Molecular Dynamics Simulation of Mg2+ and Ca2+ Ions in Water

Molecular dynamics simulations of single Mg²⁺ and Ca²⁺ ions in water have been carried out. Different ion-water potentials from the literature have been used, whereas the same water potential, a rigid simple point charged model, has been considered in all the simulations. Structural, thermodynamic, and dynamic properties have been calculated, and the results for different potentials have been compared with available experimental data. The study includes ion–water radial distribution functions, coordination numbers, solution enthalpies, hydration free energies, self-diffusion coefficients, and reorientational times of water molecules in the hydration shells.     

Na+-Ca2+ exchanger modulates Ca2+ content in intracellular Ca2+ stores in rat osteoblasts

Na+-Ca2+ exchanger (NCX) transports Ca2+ coupled with Na+ across the plasma membrane in a bi-directional mode. Ca2+ flux via NCX mediates osteogenic processes, such as formation of extracellular matrix proteins and bone nodules. However, it is not clearly understood how the NCX regulates cellular Ca2+ movements in osteogenic processes. In this study, the role of NCX in modulating Ca2+ content of intracellular stores ([Ca2+]ER) was investigated by measuring intracellular Ca2+ activity in isolated rat osteoblasts. Removal of extracellular Na+ elicited a transient increase of intracellular Ca2+ concentration ([Ca2+]i). Pretreatment of antisense oligodeoxynucleotide (AS) against NCX depressed this transient Ca2+ rise and raised the basal level of [Ca2+]i. In AS-pretreated cells, the expression and activity of alkaline phosphatase (ALP), an osteogenic marker, were decreased. However, the cell viability was not affected by AS-pretreatment. Suppression of NCX activity by the AS-pretreatment decreased ATP-activated Ca2+ release from intracellular stores and significantly enhanced Ca2+ influx via store operated calcium influx (SOCI), compared to those of S-pretreated or control cells. These results strongly suggest that NCX has a regulatory role in cellular Ca2+ pathways in osteoblasts by modulating intracellular Ca2+ content.     

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.     

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.     

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.     

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.     

天然水中钙镁离子的测定

对Ｃａ２＋、Ｍｇ２＋在含２×１０３ｍｏｌ／ＬＥＤＴＡ和１５×１０２ｍｏｌ／Ｌ硼砂载体溶液中的毛细管电泳行为进行了讨论,并在２００ｎｍ、３５℃、２０ｋＶ的优化条件下,５ｍｉｎ内对天然水中的Ｃａ２＋、Ｍｇ２＋离子进行了同时定量,为分析水质硬度提供了一种高效、快速的测定方法。     

Evidence for a random entry of Ca2+ into human red cells

Under the influence of a Gardos channel activator, NS309, acting through an increase of the channels Ca2+ sensitivity, it is found that the single population behavior of a suspension of human red cells, showing normal distributed osmotic resistance and density, after addition of NS309 in a time dependent manner changes to a two population distribution, with an increasing fraction of cells having high osmotic resistance or high density. The increase with time of the high resistance fraction can be fitted to an exponential, with a time constant corresponding to about 50 min. Since the 'remaining' cell fraction is practically unchanged, this points to a sudden random activation of the individual cells, caused by Ca2+ entry through a channel like pathway.     

Characterization of hydromedusan Ca2+-regulated photoproteins as a tool for measurement of Ca2+concentration

Calcium ion is a ubiquitous intracellular messenger, performing this function in many eukaryotic cells. To understand calcium regulation mechanisms and how disturbances of these mechanisms are associated with disease states, it is necessary to measure calcium inside cells. Ca²⁺-regulated photoproteins have been successfully used for this purpose for many years. Here we report the results of comparative studies on the properties of recombinant aequorin from Aequorea victoria, recombinant obelins from Obelia geniculata and Obelia longissima, recombinant mitrocomin from Mitrocoma cellularia, and recombinant clytin from Clytia gregaria as intracellular calcium indicators in a set of identical in vitro and in vivo experiments. Although photoproteins reveal a high degree of identity of amino acid sequences and spatial structures, and, apparently, have a common mechanism for the bioluminescence reaction, they were found to differ in the Ca²⁺ concentration detection limit, the sensitivity of bioluminescence to Mg²⁺, and the rates of the rise of the luminescence signal with a sudden change of Ca²⁺ concentration. In addition, the bioluminescence activities of Chinese hamster ovary cells expressing wild-type photoproteins also differed. The light signals of cells expressing mitrocomin, for example, slightly exceeded the background, suggesting that mitrocomin may be hardly used to detect intracellular Ca²⁺ without modifications improving its properties. On the basis of experiments on the activation of endogenous P2Y₂ receptor in Chinese hamster ovary cells by ATP, we suggest that wild-type aequorin and obelin from O. longissima are more suitable for calcium detection in cytoplasm, whereas clytin and obelin from O. geniculata can be used for calcium measurement in cell compartments with high Ca²⁺ concentration. Figure

Hydromedusan photoproteins differ in Ca²⁺ concentration detection limit, sensitivity of bioluminescence to Mg²⁺, and rates of rise of luminescence signal with a sudden change of [Ca²⁺] despite a high degree of identity of their amino acid sequences and spatial structures, and, apparently, a common mechanism for the bioluminescence reaction.     

