Simulations of calcium channel block by trivalent cations: Gd competes with permeant ions for the selectivity filter

Current through L-type calcium channels (Ca_V1.2 or dihydropyridine receptor) can be blocked by micromolar concentrations of trivalent cations like the lanthanide gadolinium (Gd³⁺). It has been proposed that trivalent block is due to ions competing for a binding site in both the open and closed configuration, but possibly with different trivalent affinities. Here, we corroborate this general view of trivalent block by computing conductance of a model L-type calcium channel. The model qualitatively reproduces the Gd³⁺ concentration dependence and the effect that substantially more Gd³⁺ is required to produce similar block in the presence of Sr²⁺ (compared to Ba²⁺) and even more in the presence of Ca²⁺. Trivalent block is explained in this model by cations binding in the selectivity filter with the charge/space competition mechanism. This is the same mechanism that in the model channel governs other selectivity properties. Specifically, selectivity is determined by the combination of ions that most effectively screen the negative glutamates of the protein while finding space in the midst of the closely packed carboxylate groups of the glutamate residues.     

Metal-ATP complexes as substrates and free metal ions as activators of the red cell calcium pump

The plasma membrane calcium pump in most mammalian cells is the basic mechanism for assuring a low cytoplasmic calcium concentration. In inside-out human red cell membrane vesicles /IOVs/ the substrate and metal specificity as well as the intracellular protein /calmodulin/ regulation of the ATP-dependent active calcium transport can be investigated $\text{in}$$\text{situ}$. In this paper we demonstrate that Me²⁺. ATP^4? /in the following MeATP/ complexes, including MgATP, MnATP, CoATP, FeATP, and NiATP, can serve as substrates for the calcium pump in IOVs. Calcium pumping is activated by the above metals, while Sr, Ba, Cu, Cd ions or the trivalent cations are ineffective in this respect. Calmodulin-stimulation of the calcium transport is present independent of the metal ions used for the activation of the pump. Based on kinetic studies we suggest that divalent metal ions interact with the red cell calcium pump at four different sites: 1./ MeATP complex is the true substrate of the pump; 2./ Ca or Sr ions activate the system by binding to the transport site/s/ and other metal ions competitively inhibit this binding; 3./ the presence of free divalent metal ions /Mg, Mn, Co, Fe, or Ni, but not Ca, Sr, Ba/ is required for activating calcium translocation; 4./ interaction with a Ca — calmodulin complex specifically stimulates calcium pumping.     

Crystal structures of Bacillus alkaline phytase in complex with divalent metal ions and inositol hexasulfate

Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general.     

Ion binding by repeat hexapeptide of elastin

Synthetic elastin hexapeptides, N-formyl-Val-Ala-Pro-Gly-Val-Gly-O-methyl, n-formyl-Ala-Pro-Gly-Val-Gly-Val-O-methyl, and N-formyl-Pro-Gly-Val-Gly-Val-Ala-O-methyl were studied in order to determine their affinity and selectivity for calcium, magnesium, strontium, sodium, and potassium ions. Of the three permutations N-forrnyl-Val-Ala-Pro-Gly-Val-Gly-O-methyl had the highest calcium affinity, k_a ～- 0.5 × 10⁶. All three hexamers displayed a selectivity toward calcium, although strontium did bind well also. The peptides could not compete effectively with water for magnesium ions and had little affinity for sodium and potassium ions. The significance of these results lies in the possible role of this hexameric sequence in calcium binding in natural elastin.     

ATP in cellular calcium-overload by trivalent metal ions

Extracellular Ca²⁺-influx induced by trivalent metal ions (Fe³⁺, Al³⁺, Cr³⁺, In³⁺, Ga³⁺, and La³⁺) in Ehrlich carcinoma cells is enhanced by ATP. This action seems to be related to the high coordination capacity of the ATP ligand that inhibits the polymerization of the solvated cations taking place at physiological pH, and consequently permits their biological activity. A general relationship between induced lipid peroxidation and increased calcium uptake was not found. These results emphasize the ATP role in the toxicity of trivalent metals, and its possible involvement, via cellular calcium overload, in a neurodegenerative process, such as Alzheimer's and Parkinson's diseases, in whose etiology the implication of aluminum and iron has been suggested.     

