IP3 Receptor Function Research Articles

The IP3 receptor-KRAP complex at the desmosomes: A new player in the apoptotic process

The IP3 receptor (IP3R) is a ubiquitously expressed Ca2+-release channel located in the endoplasmic reticulum (ER). Ca2+ signals originating from the IP3R initiate or regulate a plethora of cellular events, including cell life and death processes, e.g. exaggerated Ca2+ release from the ER to the mitochondria is a trigger for apoptosis. Recently, Cho et al. (Current Biology, 2024, DOI: 10.1016/j.cub.2024.08.057) demonstrated that in epithelial monolayers a sustained [Ca2+] elevation caused by the IP3Rs is responsible for the extrusion of adjacent apoptotic cells out of the epithelial monolayer. Interestingly, the IP3Rs involved are associated with the desmosomes via K-Ras-induced actin-interacting protein (KRAP). This study not only highlight a novel role of the IP3R in apoptosis, but also shed a new light on how KRAP -and by extension KRAP-related proteins- contribute to the regulation of IP3R activity and, more broadly, underscores the crucial role of associated proteins in determining the function of IP3Rs.

Impacts of exposure to and subsequent discontinuation of clozapine on tripartite synaptic transmission.

Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but its discontinuation leads to discontinuation syndrome/catatonia complicated by benzodiazepine-resistance and rhabdomyolysis. This study determined time-dependent effects of exposure and subsequent discontinuation of clozapine on expression of connexin43, 5-HT receptors, intracellular L-β-aminoisobutyrate (L-BAIBA) and 2nd-messengers and signalling of AMPK, PP2A and Akt in cultured astrocytes and rat frontal cortex. Intracellular L-BAIBA levels increased during clozapine exposure but immediately recovered after discontinuation. Both exposure to clozapine and L-BAIBA increased connexin43 and signalling of AMPK/Akt time-dependently, but reduced PP2A signalling, 5-HT receptor expression and IP3 level. These changes recovered within 2 weeks after discontinuation, while 5-HT receptors and IP3 transiently increased during the recovery process. L-BAIBA activated AMPK signalling, leading to attenuated PP2A signalling. Astroglial D-serine release was increased by clozapine exposure but continued to increase within 1 week after discontinuation via activation of IP3 receptor function. Clozapine discontinuation restored PP2A signalling due to decreased L-BAIBA, increased 5-HT receptor expression via probably enhanced 5-HT receptor recycling, but increased astroglial D-serine release persisted by transiently activated IP3 receptors via transiently increased IP3 level. Decreased L-BAIBA caused by clozapine discontinuation is, at least partially, involved in the transiently increased 5-HT receptor and astroglial D-serine release.

Calcium and redox signals at mitochondrial interfaces: A nanoview perspective

• The interplay of calcium and redox signals at mitochondrial interfaces is highly relevant for cell biology. • Booth and colleagues reveal a novel mechanism that links single mitochondria oxidative bursts and IP3 Receptor function. • Novel redox sensors will provide further insights into the calcium-redox interplay at ER-mitochondria-PM junctions with a nanoscale resolution.

Mibefradil alters intracellular calcium concentration by activation of phospholipase C and IP3 receptor function

Mibefradil is a tetralol derivative originally developed as an antagonist of T-type voltage-gated calcium (Ca2+) channels to treat hypertension when used at nanomolar dosage. More recently, its therapeutic application in hypertension has declined and has been instead repurposed as a treatment of cancer cell proliferation and solid tumor growth. Beyond its function as a Cav blocker, the micromolar concentration of mibefradil can stimulate a rise in [Ca2+]cyt although the mechanism is poorly known. The chanzyme TRPM7 (transient receptor potential melastanin 7), the release of intracellular Ca2+ pools, and Ca2+ influx by ORAI channels have been associated with the increase in [Ca2+]cyt triggered by mibefradil. This study aims to investigate the cellular targets and pathways associated with mibefradil’s effect on [Ca2+]cyt. To address these questions, we monitored changes in [Ca2+]cyt in the specialized mouse epithelial cells (LS8 and ALC) and the widely used HEK-293 cells by stimulating these cells with mibefradil (0.1 μM to 100 μM). We show that mibefradil elicits an increase in [Ca2+]cyt at concentrations above 10 μM (IC50 around 50 μM) and a fast Ca2+ increase capacity at 100 μM. We found that inhibiting IP3 receptors, depleting the ER-Ca2+ stores, or blocking phospholipase C (PLC), significantly decreased the capacity of mibefradil to elevate [Ca2+]cyt. Moreover, the transient application of 100 μM mibefradil triggered Ca2+ influx by store-operated Ca2+ entry (SOCE) mediated by the ORAI channels. Our findings reveal that IP3R and PLC are potential new targets of mibefradil offering novel insights into the effects of this drug.

