Free Calcium Concentration Research Articles

Interaction of calcium responsive proteins and transcriptional factors with the PHO regulon in yeasts and fungi.

Phosphate and calcium ions are nutrients that play key roles in growth, differentiation and the production of bioactive secondary metabolites in filamentous fungi. Phosphate concentration regulates the biosynthesis of hundreds of fungal metabolites. The central mechanisms of phosphate transport and regulation, mediated by the master Pho4 transcriptional factor are known, but many aspects of the control of gene expression need further research. High ATP concentration in the cells leads to inositol pyrophosphate molecules formation, such as IP3 and IP7, that act as phosphorylation status reporters. Calcium ions are intracellular messengers in eukaryotic organisms and calcium homeostasis follows elaborated patterns in response to different nutritional and environmental factors, including cross-talking with phosphate concentrations. A large part of the intracellular calcium is stored in vacuoles and other organelles forming complexes with polyphosphate. The free cytosolic calcium concentration is maintained by transport from the external medium or by release from the store organelles through calcium permeable transient receptor potential (TRP) ion channels. Calcium ions, particularly the free cytosolic calcium levels, control the biosynthesis of fungal metabolites by two mechanisms, 1) direct interaction of calcium-bound calmodulin with antibiotic synthesizing enzymes, and 2) by the calmodulin-calcineurin signaling cascade. Control of very different secondary metabolites, including pathogenicity determinants, are mediated by calcium through the Crz1 factor. Several interactions between calcium homeostasis and phosphate have been demonstrated in the last decade: 1) The inositol pyrophosphate IP3 triggers the release of calcium ions from internal stores into the cytosol, 2) Expression of the high affinity phosphate transporter Pho89, a Na+/phosphate symporter, is controlled by Crz1. Also, mutants defective in the calcium permeable TRPCa7-like of Saccharomyces cerevisiae shown impaired expression of Pho89. This information suggests that CrzA and Pho89 play key roles in the interaction of phosphate and calcium regulatory pathways, 3) Finally, acidocalcisomes organelles have been found in mycorrhiza and in some melanin producing fungi that show similar characteristics as protozoa calcisomes. In these organelles there is a close interaction between orthophosphate, pyrophosphate and polyphosphate and calcium ions that are absorbed in the polyanionic polyphosphate matrix. These advances open new perspectives for the control of fungal metabolism.

SARS-CoV-2 spike protein receptor-binding domain perturbates intracellular calcium homeostasis and impairs pulmonary vascular endothelial cells

Exposure to the spike protein or receptor-binding domain (S-RBD) of SARS-CoV-2 significantly influences endothelial cells and induces pulmonary vascular endotheliopathy. In this study, angiotensin-converting enzyme 2 humanized inbred (hACE2 Tg) mice and cultured pulmonary vascular endothelial cells were used to investigate how spike protein/S-RBD impacts pulmonary vascular endothelium. Results show that S-RBD leads to acute-to-prolonged induction of the intracellular free calcium concentration ([Ca2+]i) via acute activation of TRPV4, and prolonged upregulation of mechanosensitive channel Piezo1 and store-operated calcium channel (SOCC) key component Orai1 in cultured human pulmonary arterial endothelial cells (PAECs). In mechanism, S-RBD interacts with ACE2 to induce formation of clusters involving Orai1, Piezo1 and TRPC1, facilitate the channel activation of Piezo1 and SOCC, and lead to elevated apoptosis. These effects are blocked by Kobophenol A, which inhibits the binding between S-RBD and ACE2, or intracellular calcium chelator, BAPTA-AM. Blockade of Piezo1 and SOCC by GsMTx4 effectively protects the S-RBD-induced pulmonary microvascular endothelial damage in hACE2 Tg mice via normalizing the elevated [Ca2+]i. Comparing to prototypic strain, Omicron variants (BA.5.2 and XBB) of S-RBD induces significantly less severe cell apoptosis. Transcriptomic analysis indicates that prototypic S-RBD confers more severe acute impacts than Delta or Lambda S-RBD. In summary, this study provides compelling evidence that S-RBD could induce persistent pulmonary vascular endothelial damage by binding to ACE2 and triggering [Ca2+]i through upregulation of Piezo1 and Orai1. Targeted inhibition of ACE2-Piezo1/SOCC-[Ca2+]i axis proves a powerful strategy to treat S-RBD-induced pulmonary vascular diseases.

