Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells

Calcium (Ca²⁺) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca²⁺]_i is the use of fluorescent Ca²⁺ indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca²⁺ indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca²⁺-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca²⁺-free and Ca²⁺-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca²⁺-free and Ca²⁺-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca²⁺ sensitivity is reduced. Nevertheless, quantitative [Ca²⁺]_i recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology.

Effect of the ionic strength and prostaglandin E2 on the free Ca2+ concentration and the Ca2+ influx in human red blood cells

Human red blood cells (RBCs) were loaded with the Ca(2+)-sensitive fluorescent dye fura-2 to investigate the effects of media ionic strength and prostaglandin E2 (PGE2) on the intracellular free Ca2+ concentration ([Ca2+]i). [Ca2+]i of intact RBCs in a Ca(2+)-containing physiological (high) ionic strength (HIS) solution was 75.1 +/- 8.3 nM after 5 min incubation, increasing to 114.9 +/- 9.6 nM after 1 h. In Ca(2+)-containing low ionic strength (LIS) solutions, [Ca2+]i was significantly lower than in the Ca(2+)-containing HIS solution (p = 0.041 or 0.0385 for LIS solutions containing 200 or 250 mM sucrose, respectively), but, as in HIS solution, an increase of [Ca2+]i was seen after 1 h. In Ca(2+)-free (0 Ca2+ plus 15 microM EGTA) media, [Ca2+]i decreased (ranging from 15 to 21 nM), but were not significantly different in HIS or LIS, and did not change following 1 h incubation. The effect of the ionic strength and PGE2 on passive Ca2+ influx was investigated on ATP-depleted RBCs. Ca2+ influx was faster during the initial 10 min in comparison with the subsequent time period (10-45 min), both in HIS and LIS media, decreasing from 20.3 +/- 1.9 to 12.9 +/- 1.3 micromol/(lcells x h) in HIS, and from 36.7 +/- 5.3 to 8.6 +/- 1.2 micromol/(lcells x h) in LIS. Prostaglandin E2 (PGE2; 10(-7)-10(-11) M), dissolved in deionised water or in ethanol, did not affect [Ca2+]i in either normal or in ATP-depleted RBCs suspended in Ca(2+)-containing HIS medium. Finally, the addition of carbachol (100 microM) did not affect [Ca2+]i. The present findings suggest that stimulation of the Ca(2+)-activated K+ channel by PGE2, reported in [J. Biol. Chem. 271 (1996) 18651], cannot be mediated via increased [Ca2+]i.     

Ca2+ selectivity of the sarcoplasmic reticulum Ca2+-ATPase at the enzyme-water interface and in the Ca2+ entrance channel

The sarcoplasmic reticulum (SR) Ca(2+)-ATPase, a P-type transmembrane protein, can transport Ca(2+) from the cytoplasmic to the luminal side over other cations specifically. The proposed Ca(2+) entrance channel, composed of the main-chain carbonyl oxygen and side-chain carboxyl oxygen atoms of the amino acids, opens on the enzyme surface, just above the biphospholipid layer membrane-water interface, where Trp residues are frequently found. In this work, the physicochemical nature of Ca(2+) selectivity over Mg(2+) on the surface of the SR Ca(2+)-ATPase has been investigated using the density functional theory (DFT) method. The selection process can be regarded as the first step of the specificity of the enzyme to transport Ca(2+). Subsequently, the specificity of the entrance channel to conduct Ca(2+) over other cations has also been explored. As revealed by thermodynamic analyses, either the aromatic or the aliphatic amino acid residues distributed on the surface of Ca(2+)-ATPase have a bigger affinity to Mg(2+) than to Ca(2+), resulting in a concentration decrease of free Mg(2+) in the local region. Thus, Ca(2+) can transport into the Ca(2+)-entrance channel more easily. Whereafter, for a small quantity of Mg(2+) entering this channel accompanying the Ca(2+) current, the strong electrostatic interactions between Mg(2+) and the ligands will limit the activity of this metal ion, which facilitates the weakly bonded Ca(2+) passing through the channel at a relatively high rate, as suggested by the "sticky-pore" hypothesis. Furthermore, the corresponding theoretical investigations have demonstrated that the increase of the ligand electronegativity can enhance their discrimination between these two cations effectively.     