Ca2+ -dependent regulation of phototransduction

Photon absorption by rhodopsin triggers the phototransduction signaling pathway that culminates in degradation of cGMP, closure of cGMP-gated ion channels and hyperpolarization of the photoreceptor membrane. This process is accompanied by a decrease in free Ca(2+) concentration in the photoreceptor cytosol sensed by Ca(2+)-binding proteins that modulate phototransduction and activate the recovery phase to reestablish the photoreceptor dark potential. Guanylate cyclase-activating proteins (GCAPs) belong to the neuronal calcium sensor (NCS) family and are responsible for activating retinal guanylate cyclases (retGCs) at low Ca(2+) concentrations triggering synthesis of cGMP and recovery of the dark potential. Here we review recent structural insight into the role of the N-terminal myristoylation in GCAPs and compare it to other NCS family members. We discuss previous studies identifying regions of GCAPs important for retGC1 regulation in the context of the new structural data available for myristoylated GCAP1. In addition, we present a hypothetical model for the Ca(2+)-triggered conformational change in GCAPs and retGC1 regulation. Finally, we briefly discuss the involvement of mutant GCAP1 proteins in the etiology of retinal degeneration as well as the importance of other Ca(2+) sensors in the modulation of phototransduction.     

Molecular simulation analysis and X-ray absorption measurement of Ca2+, K+ and Cl- ions in solution

This paper presents recent advances in the use of molecular simulations and extended X-ray absorption fine structure (EXAFS) spectroscopy, which enable us to understand solvated ions in solution. We report and discuss the EXAFS spectra and related properties governing solvation processes of different ions in water and methanol. Molecular dynamics (MD) trajectories are coupled to electron scattering simulations to generate the MD-EXAFS spectra, which are found to be in very good agreement with the corresponding experimental measurements. From these simulated spectra, the ion-oxygen distances for the first hydration shell are in agreement with experiment within 0.05-0.1 A. The ionic species studied range from monovalent to divalent, positive and negative: K+, Ca2+, and Cl-. This work demonstrates that the combination of MD-EXAFS and the corresponding experimental measurement provides a powerful tool in the analysis of the solvation structure of aqueous ionic solutions. We also investigate the value of electronic structure analysis of small aqueous clusters as a benchmark to the empirical potentials. In a novel computational approach, we determine the Debye-Waller factors for Ca2+, K+, and Cl- in water by combining the harmonic analysis of data obtained from electronic structure calculations on finite ion-water clusters, providing excellent agreement with the experimental values, and discuss how they compare with results from a harmonic classical statistical mechanical analysis of an empirical potential.     

A calixarene based fluorescent Sr2+ and Ca2+ probe

A fluorescent probe, PyCalix, which has two pyrene moieties at the lower rim of a calix[4]arene fixed in the cone conformation was synthesized and its complexation behavior with alkali and alkaline earth cations was studied by fluorescence spectrometry. The compound showed intramolecular excimer emission at approximately 480 nm in the fluorescence spectra. Upon complexation with alkaline earth metal cations, a decrease of excimer emission was observed. The decrease of excimer emission was accompanied by an increase of monomer emission of pyrenes at 397 nm. The order of complexation constants of PyCalix with metal ions was Sr(+ approximately Ca2+ > Ba2+ > Mg2+ > K+ > Na+ > Cs+ for all reagents. PyCalix doped polyvinyl chloride (PVC) membrane was fabricated and our results showed that this membrane can be used for selective detection of Sr2+.     

Ca2+ Responsive Microgel-Stabilized Pickering Emulsions

As an ionic cross-linker that can change the size of poly(N-isopropylacrylamide-co-acrylic acid) microgel, Ca²⁺ is applied as a trigger to demulsify microgel-stabilized oil/water Pickering emulsions. The influence of Ca²⁺ induced intra-particle ionic cross-linking and inter-particle aggregation on the stability of microgel-stablized “Pickering” emulsion is described. At low and mediate concentration of Ca²⁺, ionic cross-linking can change the internal elasticity of the microgel, and cause the coarsening of the oil droplets. At high concentration of Ca²⁺, microgels flocculate due to the salt out effect and the emulsion is destabilized. This work provide a facile method to control the stability of the Pickering emulsions at ambient condition.     