Combined chelation of bi-functional bis-hydroxypiridinone and mono-hydroxypiridinone: Synthesis, solution and in vivo evaluation

3-Hydroxy-4-pyridinones (3,4-HP) are well known iron-chelators with applications in medicinal chemistry, mainly associated with their high affinity towards trivalent hard metal ions (e.g. M³⁺, M = Fe, Al, Ga) and use as decorporating agents in situations of metal accumulation. The polydenticity and the extra-functionality of 3,4-HP derivatives have been explored, aimed at improving the chelating efficacy and the selectivity of the interaction with specific biological receptors. However, the ideal conjugation of both features in one molecular unity usually leads to high molecular weight compounds which can have crossing-membrane limitations.Herein, a different approach is used combining a arylpiperazine-containing bis-hydroxypyridone (H₂L¹) with a biomimetic mono-hydroxypyridinone, ornithine-derivative (HL²), to assess the potential coadjuvating effect that could result from the administration of both compounds for the decorporation of hard metal ions. This work reports the results of solution and in vivo studies on their chelating efficacy either as a simple binary or a ternary system (H₂L¹:HL²:M³⁺), using potentiometric and spectrophotometric methods. The solution complexation studies with Fe(III) indicate that the solubility of the complexes is considerably increased in the ternary system, an important feature for the metal complex excretion, upon the metal sequestration. The results of the in vivo studies with ⁶⁷Ga-injected mice show differences on the biodistribution profiles of the radiotracer, upon the administration of each chelating agent, that are mainly ascribed to the differences of their extra-functional groups and lipo/hydrophilic character. However, administration of both chelating agents leads to a more steady metal mobilization, which may be attributed to an improved access to different cellular compartments.     

Molecular Tuning of an EF-Hand-like Calcium Binding Loop : Contributions of the Coordinating Side Chain at Loop Position 3

Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca²⁺ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca²⁺ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca²⁺ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca²⁺ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning. 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics. Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity. Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain. Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.     

Neutral metal chelator-sensitive protease in insect moulting fluid

Proteolytic activity in moulting fluid from the sphingid Manduca sexta has at least two pH optima; these occur at pH 7 and at pH 7·7. The latter activity is shown to be trypsin-like in that it is susceptible to inhibition by diisopropylfluorophosphate. By contrast, the peak at neutral pH consists of proteolytic activity not hitherto described in invertebrates. This activity shows little or no inhibition with diisopropylfluorophosphate or p-hydroxymercuribenzoate but is strongly inhibited by chelators such as 1,10-phenanthroline, 8-hydroxyquinoline, and EDTA. The neutral metal chelator-sensitive activity requires calcium but the inhibitor data permit the conclusion that the metal ion inhibited by the chelators belongs to the first transition series and thus cannot be calcium. The neutral protease appears to be similar to proteases previously characterized from bacteria and snake venom. In moulting fluid from Manduca, proteolytic activity in vitro is very low in the presence of 1,10-phenanthroline at every pH studied except pH 7·7; in vivo, ecdysis is inhibited in Manduca larvae fed on diet containing a sufficient level (0·02 per cent or higher) of 1,10-phenanthroline. The metal chelator-sensitive proteolytic activity appears to be an essential moulting protease in Manduca.     

Enzymatic hydrolysis of molecules associated with bacterial quorum sensing using an acyl homoserine lactonase from a novel Bacillus thuringiensis strain

N-acyl homoserine lactones are key components of quorum sensing, the bacterial communication system. This communication mechanism regulates the expression of genes, including those involved in virulence and biofilm formation. This system can be interrupted by the action of enzymes that hydrolyze the signaling molecules. In this work, we studied the enzymatic properties of a recombinant AHL-lactonase from Bacillus thuringiensis strain 147-11516, using substrates with acyl chains of different length (C4-HSL, C6-HSL, C7-HSL, C8-HSL and C10-HSL), we also investigated the effect of pH (5.0–9.0), temperature (20–70 °C), concentration of monovalent, divalent and trivalent metals ions (0.2 and 2.0 mM) and EDTA. The results showed that the recombinant AHL-lactonase had biological activity in alkaline pH conditions (8.0) and high temperature (47 % of hydrolyzed substrate at 60 °C). The recombinant AHL-lactonase has activity on substrates with different acyl chain length. However, the activity of the recombinant enzyme was decreased in the two concentrations of all metal ions evaluated but was not inhibited by EDTA. The affinity of the enzyme for all substrates tested and its performance, in the evaluated conditions, suggest that the AHL-lactonase from B. thuringiensis strain 147-11516 could be used as a strategy for disruption of the Gram-negative bacteria communication system under normal and challenging conditions.     

