J B Parys

Selective regulation of IP3-receptor-mediated Ca2+ signaling and apoptosis by the BH4 domain of Bcl-2 versus Bcl-Xl.

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.

Bax Inhibitor-1 is a novel IP3 receptor-interacting and -sensitizing protein

Dear Editor, n nBax Inhibitor-1 (BI-1) is an evolutionary conserved endoplasmic reticulum (ER)-located protein that protects against ER stress-induced apoptosis.1 This function has been closely related to its ability to permeate Ca2+ from the ER2 and to lower the steady-state [Ca2+]ER.3 BI-1 may function as an H+/Ca2+-antiporter2 or Ca2+ channel.4 Recently, BI-1 was proposed as a negative regulator of autophagy through IRE1α.5 However, recent findings indicate that BI-1 may promote autophagy.6 The latter required the presence of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). The observations were explained through BI-1-enhanced IP3R activity, which lowered steady-state [Ca2+]ER, reducing ER-mitochondrial Ca2+ transfer and decreasing mitochondrial bio-energetics.7 However, direct evidence that BI-1 binds to IP3Rs and sensitizes IP3-induced Ca2+ release (IICR) is lacking. Therefore, we studied the regulation of IP3R function by BI-1 (see Supplementary Information for Methods). We constructed a 5xMyc-BI-1-expression plasmid, allowing the detection and purification of ectopically expressed BI-1 from transfected HeLa cells using anti-Myc-agarose beads (Figure 1a). Using isoform-specific IP3R antibodies, we demonstrated the co-immunoprecipitation of IP3R1 and IP3R3 with 5xMyc-BI-1 from HeLa cell lysates. Next, we screened for the subdomain of BI-1 responsible for IP3R interaction. We found that a synthetic Flag-tagged peptide containing BI-1s Ca2+-channel pore domain (CTP1; amino acids 198–217 of human BI-1) interacted with IP3R1 (Figure 1b). Lysates not exposed to Flag-CTP1 served as negative control. Moreover, proteolytic fragments of the IP3R containing its C terminus (indicated as IP3R1-Cterm in Figure 1b) were immunoprecipitated with Flag-CTP1. These C-terminal fragments were recognized by our antibody (Rbt03) that has its epitope in the last 15 C-terminal amino acids of the IP3R1.8 These fragments include the Ca2+-channel pore of the IP3R1, indicating that the Ca2+-channel pore domain of BI-1 interacted with the Ca2+-channel pore domain of IP3R1. Next, we examined the effect of BI-1 on IP3R function. Therefore, we used BI-1−/− mouse embryonic fibroblasts (MEF) and stably and ectopically overexpressed either empty vector (RFP-only), wild-type BI-1 or BI-1D213R with a bi-cistronic C-terminal IRES-RFP reporter. BI-1D213R is a mutant, in which the Asp213 critical for BI-1-mediated Ca2+ flux is altered into an Arg and which fails to lower [Ca2+]ER.4 BI-1-mRNA expression was detected using specific primers, and similar expression levels were found for wild-type BI-1 and BI-1D213R, while no signal was observed in vector-expressing BI-1−/− MEF cells (inset Figure 1c). Wild-type BI-1, but not BI-1D213R, overexpression significantly improved cell survival after thapsigargin exposure, an irreversible SERCA inhibitor, which kills cells through ER stress (empty vector: 33.65±4.48% wild-type BI-1: 44.39±5.31%* BI-1D213R: 34.14±4.19% surviving cells after 48u2009h, 20u2009nM thapsigargin normalized to vehicle-treated cells expressing empty vector. Mean±S.E.M. of four pooled experiments done in triplicates is shown, *P<0.05 Students t-test). These data indicate that BI-1s Ca2+-flux properties are essential for BI-1s anti-apoptotic function. Next, we analyzed the direct effect of ectopically expressed BI-1 on IP3R function in the absence of endogenous BI-1 (Figure 1c). We used a unidirectional 45Ca2+-flux assay in saponin-permeabilized BI-1−/− MEF cells, allowing direct ER access and an accurate analysis of IP3R function in the absence of plasmalemmal Ca2+ fluxes, SERCA activity or mitochondrial Ca2+ uptake.8 Cells ectopically overexpressing BI-1 displayed a sensitized IICR and concomitant decrease in EC50 from 3.57u2009μM to 2.25u2009μM IP3. To exclude that Ca2+ flux mediated by BI-1 indirectly sensitized IP3Rs through Ca2+-induced Ca2+ release, we examined the effect of BI-1D213R overexpression on IP3R function. BI-1D213R also sensitized IICR and concomitantly decreased the EC50 from 3.57u2009μM to 1.98u2009μM IP3. This correlates with the ability of BI-1D213R to co-immunoprecipitate with IP3Rs (Figure 1a). Collectively, these data indicate a direct sensitizing effect of BI-1 on IP3Rs, which may contribute to a decrease in steady-state [Ca2+]ER and mitochondrial bioenergetics and subsequent induction of basal autophagy. n n n nFigure 1 n n(a) Interaction of 5xMyc-BI-1 and 5xMyc-BI-1D213R with IP3R channels. BI-1 and BI-1D213R were expressed as 5xMyc-tagged fusion proteins. The empty 5xMyc vector was used as negative control. The vectors were transfected into HeLa cells for 2 days allowing ...