IP3R1 regulates calcium balance in porcine oocyte maturation and early embryonic development

The dynamic balance of Ca2+ in oocytes promotes the recovery of the meiotic arrest phase, consequently promoting oocyte maturation. Hence, the analysis of the maintenance and role of calcium homeostasis in oocytes has important guiding significance for obtaining high-quality eggs and maintaining the development of preimplantation embryos. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are calcium channel proteins that regulate the dynamic balance between the endoplasmic reticulum (ER) and mitochondrial Ca2+. Nevertheless, the expression and role of IP3R in normal pig oocytes have not been reported, and other studies have focused on the role of IP3R in damaged cells. The purpose of this study was to investigate the potential role of IP3R in regulating calcium homeostasis in oocyte maturation and early embryonic development. Our results showed that IP3R1 is stably expressed at different stages of porcine oocyte meiosis, IP3R1 gradually converges to the cortex, and cortical clusters are formed in MII stages. The loss of IP3R1 activity contributeds to the failure of porcine oocyte maturation and cumulus cell expansion, as well as the obstruction of polar body excretion. Further analysis showed that IP3R1 plays an important role in affecting calcium balance by regulating the IP3R1-GRP75-VDAC1 channel between mitochondria and the endoplasmic reticulum (ER) during porcine oocyte maturation. Inhibiting IP3R1 expression-induced ER dysfunction, contributeding to ER calcium concentration ([Ca2+]ER) release outwards into mitochondria and causing mitochondrial free calcium concentration ([Ca2+]m) overload and mitochondrial oxidative stress, which was confirmed by the increase in the level of reactive oxygen species (ROS) and apoptosis. Thereby, IP3R1 plays an important role in affecting calcium balance by regulating the IP3R1-GRP75 -VDAC1 channel between mitochondria and the ER during porcine oocyte maturation, inhibiting IP3R1 expression-induced calcium overload and mitochondrial oxidative stress, and increasing ROS levels and apoptosis.

Evaluation of a novel microfluidic chip-like device for purifying bovine frozen-thawed semen for in vitro fertilization

The aim of this study was to validate a novel sperm purification device, the VetCount™ Harvester, for use in bovine in vitro fertilization (IVF). The device's performance was compared to BoviPure™ gradient centrifugation, a commercially available and accepted routine technique. Semen quality parameters were assessed for frozen-thawed semen from six different bulls (n = 6) following sperm purification. For each bull two semen subsamples were purified utilizing BoviPure™ gradient centrifugation and the VetCount™ Harvester, including a third subsample as untreated control. Both treatments significantly increased the proportion of progressively motile sperm cells (84.4 ± 14.1% and 85.1 ± 7.8%, respectively) compared to the untreated semen (41.9 ± 18.8%). BoviPure™ gradient and VetCount™ Harvester selected predominantly viable acrosome intact (VAI) sperm cells with low membrane fluidity and low free intracellular calcium concentration [Ca2+]i (76.5 ± 4.4% and 78.6 ± 6.0%). Normalizing [Ca2+]i of VAI sperm cells (non-treated semen: [Ca2+]i = 1) VetCount™ Harvester purified spermatozoa (0.67 ± 0.10) showed significantly lower [Ca2+]i than BoviPure™ treated sperm (0.84 ± 0.14; P < 0.05). Subsequently, the fertilizing ability of the spermatozoa was evaluated performing a competitive fertilization assay. Sperm cells from both treatment groups were fluorescently labelled using different dyes and added in equal amounts to in vitro matured oocytes. After 18 h co-incubation, the origin of the fertilizing sperm cell was evaluated via fluorescence microscopy. In two bulls, VetCount™ Harvester selected sperm that fertilized significantly more oocytes then BoviPure™ treated sperm, in another bull it was the opposite. For three bulls no difference was observed.We conclude that the VetCount™ Harvester selects a high-quality, fertile sperm fraction from frozen-thawed bull semen. However, some considerations have to be kept in mind for the direct use of the isolated sperm fraction in IVF.