A near-infrared fluorescent calcium probe: a new tool for intracellular multicolour Ca2+ imaging

We report a novel near-infrared fluorescent calcium probe (KFCA), which has good optical properties such as intense NIR fluorescence emission (670 nm, QY: 0.24), excellent ON/OFF ratio (120-fold), and good wavelength-compatibility with visible-light-emissive fluorophores (Fluo-4, DsRed2), and which is applicable for real-time dual-colour intracellular Ca(2+) imaging.     

Molecular dynamics simulations of the Ca2+-pump: a structural analysis

We report large scale molecular dynamics computer simulations, ～100 ns, of the ion pump Ca(2+)-ATPase immersed in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. The structure simulated here, E1, one of the several conformations resolved using X-ray diffraction techniques, hosts two Ca(2+)-ions in the hydrophobic domain. Our results indicate that protonated residues lead to stronger ion-residue interactions, supporting previous conclusions regarding the sensitivity of the Ca(2+) behaviour to the protonated state of the amino acid binding sites. We also investigate how the protein perturbs the bilayer structure. We show that the POPC bilayer is ～12% thinner than the pure bilayer, near the protein surface. This perturbation decays exponentially with the distance from the protein with a characteristic decay length of 0.8 nm. We find that the projected area per lipid also decreases near the protein. Using an analytical model we show that this change in the area is only apparent and it can be explained by considering the local curvature of the membrane. Our results indicate that the real area per lipid near the protein is not significantly modified with respect to the pure bilayer result. Further our results indicate that the local deformation of the membrane around the protein might be compatible with the enhanced protein activity observed in experiments over a narrow range of membrane thicknesses.     

Time-of-flight secondary ion mass spectrometry (TOF-SIMS):--versatility in chemical and imaging surface analysis

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has emerged as one of the most important and versatile surface analytical techniques available for advanced materials research. This arises from its excellent mass resolution, sensitivity and high spatial resolution providing both chemical and distributional (laterally and depth) information for a wide variety of materials and applications. Understanding the various modes of operation and the information that each provides is crucial to the analyst in order to optimise the type of data that is obtained. New developments in primary ion sources and the application of multivariate analysis techniques, which can only extend the versatility and applicability of the technique, are also discussed.     

Effect of the calcium-binding protein regucalcin on the Ca2+ transport system in rat liver microsomes: the protein stimulates Ca2+ release

The effect of the calcium-binding protein regucalcin on the Ca2+ transport system in the liver microsomes from fed rats was investigated. Ca2+ transport was assayed by the method of Millipore filtration to estimate microsomal 45Ca2+ accumulation following addition of 10 mM adenosine triphosphate (ATP). 45Ca2+ uptake was retarded by the presence of regucalcin (1.0-4.0 microM). This retardation was remarkable at 1 min after regucalcin addition, while appreciable retardation was no longer seen at 5 min. Regucalcin (2.0 microM)-induced retardation of 45Ca2+ uptake was prevented by the presence of calmodulin (5 micrograms/ml). Calmodulin alone (1 and 5 micrograms/ml) caused a significant increase in 45Ca2+ uptake at 3 min after the start of incubation. Also, regucalcin (2.0 microM)-induced retardation of 45Ca2+ uptake was completely blocked by the presence of a Ca2(+)-trapping agent, oxalate (3 mM). On the other hand, 45Ca2+, which accumulated in microsomes during 5 min after ATP addition, was markedly released by the addition of regucalcin. This release was dose-dependent (0.5-4.0 microM). Guanosine triphosphate (GTP; 10-100 microM) caused a significant release of 45Ca2+ from the microsomes. The presence of regucalcin (2.0 microM) further enhanced the GTP effect. Regucalcin (2.0 microM)-induced release of 45Ca2+ was not blocked by the presence of the protein thiol-protecting agent dithiothreitol (0.1 mM). The presence of oxalate (3 mM) completely blocked the effect of regucalcin on 45Ca2+ release from the microsomes. These results indicate that regucalcin stimulates Ca2+ release from liver microsomes, and that the protein retards the microsomal Ca2+ uptake. The present study suggests that regucalcin can regulate the Ca2+ transport system in rat liver microsomes.     