Endogenously Bound Calmodulin Is Essential for the Function of the Inositol 1,4,5-Trisphosphate Receptor

Calmodulin (CaM) is a ubiquitous Ca2+ sensor protein that plays an important role in regulating a large number of Ca2+ channels, including the inositol 1,4,5-trisphosphate receptor (IP3R). Despite many efforts, the exact mechanism by which CaM regulates the IP3R still remains elusive. Here we show, using unidirectional 45Ca2+ flux experiments on permeabilized L15 fibroblasts and COS-1 cells, that endogenously bound CaM is essential for the proper activation of the IP3R. Removing endogenously bound CaM by titration with a high affinity (pm) CaM-binding peptide derived from smooth muscle myosin light-chain kinase (MLCK peptide) strongly inhibited IP3-induced Ca2+ release. This inhibition was concentration- and time-dependent. Removing endogenously bound CaM affected the maximum release capacity but not its sensitivity to IP3. A mutant peptide with a strongly reduced affinity for CaM did not affect inhibited IP3-induced Ca2+ release. Furthermore, the inhibition by the MLCK peptide was fully reversible. Re-adding exogenous CaM, but not CaM1234, reactivated the IP3R. These data suggest that, by using a specific CaM-binding peptide, we removed endogenously bound CaM from a high affinity CaM-binding site on the IP3R, and this resulted in a complete loss of the IP3R activity. Our data support a new model whereby CaM is constitutively associated with the IP3R and functions as an essential subunit for proper functioning of the IP3R.

Control of the Ca2+ Release Induced by myo-Inositol Trisphosphate and the Implication in Signal Transduction

Inositol-1,4,5-trisphosphate (InsP3) is a diffusible messenger formed within the cell in response to external stimuli. It mobilizes Ca2+ from those nonmitochondrial Ca2+ pools that express the InsP3 receptor (InsP3R), a specific Ca2+-release channel (Berridge and Irvine, 1989; Berridge, 1993). The nonmitochondrial pools were originally classified as InsP3-sensitive and InsP3insensitive. Recent evidence suggests that the InsP3-sensitive Ca2+ pool is much larger than hitherto expected (Bird et al., 1992) and that InsP3-insensitive Ca2+ pools can artifactually be formed during the permeabilization procedure (Hajnoczky et al., 1994). Under conditions of very mild permeabilization, 95% of the nonmitochondrial Ca2+ pools can be InsP3-sensitive, e.g., in A7r5 smooth muscle cells (Missiaen et al., 1992b).

BAX inhibitor-1 is a Ca(2+) channel critically important for immune cell function and survival

The endoplasmic reticulum (ER) serves as the major intracellular Ca2+ store and has a role in the synthesis and folding of proteins. BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is a Ca2+ leak channel also implicated in the response against protein misfolding, thereby connecting the Ca2+ store and protein-folding functions of the ER. We found that BI-1-deficient mice suffer from leukopenia and erythrocytosis, have an increased number of splenic marginal zone B cells and higher abundance and nuclear translocation of NF-κB (nuclear factor-κ light-chain enhancer of activated B cells) proteins, correlating with increased cytosolic and ER Ca2+ levels. When put into culture, purified knockout T cells and even more so B cells die spontaneously. This is preceded by increased activity of the mitochondrial initiator caspase-9 and correlated with a significant surge in mitochondrial Ca2+ levels, suggesting an exhausted mitochondrial Ca2+ buffer capacity as the underlying cause for cell death in vitro. In vivo, T-cell-dependent experimental autoimmune encephalomyelitis and B-cell-dependent antibody production are attenuated, corroborating the ex vivo results. These results suggest that BI-1 has a major role in the functioning of the adaptive immune system by regulating intracellular Ca2+ homeostasis in lymphocytes.

CHAPTER 29 – Electromechanical and Pharmacomechanical Coupling in Vascular Smooth Muscle Cells

This chapter focuses on the various Ca 2+ entry pathways and intracellular Ca 2+ release mechanisms that contribute to the increase in [Ca 2+ ] i , triggering the contraction of vascular smooth muscle. The L type—long-lasting or high voltage activated; single channel conductance 20–28 Ps—is the most predominant Ca 2+ channel in smooth muscle cells, although the T type—transient or low voltage activated; single channel conductance 7–15 pS—has also been observed in a number of smooth muscle cell preparations. Expression of the L-type Ca 2+ channel depends on the differentiated state of vascular smooth muscle cells, as the current is decreased significantly in dedifferentiated A7r5 cells and increased upon differentiation with retinoic acid. L-type voltage-dependent Ca 2+ channels are modulated not only by vasoactive agonists, but also by extracellular pH, PO 2 , and blood pressure. Vasoactive agonists also activate additional Ca 2+ entry pathways, either directly or via generation of second messengers, or via depletion of IP3-sensitive Ca 2+ stores. IP 3 R seems to be the major pathway for Ca 2+ release during pharmacomechanical coupling. Smooth muscle cells also express ryanodine receptors and they also contribute to the shaping of the intracellular Ca 2+ signal. There are three different types of IP 3 R, and it is becoming increasingly evident that functional differences exist between these isoforms, including differences in redox sensitivity, in ATP sensitivity, and possibly also in Ca 2+ sensitivity.