2+)-induced Ca(2+) release. The data strongly suggest that defects in this mechanism may account for the altered Ca(2+) signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.