Blockage of spontaneous Ca2+ oscillation causes cell death in intraerythrocitic Plasmodium falciparum

Malaria remains one of the world's most important infectious diseases and is responsible for enormous mortality and morbidity. Resistance to antimalarial drugs is a challenging problem in malaria control. Clinical malaria is associated with the proliferation and development of Plasmodium parasites in human erythrocytes. Especially, the development into the mature forms (trophozoite and schizont) of Plasmodium falciparum (P. falciparum) causes severe malaria symptoms due to a distinctive property, sequestration which is not shared by any other human malaria. Ca(2+) is well known to be a highly versatile intracellular messenger that regulates many different cellular processes. Cytosolic Ca(2+) increases evoked by extracellular stimuli are often observed in the form of oscillating Ca(2+) spikes (Ca(2+) oscillation) in eukaryotic cells. However, in lower eukaryotic and plant cells the physiological roles and the molecular mechanisms of Ca(2+) oscillation are poorly understood. Here, we showed the observation of the inositol 1,4,5-trisphospate (IP(3))-dependent spontaneous Ca(2+) oscillation in P. falciparum without any exogenous extracellular stimulation by using live cell fluorescence Ca(2+) imaging. Intraerythrocytic P. falciparum exhibited stage-specific Ca(2+) oscillations in ring form and trophozoite stages which were blocked by IP(3) receptor inhibitor, 2-aminoethyl diphenylborinate (2-APB). Analyses of parasitaemia and parasite size and electron micrograph of 2-APB-treated P. falciparum revealed that 2-APB severely obstructed the intraerythrocytic maturation, resulting in cell death of the parasites. Furthermore, we confirmed the similar lethal effect of 2-APB on the chloroquine-resistant strain of P. falciparum. To our best knowledge, we for the first time showed the existence of the spontaneous Ca(2+) oscillation in Plasmodium species and clearly demonstrated that IP(3)-dependent spontaneous Ca(2+) oscillation in P. falciparum is critical for the development of the blood stage of the parasites. Our results provide a novel concept that IP(3)/Ca(2+) signaling pathway in the intraerythrocytic malaria parasites is a promising target for antimalarial drug development.     

Cyclic AMP and calcium interplay as second messengers in melatonin-dependent regulation of Plasmodium falciparum cell cycle

The host hormone melatonin increases cytoplasmic Ca(2+) concentration and synchronizes Plasmodium cell cycle (Hotta, C.T., M.L. Gazarini, F.H. Beraldo, F.P. Varotti, C. Lopes, R.P. Markus, T. Pozzan, and C.R. Garcia. 2000. Nat. Cell Biol. 2:466-468). Here we show that in Plasmodium falciparum melatonin induces an increase in cyclic AMP (cAMP) levels and cAMP-dependent protein kinase (PKA) activity (40 and 50%, respectively).When red blood cells infected with P. falciparum are treated with cAMP analogue adenosine 3',5'-cyclic monophosphate N6-benzoyl/PKA activator (6-Bz-cAMP) there is an alteration of the parasite cell cycle. This effect appears to depend on activation of PKA (abolished by the PKA inhibitors adenosine 3',5'-cyclic monophosphorothioate/8 Bromo Rp isomer, PKI [cell permeable peptide], and H89). An unexpected cross talk was found to exist between the cAMP and the Ca(2+)-dependent signaling pathways. The increases in cAMP by melatonin are inhibited by blocker of phospholipase C U73122, and addition of 6-Bz-cAMP increases cytosolic Ca(2+) concentration, through PKA activation.These findings suggest that in Plasmodium a highly complex interplay exists between the Ca(2+) and cAMP signaling pathways, but also that the control of the parasite cell cycle by melatonin requires the activation of both second messenger controlled pathways.     

Calcium transport and compartment analysis of free and exchangeable calcium in Plasmodium falciparum-infected red blood cells.