Effect of BCL-2 on Harringtonine-induced apoptosis and intracellular Ca2+ mobilization in human leukemia HL-60 cells

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb Cephalotaxus hainanensis Li, has been used in the clinical treatment of human glanulocytic leukemia and chromic myelocytic leukemia. In this study, we investigated the effect of Bcl-2 on HT-induced apoptosis and Ca²⁺ mobilization in human leukemia HL-60 cells. 1 g/ml HT induced the apoptosis of HL-60/Neo cells in a time-dependent manner; while 1 g/ml HT failed to induce the apoptosis of HL-60/ Bcl-2 cells. HT-, A23187- (a Ca²⁺ ionophore), Carbonyl cyanide m-chlorophenylhydrazone (CCCP,a specific releaser of Ca²⁺ from mitochondria) and thapsigargin- (an inhibitor of endoplasmic reticulum Ca^2+> -ATPase) induced changes in [Ca²⁺]_i were monitored by using Fluo 3-AM with confocal laser scanning microscopy. The results demonstrated that HL-60 cells with enforced expression of Bcl-2 (HL-60/Bcl-2 cells) had increased Ca²⁺ permeability and increased intracellular Ca²⁺ store in comparison with HL-60 cells with negative control vectors (HL-60/Neo cells), suggesting that Bcl-2 might prevent HT-induced apoptosis by increased Ca²⁺ permeability and increased intracellular Ca²⁺ buffering capacity.     

Modulator binding protein antagonizes activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport of red blood cell membranes.

Red blood cells contain a protein that activates membrane-bound (Ca2+ + Mg2+)-ATPase and Ca2+ transport. The red blood cell activator protein is similar to a modulator protein that stimulates cyclic AMP phosphodiesterase. Wang and Desai [Journal of Biological Chemistry 252:4175--4184, 1977] described a modulator-binding protein that antagonizes the activation of cyclic AMP phosphodiesterase by modulator protein. In the present work, modulator-binding protein was shown to antagonize the activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport by red blood cell activator protein. The results further demonstrate the similarity between the activator protein from human red blood cells and the modulator protein from bovine brain.     

Safranal Induces Vasorelaxation by Inhibiting Ca2+ Influx and Na+/Ca2+ Exchanger in Isolated Rat Aortic Rings

Introduction: Safranal, which endows saffron its unique aroma, causes vasodilatation and has a hypotensive effect in animal studies, but the mechanisms of these effects are unknown. In this study, we investigated the mechanisms of safranal vasodilation. Methods: Isolated rat endothelium-intact or -denuded aortic rings were precontracted with phenylephrine and then relaxed with safranal. To further assess the involvement of nitric oxide, prostaglandins, guanylate cyclase, and phospholipase A₂ in safranal-induced vasodilation, aortic rings were preincubated with L-NAME, indomethacin, methylene blue, or quinacrine, respectively, then precontracted with phenylephrine, and safranal concentration–response curves were established. To explore the effects of safranal on Ca²⁺ influx, phenylephrine and CaCl₂ concentration–response curves were established in the presence of safranal. Furthermore, the effect of safranal on aortic rings in the presence of ouabain, a Na⁺-K⁺ ATPase inhibitor, was studied to explore the contribution of Na⁺/Ca²⁺ exchanger to this vasodilation. Results: Safranal caused vasodilation in endothelium-intact and endothelium-denuded aortic rings. The vasodilation was not eliminated by pretreatment with L-NAME, indomethacin, methylene blue, or quinacrine, indicating the lack of a role for NO/cGMP. Safranal significantly inhibited the maximum contractions induced by phenylephrine, or by CaCl₂ in Ca²⁺-free depolarizing buffer. Safranal also relaxed contractions induced by ouabain, but pretreatment with safranal totally abolished the development of ouabain contractions. Discussion/Conclusion: Inhibition of Na⁺-K⁺ ATPase by ouabain leads to the accumulation of Na⁺ intracellularly, forcing the Na⁺/Ca²⁺ exchanger to work in reverse mode, thus causing a contraction. Inhibition of the development of this contraction by preincubation with safranal indicates that safranal inhibited the Na⁺/Ca²⁺ exchanger. We conclude that safranal vasodilation is mediated by the inhibition of calcium influx from extracellular space through L-type Ca²⁺ channels and by the inhibition of the Na⁺/Ca²⁺ exchanger.     

Changes in the intracellular Ca2+ content in human red blood cells in the presence of glycerol

Changes of the intracellular Ca2+ content in human red blood cells (RBCs) in glycerol-containing solutions and after freeze-thawing the cells with glycerol and subsequent deglycerolization were investigated with the Ca2+-sensitive fluorescent dye fluo-4 using fluorescence microscopy. In the glycerol-containing solutions the Ca2+ content increased when compared with a physiological medium (Hepes buffered saline solution (HBSS)). This effect was most likely a result of an inhibition of the Ca2+ pump. After inhibiting the Ca2+ pump using o-vanadate, the Ca2+ uptake was not significantly different in the cells in glycerol-containing and physiological medium. Freeze-thawing and deglycerolization of RBCs resulted in a more pronounced increase in the Ca2+ content. Also in this case, the Ca2+ pump seemed to play a major role.