The endoplasmic reticulum pool of Bcl-xL prevents cell death through IP3R-dependent calcium release.

Apoptosis plays a role in cell homeostasis in both normal development and disease. Bcl-xL, a member of the Bcl-2 family of proteins, regulates the intrinsic mitochondrial pathway of apoptosis. It is overexpressed in several cancers.

Loss of INPP5K attenuates IP-induced Ca responses in the glioblastoma cell line U-251 MG cells.

INPP5K (inositol polyphosphate 5-phosphatase K) is an endoplasmic reticulum (ER)-resident enzyme that acts as a phosphoinositide (PI) 5-phosphatase, capable of dephosphorylating various PIs including PI 4,5-bisphosphate (PI(4,5)P), a key phosphoinositide found in the plasma membrane. Given its ER localization and substrate specificity, INPP5K may play a role in ER-plasma membrane contact sites. Furthermore, PI(4,5)P serves as a substrate for phospholipase C, an enzyme activated downstream of extracellular agonists acting on Gq-coupled receptors or tyrosine-kinase receptors, leading to IP production and subsequent release of Ca from the ER, the primary intracellular Ca storage organelle.

The alkalinizing, lysosomotropic agent ML-9 induces a pH-dependent depletion of ER Ca stores in cellulo.

ML-9 elicits a broad spectrum of effects in cells, including inhibition of myosin light chain kinase, inhibition of store-operated Ca entry and lysosomotropic actions that result in prostate cancer cell death. Moreover, the compound also affects endoplasmic reticulum (ER) Ca homeostasis, although the underlying mechanisms remain unclear. We found that ML-9 provokes a rapid mobilization of Ca from ER independently of IPRs or TMBIM6/Bax Inhibitor-1, two ER Ca-leak channels.

Synthesis and Characterization of Store-Operated Calcium Entry Inhibitors Active in the Submicromolar Range.

The store-operated calcium entry, better known as SOCE, forms the main Ca influx pathway in non-excitable cells, especially in leukocytes, where it is required for cell activation and the immune response. During the past decades, several inhibitors were developed, but they lack specificity or efficacy. From the non-specific SOCE inhibitor 2-aminoethyl diphenylborinate (2-APB), we synthetized 16 new analogues by replacing/modifying the phenyl groups.

APOL1 C-Terminal Variants May Trigger Kidney Disease through Interference with APOL3 Control of Actomyosin.

The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB).

Bcl-2 and IP compete for the ligand-binding domain of IPRs modulating Ca signaling output.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca dynamics by directly targeting and inhibiting the IP receptor (IPR), a major intracellular Ca-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IPR activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IPR function provoked by low concentration of agonist or IP, while fails to attenuate against high concentration of agonist or IP. Surprisingly, Bcl-2 remained capable of inhibiting IPR1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.

Ion and pH Sensitivity of a TMBIM Ca Channel.

The anti-apoptotic transmembrane Bax inhibitor motif (TMBIM) containing protein family regulates Ca homeostasis, cell death, and the progression of diseases including cancers. The recent crystal structures of the TMBIM homolog BsYetJ reveal a conserved Asp171-Asp195 dyad that is proposed in regulating a pH-dependent Ca translocation. Here we show that BsYetJ mediates Ca fluxes in permeabilized mammalian cells, and its interaction with Ca is sensitive to protons and other cations.

The ER Stress Inducer l-Azetidine-2-Carboxylic Acid Elevates the Levels of Phospho-eIF2α and of LC3-II in a Ca-Dependent Manner.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to reduce protein load and restore homeostasis, including via induction of autophagy. We used the proline analogue l-azetidine-2-carboxylic acid (AZC) to induce ER stress, and assessed its effect on autophagy and Ca homeostasis. Treatment with 5 mM AZC did not induce poly adenosine diphosphate ribose polymerase (PARP) cleavage while levels of binding immunoglobulin protein (BiP) and phosphorylated eukaryotic translation initiation factor 2α (eIF2α) increased and those of activating transcription factor 6 (ATF6) decreased, indicating activation of the protein kinase RNA-like ER kinase (PERK) and the ATF6 arms of the UPR but not of apoptosis.

Extracellular and ER-stored Ca contribute to BIRD-2-induced cell death in diffuse large B-cell lymphoma cells.

The anti-apoptotic protein Bcl-2 is upregulated in several cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). In a subset of these cancer cells, Bcl-2 blocks Ca-mediated apoptosis by suppressing the function of inositol 1,4,5-trisphosphate (IP) receptors (IPRs) located at the endoplasmic reticulum (ER). A peptide tool, called Bcl-2/IP receptor disruptor-2 (BIRD-2), was developed to disrupt Bcl-2/IPR complexes, triggering pro-apoptotic Ca signals and killing Bcl-2-dependent cancer cells.

The mycotoxin phomoxanthone A disturbs the form and function of the inner mitochondrial membrane.

Mitochondria are cellular organelles with crucial functions in the generation and distribution of ATP, the buffering of cytosolic Ca and the initiation of apoptosis. Compounds that interfere with these functions are termed mitochondrial toxins, many of which are derived from microbes, such as antimycin A, oligomycin A, and ionomycin. Here, we identify the mycotoxin phomoxanthone A (PXA), derived from the endophytic fungus Phomopsis longicolla, as a mitochondrial toxin.

Resveratrol-induced autophagy is dependent on IPRs and on cytosolic Ca.

Previous work revealed that intracellular Ca signals and the inositol 1,4,5-trisphosphate (IP) receptors (IPR) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IPRs and Ca signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells.