Role of Calcium-Activated Potassium Channels in Proliferation, Migration and Invasion of Human Chronic Myeloid Leukemia K562 Cells.

Calcium-activated potassium channels (KCa) are important participants in calcium signaling pathways due to their ability to be activated by an increase in intracellular free calcium concentration. KCa channels are involved in the regulation of cellular processes in both normal and pathophysiological conditions, including oncotransformation. Previously, using patch-clamp, we registered the KCa currents in the plasma membrane of human chronic myeloid leukemia K562 cells, whose activity was controlled by local Ca2+ entry via mechanosensitive calcium-permeable channels. Here, we performed the molecular and functional identification of KCa channels and have uncovered their role in the proliferation, migration and invasion of K562 cells. Using a combined approach, we identified the functional activity of SK2, SK3 and IK channels in the plasma membrane of the cells. Selective SK and IK channel inhibitors, apamin and TRAM-34, respectively, reduced the proliferative, migratory and invasive capabilities of human myeloid leukemia cells. At the same time, the viability of K562 cells was not affected by KCa channel inhibitors. Ca2+ imaging showed that both SK and IK channel inhibitors affect Ca2+ entry and this could underlie the observed suppression of pathophysiological reactions of K562 cells. Our data imply that SK/IK channel inhibitors could be used to slow down the proliferation and spreading of chronic myeloid leukemia K562 cells that express functionally active KCa channels in the plasma membrane.

Nanoscale thermal control of a single living cell enabled by diamond heater-thermometer

We report a new approach to controllable thermal stimulation of a single living cell and its compartments. The technique is based on the use of a single polycrystalline diamond particle containing silicon-vacancy (SiV) color centers. Due to the presence of amorphous carbon at its intercrystalline boundaries, such a particle is an efficient light absorber and becomes a local heat source when illuminated by a laser. Furthermore, the temperature of such a local heater is tracked by the spectral shift of the zero-phonon line of SiV centers. Thus, the diamond particle acts simultaneously as a heater and a thermometer. In the current work, we demonstrate the ability of such a Diamond Heater-Thermometer (DHT) to locally alter the temperature, one of the numerous parameters that play a decisive role for the living organisms at the nanoscale. In particular, we show that the local heating of 11–12 °C relative to the ambient temperature (22 °C) next to individual HeLa cells and neurons, isolated from the mouse hippocampus, leads to a change in the intracellular distribution of the concentration of free calcium ions. For individual HeLa cells, a long-term (about 30 s) increase in the integral intensity of Fluo-4 NW fluorescence by about three times is observed, which characterizes an increase in the [Ca2+]cyt concentration of free calcium in the cytoplasm. Heating near mouse hippocampal neurons also caused a calcium surge—an increase in the intensity of Fluo-4 NW fluorescence by 30% and a duration of ~ 0.4 ms.

Effects of pH and Ionic Strength in Calcium on the Stability and Aeration Characteristics of Dairy Emulsion.

The effects of different pH levels and ionic strength in calcium on the stability and aeration characteristics of dairy emulsions were investigated in this study. The results revealed that the stability and aeration characteristics of the emulsion were enhanced as the pH value increased from 6.5 to 7.0 and were optimal within the pH of 6.8~7.0, while the concentration of free calcium ions (Ca2+) was 2.94~3.22 mM. With the pH subsequently fixed at 6.8 and 7.0, when the addition of CaCl2 was increased to 2.00 mM (free Ca2+ strength > 4.11 mM), stability and aeration characteristics reduced significantly, including the flocculation of fat globules, an increase in particle size, and a decrease in the zeta potential and viscosity of the O/W emulsion, all leading to an increase in interfacial protein mass and decreased overrun and foam firmness. Overall, the results indicated that pH changes and CaCl2 addition significantly influenced the stability and aeration characteristics of dairy emulsions, by influencing free Ca2+ strength, which is an important factor in determining the quality of dairy emulsions.

MICU1 occludes the mitochondrial calcium uniporter in divalent-free conditions.

