Antiapoptotic B-cell lymphoma 2 (Bcl-2) targets the inositol 1,4,5-trisphosphate receptor (IP sub(3)R) via its BH4 domain, thereby suppressing IP sub(3)R Ca super(2+)-flux properties and protecting ...against Ca super(2+)-dependent apoptosis. Here, we directly compared IP sub(3)R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound the modulatory domain of IP sub(3)R nor inhibited IP sub(3)-induced Ca super(2+) 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 IP sub(3)R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP sub(3)R binding and inhibition. This difference in IP sub(3)R 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 IP sub(3)Rs. In agreement with the IP sub(3)R-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 IP sub(3)R, 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 IP sub(3)Rs and Ca super(2+)-driven apoptosis by these functional domains. Mutating this residue affects the function of Bcl-2 in Ca super(2+) signaling and apoptosis.
The type-3 inositol 1,4,5-trisphosphate (IP3) receptor, in contrast to the type-1 IP3 receptor (IP3R), is not stimulated by sulfhydryl oxidation and is less sensitive to adenosine 5'-triphosphate. In ...the present study we compared the effect of pH on the Ca2+ release induced by IP3 and cytosolic Ca2+ between IP3R3-expressing 16HBE14o- cells and IP3R1-expressing A7r5 cells. Changing pH from 6.8 to 7.5 decreased the IP3 concentration required for half-maximal stimulation of IP3R3 (EC50) 10.7-fold (from 2.14 to 0.20 microM). Similar alkalinization decreased the IP3 concentration (EC50) for stimulation of IP3R1 only 2.5-fold (from 0.87 to 0.35 microM). IP3R1 is therefore the more sensitive isoform at pH 6.8, while IP3R3 is more sensitive at pH 7.5. Stimulation and inhibition of IP3R1 and -3 by low and high cytosolic Ca2+ respectively was observed at both pH 6.8 and 7.5. Increasing H+ shifted the Ca2+-activation curve of IP3R1 towards higher Ca2+ but did not affect the Ca2+ dependence of IP3R3. We conclude that IP3R1 and -3 differ markedly in their response to protons.
Calmodulin (CaM) is a ubiquitous Ca 2+ -sensor protein that plays an important role in regulating a large number of Ca 2+ channels, including the inositol 1,4,5-trisphosphate receptor (IP 3 R). CaM ...binds to the IP 3 R at Ca 2+ -dependent as well as at Ca 2+ -independent interaction sites. In this study, we have investigated the Ca 2+ -independent CaM-binding site for its role in the regulation of the Ca 2+ -dependent bell-shaped activation curve of the IP 3 R. Suramin, a polysulfonated napthylurea, displaced CaM in both the presence and the absence of Ca 2+ . Suramin competed with CaM for binding to different peptides representing the previously identified CaM-binding sites on
IP 3 R1. By interacting with the N-terminal Ca 2+ -independent CaM-binding site, suramin mimicked the functional effect of CaM and induced an allosteric but competitive inhibition
of IP 3 binding. Therefore, suramin also potently inhibited IP 3 -induced Ca 2+ release (IICR) from permeabilized cells predominantly expressing IP 3 R1 (L15 fibroblasts) or IP 3 R3 (Lvec fibroblasts), even though the IP 3 R3 does not contain Ca 2+ -dependent CaM-binding sites. Furthermore, we have found that CaM 1234 , a CaM mutated in its four EF hands, inhibited IICR in a Ca 2+ -dependent way with the same potency as CaM. We conclude that CaM inhibits IICR via the N-terminal binding site. The inhibition
requires Ca 2+ but CaM itself is not the Ca 2+ sensor for the inhibition of the IP 3 R.