Calcium (Ca2+) is indispensable for normal development of the various stages of the asexual erythrocytic cycle of malaria parasites. However, the mechanisms involved in Ca2+ uptake, compartmentalization and cellular regulation are poorly understood. To clarify some of these issues, we have measured total, exchangeable, and free Ca2+ in normal red cells (RBCs) and Plasmodium falciparum (FCR-3)-infected cells (IRBCs) as a function of parasite development. All three forms of Ca2+ were found to be substantially higher in IRBCs than in RBCs, and to increase with parasite maturation up to the trophozoite stage and decline thereafter. Exchangeable and free [Ca2+] in host cell and parasite compartments were determined by selectively lysing IRBCs with Sendai virus, and estimating these parameters in the lysate (host cytosol) and the pellet (parasite cytosol). Levels of both exchangeable and free [Ca2+] were found to be higher in host cytosol than in parasite cytosol. The Ca2+ gradient across the parasite membrane can be maintained by the pH gradient across this membrane by means of a Ca2+/H+ antiporter. Host cytosol free [Ca2+] reached levels known to produce structural, physiological and biochemical changes in RBCs, and could account for similar features normally seen in malaria-infected red cells. Uptake of Ca2+ into IRBCs was nonsaturable and substantially faster than the saturable Ca2+ uptake into RBCs. The rate of Ca2+ uptake across the parasite membrane was even faster suggesting that the rate-limiting step in uptake into intact IRBCs is the translocation of Ca2+ across the host cell membrane.     

Calcium release in HSY cells conforms to a steady-state mechanism involving regulation of the inositol 1,4,5-trisphosphate receptor Ca2+ channel by luminal [Ca2+]

In many cell types, low concentrations of inositol 1,4,5-trisphosphate (IP3) release only a portion of the intracellular IP3-sensitive Ca2+ store, a phenomenon known as "quantal" Ca2+ release. It has been suggested that this effect is a result of reduced activity of the IP3- dependent Ca2+ channel with decreasing calcium concentration within the IP3-sensitive store ([Ca2+]s). To test this hypothesis, the properties of IP3-dependent Ca2+ release in single saponin-permeabilized HSY cells were studied by monitoring [Ca2+]s using the Ca(2+)-sensitive fluorescent dye mag-fura-2. In permeabilized cells, blockade of the sarco/ER Ca(2+)-ATPase pump in stores partially depleted by IP3 induced further Ca2+ release via an IP3-dependent route, indicating that Ca2+ entry via the sarco/ER Ca(2+)-ATPase pump had been balanced by Ca2+ loss via the IP3-sensitive channel before pump inhibition. IP3- dependent Mn2+ entry, monitored via quenching of luminal mag-fura-2 fluorescence, was readily apparent in filled stores but undetectable in Ca(2+)-depleted stores, indicating markedly reduced IP3-sensitive channel activity in the latter. Also consistent with reduced responsiveness of Ca(2+)-depleted stores to IP3, the initial rate of refilling of these stores was unaffected by the presence of 0.3 microM IP3, a concentration that was clearly effective in eliciting Ca2+ release from filled stores. Analysis of the rate of Ca2+ release at various IP3 concentrations indicated a significant shift of the IP3 dose response toward higher [IP3] with decreasing [Ca2+]s. We conclude that IP3-dependent Ca2+ release in HSY cells is a steady-state process wherein Ca2+ efflux via the IP3 receptor Ca2+ channel is regulated by [Ca2+]s, apparently via changes in the sensitivity of the channel to IP3.     

Annexin 7 mobilizes calcium from endoplasmic reticulum stores in brain

Mobilization of intracellular calcium from inositol-1,4,5-triphosphate (IP3)-sensitive endoplasmic reticulum (ER) stores plays a prominent role in brain function. Mice heterozygous for the annexin A7 (Anx7) gene have a profound reduction in IP3 receptor function in pancreatic islets along with defective insulin secretion. We examined IP3-sensitive calcium pools in the brains of Anx7 (+/-) mice by utilizing ATP/Mg(2+)-dependent (45)Ca(2+) uptake into brain membrane preparations and tissue sections. Although the Anx7 (+/-) mouse brain displayed similar levels of IP3 binding sites and thapsigargin-sensitive (45)Ca(2+) uptake as that seen in wild-type mouse brain, the Anx7 (+/-) mouse brain Ca(2+) pools showed markedly reduced sensitivity to IP3. A potent and saturable Ca(2+)-releasing effect of recombinant ANX7 protein was demonstrated in mouse and rat brain membrane preparations, which was additive with that of IP3. We propose that ANX7 mobilizes Ca(2+) from an endoplasmic reticulum-like pool, which can be recruited to enhance IP3-mediated Ca(2+) release.     

Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival

Communication between the endoplasmic reticulum (ER) and mitochondrion is important for bioenergetics and cellular survival. The ER supplies Ca(2+) directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). We found here that the ER protein sigma-1 receptor (Sig-1R), which is implicated in neuroprotection, carcinogenesis, and neuroplasticity, is a Ca(2+)-sensitive and ligand-operated receptor chaperone at MAM. Normally, Sig-1Rs form a complex at MAM with another chaperone, BiP. Upon ER Ca(2+) depletion or via ligand stimulation, Sig-1Rs dissociate from BiP, leading to a prolonged Ca(2+) signaling into mitochondria via IP3Rs. Sig-1Rs can translocate under chronic ER stress. Increasing Sig-1Rs in cells counteracts ER stress response, whereas decreasing them enhances apoptosis. These results reveal that the orchestrated ER chaperone machinery at MAM, by sensing ER Ca(2+) concentrations, regulates ER-mitochondrial interorganellar Ca(2+) signaling and cell survival.     

Cyclosporin A inhibits inositol 1,4,5-trisphosphate-dependent Ca2+ signals by enhancing Ca2+ uptake into the endoplasmic reticulum and mitochondria

Cytosolic Ca(2+) ([Ca(2+)](i)) oscillations may be generated by the inositol 1,4,5-trisphosphate receptor (IP(3)R) driven through cycles of activation/inactivation by local Ca(2+) feedback. Consequently, modulation of the local Ca(2+) gradients influences IP(3)R excitability as well as the duration and amplitude of the [Ca(2+)](i) oscillations. In the present work, we demonstrate that the immunosuppressant cyclosporin A (CSA) reduces the frequency of IP(3)-dependent [Ca(2+)](i) oscillations in intact hepatocytes, apparently by altering the local Ca(2+) gradients. Permeabilized cell experiments demonstrated that CSA lowers the apparent IP(3) sensitivity for Ca(2+) release from intracellular stores. These effects on IP(3)-dependent [Ca(2+)](i) signals could not be attributed to changes in calcineurin activity, altered ryanodine receptor function, or impaired Ca(2+) fluxes across the plasma membrane. However, CSA enhanced the removal of cytosolic Ca(2+) by sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), lowering basal and inter-spike [Ca(2+)](i). In addition, CSA stimulated a stable rise in the mitochondrial membrane potential (DeltaPsi(m)), presumably by inhibiting the mitochondrial permeability transition pore, and this was associated with increased Ca(2+) uptake and retention by the mitochondria during a rise in [Ca(2+)](i). We suggest that CSA suppresses local Ca(2+) feedback by enhancing mitochondrial and endoplasmic reticulum Ca(2+) uptake, these actions of CSA underlie the lower IP(3) sensitivity found in permeabilized cells and the impaired IP(3)-dependent [Ca(2+)](i) signals in intact cells. Thus, CSA binding proteins (cyclophilins) appear to fine tune agonist-induced [Ca(2+)](i) signals, which, in turn, may adjust the output of downstream Ca(2+)-sensitive pathways.     

The digestive food vacuole of the malaria parasite is a dynamic intracellular Ca2+ store

The acidic food vacuole of Plasmodium falciparum has been the subject of intense scientific investigation in the 40 years since its role in the digestion of host hemoglobin was first suggested. This proposed role has important implications for the complex host-parasite inter-relationship and also for the mode of action of several of the most effective antimalarial drugs. In addition, adaptive changes in the physiology of this organelle are implicated in drug resistance. Here we show that in addition to these functions, the digestive food vacuole of the malaria parasite is a dynamic internal store for free Ca2+, a role hitherto unsuspected. With the aid of live-cell laser scanning confocal imaging, spatiotemporal studies revealed that maintenance of elevated free Ca2+ in the digestive food vacuole (relative to cytosolic levels) is achieved by a thapsigargin (and cyclopiazonic acid)-sensitive Ca2+-pump in cooperation with a H+-dependent Ca2+ transporter. Redistribution of free cytosolic and vacuolar Ca2+ during parasite growth also suggests that vacuolar Ca2+ plays an essential role in parasite morphogenesis. These data imply that the digestive food vacuole of the malaria parasite is functionally akin to the vacuole of plants (tonoplast) and the small electron-dense granules of some parasites (acidocalcisomes) whereby H+-coupled Ca2+ transport is involved in ion transport, Ca2+ homeostasis, and signal transduction. These findings have significant implications for parasite development, antimalarial drug action, and mechanisms of drug resistance.     