Cooperative adsorption of critical metal ions using archaeal poly-γ-glutamate

Antimony, beryllium, chromium, cobalt (Co), gallium (Ga), germanium, indium (In), lithium, niobium, tantalum, the platinoids, the rare-earth elements (including dysprosium, Dy), and tungsten are generally regarded to be critical (rare) metals, and the ions of some of these metals are stabilized in acidic solutions. We examined the adsorption capacities of three water-soluble functional polymers, namely archaeal poly-γ-glutamate (L-PGA), polyacrylate (PAC), and polyvinyl alcohol (PVA), for six valuable metal ions (Co²⁺, Ni²⁺, Mn²⁺, Ga³⁺, In³⁺, and Dy³⁺). All three polymers showed apparently little or no capacity for divalent cations, whereas L-PGA and PAC showed the potential to adsorb trivalent cations, implying the beneficial valence-dependent selectivity of anionic polyelectrolytes with multiple carboxylates for metal ions. PVA did not adsorb metal ions, indicating that the crucial role played by carboxyl groups in the adsorption of crucial metal ions cannot be replaced by hydroxyl groups under the conditions. In addition, equilibrium studies using the non-ideal competitive adsorption model indicated that the potential for L-PGA to be used for the removal (or collection) of water-soluble critical metal ions (e.g., Ga³⁺, In³⁺, and Dy³⁺) was far superior to that of any other industrially-versatile PAC materials.     

Lanthanide ions are agonists of transient gene expression in rice protoplasts and act in synergy with ABA to increase Em gene expression

Previous work has shown that in rice suspension cells, NaCl at 0.4 M can induce Em gene expression and act synergistically with ABA, possibly by potentiating the ABA response pathway through a rate-limiting intermediate (R.M. Bostock and R.S. Quatrano (1992) Plant Physiol., 98, 1356–1363). Since calcium is an intermediate in ABA regulation of stomatal closure, we tested the effect of calcium changes on ABA-inducible Em gene expression in transiently transformed rice protoplasts. We show that calcium is required for ABA-inducible Em-GUS expression and can act in synergy with ABA. The trivalent ions lanthanum, gadolinium, and aluminum, which are known to interact with calcium- and other signaling pathways, can act at sub-millimolar concentrations to increase GUS reporter gene expression driven by several promoters in transiently transformed rice protoplasts. This effect is not specific for the ABA-inducible Em promoter, but is synergistic with ABA. The lanthanum synergy with ABA does not require calcium. In rice suspension cells, lanthanum alone does not induce Em gene expression, in contrast to transiently transformed protoplasts, yet can act synergistically with ABA to effectively increase the sensitivity to ABA greater than tenfold. Trivalent ions may be a useful tool to study the regulation of gene expression. The possible effects of trivalent ions on ABA signal transduction and gene expression are discussed.     

Exchange Properties of Isolated Cell Walls of Lemna minor L

From our theoretical treatment which is an extension of the classical Donnan theory, we have estimated the rational selectivity coefficients of the carboxylic groups of the walls during exchanges of divalent ions against monovalent ones (i.e. calcium and potassium or calcium and sodium ions). These coefficients express the interactions between the different ions, those between the counter-ions and the ionized groups of the wall, and the influence of water. These quantitative values are consistent with the great affinity of the carboxylic groups for the calcium ions. They vary with the experimental conditions, showing a purely physicochemical mechanism of “regulation” of the exchanges in the cells walls of Lemna minor L.     

The action of certain polyvalent cations on the voltage-clamped lobster axon

Calcium appears to be an essential participant in axon excitation processes. Many other polyvalent metal ions have calcium-like actions on axons. We have used the voltage-clamped lobster giant axon to test the effect of several of these cations on the position of the peak initial (sodium) and steady-state (potassium) conductance vs. voltage curves on the voltage axis as well as on the rate parameters for excitation processes. Among the alkaline earth metals, Mg⁺² is a very poor substitute for Ca⁺², while Ba⁺² behaves like "high calcium" when substituted for Ca⁺² on a mole-for-mole basis. The transition metal ions, Ni⁺², Co⁺², and Cd⁺² also act like high calcium when substituted mole-for-mole. Among the trivalent ions, La⁺³ is a very effective Ca⁺² replacement. Al⁺³ and Fe⁺³ are extremely active and seem to have some similar effects. Al⁺³ is effective at concentrations as low as 10^-5 M. The data suggest that many of these ions may interact with the same cation-binding sites on the axon membrane, and that the relative effects on the membrane conductance and rate parameters depend on the relative binding constants of the ions. The total amount of Na⁺ transferred during a large depolarizing transient is nearly independent of the kind or amount of polyvalent ion applied.     