Mitochondrial Ca2+uptake is mediated by the mitochondrial uniporter complex (mtCU) that includes a tetramer of the pore-forming subunit, MCU, a scaffold protein, EMRE, and the EF-hand regulatory subunit, MICU1 either homodimerized or heterodimerized with MICU2/3. MICU1 has been proposed to regulate Ca2+ uptake via the mtCU by physically occluding the pore and preventing Ca2+ flux at resting cytoplasmic [Ca2+] (free calcium concentration) and to increase Ca2+ flux at high [Ca2+] due to cooperative activation of MICUs EF-hands. However, mtCU and MICU1 functioning when its EF-hands are unoccupied by Ca2+ is poorly studied due to technical limitations. To overcome this barrier, we have studied the mtCU in divalent-free conditions by assessing the Ru265-sensitive Na+influx using fluorescence-based measurement of mitochondrial matrix [Na+] (free sodium concentration) rise and the ensuing depolarization and swelling. We show an increase in all these measures of Na+ uptake in MICU1KO cells as compared to wild-type (WT) and rescued MICU1KO HEK cells. However, mitochondria in WT cells and MICU1 stable-rescued cells still allowed some Ru265-sensitive Na+ influx that was prevented by MICU1 in excess upon acute overexpression. Thus, MICU1 restricts the cation flux across the mtCU in the absence of Ca2+, but even in cells with high endogenous MICU1 expression such as HEK, some mtCU seem to lack MICU1-dependent gating. We also show rearrangement of the mtCU and altered number of functional channels in MICU1KO and different rescues, and loss of MICU1 during mitoplast preparation, that together might have obscured the pore-blocking function of MICU1 in divalent-free conditions in previous studies.

An increase in the cytosolic concentration of free calcium ions activates the neutrophil NADPH-oxidase provided that the free fatty acid receptor 2 has been allosterically modulated

Signals generated by free fatty acid receptor 2 (FFA2R) can activate the neutrophil NADPH-oxidase without involvement of any orthosteric FFA2R agonist. The initiating signals may be generated by P2Y2R, the receptor for ATP. An FFA2R specific allosteric modulator (PAM; Cmp58) was required for this response and used to investigate the mechanism by which signals generated by ATP/P2Y2R activate an FFA2R dependent process. The P2Y2R induced signal that together with the modulated FFA2R activates neutrophils, was generated downstream of the Gαq containing G protein coupled to P2Y2R. A rise in the cytosolic concentration of ionized calcium ([Ca2+]i) was hypothesized to be the important signal. The hypothesis gained support from the finding that the modulator transferred the neutrophils to a Ca2+sensitive state. The rise in [Ca2+]i induced by the Ca2+ specific ionophore ionomycin, activated the neutrophils provided that an allosteric modulator was bound to FFA2R. The activity of the superoxide generating NADPH-oxidase induced by ionomycin was rapidly terminated and the FFA2Rs could then no longer be activated by the FFA2R agonist propionate or by the signal generated by ATP/P2Y2R. The non-responding state of FFA2R was, however, revoked by a cross-activating allosteric FFA2R modulator. The [Ca2+]i mediated activation of neutrophils with their FFA2Rs allosterically modulated, represent a unique regulatory receptor crosstalk mechanism by which the activation potency of a G protein coupled receptor is controlled by a receptor-crosstalk signaling system operating from the cytosolic side of the plasma membrane.

Novel C-F-S-H/PCE nanocomposites with high-efficiency seeding effect on the hydration of Portland cement

Novel C-F-S-H/PCE (calcium-ferrite-silicate-hydrates/polycarboxylate ether) nanocomposites were synthesized successfully by incorporating Fe3+ during the synthesis of C-S-H/PCE. The results of isothermal heat flow calorimetry and compressive strength tests indicated that C-F-S-H/PCE nanocomposites showed high-efficiency seeding effect on early cement hydration due to the higher absorption of PCE and smaller particle size of nano-seeds than those of traditional C-S-H/PCE nanocomposites. And the best seeding effect of C-F-S-H/PCE nanocomposites was identified when the Ca/Si and Fe/Si were 1.0 and 0.03 respectively. Moreover, the incorporation of Fe3+ did not affect the concentration of free calcium in the C-F-S-H/PCE suspension whose morphologies were composed of irregular globules and network gel. Additionally, the incorporated Fe3+ made the semi-crystalline C-S-H structure more amorphous though it was not a direct factor that affected the seeding effect of the nanocomposites. All in all, this study provided a new idea from modified seeding nanocomposites for low-CO2 cement and a basis for the production of nano-seeds based on Fe-rich wastes.