The gametocyte-activating factor xanthurenic acid stimulates an increase in membrane-associated guanylyl cyclase activity in the human malaria parasite Plasmodium falciparum

Sex is an obligate step in the life cycle of the malaria parasite and occurs in the midgut of the mosquito vector. With both Plasmodium falciparum and Plasmodium berghei, the tryptophan metabolite xanthurenic acid induces the release of motile male gametes from red blood cells (exflagellation), a prerequisite for fertilization. The addition of cGMP or phosphodiesterase inhibitors to cultures of mature gametocytes has also been shown to stimulate exflagellation. Here, we demonstrate that there is a guanylyl cyclase activity associated with mature P. falciparum gametocyte membrane preparations, which is dependent on the presence of Mg(2+)/Mn(2+) but is inhibited by Ca(2+). Significantly, this activity is increased on addition of xanthurenic acid. In contrast, a xanthurenic acid precursor (3-hydroxykynurenine), which is not an inducer of exflagellation, does not induce this guanylyl cyclase activity. These results therefore suggest that xanthurenic acid-induced exflagellation may be mediated by activation of the parasite cGMP signalling pathway.     

Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.     

IP3 receptor activity is differentially regulated in endoplasmic reticulum subdomains during oocyte maturation

Fertilization competency results from hormone-induced remodeling of oocytes into eggs. The signaling pathways that effect this change exemplify bistability, where brief hormone exposure irrevocably switches cell fate. In Xenopus, changes in Ca(2+) signaling epitomize such remodeling: The reversible Ca(2+) signaling phenotype of oocytes rapidly adapts to support irreversible propagation of the fertilization Ca(2+) wave. Here, we simultaneously resolved IP(3) receptor (IP(3)R) activity with endoplasmic reticulum (ER) structure to optically dissect the functional architecture of the Ca(2+) release apparatus underpinning this reorganization. We show that changes in Ca(2+) signaling correlate with IP(3)R redistribution from specialized ER substructures called annulate lamellae (AL), where Ca(2+) release activity is attenuated, into IP(3)R-replete patches in the cortical ER of eggs that support the fertilization Ca(2+) wave. These data show: first, that IP(3)R sensitivity is regulated with high spatial acuity even between contiguous ER regions; and second, that drastic reorganization of Ca(2+) signaling dynamics can be driven by subcellular redistribution in the absence of changes in channel number or molecular or familial Ca(2+) channel diversity. Finally, these results define a novel role for AL in Ca(2+) signaling. Because AL are prevalent in other scenarios of rapid cell division, further studies of their impact on Ca(2+) signaling are warranted.     

Mitochondria suppress local feedback activation of inositol 1,4, 5-trisphosphate receptors by Ca2+.

The concerted action of inositol 1,4,5-trisphosphate (IP3) and Ca2+ on the IP3 receptor Ca2+ release channel (IP3R) is a fundamental step in the generation of cytosolic Ca2+ oscillations and waves, which underlie Ca2+ signaling in many cells. Mitochondria appear in close association with regions of endoplasmic reticulum (ER) enriched in IP3R and are particularly responsive to IP3-induced increases of cytosolic Ca2+ ([Ca2+]c). To determine whether feedback regulation of the IP3R by released Ca2+ is modulated by mitochondrial Ca2+ uptake, the interactions between ER and mitochondrial Ca2+ pools were examined by fluorescence imaging of compartmentalized Ca2+ indicators in permeabilized hepatocytes. IP3 decreased luminal ER Ca2+ ([Ca2+]ER), and this was paralleled by an increase in mitochondrial matrix Ca2+ ([Ca2+]m) and activation of Ca2+-sensitive mitochondrial metabolism. Remarkably, the decrease in [Ca2+]ER evoked by submaximal IP3 was enhanced when mitochondrial Ca2+ uptake was blocked with ruthenium red or uncoupler. Moreover, subcellular regions that were relatively deficient in mitochondria demonstrated greater sensitivity to IP3 than regions of the cell with a high density of mitochondria. These data demonstrate that Ca2+ uptake by the mitochondria suppresses the local positive feedback effects of Ca2+ on the IP3R, giving rise to subcellular heterogeneity in IP3 sensitivity and IP3R excitability. Thus, mitochondria can play an important role in setting the threshold for activation and establishing the subcellular pattern of IP3-dependent [Ca2+]c signaling.     