Phosphorylation-dependent metal binding by α-synuclein peptide fragments

α-Synuclein (α-syn) is the major protein component of the insoluble fibrils that make up Lewy bodies, the hallmark lesions of Parkinson’s disease. Its C-terminal region contains motifs of charged amino acids that potentially bind metal ions, as well as several identified phosphorylation sites. We have investigated the metal-binding properties of synthetic model peptides and phosphopeptides that correspond to residues 119–132 of the C-terminal, polyacidic stretch of human α-syn, with the sequence Ac-Asp-Pro-Asp-Asn-Glu-Ala-Tyr-Glu-Met-Pro-Ser-Glu-Glu-Gly (α-syn119–132). The peptide pY125 replaces tyrosine with phosphotyrosine, whereas pS129 replaces serine with phosphoserine. By using Tb³⁺ as a luminescent probe of metal binding, we find a marked selectivity of pY125 for Tb³⁺ compared with pS129 and α-syn119–132, a result confirmed by isothermal titration calorimetry. Truncated or alanine-substituted peptides show that the phosphoester group on tyrosine provides a metal-binding anchor that is supplemented by carboxylic acid groups at positions 119, 121, and 126 to establish a multidentate ligand, while two glutamic acid residues at positions 130 and 131 contribute to binding additional Tb³⁺ ions. The interaction of other metal ions was investigated by electrospray ionization mass spectrometry, which confirmed that pY125 is selective for trivalent metal ions over divalent metal ions, and revealed that Fe³⁺ and Al³⁺ induce peptide dimerization through metal ion cross-links. Circular dichroism showed that Fe³⁺ can induce a partially folded structure for pY125, whereas no change was observed for pS129 or the unphosphorylated analog. The results of this study show that the type and location of a phosphorylated amino acid influence a peptide’s metal-binding specificity and affinity as well as its overall conformation. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Application of Polypyrrole Multi-Walled Carbon Nanotube Composite Layer for Detection of Mercury,Lead and Iron Ions Using Surface Plasmon Resonance Technique

Polypyrrole multi-walled carbon nanotube composite layers were used to modify the gold layer to measure heavy metal ions using the surface plasmon resonance technique. The new sensor was fabricated to detect trace amounts of mercury (Hg), lead (Pb), and iron (Fe) ions. In the present research, the sensitivity of a polypyrrole multi-walled carbon nanotube composite layer and a polypyrrole layer were compared. The application of polypyrrole multi-walled carbon nanotubes enhanced the sensitivity and accuracy of the sensor for detecting ions in an aqueous solution due to the binding of mercury, lead, and iron ions to the sensing layer. The Hg ion bonded to the sensing layer more strongly than did the Pb and Fe ions. The limitation of the sensor was calculated to be about 0.1 ppm, which produced an angle shift in the region of 0.3° to 0.6°.     

Metallothionein synthesis and its induction mechanism: Correlation with metal ion electronic configurations and softness parameters

An examination of metallothionein induction by toxic metal ions reveals that induction is especially prominent by ions with the electronic configurations (n − 1)d⁸, (n − 1)d⁹, (n − 1)d¹⁰, and ns²(n − 1)d¹⁰. These electronic configurations are also those of both the ‘softest’ and many of the most toxic of the metal ions. The induction process for this protein appears to be one capable of sensing the electronic configurations of these species through the formation of a trans acting induction complex. The relative ability of toxic heavy metals to induce metallothionein is found to be correlated inversely with their softness parameters, σ_p. Examination of the acceptor properties of these inducing ions suggests that an SH or SeH group (soft base) is the critical reactant site for these ions, as these two species form stable bonds with ions that have such electronic configurations. The involvement of SH or SeH in the initial step of the induction process, i.e. as a component of the trans acting element, in a reaction with ions of such electronic configurations would provide the cell with an appropriate response to the presence of species capable of depleting its supply of glutathione, cysteine t-RNA, selenocysteine t-RNA and similar essential species containing SH or SeH. The enchancement of metal ion toxicity in states of selenium deficiency suggests that an SeH containing molecule participates in this step. Two general mechanisms, based on the reaction of inducing metals with selenocysteine t-RNA, are suggested for the initial step in the induction process. The problem of species which are expected on the basis of their electronic configurations to induced MT, but which have not yet been shown to do so is apparently connected with the attempt to use non-labile complexes or extensively hydrolyzed or insoluble compounds as the inducing species.     