A microfluidic-based system for analysis of single cells based on Ca2+ flux

A microfluidic format-based system has been developed for in situ monitoring of the calcium flux response to agonists using Chinese hamster ovary (CHO) cells. The assay is based on measuring the fluorescent intensity of the calcium-sensitive indicator, Fluo-4 AM, and was performed in a modified glass chip channel, whose surface was functionalised using a silanisation method with 3-aminopropyltriethoxysilane (APTS) (enabling the cells to be immobilised on the channel surface). CHO cells calcium flux response was measured for different agonists over a range of concentrations. Cells and reagents were introduced into the chip in a continuous flow as a series of plugs in a given sequence.     

Factors governing the substitution of La3+ for Ca2+ and Mg2+ in metalloproteins: a DFT/CDM study

Trivalent lanthanide cations are extensively being used in biochemical experiments to probe various dication-binding sites in proteins; however, the factors governing the binding specificity of lanthanide cations for these binding sites remain unclear. Hence, we have performed systematic studies to evaluate the interactions between La3+ and model Ca2+ - and Mg2+ -binding sites using density functional theory combined with continuum dielectric methods. The calculations reveal the key factors and corresponding physical bases favoring the substitution of trivalent lanthanides for divalent Ca2+ and Mg2+ in holoproteins. Replacing Ca2+ or Mg2+ with La3+ is facilitated by (1) minimizing the solvent exposure and the flexibility of the metal-binding cavity, (2) freeing both carboxylate oxygen atoms of Asp/Glu side chains in the metal-binding site so that they could bind bidentately to La3+, (3) maximizing the number of metal-bound carboxylate groups in buried sites, but minimizing the number of metal-bound carboxylate groups in solvent-exposed sites, and (4) including an Asn/Gln side chain for sites lined with four Asp/Glu side chains. In proteins bound to both Mg2+ and Ca2+, La3+ would prefer to replace Ca2+, as compared to Mg2+. A second Mg2+-binding site with a net positive charge would hamper the Mg2+ --> La3+ exchange, as compared to the respective mononuclear site, although the La3+ substitution of the first native metal is more favorable than the second one. The findings of this work are in accord with available experimental data.     

Switching Ca2+/Ba2+ to Ba2+/Ca2+ Potentiometric Selectivities of Podands with Phosphoryl-containing Terminal Groups: A Molecular Modelling Study

It has been shown experimentally that theCa²⁺/Ba²⁺ potentiometric selectivity ofphosphoryl-containing podandR–-O–-(CH₂–-CH₂–-O)_nndash;-R,R = –-C₆H₄–-P(O)Ph₂, n = 3 (I),switches to Ba²⁺/Ca²⁺ when the ligand containsthe longer polyether chain, n = 5 (II). Here, we reportmolecular dynamics and free energy perturbation simulationsperformed using the amber 4.1 program on the complexesL ^.M²⁺ (M²⁺ = Ca²⁺, Sr²⁺ andBa²⁺, L = I and II) in the gas phase inorder to gain a microscopic insight into structural and energy bindingproperties of podands as a function of n. Mixed QM/MM (PM3/ amber) calculations were performed toanalyse the role of polarisation effects on the complexation selectivityof podands. It isshown that an increase of n does not affect the interactions ofM²⁺ with phosphine oxide groups,but leads to less efficient interactions of small cations with the polyether chain. Calculatedpotentiometric selectivities of I (Ca²⁺ > Ba²⁺)and II (Ba²⁺ > Ca²⁺) are in agreement with the experimental data.     