Ethanol Disrupts Hormone-Induced Calcium Signaling in Liver.

Receptor-coupled phospholipase C (PLC) is an important target for the actions of ethanol. In the ex vivo perfused rat liver, concentrations of ethanol >100 mM were required to induce a rise in cytosolic calcium (Ca2+) suggesting that these responses may only occur after binge ethanol consumption. Conversely, pharmacologically achievable concentrations of ethanol (≤30 mM) decreased the frequency and magnitude of hormone-stimulated cytosolic and nuclear Ca2+ oscillations and the parallel translocation of protein kinase C-β to the membrane. Ethanol also inhibited gap junction communication resulting in the loss of coordinated and spatially organized intercellular Ca2+ waves in hepatic lobules. Increasing the hormone concentration overcame the effects of ethanol on the frequency of Ca2+ oscillations and amplitude of the individual Ca2+ transients; however, the Ca2+ responses in the intact liver remained disorganized at the intercellular level, suggesting that gap junctions were still inhibited. Pretreating hepatocytes with an alcohol dehydrogenase inhibitor suppressed the effects of ethanol on hormone-induced Ca2+ increases, whereas inhibiting aldehyde dehydrogenase potentiated the inhibitory actions of ethanol, suggesting that acetaldehyde is the underlying mediator. Acute ethanol intoxication inhibited the rate of rise and the magnitude of hormone-stimulated production of inositol 1,4,5-trisphosphate (IP3), but had no effect on the size of Ca2+ spikes induced by photolysis of caged IP3. These findings suggest that ethanol inhibits PLC activity, but does not affect IP3 receptor function. We propose that by suppressing hormone-stimulated PLC activity, ethanol interferes with the dynamic modulation of [IP3] that is required to generate large, amplitude Ca2+ oscillations.

Disease-associated mutations in inositol 1,4,5-trisphosphate receptor subunits impair channel function

The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which form tetrameric channels, play pivotal roles in regulating the spatiotemporal patterns of intracellular calcium signals. Mutations in IP3Rs have been increasingly associated with many debilitating human diseases such as ataxia, Gillespie syndrome, and generalized anhidrosis. However, how these mutations affect IP3R function, and how the perturbation of as-sociated calcium signals contribute to the pathogenesis and severity of these diseases remains largely uncharacterized. Moreover, many of these diseases occur as the result of autosomal dominant inheritance, suggesting that WT and mutant subunits associate in heterotetrameric channels. How the in-corporation of different numbers of mutant subunits within the tetrameric channels affects its activities and results in different disease phenotypes is also unclear. In this report, we investigated representative disease-associated missense mutations to determine their effects on IP3R channel activity. Additionally, we designed concatenated IP3R constructs to create tetrameric channels with a predefined subunit composition to explore the functionality of heteromeric channels. Using calcium imaging techniques to assess IP3R channel function, we observed that all the mutations studied resulted in severely attenuated Ca2+ release when expressed as homotetramers. However, some heterotetramers retained varied degrees of function dependent on the composition of the tetramer. Our findings suggest that the effect of mutations depends on the location of the mutation in the IP3R structure, as well as on the stoichiometry of mutant subunits assembled within the tetrameric channel. These studies provide insight into the pathogenesis and penetrance of these devastating human diseases.

Inositol 1,4,5-Trisphosphate Receptor Type 3 Regulates Neuronal Growth Cone Sensitivity to Guidance Signals.