Proteomic analysis of calcium-dependent secretion in Toxoplasma gondii

Toxoplasma gondii is an intracellular protozoan parasite that invades a wide range of nucleated cells. In the course of intracellular parasitism, the parasite releases a large variety of proteins from three secretory organelles, namely, micronemes, rhoptries and dense granules. Elevation of intracellular Ca(2+) in the parasite causes microneme discharge, and microneme secretion is essential for the invasion. In this study, we performed a proteomic analysis of the Ca(2+)-dependent secretion to evaluate the protein repertoire. We found that Ca(2+)-mobilising agents, such as thapsigargin, NH(4)Cl, ethanol and a Ca(2+) ionophore, A23187, promoted the secretion of the parasite proteins. The proteins, artificially secreted by A23187, were used in a comparative proteomic analysis by 2-DE followed by PMF analysis and/or N-terminal sequencing. Major known microneme proteins (MICs), such as MIC2, MIC4, MIC6 and MIC10 and apical membrane antigen 1 (AMA1), were identified, indicating that the proteomic analysis worked accurately. Interestingly, new members of secretory proteins, namely rhoptry protein 9 (ROP9) and Toxoplasma SPATR (TgSPATR), which was a homologue of a Plasmodium secreted protein with an altered thrombospondin repeat (SPATR), were detected in Ca(2+)-dependent secretion. Thus, we succeeded in detecting Ca(2+)-dependent secretory proteins in T. gondii, which contained novel secretory proteins.     

IP3 receptor-dependent Ca2+ release modulates excitation-contraction coupling in rabbit ventricular myocytes

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-dependent Ca(2+) signaling exerts positive inotropic, but also arrhythmogenic, effects on excitation-contraction coupling (ECC) in the atrial myocardium. The role of IP(3)R-dependent sarcoplasmic reticulum (SR) Ca(2+) release in ECC in the ventricular myocardium remains controversial. Here we investigated the role of this signaling pathway during ECC in isolated rabbit ventricular myocytes. Immunoblotting of proteins from ventricular myocytes showed expression of both type 2 and type 3 IP(3)R at levels approximately 3.5-fold less than in atrial myocytes. In permeabilized myocytes, direct application of IP(3) (10 microM) produced a transient 21% increase in the frequency of Ca(2+) sparks (P < 0.05). This increase was accompanied by a 13% decrease in spark amplitude (P < 0.05) and a 7% decrease in SR Ca(2+) load (P < 0.05) and was inhibited by IP(3)R antagonists 2-aminoethoxydiphenylborate (2-APB; 20 microM) and heparin (0.5 mg/ml). In intact myocytes endothelin-1 (100 nM) was used to stimulate IP(3) production and caused a 38% (P < 0.05) increase in the amplitude of action potential-induced (0.5 Hz, field stimulation) Ca(2+) transients. This effect was abolished by the IP(3)R antagonist 2-APB (2 microM) or by using adenoviral expression of an IP(3) affinity trap that buffers cellular IP(3). Together, these data suggest that in rabbit ventricular myocytes IP(3)R-dependent Ca(2+) release has positive inotropic effects on ECC by facilitating Ca(2+) release through ryanodine receptor clusters.     

Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells

The mechanism of action of the oncogene bcl-2, a key regulator of the apoptotic process, is still debated. We have employed organelle-targeted chimeras of the Ca(2+)-sensitive photoprotein, aequorin, to investigate in detail the effect of Bcl-2 overexpression on intracellular Ca(2+) homeostasis. In the ER and the Golgi apparatus, Bcl-2 overexpression increases the Ca(2+) leak (while leaving Ca(2+) accumulation unaffected), hence reducing the steady-state [Ca(2+)] levels. As a direct consequence, the [Ca(2+)] increases caused by inositol 1,4,5 trisphosphate (IP3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria. Bcl-2 overexpression also reduced the rate of Ca(2+) influx activated by Ca(2+) store depletion, possibly by an adaptive downregulation of this pathway. By interfering with Ca(2+)-dependent events at multiple intracellular sites, these effects of Bcl-2 on intracellular Ca(2+) homeostasis may contribute to the protective role of this oncogene against programmed cell death.     

Calsequestrin accumulation in rough endoplasmic reticulum promotes perinuclear Ca2+ release