Особенности постлучевых нарушений аккумуляции ионов кальция во внутриклеточных депо тромбоцитов в различные сроки постлучевого периода

Calcium ions are an important intracellular regulator; an increase in their concentration in the cytoplasm plays a key role in determining the functional activity of platelets associated with platelet aggregation and the release reaction. The main way to reduce the concentration of free calcium ions in the platelet cytoplasm is the activation of Ca2+-ATPases of intracellular depositing structures and the plasma membrane, which reduce the cytoplasmic content of calcium due to its reuptake in the depot (resequestration) and the release of Ca2+ from the cell. Platelets express two Ca2+-ATPase isoforms in different depositor structures (SERCA2b and SERCA3) and two plasma membrane Ca2+-ATPase isoforms (PMCA1b and 4b). The SERCA2b isoform, which is Ca2+-ATPase of the dense tubular system, is inhibited by thapsigargin. The SERCA3 isoform present in acidic organelles has low sensitivity to thapsigargin, but high sensitivity to tBHQ (2,5-di-(tert-butyl)-1,4-hydroquinone). When stimulated with micromolar concentrations of thapsigargin and ionomycin, which cause complete depletion of the calcium depots of the tubular system and acid organelles, differences in the levels of accumulated calcium in the norm and in the post-radiation period are found in a calcium-free medium: an increase in calcium concentration by 1.4 times is noted on the 3rd day. In the structures of the tubular system of platelets, under the action of nmolar concentrations of thapsigargin, which inhibit SER-CA2b Ca2+-ATPase of tubular systems, a change in the content of deposited calcium in irradiated animals was revealed with an increase on the 3rd day. Under conditions of inhibition of Ca2+-ATPase SERCA3 in a calcium-free medium in platelets on the 3rd day after irradiation at a dose of 1 Gy, a more intense release of calcium ions was also noted compared to the control. On the 10th, 30th, 90th day, there were no statistically significant differences in these indicators. In the calcium-containing medium, a more significant intake of calcium ions from the outside into the cytoplasm was observed due to the activation of depot-controlled mechanisms in the post-radiation period, especially on the 3rd and 10th days.

Isotherm kinetics of PIP2 bound gelsolin inactivation

Actin monomers (G-actin) and filaments (F-actin) have dynamical rearrangement thus manage cellular motility, division and transport processes. The gelsolin (GSN) regulates the remodeling of cytoskeleton. After the activation of GSN by calcium ions, it can sever actin filaments then capped at its barbed end. In the cytoplasm, GSN manages the cellular motions and morphology. Phosphatidylinositol 4,5-bisphosphate (PIP2) is involved in signal transduction and the regulation of the actin cytoskeleton by regulation of actin-binding proteins. GSN can bind to PIP2 and thus can be localized in the near of the plasma membrane and released from the end of F-actin. We test here with isoperibol calorimetry the enthalpy change, within the interplay between GSN and F-actin under nano-, micro- and millimolar calcium concentrations and express the importance of PIP2 binding for the inactivation of GSN. As we have demonstrated here that PIP2 binding stabilizes the structure of gelsolin and reduces its actin monomer binding activity under nanomolar calcium as the typical cytoplasmic calcium concentration of resting cells. The gelsolin shows partial activity under micromolar and total activity with strong responses under millimolar calcium. If gelsolin-capped filaments point at the plasma membrane helps the binding between gelsolin and PIP2, and hence, filament uncapping in case of resting cells. We presume that the low free calcium concentration keeps on the structure of gelsolin which is able to bind actin within the cooperativity of actin bound calcium. Gelsolin can help to manage monomer pool far from the membrane and it can be linked to a basic sensory mechanism which drives the direction of filament growth in the near of the membrane.