SummaryDuring neurodevelopment, the growth cone deciphers directional information from extracellular guidance cues presented as shallow concentration gradients via signal amplification. However, it remains unclear how the growth cone controls this amplification process during its navigation through an environment in which basal cue concentrations vary widely. Here, we identified inositol 1,4,5-trisphosphate (IP3) receptor type 3 as a regulator of axonal sensitivity to guidance cues in vitro and in vivo. Growth cones lacking the type 3 subunit are hypersensitive to nerve growth factor (NGF), an IP3-dependent attractive cue, and incapable of turning toward normal concentration ranges of NGF to which wild-type growth cones respond. This is due to globally, but not asymmetrically, activated Ca2+ signaling in the hypersensitive growth cones. Remarkably, lower NGF concentrations can polarize growth cones for turning if IP3 receptor type 3 is deficient. These data suggest a subtype-specific IP3 receptor function in sensitivity adjustment during axon navigation.

IP3 Receptor Plasticity Underlying Diverse Functions.

In the body, extracellular stimuli produce inositol 1,4,5-trisphosphate (IP3), an intracellular chemical signal that binds to the IP3 receptor (IP3R) to release calcium ions (Ca2+) from the endoplasmic reticulum. In the past 40 years, the wide-ranging functions mediated by IP3R and its genetic defects causing a variety of disorders have been unveiled. Recent cryo-electron microscopy and X-ray crystallography have resolved IP3R structures and begun to integrate with concurrent functional studies, which can explicate IP3-dependent opening of Ca2+-conducting gates placed ∼90 Å away from IP3-binding sites and its regulation by Ca2+. This review highlights recent research progress on the IP3R structure and function. We also propose how protein plasticity within IP3R, which involves allosteric gating and assembly transformations accompanied by rapid and chronic structural changes, would enable it to regulate diverse functions at cellular microdomains in pathophysiological states.

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

Bcl-2 proteins haveemerged 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.

Spontaneous Ca2+ Influx in Drosophila Pupal Neurons Is Modulated by IP3-Receptor Function and Influences Maturation of the Flight Circuit.

Inositol 1,4,5-trisphosphate receptors (IP3R) are Ca2+ channels on the neuronal endoplasmic reticulum (ER) membrane. They are gated by IP3, produced upon external stimulation and activation of G protein-coupled receptors on the plasma membrane (PM). IP3-mediated Ca2+ release, and the resulting depletion of the ER store, triggers entry of extracellular Ca2+ by store-operated Ca2+ entry (SOCE). Mutations in IP3R attenuate SOCE. Compromised IP3R function and SOCE during pupal development of Drosophila leads to flight deficits and mimics suppression of neuronal activity during pupal or adult development. To understand the effect of compromised IP3R function on pupal neuronal calcium signaling, we examined the effects of mutations in the IP3R gene (itpr) on Ca2+ signals in cultured neurons derived from Drosophila pupae. We observed increased spontaneous Ca2+ influx across the PM of isolated pupal neurons with mutant IP3R and also a loss of SOCE. Both spontaneous Ca2+ influx and reduced SOCE were reversed by over-expression of dOrai and dSTIM, which encode the SOCE Ca2+ channel and the ER Ca2+-sensor that regulates it, respectively. Expression of voltage-gated Ca2+ channels (cac, Ca-α1D and Ca-αT) was significantly reduced in itpr mutant neurons. However, expression of trp mRNAs and transient receptor potential (TRP) protein were increased, suggesting that TRP channels might contribute to the increased spontaneous Ca2+ influx in neurons with mutant IP3R. Thus, IP3R/SOCE modulates spontaneous Ca2+ influx and expression of PM Ca2+ channels in Drosophila pupal neurons. Spontaneous Ca2+ influx compensates for the loss of SOCE in Drosophila itpr mutant neurons.