The Ca(2+)- and calmodulin-dependent phosphatase calcineurin was reported to interact with the inositol 1,4,5-trisphosphate receptor (IP(3)R) and the ryanodine receptor (RyR) and to modulate their ...phosphorylation status and activity. However, controversial data on the molecular mechanisms involved and on the functional relevance of calcineurin for these channel-complexes have been described. Hence, we will focus on the functional importance of calcineurin for IP(3)R and RyR function and on the different mechanisms by which Ca(2+)-dependent dephosphorylation can affect the gating of those intracellular Ca(2+)-release channels. Since many studies made use of immunosuppressive drugs that are inhibiting calcineurin activity, we will also have to take the different side effects of these drugs into account for the proper interpretation of the effects of calcineurin on intracellular Ca(2+)-release channels. In addition, it became recently known that various other phosphatases and kinases can associate with these channels, thereby forming macromolecular complexes. The relevance of these enzymes for IP(3)R and RyR functioning will be reviewed since in some cases they could interfere with the effects ascribed to calcineurin. Finally, we will discuss the downstream effects of calcineurin on the regulation of the expression levels of intracellular Ca(2+)-release channels as well as the relation between IP(3)R- and RyR-mediated Ca(2+) release and calcineurin-dependent gene expression.
Disrupting inositol 1,4,5-trisphosphate (IP sub(3)) receptor (IP sub(3)R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP sub(3)R-derived peptide (TAT-IDP ...super(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 Ca super(2+) signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP sub(3)R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDP super(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 super(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 super(S) to promote IP sub(3)R-mediated Ca super(2+) release. Although total IP sub(3)R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP sub(3)R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDP super(S)-induced Ca super(2+) rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP sub(3)R2-protein level, but not with IP sub(3)R1-, IP sub(3)R3- or total IP sub(3)R-expression levels. Inhibiting or knocking down IP sub(3)R2 activity in SU-DHL-4-reduced TAT-IDP super(S)-induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP sub(3)R2 and to promote IP sub(3)-induced pro-apoptotic Ca super(2+) 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 IP sub(3)R hyperactivity. In particular, cancer cells expressing high levels of IP sub(3)R2 are addicted to IP sub(3)R/Bcl-2 complex formation and disruption of these complexes using peptide tools results in pro-apoptotic Ca super(2+) signaling and cell death.
The N-terminal 1–225 amino acids (aa) of the type 1 inositol 1,4,5-trisphosphate receptor (IP
3R1) function as a suppressor/coupling domain. In this study we used IP
3R-deficient B-lymphocytes to ...investigate the effects of modifications in this domain on IP
3 binding and Ca
2+-release activity. Although the N-terminal 1–225 aa of IP
3R3 had the same role as in IP
3R1, the suppression of IP
3 binding for IP
3R1 was lost when the suppressor/coupling domains were exchanged between the two isoforms. Resulting chimeric receptors showed a higher sensitivity to IP
3-induced activation (IICR). Deletion of 11 aa in IP
3R1 (Δ76–86-IP
3R1) or replacing aa 76–86 of the IP
3R1 in the suppressor/coupling domain by 13 aa of IP
3R3 (75–87 T3-IP
3R1) also resulted in increased IP
3 binding and sensitivity of IICR. These residues constitute the only part of the suppressor/coupling domain that is strikingly different between the two isoforms. Expression of Δ76–86-IP
3R1 and of 75–87 T3-IP
3R1 increased the propensity of cells to undergo staurosporine-induced apoptosis, but had no effect on the Ca
2+ content in the endoplasmic reticulum. In the cell model used, our observations suggest that the sensitivity of the Ca
2+-release activity of IP
3R1 to IP
3 influences the sensitivity of the cells to apoptotic stimuli and that the suppressor/coupling domain may have an anti-apoptotic function by attenuating the sensitivity of IICR.
