GAP45 Phosphorylation Controls Assembly of the Toxoplasma Myosin XIV Complex

Toxoplasma gondii motility is powered by the myosin XIV motor complex, which consists of the myosin XIV heavy chain (MyoA), the myosin light chain (MLC1), GAP45, and GAP50, the membrane anchor of the complex. MyoA, MLC1, and GAP45 are initially assembled into a soluble complex, which then associates with GAP50, an integral membrane protein of the parasite inner membrane complex. While all proteins in the myosin XIV motor complex are essential for parasite survival, the specific role of GAP45 remains unclear. We demonstrate here that final assembly of the motor complex is controlled by phosphorylation of GAP45. This protein is phosphorylated on multiple residues, and by using mass spectroscopy, we have identified two of these, Ser¹⁶³ and Ser¹⁶⁷. The importance of these phosphorylation events was determined by mutation of Ser¹⁶³ and Ser¹⁶⁷ to Glu and Ala residues to mimic phosphorylated and nonphosphorylated residues, respectively. Mutation of Ser¹⁶³ and Ser¹⁶⁷ to either Ala or Glu residues does not affect targeting of GAP45 to the inner membrane complex or its association with MyoA and MLC1. Mutation of Ser¹⁶³ and Ser¹⁶⁷ to Ala residues also does not affect assembly of the mutant GAP45 protein into the myosin motor complex. Mutation of Ser¹⁶³ and Ser¹⁶⁷ to Glu residues, however, prevents association of the MyoA-MLC1-GAP45 complex with GAP50. These observations indicate that phosphorylation of Ser¹⁶³ and Ser¹⁶⁷ in GAP45 controls the final step in assembly of the myosin XIV motor complex.     

Plasticity between MyoC- and MyoA-Glideosomes: An Example of Functional Compensation in Toxoplasma gondii Invasion

The glideosome is an actomyosin-based machinery that powers motility in Apicomplexa and participates in host cell invasion and egress from infected cells. The central component of the glideosome, myosin A (MyoA), is a motor recruited at the pellicle by the acylated gliding-associated protein GAP45. In Toxoplasma gondii, GAP45 also contributes to the cohesion of the pellicle, composed of the inner membrane complex (IMC) and the plasma membrane, during motor traction. GAP70 was previously identified as a paralog of GAP45 that is tailored to recruit MyoA at the apical cap in the coccidian subgroup of the Apicomplexa. A third member of this family, GAP80, is demonstrated here to assemble a new glideosome, which recruits the class XIV myosin C (MyoC) at the basal polar ring. MyoC shares the same myosin light chains as MyoA and also interacts with the integral IMC proteins GAP50 and GAP40. Moreover, a central component of this complex, the IMC-associated protein 1 (IAP1), acts as the key determinant for the restricted localization of MyoC to the posterior pole. Deletion of specific components of the MyoC-glideosome underscores the installation of compensatory mechanisms with components of the MyoA-glideosome. Conversely, removal of MyoA leads to the relocalization of MyoC along the pellicle and at the apical cap that accounts for residual invasion. The two glideosomes exhibit a considerable level of plasticity to ensure parasite survival.     

Subcellular location, phosphorylation and assembly into the motor complex of GAP45 during Plasmodium falciparum schizont development

An actomyosin motor complex assembled below the parasite's plasma membrane drives erythrocyte invasion by Plasmodium falciparum merozoites. The complex is comprised of several proteins including myosin (MyoA), myosin tail domain interacting protein (MTIP) and glideosome associated proteins (GAP) 45 and 50, and is anchored on the inner membrane complex (IMC), which underlies the plasmalemma. A ternary complex of MyoA, MTIP and GAP45 is formed that then associates with GAP50. We show that full length GAP45 labelled internally with GFP is assembled into the motor complex and transported to the developing IMC in early schizogony, where it accumulates during intracellular development until merozoite release. We show that GAP45 is phosphorylated by calcium dependent protein kinase 1 (CDPK1), and identify the modified serine residues. Replacing these serine residues with alanine or aspartate has no apparent effect on GAP45 assembly into the motor protein complex or its subcellular location in the parasite. The early assembly of the motor complex suggests that it has functions in addition to its role in erythrocyte invasion.     