Effects of Ca2+ and V5+ on glucose-6-phosphatase activity in rat liver microsomes: the Ca2+ effect is reversed by regucalcin

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on glucose-6-phosphatase in the microsomes of rat liver was investigated. Addition of Ca2+ up to 2.5 microM to the enzyme reaction mixture caused a significant increase of glucose-6-phosphatase activity in hepatic microsomes, while Ni2+, Zn2+, Cd2+, Cu2+, Mn2+ and Co2+ (20 microM) did not have an appreciable effect. Vanadate (V5+) markedly inhibited the enzyme activity; a significant inhibitory effect was seen at 10 microM V5+. The Ca2+-induced increase of glucose-6-phosphatase activity was reversed by the presence of regucalcin; the effect was complete at 1.0 microM of the protein. Regucalcium had no effect on the basal activity of the enzyme. Meanwhile, the inhibitory effect of V5+ (10-100 microM) on glucose-6-phosphatase was not appreciably blocked by the presence of regucalcin (up to 2.0 microM). The present data suggest that hepatic microsomal glucose-6-phosphatase is uniquely regulated by Ca2+ and V5+, of various metals, and that the Ca2+ effect is reversed by regucalcin. The present study supports the view that regucalcin plays an important role as a regulatory protein in liver cell function related to Ca2+.     

新型钙荧光探针在H2O2对HL-60胞浆钙影响研究中的应用

采用H2O2、黄嘌呤和黄嘌呤氧化酶、H2O2/抗坏血酸/Fe^2＋（或H2O2/Fe^＋）、H2O2和辣根过氧化酶（HRP）等不同活性氧产生体系,对自行合成的胞浆特异性钙荧光探针STDIn的活性氧耐受性进行了考察.以STDIn-AM为探针标记HL-60细胞,激光共聚焦技术检测单细胞内Ca^2＋-STDIn荧光强度的变化;探讨了不同浓度的H2O2对胞内Ca^2＋浓度变化的影响,从膜结构和生物信号传导的角度研究了氧自由基对培养细胞的生物效应.     

Kinetics of Ca2+ complexation with some carbohydrates in aqueous solutions

For solutions of four saccharides in water with alkaline-earth chlorides added ultrasonic attenuation spectra between 100 kHz and 2 GHz are reported and compared to those for carbohydrate solutions without salt. Calcium chloride does not alter the relaxation times in the spectra of D-glucose and D+-maltose solutions, reflecting the exocyclic hydroxymethyl group rotation, a saccharide-saccharide association, and, with the disaccharide, also motions of both rings of a molecule relative to one another. The spectra of D-xylose and D-fructose solutions are substantially changed by the salts. With both saccharides an additional term with relaxation time around some nanoseconds exists which is assigned to a rearrangement of a carbohydrate-cation complex. Other relaxation terms of these saccharide solutions are also subject to noticeable changes by the salt, indicating specific carbohydrate-cation interactions. The ultrasonic spectra show that such interactions may exist also with carbohydrates which do not display the particular hydroxyl group sequences that are considered to promote complexation with cations.     