Calcium-ion-induced stabilization of the protease from Bacillus cereus WQ9-2 in aqueous hydrophilic solvents: effect of calcium ion binding on the hydration shell and intramolecular interactions

The neutral protease WQ from Bacillus cereus is stable in various aqueous organic mixtures, with the exception of those containing acetonitrile (ACN) and dimethylformamide (DMF). The stability of the enzyme in aqueous hydrophilic solvents was dramatically enhanced with the addition of calcium ions, with the degree of improvement in the half-life relative to different solutions ranging from fourfold to more than 70-fold. Studies of the kinetic constants showed that calcium ions induced slight conformational changes in the active site of the enzyme in aqueous ACN. We investigated the molecular mechanisms underlying this stabilizing effect by employing a combination of biophysical techniques and molecular dynamics simulation. In aqueous ACN, the intrinsic fluorescence and circular dichroism analysis demonstrated that the addition of calcium ions induced a relatively compact conformation and maintained both the native-like microenvironment near the tryptophan residues and the secondary structure. Alternatively, homology modeling confirmed the location of four calcium-ion-binding sites in the enzyme, and molecular dynamics simulation revealed that three other calcium ions were bound to the surface of the enzyme. Calcium ions, known as a type of kosmotrope, can strongly bond with water molecules, thus aiding in the formation of the regional hydration shell required for the maintenance of enzyme activity. In addition, the introduction of calcium ions resulted in the formation of additional ionic interactions, providing propitious means for protein stabilization. Thus, the stronger intramolecular interactions were also expected to contribute partially to the enhanced stability of the enzyme in an aqueous organic solvent.     

Affinity and stoichiometry of calcium binding by arsenazo III

Arsenazo III forms a 1:1 complex with calcium. The affinity constant of arsenazo III for calcium (pK_Ca) has been determined by titrating purified arsenazo III with standard calcium solutions. The method of evaluation used allows one to determine correct pK_Ca values even in the presence of micromolar amounts of contaminating calcium. The pK_Ca is influenced by the following factors: (a) in the neutral pH range the apparent pK_Ca increases strongly with pH; (b) alkali ions bind weakly to arsenazo III and millimolar concentrations cause a decrease in the apparent pK_Ca; (c) the magnesium affinity of arsenazo III, although much lower than the calcium affinity, increases strongly with pH in the neutral range (at pH 7.0 the calcium affinity of arsenazo III is not appreciably altered by up to 2 mm magnesium); (d) strontium and barium form weaker complexes with arsenazo III than calcium, but much stronger complexes than magnesium; (e) the apparent pK_Ca decreases with increasing buffer concentration in the millimolar range. The pK_Ca of arsenazo III is so high that, unless the arsenazo III concentration greatly exceeds the calcium concentration, a considerable fraction of the total arsenazo III is in the calcium complexed form. Because of this, arsenazo III responds nonlinearly to all but the lowest calcium concentrations; however, quantitation of the calcium concentration can readily be done from the mass action law provided that the pK_Ca is determined under the actual experimental conditions. Arsenazo III is a reliable calcium indicator if the experimental conditions, particularly pH, are well controlled.     

One-step production of biodiesel from Jatropha oil with high-acid value in ionic liquids

Catalytic conversion of un-pretreated Jatropha oil with high-acid value (13.8 mg KOH/g) to biodiesel was studied in ionic liquids (ILs) with metal chlorides. Several commercial ILs were used to catalyze the esterification of oleic acid. It was found that 1-butyl-3-methylimidazolium tosylate ([BMIm][CH₃SO₃]; a Brønsted acidic IL) had the highest catalytic activity with 93% esterification rate for oleic acid at 140 °C but only 12% biodiesel yield at 120 °C. When FeCl₃ was added to [BMIm][CH₃SO₃], a maximum biodiesel yield of 99.7% was achieved at 120 °C. Because metal ions in ILs supplied Lewis acidic sites, and more of the sites could be provided by trivalent metallic ions than those of bivalent ones. It was also found that the catalytic activity with bivalent metallic ions increased with atomic radius. Mixture of [BMIm][CH₃SO₃] and FeCl₃ was easily separated from products for reuse to avoid producing pollutants.