Calcium-dependent phosphorylation alters class XIVa myosin function in the protozoan parasite Toxoplasma gondii

Class XIVa myosins comprise a unique group of myosin motor proteins found in apicomplexan parasites, including those that cause malaria and toxoplasmosis. The founding member of the class XIVa family, Toxoplasma gondii myosin A (TgMyoA), is a monomeric unconventional myosin that functions at the parasite periphery to control gliding motility, host cell invasion, and host cell egress. How the motor activity of TgMyoA is regulated during these critical steps in the parasite''s lytic cycle is unknown. We show here that a small-molecule enhancer of T. gondii motility and invasion (compound 130038) causes an increase in parasite intracellular calcium levels, leading to a calcium-dependent increase in TgMyoA phosphorylation. Mutation of the major sites of phosphorylation altered parasite motile behavior upon compound 130038 treatment, and parasites expressing a nonphosphorylatable mutant myosin egressed from host cells more slowly in response to treatment with calcium ionophore. These data demonstrate that TgMyoA undergoes calcium-dependent phosphorylation, which modulates myosin-driven processes in this important human pathogen.     

Calcium signaling and the lytic cycle of the Apicomplexan parasite Toxoplasma gondii

Toxoplasma gondii has a complex life cycle involving different hosts and is dependent on fast responses, as the parasite reacts to changing environmental conditions. T. gondii causes disease by lysing the host cells that it infects and it does this by reiterating its lytic cycle, which consists of host cell invasion, replication inside the host cell, and egress causing host cell lysis. Calcium ion (Ca²⁺) signaling triggers activation of molecules involved in the stimulation and enhancement of each step of the parasite lytic cycle. Ca²⁺ signaling is essential for the cellular and developmental changes that support T. gondii parasitism.The characterization of the molecular players and pathways directly activated by Ca²⁺ signaling in Toxoplasma is sketchy and incomplete. The evolutionary distance between Toxoplasma and other eukaryotic model systems makes the comparison sometimes not informative. The advent of new genomic information and new genetic tools applicable for studying Toxoplasma biology is rapidly changing this scenario. The Toxoplasma genome reveals the presence of many genes potentially involved in Ca²⁺ signaling, even though the role of most of them is not known. The use of Genetically Encoded Calcium Indicators (GECIs) has allowed studies on the role of novel calcium-related proteins on egress, an essential step for the virulence and dissemination of Toxoplasma. In addition, the discovery of new Ca²⁺ players is generating novel targets for drugs, vaccines, and diagnostic tools and a better understanding of the biology of these parasites.     

Purification of tropomyosin, paramyosin, actin, tubulin, troponin and kinases for chemiproteomics and its application to different scientific fields

Background

p-aminobenzamidine (p-ABA) is used as a ligand in the purification of many serine proteases and in their removal from heterogeneous samples. Moreover, p-ABA has a potent ability to bind Ca²⁺-binding proteins. The binding ability and use of p-ABA in purification processes is still not fully understood.

Methodology/Principal Findings

A p-Aminobenzamidine (p-ABA) ligand enabled the purification of the panallergenic proteins tropomyosin and paramyosin, as well as actin, tubulin, troponin and several kinases and annexins, with variable specificity depending on the tissue source and slight modifications to the purification process. The high affinity of p-ABA to tropomyosin, paramyosin, actin, troponin and myosin is calcium-dependent, since calcium regulates the function of these proteins. In addition, p-ABA probably simulates phosphorylated serine and therefore purified appropriate kinases. Because p-ABA binds to calcium-dependent proteins, and probably those with binding sites containing serine, it is not a suitable inhibitor of proteolysis during the purification of such proteins. p-ABA is widely used to inhibit proteases during protein purification processes, but it is used in columns here to purify non-protease proteins. Two strategies were applied; the first was the inactivation of proteases that were not of interest using protease inhibitors. The second strategy employed was the use of a Ca²⁺ wash solution to remove calcium-dependent proteins. The removal of calcium-dependent proteins from rabbit hind muscle pointed out even more selective purification. It is possible to obtain two purified samples: a) calcium dependent proteins and b) calcium independent proteins. Moreover, p-ABA may be useful as a model to study processes involving the phosphorylation of serine.