A Review on the Effect of Calcium Sequestering Salts on Casein Micelles: From Model Milk Protein Systems to Processed Cheese.

Phosphates and citrates are calcium sequestering salts (CSS) most commonly used in the manufacture of processed cheese, either singly or in mixtures. Caseins are the main structure forming elements in processed cheese. Calcium sequestering salts decrease the concentration of free calcium ions by sequestering calcium from the aqueous phase and dissociates the casein micelles into small clusters by altering the calcium equilibrium, thereby resulting in enhanced hydration and voluminosity of the micelles. Several researchers have studied milk protein systems such as rennet casein, milk protein concentrate, skim milk powder, and micellar casein concentrate to elucidate the influence of calcium sequestering salts on (para-)casein micelles. This review paper provides an overview of the effects of calcium sequestering salts on the properties of casein micelles and consequently the physico-chemical, textural, functional, and sensorial attributes of processed cheese. A lack of proper understanding of the mechanisms underlying the action of calcium sequestering salts on the processed cheese characteristics increases the risk of failed production, leading to the waste of resources and unacceptable sensorial, appearance, and textural attributes, which adversely affect the financial side of processors and customer expectations.

СОДЕРЖАНИЕ ИОНИЗИРОВАННОГО КАЛЬЦИЯ В РОТОВОЙ ЖИДКОСТИ У ДЕТЕЙ В ГОРОДЕ С ЭКОТОКСИКАНТАМИ

The study of the pathogenesis of enamel demineralization in children living in a city with ecotoxicants is an urgent task of modern dentistry. We examined 183 schoolchildren aged 7 and 12 in Ufa. The children were divided into groups depending on where they live. The first group included 94 children from an area located in an industrial district with an environmentally unfavorable situation. The second group (control) included 89 schoolchildren living in an ecologically good district of the city of Ufa. We studied the content of free calcium in the saliva of schoolchildren in two districts of the city of Ufa. When determining the activity of caries according to Vinogradova T.F. (I, II, III degree) among 7 and 12-year-old children, we did not reveal a high correlation between the district where the children live and the concentration of free calcium in the oral fluid. In the mixed saliva of 7-year-old schoolchildren of the first and second groups, the concentration of free calcium was 0.645 ± 0.018 and 0.638 ± 0.011 mm/l (p &gt; 0.05), in 12-year-olds it was 0.603 ± 0.050 and 0.597 ± 0.040 mm/l (p &gt; 0.05). In the industrial district, we noted the maximum increase in free calcium at the III degree of caries activity. This indicator was 0.851 ± 0.006 mmol/l (p &lt; 0.001) in 7-year-old children, and 0.760 ± 0.005 mmol/l in 12-year-old schoolchildren (p &lt; 0.001). While in the district without ecotoxicants, free calcium in 7-year-old schoolchildren was 0.846 ± 0.026 mmol/l (p &lt; 0.001), and in 12-year-old children it was 0.774 ± 0.010 mmol/l (p &lt; 0.001). Thus, the third degree of caries activity is highly correlated with an increase in the concentration of free calcium in saliva, which is proved by studies of oral fluid in schoolchildren from different districts of Ufa.

Delineating Ultrafast Structural Dynamics of a Green-Red Fluorescent Protein for Calcium Sensing.

Fluorescent proteins (FPs) are indispensable tools for noninvasive bioimaging and sensing. Measuring the free cellular calcium (Ca2+) concentrations in vivo with genetically encodable FPs can be a relatively direct measure of neuronal activity due to the complex signaling role of these ions. REX-GECO1 is a recently developed red-green emission and excitation ratiometric FP-based biosensor that achieves a high dynamic range due to differences in the chromophore response to light excitation with and without calcium ions. Using steady-state electronic measurements (UV/Visible absorption and emission), along with time-resolved spectroscopic techniques including femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS), the potential energy surfaces of these unique biosensors are unveiled with vivid details. The ground-state structural characterization of the Ca2+-free biosensor via FSRS reveals a more spacious protein pocket that allows the chromophore to efficiently twist and reach a dark state. In contrast, the more compressed cavity within the Ca2+-bound biosensor results in a more heterogeneous distribution of chromophore populations that results in multi-step excited state proton transfer (ESPT) pathways on the sub-140 fs, 600 fs, and 3 ps timescales. These results enable rational design strategies to enlarge the spectral separation between the protonated/deprotonated forms and the Stokes shift leading to a larger dynamic range and potentially higher fluorescence quantum yield, which should be broadly applicable to the calcium imaging and biosensor communities.