DSB显著提高羧酸盐驱油体系抗钙镁离子能力的研究

测定了工业品级的天然混合长链烷基羧酸盐(SDC)以及与3-(N,N-二甲基十二烷基胺)-2-羟基-丙磺酸(DSB)复配驱油体系的界面张力(ITFmin), 分别得出其抗钙镁离子的能力为400和5000 mg/L. 选择试剂级十二烷基羧酸钠与DSB复配, 测定了不同配比溶液的表面张力值和临界胶束浓度cmc, 结合长链烷基脂肪酸与钙离子的溶度积Ksp, 分析了对不溶性长链烷基羧酸盐形成的影响. 根据现场驱油体系配方, 计算了两者在溶液中的摩尔配比为4:1时的十二烷基羧酸盐在胶束中的摩尔分数xm1为0.51, 相互作用参数βm值为-3.11, 反映了两者有较强的相互作用. 采用量子化学方法, 对由1个十二烷基羧酸分子、1个DSB分子及1个二价钙离子组成的模型复合物进行了能量计算和电荷分布计算, 得出在长链烷基羧酸盐和DSB两者混合胶束的界面层中存在负电荷空穴, 提出二价金属离子被络合的模型, 合理地解释了实验事实.     

A highly selective fluorescent sensor for Ca2+ and Sr2+ based on diarylethene with a furan-carbohydrazide unit

A fluorescent sensor based on diarylethene has been designed and synthesized. The sensor not only exhibited excellent photochromic properties, but also has distinguishing ability for Ca²⁺ and Sr²⁺ from other metal ions. Upon addition of Ca²⁺ and Sr²⁺, its emission intensity enhanced 27-fold and 24-fold respectively, accompanied by the emission peak shifted from 439 nm to 484 nm for Ca²⁺, and to 479 nm for Sr²⁺. The 1:1 stoichiometry between the sensor and the two ions was confirmed by Job's plot and HRMS. The LOD for Ca²⁺ and Sr²⁺ was determined to be 9.4 × 10^?8 mol L^?1 and 7.2 × 10^?8 mol L^?1, respectively. Furthermore, the sensor was applied in the detection of Ca²⁺ and Sr²⁺ in practical samples successfully.     

Ion-selective electrode for measuring low Ca2+ concentrations in the presence of high K+, Na+ and Mg2+ background

In this work, ion-selective electrodes for calcium ion were investigated. Two ionophores were used in the membranes: ETH 1001 and ETH 129. An internal filling solution buffered for primary ion was used that allowed the lower detection limit to be decreased down to 10^−8.8 M. Theoretical and experimental electrode characteristics pertaining to both primary and interfering ions are discussed. Better behavior was obtained with the electrode prepared with ETH 129 in the membrane. This electrode would be the most likely candidate for obtaining a low Ca²⁺ detection limit in measurements performed with high K⁺, Na⁺, Mg²⁺ background, which is found inside the cells of living organisms, for example. The potentiometric response of the electrode in solutions containing main and interfering ions is in good agreement with simulated curves obtained using the Nernst–Planck–Poisson (NPP) model.     

Understanding the solubilization of Ca acetylide with a new computational model for ionic pairs

The unique reactivity of the acetylenic unit in DMSO gives rise to ubiquitous synthetic methods. We theoretically consider CaC₂ solubility and protolysis in DMSO and formulate a strategy for CaC₂ activation in solution-phase chemical transformations. For this, we use a new strategy for the modeling of ionic compounds in strongly coordinating solvents combining Born–Oppenheimer molecular dynamics with the DFTB3-D3(BJ) Hamiltonian and static DFT computations at the PBE0-D3(BJ)/pob-TZVP-gCP level. We modeled the thermodynamics of CaC₂ protolysis under ambient conditions, taking into account its known heterogeneity and considering three polymorphs of CaC₂. We give a theoretical basis for the existence of the elusive intermediate HC C–Ca–OH and show that CaC₂ insolubility in DMSO is of thermodynamic nature. We confirm the unique role of water and specific properties of DMSO in CaC₂ activation and explain how the activation is realized. The proposed strategy for the utilization of CaC₂ in sustainable organic synthesis is outlined.

Constructing the carbon framework from a carbon-neutral source: a new computational model for ionic pairs in solution based on DFTB MD and DFT helps to propose a strategy for sustainable organic transformations with solid CaC₂.