Conclusion

A p-Aminobenzamidine (p-ABA) ligand enabled the purification of non-protease proteins, with variable specificity depending on the tissue source and slight modifications to the purification process. The method is applicable to various scientific branches, but is especially practical for medicinal applications.     

New therapeutic targets in Alzheimer's disease: brain deregulation of calcium and zinc

The molecular determinants of Alzheimer''s (AD) disease are still not completely known; however, in the past two decades, a large body of evidence has indicated that an important contributing factor for the disease is the development of an unbalanced homeostasis of two signaling cations: calcium (Ca²⁺) and zinc (Zn²⁺). Both ions serve a critical role in the physiological functioning of the central nervous system, but their brain deregulation promotes amyloid-β dysmetabolism as well as tau phosphorylation. AD is also characterized by an altered glutamatergic activation, and glutamate can promote both Ca²⁺ and Zn²⁺ dyshomeostasis. The two cations can operate synergistically to promote the generation of free radicals that further intracellular Ca²⁺ and Zn²⁺ rises and set the stage for a self-perpetuating harmful loop. These phenomena can be the initial steps in the pathogenic cascade leading to AD, therefore, therapeutic interventions aiming at preventing Ca²⁺ and Zn²⁺ dyshomeostasis may offer a great opportunity for disease-modifying strategies.     

Artemisinin induces calcium-dependent protein secretion in the protozoan parasite Toxoplasma gondii

Intracellular calcium controls several crucial cellular events in apicomplexan parasites, including protein secretion, motility, and invasion into and egress from host cells. The plant compound thapsigargin inhibits the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), resulting in elevated calcium and induction of protein secretion in Toxoplasma gondii. Artemisinins are natural products that show potent and selective activity against parasites, making them useful for the treatment of malaria. While the mechanism of action is uncertain, previous studies have suggested that artemisinin may inhibit SERCA, thus disrupting calcium homeostasis. We cloned the single-copy gene encoding SERCA in T. gondii (TgSERCA) and demonstrate that the protein localizes to the endoplasmic reticulum in the parasite. In extracellular parasites, TgSERCA partially relocalized to the apical pole, a highly active site for regulated secretion of micronemes. TgSERCA complemented a calcium ATPase-defective yeast mutant, and this activity was inhibited by either thapsigargin or artemisinin. Treatment of T. gondii with artemisinin triggered calcium-dependent secretion of microneme proteins, similar to the SERCA inhibitor thapsigargin. Artemisinin treatment also altered intracellular calcium in parasites by increasing the periodicity of calcium oscillations and inducing recurrent, strong calcium spikes, as imaged using Fluo-4 labeling. Collectively, these results demonstrate that artemisinin perturbs calcium homeostasis in T. gondii, supporting the idea that Ca²⁺-ATPases are potential drug targets in parasites.     

Anthrax lethal toxin suppresses murine cardiomyocyte contractile function and intracellular Ca2+ handling via a NADPH oxidase-dependent mechanism

Objectives

Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca²⁺ properties.

Methods

Murine cardiomyocyte contractile function and intracellular Ca²⁺ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca²⁺ decay rate. Stress signaling and Ca²⁺ regulatory proteins were assessed using Western blot analysis.

Results

In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca²⁺ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca²⁺ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca²⁺ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca²⁺ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.

Conclusions

Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca²⁺ through a NADPH oxidase-dependent mechanism.     