IP3 Receptor Determines Increased Basal Mitochondrial Calcium And Autophagy Regulation In Mdx Skeletal Muscle

Several reports describe that IP3 receptor (IP3R) is essential for efficient mitochondrial respiration and maintenance of cellular bioenergetics. This is due to the participation of this receptor in calcium transfer from the ER to the mitochondria. Changes in IP3R function can compromise mitochondrial function, changing ATP production and finally altering autophagy. Nevertheless until now there is no detailed study of the IP3R/calcium/autophagy axis in DMD. Our aim was to investigate the participation of IP3R in the regulation of mitochondrial calcium and autophagy in fibers from a DMD animal model, mdx. We analyzed protein levels of LC3, p62, atg5, beclin and bcl‐2 in FDB muscle of mdx (5–7 weeks), electroporated with a short‐hairpin for IP3R. We also evaluated the calcium transfer with mitochondria‐targeted probes that can be electroporated and also mitochondrial potential with TMRE in skeletal muscle fibers. The basal levels of expression of LC3II are diminished in mdx fibers compared with controls. This result correlates with an increased expression of p62 in mdx fibers. We also found differences in other autophagy proteins like atg5, beclin1 and Bcl‐2. When we performed the knockdown for IP3R we observed differences in the expression of almost all of the proteins analyzed. The expression of LC3II was increased in the electroporated mdx fibers with a decrease in p62 expression. We also confirmed that basal mitochondrial calcium level was increased in mdx fibers. Moreover, when we decreased the expression of IP3R, the basal mitochondrial calcium levels returned to normal. Similar result was obtained when we measured the mitochondrial potential in mdx fibers. Collectively, these results suggest that IP3R regulates mitochondrial calcium flux in adult skeletal muscle and is responsible for the increased mitochondrial calcium in mdx fibers. Moreover, knockdown of IP3R can regulate the expression levels of proteins associated with autophagy. Finally, we propose that some inhibitors of the IP3 pathway can be used as a new treatment for the muscle weakness observed in DMD patientsSupport or Funding InformationFONDECYT 3140491(DV), 3140532(FW), 1151293(EJ), ACT‐111 (EJ, SL), FONDAP 15130011(SL)

Analyzing and Quantifying the Gain-of-Function Enhancement of IP3 Receptor Gating by Familial Alzheimer's Disease-Causing Mutants in Presenilins.

Familial Alzheimer’s disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca2+ release channels resulting in enhanced IP3R channel gating in an amyloid beta (Aβ) production-independent manner. This gain-of-function enhancement of IP3R activity is considered to be the main reason behind the upregulation of intracellular Ca2+ signaling in the presence of optimal and suboptimal stimuli and spontaneous Ca2+ signals observed in cells expressing mutant PS. In this paper, we employed computational modeling of single IP3R channel activity records obtained under optimal Ca2+ and multiple IP3 concentrations to gain deeper insights into the enhancement of IP3R function. We found that in addition to the high occupancy of the high-activity (H) mode and the low occupancy of the low-activity (L) mode, IP3R in FAD-causing mutant PS-expressing cells exhibits significantly longer mean life-time for the H mode and shorter life-time for the L mode, leading to shorter mean close-time and hence high open probability of the channel in comparison to IP3R in cells expressing wild-type PS. The model is then used to extrapolate the behavior of the channel to a wide range of IP3 and Ca2+ concentrations and quantify the sensitivity of IP3R to its two ligands. We show that the gain-of-function enhancement is sensitive to both IP3 and Ca2+ and that very small amount of IP3 is required to stimulate IP3R channels in the presence of FAD-causing mutant PS to the same level of activity as channels in control cells stimulated by significantly higher IP3 concentrations. We further demonstrate with simulations that the relatively longer time spent by IP3R in the H mode leads to the observed higher frequency of local Ca2+ signals, which can account for the more frequent global Ca2+ signals observed, while the enhanced activity of the channel at extremely low ligand concentrations will lead to spontaneous Ca2+ signals in cells expressing FAD-causing mutant PS.