Intracellular free calcium concentration and oxidative damage in erythrocytes from workers occupationally exposed to organophosphate pesticides

Intracellular free calcium concentration and oxidative damage in erythrocytes from workers occupationally exposed to organophosphate pesticides

Effect of Buffers with Multiple Binding Sites on Calcium Waves

The existence and properties of intracellular waves of increased free cytoplasmic calcium concentration (calcium waves) are strongly affected by the binding and unbinding of calcium ions to a multitude of different buffers in the cell. These buffers can be mobile or immobile and, in general, have multiple binding sites that are not independent. Previous theoretical studies have focused on the case when each buffer molecule binds a single calcium ion. In this study, we analyze how calcium waves are affected by calcium buffers with two non-independent binding sites, and show that the interactions between the calcium binding sites can result in the emergence of new behaviors. In particular, for certain combinations of kinetic parameters, the profiles of buffer molecules with one calcium ion bound can be non-monotone.

CNTnC and fYTnC2, Genetically Encoded Green Calcium Indicators Based on Troponin C from Fast Animals

NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca2+ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively.

Binary-FRET reveals transient excited-state structure associated with activity-dependent CaMKII - NR2B binding and adaptation

Synaptic functions are mediated and modulated by a coordinated choreography of protein conformational changes and interactions in response to intracellular calcium dynamics. Time-lapse Förster resonance energy transfer can be used to study the dynamics of both conformational changes and protein-protein interactions simultaneously under physiological conditions if two resonance energy transfer reactions can be multiplexed. Binary-FRET is a technique developed to independently monitor the dynamics of calcium-calmodulin dependent protein kinase-II catalytic-domain pair separation in the holoenzyme, and its role in establishing activity-dependent holoenzyme affinity for the NR2B binding fragment of the N-methyl-D-aspartate receptor. Here we show that a transient excited-state intermediate exists where paired catalytic-domains in the holoenzyme first separate prior to subsequent NR2B association. Additionally, at non-saturating free calcium concentrations, our multiplexed approach reveals that the holoenzyme exhibits a biochemical form of plasticity, calcium dependent adaptation of T-site ligand binding affinity.

Insulin Diminishes Superoxide Increase in Cytosol and Mitochondria of Cultured Cortical Neurons Treated with Toxic Glutamate.

Glutamate excitotoxicity is involved in the pathogenesis of many disorders, including stroke, traumatic brain injury, and Alzheimer’s disease, for which central insulin resistance is a comorbid condition. Neurotoxicity of glutamate (Glu) is primarily associated with hyperactivation of the ionotropic N-methyl-D-aspartate receptors (NMDARs), causing a sustained increase in intracellular free calcium concentration ([Ca2+]i) and synchronous mitochondrial depolarization and an increase in intracellular superoxide anion radical (O2–•) production. Recently, we found that insulin protects neurons against excitotoxicity by decreasing the delayed calcium deregulation (DCD). However, the role of insulin in O2–• production in excitotoxicity still needs to be clarified. The present study aims to investigate insulin’s effects on glutamate-evoked O2–• generation and DCD using the fluorescent indicators dihydroethidium, MitoSOX Red, and Fura-FF in cortical neurons. We found a linear correlation between [Ca2+]i and [O2–•] in primary cultures of the rat neuron exposed to Glu, with insulin significantly reducing the production of intracellular and mitochondrial O2–• in the primary cultures of the rat neuron. MK 801, an inhibitor of NMDAR-gated Ca2+ influx, completely abrogated the glutamate effects in both the presence and absence of insulin. In experiments in sister cultures, insulin diminished neuronal death and O2 consumption rate (OCR).