KCa2 channels activation prevents [Ca2+]i deregulation and reduces neuronal death following glutamate toxicity and cerebral ischemia

Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca²⁺]_i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca²⁺]_i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (K_Ca2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of K_Ca2.2 channels within 3 h after the onset of glutamate exposure. Activation of K_Ca2 channels preserved K_Ca2 expression and significantly reduced pathological increases in [Ca²⁺]_i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for K_Ca2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.     

Calcium Spiking Patterns and the Role of the Calcium/Calmodulin-Dependent Kinase CCaMK in Lateral Root Base Nodulation of Sesbania rostrata

Nodulation factor (NF) signal transduction in the legume-rhizobium symbiosis involves calcium oscillations that are instrumental in eliciting nodulation. To date, Ca²⁺ spiking has been studied exclusively in the intracellular bacterial invasion of growing root hairs in zone I. This mechanism is not the only one by which rhizobia gain entry into their hosts; the tropical legume Sesbania rostrata can be invaded intercellularly by rhizobia at cracks caused by lateral root emergence, and this process is associated with cell death for formation of infection pockets. We show that epidermal cells at lateral root bases respond to NFs with Ca²⁺ oscillations that are faster and more symmetrical than those observed during root hair invasion. Enhanced jasmonic acid or reduced ethylene levels slowed down the Ca²⁺ spiking frequency and stimulated intracellular root hair invasion by rhizobia, but prevented nodule formation. Hence, intracellular invasion in root hairs is linked with a very specific Ca²⁺ signature. In parallel experiments, we found that knockdown of the calcium/calmodulin-dependent protein kinase gene of S. rostrata abolished nodule development but not the formation of infection pockets by intercellular invasion at lateral root bases, suggesting that the colonization of the outer cortex is independent of Ca²⁺ spiking decoding.     

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca²⁺ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP₃Rs. Blockade of Ca²⁺-induced Ca²⁺ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca²⁺ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca²⁺ overload whereas IP₃R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca²⁺ release through RyRs contributes to cytosolic Ca²⁺ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes.     

Functional dissection of the apicomplexan glideosome molecular architecture

The glideosome of apicomplexan parasites is an actin- and myosin-based machine located at the pellicle, between the plasma membrane (PM) and inner membrane complex (IMC), that powers parasite motility, migration, and host cell invasion and egress. It is composed of myosin A, its light chain MLC1, and two gliding-associated proteins, GAP50 and GAP45. We identify GAP40, a polytopic protein of the IMC, as an additional glideosome component and show that GAP45 is anchored to the PM and IMC via its N- and C-terminal extremities, respectively. While the C-terminal region of GAP45 recruits MLC1-MyoA to the IMC, the N-terminal acylation and coiled-coil domain preserve pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress. The orthologous Plasmodium falciparum GAP45 can fulfill this dual function, as shown by transgenera complementation, whereas the coccidian GAP45 homolog (designated here as) GAP70 specifically recruits the glideosome to the apical cap of the parasite.     

An inhibitory effect of extracellular Ca2+ on Ca2+-dependent exocytosis

Aim

Neurotransmitter release is elicited by an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The action potential triggers Ca²⁺ influx through Ca²⁺ channels which causes local changes of [Ca²⁺]_i for vesicle release. However, any direct role of extracellular Ca²⁺ (besides Ca²⁺ influx) on Ca²⁺-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.

Results

Using photolysis of caged Ca²⁺ and caffeine-induced release of stored Ca²⁺, we found that extracellular Ca²⁺ inhibited exocytosis following moderate [Ca²⁺]_i rises (2–3 µM). The IC₅₀ for extracellular Ca²⁺ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca²⁺ concentration ([Ca²⁺]_o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca²⁺]_o. The calcimimetics Mg²⁺, Cd²⁺, G418, and neomycin all inhibited exocytosis. The extracellular Ca²⁺-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.

Conclusion/Significance

As an extension of the classic Ca²⁺ hypothesis of synaptic release, physiological levels of extracellular Ca²⁺ play dual roles in evoked exocytosis by providing a source of Ca²⁺ influx, and by directly regulating quantal size and release probability in neuronal cells.     

Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel

Increasing evidence suggests that changes in cytosolic Ca²⁺ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca²⁺ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca²⁺-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca²⁺ dependent. CDPK exhibits a Ca²⁺-induced electrophoretic mobility shift and its Ca²⁺-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPKα cross-react with CDPK. Micromolar Ca²⁺ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca²⁺-dependent protein phosphatase calcineurin enhances the Ca²⁺-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K⁺ channel KAT1 protein in a Ca²⁺-dependent manner. These results suggest that CDPK may be an important component of Ca²⁺ signaling in guard cells.     

The motility of a human parasite, Toxoplasma gondii, is regulated by a novel lysine methyltransferase

Protozoa in the phylum Apicomplexa are a large group of obligate intracellular parasites. Toxoplasma gondii and other apicomplexan parasites, such as Plasmodium falciparum, cause diseases by reiterating their lytic cycle, comprising host cell invasion, parasite replication, and parasite egress. The successful completion of the lytic cycle requires that the parasite senses changes in its environment and switches between the non-motile (for intracellular replication) and motile (for invasion and egress) states appropriately. Although the signaling pathway that regulates the motile state switch is critical to the pathogenesis of the diseases caused by these parasites, it is not well understood. Here we report a previously unknown mechanism of regulating the motility activation in Toxoplasma, mediated by a protein lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). AKMT depletion greatly inhibits activation of motility, compromises parasite invasion and egress, and thus severely impairs the lytic cycle. Interestingly, AKMT redistributes from the apical complex to the parasite body rapidly in the presence of egress-stimulating signals that increase [Ca²⁺] in the parasite cytoplasm, suggesting that AKMT regulation of parasite motility might be accomplished by the precise temporal control of its localization in response to environmental changes.     

Sulfhydryl modification induces calcium entry through IP₃-sensitive store-operated pathway in activation-dependent human neutrophils

As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT₁MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca²⁺]_c) without external calcium, followed by a second [Ca²⁺]_c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca²⁺]_c change of Ca²⁺-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca²⁺]_c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP₃) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP₃-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner.     