DPB162-AE, an inhibitor of store-operated Ca entry, can deplete the endoplasmic reticulum Ca store.

Store-operated Ca entry (SOCE), an important Ca signaling pathway in non-excitable cells, regulates a variety of cellular functions. To study its physiological role, pharmacological tools, like 2-aminoethyl diphenylborinate (2-APB), are used to impact SOCE. 2-APB is one of the best characterized SOCE inhibitors.

The BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as an evolutionary conserved inhibitor of IP receptor channels.

Ca signalling plays an important role in various physiological processes in vertebrates. In mammals, the highly conserved anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein is an important modulator of the inositol 1,4,5-trisphosphate receptor (IPR), i.e.

The selective Bcl-2 inhibitor venetoclax, a BH3 mimetic, does not dysregulate intracellular Ca signaling.

Anti-apoptotic B cell-lymphoma-2 (Bcl-2) proteins are emerging as therapeutic targets in a variety of cancers for precision medicines, like the BH3-mimetic drug venetoclax (ABT-199), which antagonizes the hydrophobic cleft of Bcl-2. However, the impact of venetoclax on intracellular Ca homeostasis and dynamics in cell systems has not been characterized in detail. Here, we show that venetoclax did not affect Ca-transport systems from the endoplasmic reticulum (ER) in permeabilized cell systems.

The trans-membrane domain of Bcl-2α, but not its hydrophobic cleft, is a critical determinant for efficient IP3 receptor inhibition.

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.

Ca(2+) signalling in human proximal tubular epithelial cells deficient for cystinosin.

Nephropathic cystinosis is an autosomal recessive lysosomal storage disorder caused by loss-of-function mutations in the CTNS gene coding for the lysosomal cystine transporter, cystinosin. Recent studies have demonstrated that, apart from cystine accumulation in the lysosomes, cystinosin-deficient cells, especially renal proximal tubular epithelial cells are characterized by abnormal vesicle trafficking and endocytosis, possible lysosomal dysfunction and perturbed intracellular signalling cascades. It is therefore possible that Ca(2+) signalling is disturbed in cystinosis, as it has been demonstrated for other disorders associated with lysosomal dysfunction, such as Gaucher, Niemann-Pick type C and Alzheimer's diseases.

Measurement of intracellular Ca2+ release in intact cells using 45Ca2+.

This protocol describes a technique using (45)Ca(2+) to measure the release of Ca(2+) from the intracellular stores in monolayers of intact cells cultured in 12-well 4-cm(2) clusters. The (45)Ca(2+)-flux technique described here can only be applied to cell types that adhere to plastic. We describe the loading of the stores with (45)Ca(2+), and the subsequent (45)Ca(2+) efflux.

Measurement of intracellular Ca2+ release in permeabilized cells using 45Ca2+.

This protocol describes a technique to measure Ca(2+) release from the nonmitochondrial intracellular Ca(2+) stores in monolayers of saponin-permeabilized cells cultured in 12-well 4-cm(2) clusters. The (45)Ca(2+)-flux technique described here can only be applied to cell types that still adhere to the plastic after exposing them to saponin. We describe the permeabilization procedure, the loading of the nonmitochondrial Ca(2+) stores with (45)Ca(2+), and the subsequent (45)Ca(2+) efflux.

Measurement of intracellular Ca2+ release in intact and permeabilized cells using 45Ca2+.

Ca(2+) is an important ion that controls almost every function in a cell. Activator Ca(2+) can be released from intracellular Ca(2+) stores, and there are various ways to study this release. Here, we introduce a technique that uses radioactive (45)Ca(2+) to quantitatively measure the unidirectional release of Ca(2+) from the nonmitochondrial Ca(2+) stores in monolayers of cultured cells.

Differential effects of bitter compounds on the taste transduction channels TRPM5 and IP3 receptor type 3.

Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a Ca(2+)-activated nonselective cation channel involved in the transduction of sweet, bitter, and umami tastes. We previously showed that TRPM5 is a locus for the modulation of taste perception by temperature changes, and by quinine and quinidine, 2 bitter compounds that suppress gustatory responses. Here, we determined whether other bitter compounds known to modulate taste perception also affect TRPM5.

Alzheimer's disease-associated peptide Aβ42 mobilizes ER Ca(2+) via InsP3R-dependent and -independent mechanisms.

Dysregulation of Ca(2+) homeostasis is considered to contribute to the toxic action of the Alzheimer's disease (AD)-associated amyloid-β-peptide (Aβ). Ca(2+) fluxes across the plasma membrane and release from intracellular stores have both been reported to underlie the Ca(2+) fluxes induced by Aβ42. Here, we investigated the contribution of Ca(2+) release from the endoplasmic reticulum (ER) to the effects of Aβ42 upon Ca(2+) homeostasis and the mechanism by which Aβ42 elicited these effects.

Alpha-helical destabilization of the Bcl-2-BH4-domain peptide abolishes its ability to inhibit the IP3 receptor.

The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the primary Ca(2+)-release channel in the endoplasmic reticulum (ER). Bcl-2 can thereby reduce pro-apoptotic IP3R-mediated Ca(2+) release from the ER.

Bax Inhibitor-1-mediated Ca2+ leak is decreased by cytosolic acidosis.

Bax Inhibitor-1 (BI-1) is an evolutionarily conserved six-transmembrane domain endoplasmic reticulum (ER)-localized protein that protects against ER stress-induced apoptotic cell death. This function is closely connected to its ability to lower steady-state ER Ca2+ levels. Recently, we elucidated BI-1's Ca(2+)-channel pore in the C-terminal part of the protein and identified the critical amino acids of its pore.