We have characterised the functional regulation of the type-3 ryanodine receptor by the 12 kDa FK506-binding protein. Wild-type type-3 ryanodine receptor and mutant type-3 ryanodine receptor in which ...the critical valine at position 2322 in the central 12 kDa FK506-binding protein binding site was substituted by aspartate, were stably expressed in human embryonic kidney cells. In contrast to the wild-type receptor, the mutant receptor was strongly impaired in binding to immobilised glutathione S-transferase 12 kDa FK506-binding protein. Caffeine-induced 45Ca2+-efflux was markedly increased in cells expressing mutant type-3 ryanodine receptor whereas the maximal-releasable Ca2+ was not affected. Confocal Ca2+ imaging provided clear evidence for a much higher sensitivity of the mutant receptor, which showed global Ca2+ release at about 20-fold lower caffeine concentrations than the wild-type receptor. Spontaneous Ca2+ sparks were observed in both wild-type- and mutant-expressing cells but the number of sparking cells was about 1.5-fold higher in the mutant group, suggesting that the degree of FK506 binding controls the stability of the closed state of ryanodine receptor channels. Furthermore, overexpression of 12 kDa FK506-binding protein decreased the number of sparking cells in the wild-type-expressing cells whereas it did not affect the number of sparking cells in cells expressing the mutant receptor. Concerning spark properties, the amplitude and duration of Ca2+ sparks mediated by mutant channels were significantly reduced in comparison to wild-type channels. This suggests that functional coupling between different mutant type-3 ryanodine receptor channels in a cluster is impaired. Our findings show for the first time that the central binding site for the 12 kDa FK506-binding protein of type-3 ryanodine receptor, encompassing the critical valine proline motif, plays a crucial role in the modulation of the Ca2+ release properties of the type-3 ryanodine receptor channel, including the regulation of both global Ca2+ responses and spontaneous Ca2+ sparks.
Intracellular calcium release is a fundamental signaling mechanism in all eukaryotic cells. The ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP
3R) are intracellular calcium ...release channels. Both channels can be regulated by calcium and calmodulin (CaM). In this review we will first discuss the role of calcium as an activator and inactivator of the IP
3R, concluding that calcium is the most important regulator of the IP
3R. In the second part we will further focus on the role of CaM as modulator of the IP
3R, using results of the voltage-dependent Ca
2+ channels and the RyR as reference material. Here we conclude that despite the fact that different CaM-binding sites have been characterized, their function for the IP
3R remains elusive. In the third part we will discuss the possible functional role of CaM in IP
3-induced Ca
2+ release (IICR) by direct and indirect mechanisms. Special attention will be given to the Ca
2+-binding proteins (CaBPs) that were shown to activate the IP
3R in the absence of IP
3.
The immunophilin FKBP12 associates with intracellular Ca 2+ channels and this interaction can be disrupted by the immunosuppressant FK506. We have investigated the effect of FK506 on
Ca 2+ release ...and Ca 2+ uptake in permeabilized cell types.
Changes in medium free Ca 2+ were detected by the fluorescent Ca 2+ indicator fluo-3 in digitonin-permeabilized SH-SY5Y human neuroblastoma cells, DT40 and R23-11 (i.e. triple inositol 1,4,5-trisphosphate
(IP 3 ) receptor knockout cells) chicken B lymphocytes and differentiated and undifferentiated BC 3 H1 skeletal muscle cells. 45 Ca 2+ fluxes were studied in saponin-permeabilized A7r5 rat smooth muscle cells.
Addition of FK506 to permeabilized SH-SY5Y cells led to a sustained elevation of the medium Ca 2+ corresponding to â¼30% of the Ca 2+ ionophore A23187-induced Ca 2+ rise. This rise in Ca 2+ was not dependent on mitochondrial activity.
This FK506-induced Ca 2+ rise was related to the inhibition of the sarcoplasmic/endoplasmic reticulum Ca 2+ -Mg 2+ -ATPase (SERCA) Ca 2+ pump. Oxalate-facilitated 45 Ca 2+ uptake in SH-SY5Y microsomes was inhibited by FK506 with an IC 50 of 19 μ m .
The inhibition of the SERCA Ca 2+ pump was not specific since several macrocyclic lactone compounds (ivermectin > FK506, ascomycin and rapamycin) were able
to inhibit Ca 2+ uptake activity.
FK506 (10 μ m ) did not affect IP 3 -induced Ca 2+ release in permeabilized SH-SY5Y and A7r5 cells, but enhanced caffeine-induced Ca 2+ release via the ryanodine receptor (RyR) in differentiated BC 3 H1 cells.
In conclusion, FK506 inhibited active Ca 2+ uptake by the SERCA Ca 2+ pump; in addition, FK506 enhanced intracellular Ca 2+ release through the RyR, but it had no direct effect on IP 3 -induced Ca 2+ release.
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