A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells

Inactivation of inward-rectifying K⁺ channels (I_K,in) by a rise in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca²⁺]_i action on I_K,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca²⁺]_i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recording I_K,in under a voltage clamp and [Ca²⁺]_i by Fura-2 fluorescence ratiophotometry. I_K,in inactivation correlated positively with [Ca²⁺]_i and indicated a K_i of 329 ± 31 nm with cooperative binding of four Ca²⁺ ions per channel. I_K,in was promoted by the Ca²⁺ channel antagonists Gd³⁺ and calcicludine, both of which suppressed the [Ca²⁺]_i rise, but the [Ca²⁺]_i rise was unaffected by the K⁺ channel blocker Cs⁺. We also found that ryanodine, an antagonist of intracellular Ca²⁺ channels that mediate Ca²⁺-induced Ca²⁺ release, blocked the [Ca²⁺]_i rise, and Mn²⁺ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca²⁺]_i control of I_K,in and to roles for discrete Ca²⁺ flux pathways in feedback control of the K⁺ channels by membrane voltage.Ca²⁺ underlies many fundamental regulatory processes in plants, including adaptive responses to abiotic environmental stress (Knight et al., 1996; Russell et al., 1996; McAinsh et al., 1997) and programmed cell death evoked by pathogen attack (Low and Merida, 1996; Hammondkosack and Jones, 1997). Coordination of changes in [Ca²⁺]_i and its integration with downstream response elements are central in coupling stimulus input to cellular response in these processes.In stomatal guard cells, the best characterized higher-plant cell model, major downstream targets of [Ca²⁺]_i and their roles in stomatal function have been identified. Increasing [Ca²⁺]_i is known to inactivate I_K,in and to activate Cl⁻ channels, events that bias plasma membrane transport for net efflux of osmotically active solute and a loss of turgor, which drives stomatal closure (Blatt and Grabov, 1997). Furthermore, changes in [Ca²⁺]_i are associated with ABA, CO₂, and the growth hormone auxin (Blatt and Grabov, 1997; McAinsh et al., 1997). These [Ca²⁺]_i signals have been observed to oscillate (McAinsh et al., 1995; Webb et al., 1996), characteristics that may constitute “Ca²⁺ signatures” to encode specific downstream responses (Berridge, 1996). Yet, despite the evidence for [Ca²⁺]_i signaling in guard cells, surprisingly little detail is known about the link between [Ca²⁺]_i changes and ion channel activity at the plasma membrane or about the mechanisms mediating such [Ca²⁺]_i changes. To our knowledge, in no instance have the characteristics of ion channel regulation by Ca²⁺ been quantified directly in any higher-plant cell.We recently described the coupling of membrane voltage to [Ca²⁺]_i, demonstrating that hyperpolarization, whether under a voltage clamp or in the presence of low [K⁺]_o, evoked [Ca²⁺]_i increases in guard cells, and that the voltage threshold for [Ca²⁺]_i rise was profoundly altered by ABA (Grabov and Blatt, 1998). Our observations indicated a link to Ca²⁺ influx across the plasma membrane and raised questions about the efficacy of [Ca²⁺]_i in inactivating I_K,in and about the contributions of intracellular Ca²⁺ release to the [Ca²⁺]_i signal. We have used membrane voltage to experimentally manipulate [Ca²⁺]_i and report that I_K,in is strongly dependent on [Ca²⁺]_i, consistent with a cooperative binding of four Ca²⁺ ions to effect inactivation. Additional experiments indicate that voltage-evoked [Ca²⁺]_i increases depend both on Ca²⁺ influx and on release of Ca²⁺ from intracellular stores. These results underscore the role of [Ca²⁺]_i as a high-gain “switch” in the control of I_K,in, and implicate [Ca²⁺]_i in feedback control linking membrane voltage to the activity of the K⁺ channels.     

The effect of OPA1 on mitochondrial Ca²⁺ signaling

The dynamin-related GTPase protein OPA1, localized in the intermembrane space and tethered to the inner membrane of mitochondria, participates in the fusion of these organelles. Its mutation is the most prevalent cause of Autosomal Dominant Optic Atrophy. OPA1 controls the diameter of the junctions between the boundary part of the inner membrane and the membrane of cristae and reduces the diffusibility of cytochrome c through these junctions. We postulated that if significant Ca²⁺ uptake into the matrix occurs from the lumen of the cristae, reduced expression of OPA1 would increase the access of Ca²⁺ to the transporters in the crista membrane and thus would enhance Ca²⁺ uptake. In intact H295R adrenocortical and HeLa cells cytosolic Ca²⁺ signals evoked with K⁺ and histamine, respectively, were transferred into the mitochondria. The rate and amplitude of mitochondrial [Ca²⁺] rise (followed with confocal laser scanning microscopy and FRET measurements with fluorescent wide-field microscopy) were increased after knockdown of OPA1, as compared with cells transfected with control RNA or mitofusin1 siRNA. Ca²⁺ uptake was enhanced despite reduced mitochondrial membrane potential. In permeabilized cells the rate of Ca²⁺ uptake by depolarized mitochondria was also increased in OPA1-silenced cells. The participation of Na⁺/Ca²⁺ and Ca²⁺/H⁺ antiporters in this transport process is indicated by pharmacological data. Altogether, our observations reveal the significance of OPA1 in the control of mitochondrial Ca²⁺ metabolism.     

Big domains are novel Ca²+-binding modules: evidences from big domains of Leptospira immunoglobulin-like (Lig) proteins

Background

Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca²⁺. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca²⁺ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold.

Principal Findings

We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9^th (Lig A9) and 10^th repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca²⁺ with dissociation constants of 2–4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold.

Conclusions

We demonstrate that the Lig are Ca²⁺-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca²⁺. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca²⁺-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca²⁺ binding.