Molecular mechanisms underlying Ca(2+) regulation by perinuclear endoplasmic/sarcoplasmic reticulum (ER/SR) cisternae in cardiomyocytes remain obscure. To investigate the mechanisms of changes in cardiac calsequestrin (CSQ2) trafficking on perinuclear Ca(2+) signaling, we manipulated the subcellular distribution of CSQ2 by overexpression of CSQ2-DsRed, which specifically accumulates in the perinuclear rough ER. Adult ventricular myocytes were infected with adenoviruses expressing CSQ2-DsRed, CSQ2-WT, or empty vector. We found that perinuclear enriched CSQ2-DsRed, but not normally distributed CSQ2-WT, enhanced nuclear Ca(2+) transients more potently than cytosolic Ca(2+) transients. Overexpression of CSQ2-DsRed produced more actively propagating Ca(2+) waves from perinuclear regions than did CSQ2-WT. Activities of the SR/ER Ca(2+)-ATPase and ryanodine receptor type 2, but not inositol 1,4,5-trisphosphate receptor type 2, were required for the generation of these perinuclear initiated Ca(2+) waves. In addition, CSQ2-DsRed was more potent than CSQ2-WT in inducing cellular hypertrophy in cultured neonatal cardiomyocytes. Our data demonstrate for the first time that CSQ2 retention in the rough ER/perinuclear region promotes perinuclear Ca(2+) signaling and predisposes to ryanodine receptor type 2-mediated Ca(2+) waves from CSQ2-enriched perinuclear compartments and myocyte hypotrophy. These findings provide new insights into the mechanism of CSQ2 in Ca(2+) homeostasis, suggesting that rough ER-localized Ca(2+) stores can operate independently in raising levels of cytosolic/nucleoplasmic Ca(2+) as a source of Ca(2+) for Ca(2+)-dependent signaling in health and disease.     

Plants, symbiosis and parasites: a calcium signalling connection

A unique family of protein kinases has evolved with regulatory domains containing sequences that are related to Ca(2+)-binding EF-hands. In this family, the archetypal Ca(2+)-dependent protein kinases (CDPKs) have been found in plants and some protists, including the malarial parasite, Plasmodium falciparum. Recent genetic evidence has revealed isoform-specific functions for a CDPK that is essential for Plasmodium berghei gametogenesis, and for a related chimeric Ca(2+) and calmodulin-dependent protein kinase (CCaMK) that is essential to the formation of symbiotic nitrogen-fixing nodules in plants. In Arabidopsis thaliana, the analysis of 42 isoforms of CDPK and related kinases is expected to delineate Ca(2+) signalling pathways in all aspects of plant biology.     

A new mode of Ca2+ signaling by G protein-coupled receptors: gating of IP3 receptor Ca2+ release channels by Gbetagamma

The most common form of Ca(2+) signaling by Gq-coupled receptors entails activation of PLCbeta2 by Galphaq to generate IP(3) and evoke Ca(2+) release from the ER. Another form of Ca(2+) signaling by G protein-coupled receptors involves activation of Gi to release Gbetagamma, which activates PLCbeta1. Whether Gbetagamma has additional roles in Ca(2+) signaling is unknown. Introduction of Gbetagamma into cells activated Ca(2+) release from the IP(3) Ca(2+) pool and Ca(2) oscillations. This can be due to activation of PLCbeta1 or direct activation of the IP(3)R by Gbetagamma. We report here that Gbetagamma potently activates the IP(3) receptor. Thus, Gbetagamma-triggered [Ca(2+)](i) oscillations are not affected by inhibition of PLCbeta. Coimmunoprecipitation and competition experiments with Gbetagamma scavengers suggest binding of Gbetagamma to IP(3) receptors. Furthermore, Gbetagamma inhibited IP(3) binding to IP(3) receptors. Notably, Gbetagamma activated single IP(3)R channels in native ER as effectively as IP(3). The physiological significance of this form of signaling is demonstrated by the reciprocal sensitivity of Ca(2+) signals evoked by Gi- and Gq-coupled receptors to Gbetagamma scavenging and PLCbeta inhibition. We propose that gating of IP(3)R by Gbetagamma is a new mode of Ca(2+) signaling with particular significance for Gi-coupled receptors.     

New insights into the molecular mechanisms of store-operated Ca2+ signaling in T cells

The activation of Ca(2+) entry through store-operated channels by agonists that deplete Ca(2+) from the endoplasmic reticulum (ER) is an ubiquitous signaling mechanism, the molecular basis of which has remained elusive for the past 20 years. In T lymphocytes, store-operated Ca(2+)-release-activated Ca(2+) (CRAC) channels constitute the sole pathway for Ca(2+) entry following antigen-receptor engagement, and their function is essential for driving the program of gene expression that underlies T-cell activation by antigen. The first molecular components of this pathway have recently been identified: stromal interaction molecule 1 (STIM1), the ER Ca(2+) sensor, and Orai1, a pore-forming subunit of the CRAC channel. Recent work shows that CRAC channels are activated in a complex fashion that involves the co-clustering of STIM1 in junctional ER directly opposite Orai1 in the plasma membrane. These studies reveal an abundance of sites where Ca(2+) signaling might be controlled to modulate the activity of T cells during the immune response.