Modeling astrocyte-neuron interactions in a tripartite synapse

Glial cells (microglia, oligodendrocytes, and especially astrocytes) play a critical role in the central nervous system by affecting in various ways the neuronal single cell level interactions as well as connectivity and communication at the network level, both in the developing and mature brain. Numerous studies (see, e.g., [1-3]) indicate an important modulatory role of astrocytes in brain homeostasis but most specifically in neuronal metabolism, plasticity, and survival. Astrocytes are also known to play an important role in many neurological disorders and neurodegenerative diseases. It is therefore important in the light of recent evidence to assess how the astrocytes interact with neurons, both in situ and in silico. The integration of biological knowledge into computational models is becoming increasingly important to help understand the role of astrocytes both in health and disease. We have previously addressed the role of transmitters and amyloid-beta peptide on calcium signals in rat cortical astrocytes [4]. In this work, we extend the work by using a modified version of the previously developed model [5] for astrocyte-neuron interactions in a tripartite synapse to explore the effects of various pre- and postsynaptic as well as extrasynaptic mechanisms on neuronal activity. We consider extending the model to include various additional mechanisms, such as the role of IP3 receptor function, recycling of neurotransmitters, K+ buffering by the Na+/K+ pump, and retrograde signaling by endocannabinoids. The improved tripartite synapse model for astrocyte-neuron interactions will provide an essential modeling tool for facilitating studies of local network dynamics in the brain. The model may also serve as an important step toward understanding mechanisms behind induction and maintenance of plastic changes in the brain.

Loss of IP3 receptor function in neuropeptide secreting neurons leads to obesity in adult Drosophila

BackgroundIntracellular calcium signaling regulates a variety of cellular and physiological processes. The inositol 1,4,5 trisphosphate receptor (IP3R) is a ligand gated calcium channel present on the membranes of endoplasmic reticular stores. In previous work we have shown that Drosophila mutants for the IP3R (itpr ku ) become unnaturally obese as adults with excessive storage of lipids on a normal diet. While the phenotype manifests in cells of the fat body, genetic studies suggest dysregulation of a neurohormonal axis.ResultsWe show that knockdown of the IP3R, either in all neurons or in peptidergic neurons alone, mimics known itpr mutant phenotypes. The peptidergic neuron domain includes, but is not restricted to, the medial neurosecretory cells as well as the stomatogastric nervous system. Conversely, expression of an itpr + cDNA in the same set of peptidergic neurons rescues metabolic defects of itpr ku mutants. Transcript levels of a gene encoding a gastric lipase CG5932 (magro), which is known to regulate triacylglyceride storage, can be regulated by itpr knockdown and over-expression in peptidergic neurons. Thus, the focus of observed itpr mutant phenotypes of starvation resistance, increased body weight, elevated lipid storage and hyperphagia derive primarily from peptidergic neurons.ConclusionsThe present study shows that itpr function in peptidergic neurons is not only necessary but also sufficient for maintaining normal lipid metabolism in Drosophila. Our results suggest that intracellular calcium signaling in peptidergic neurons affects lipid metabolism by both cell autonomous and non-autonomous mechanisms.

The essential roles of protein–protein interaction in sigma-1 receptor functions

The essential roles of protein–protein interaction in sigma-1 receptor functions

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

Dear Editor, Bax 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-1's 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 48 h, 20 nM 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 Student's t-test). These data indicate that BI-1's Ca2+-flux properties are essential for BI-1's 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 3.57 μM to 2.25 μM 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 3.57 μM to 1.98 μM 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. Figure 1 (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 ...

Protein S‐glutathionylation enhances Ca2+‐induced Ca2+ release via the IP3 receptor in cultured aortic endothelial cells

Key points In non‐excitable cells, oxidative stress increases inositol 1,4,5‐trisphosphate (IP3) receptor (IP3R) activity, which can cause Ca2+ oscillations under basal conditions and enhance agonist‐stimulated changes in cytosolic free Ca2+ concentration. Protein S‐glutathionylation, the reversible modification of cysteine thiols by glutathione, is elevated in response to oxidative stress, but the consequence of glutathionylation for IP3R function is not known. In this study we provide evidence that Ca2+‐induced Ca2+‐release (CICR) via the IP3R is enhanced by oxidant‐induced glutathionylation in cultured aortic endothelial cells. Our results suggest glutathionylation may represent a fundamental mechanism for regulating IP3R activity during physiological redox signalling and during pathological oxidative stress. Abstract In non‐excitable cells, thiol‐oxidizing agents have been shown to evoke oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) by increasing the sensitivity of the inositol 1,4,5‐trisphosphate (IP3) receptor (IP3R) to IP3. Although thiol modification of the IP3R is implicated in this response, the molecular nature of the modification(s) responsible for changes in channel activity is still not well understood. Diamide is a chemical oxidant that selectively converts reduced glutathione (GSH) to its disulfide (GSSG) and promotes the formation of protein–glutathione (P‐SSG) mixed disulfide, i.e. glutathionylation. In the present study, we examined the effect of diamide, and the model oxidant hydrogen peroxide (H2O2), on oscillations in [Ca2+]i in fura‐2‐loaded bovine (BAECs) and human (HAECs) aortic endo‐thelial cells using time‐lapse fluorescence video microscopy. In the absence of extracellular Ca2+, acute treatment with either diamide or H2O2 increased the number of BAECs exhibiting asynchronous Ca2+ oscillations, whereas HAECs were unexpectedly resistant. Diamide pretreatment increased the sensitivity of HAECs to histamine‐stimulated Ca2+ oscillations and BAECs to bradykinin‐stimulated Ca2+ oscillations. Moreover, in both HAECs and BAECs, diamide dramatically increased both the rate and magnitude of the thapsigargin‐induced Ca2+ transient suggesting that Ca2+‐induced Ca2+ release (CICR) via the IP3R is enhanced by glutathionylation. Similar to diamide, H2O2 increased the sensitivity of HAECs to both histamine and thapsigargin. Lastly, biochemical studies showed that glutathionylation of native IP3R1 is increased in cells challenged with H2O2. Collectively our results reveal that thiol‐oxidizing agents primarily increase the sensitivity of the IP3R to Ca2+, i.e. enhanced CICR, and suggest that glutathionylation may represent a fundamental mechanism for regulating IP3R activity during physiological redox signalling and during pathologicalical oxidative stress.

Melatonin and IP3-induced Ca2+ Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

STIM1, but not STIM2, is required for proper agonist-induced Ca 2+ signaling

The stromal interaction molecules STIM1 and STIM2 sense a decreasing Ca 2+ concentration in the lumen of the endoplasmic reticulum and activate Ca 2+ channels in the plasma membrane. In addition, at least 2 reports suggested that STIM1 may also interact with the inositol 1,4,5-trisphosphate (IP 3) receptor. Using embryonic fibroblasts from Stim1 −/−, Stim2 −/− and wild-type mice, we now tested the hypothesis that STIM1 and STIM2 would also regulate the IP 3 receptor. We investigated whether STIM1 or STIM2 would be the luminal Ca 2+ sensor that controls the loading dependence of the IP 3-induced Ca 2+ release. Partial emptying of the stores in plasma-membrane permeabilized cells resulted in an increased EC 50 and a decreased Hill coefficient for IP 3-induced Ca 2+ release. This effect occurred both in the presence and absence of STIM proteins, indicating that these proteins were not the luminal Ca 2+ sensor for the IP 3 receptor. Although Stim1 −/− cells displayed a normal IP 3-receptor function, agonist-induced Ca 2+ release was reduced. This finding suggests that the presence of STIM1 is required for proper agonist-induced Ca 2+ signaling. Our data do not provide experimental evidence for the suggestion that STIM proteins would directly control the function of the IP 3 receptor.

TRPC3 Controls Agonist-stimulated Intracellular Ca2+ Release by Mediating the Interaction between Inositol 1,4,5-Trisphosphate Receptor and RACK1

Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-mediated intracellular Ca(2+) release and associated with phosphatidylinositol 4,5-bisphosphate hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with receptor for activated C-kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also the interaction of IP(3)R with RACK1 and IP(3)-dependent intracellular Ca(2+) release. We show that TRPC3 interacts with RACK1 via N-terminal residues Glu-232, Asp-233, Glu-240, and Glu-244. Carbachol (CCh) stimulation of HEK293 cells expressing wild type TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP(3)R complex and increased surface expression of TRPC3 and Ca(2+) entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP(3)R complex as well as increased surface expression of TRPC3 and receptor-operated Ca(2+) entry were also attenuated. Importantly, CCh-induced intracellular Ca(2+) release was significantly reduced as was RACK1-IP(3)R association without any change in thapsigargin-stimulated Ca(2+) release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP(3)R association and decreased CCh-stimulated Ca(2+) entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca(2+) release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca(2+) release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP(3)R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca(2+) release by mediating interaction between IP(3)R and RACK1.