The effect of metal cations on the phase behavior and hydration characteristics of phospholipid membranes

To characterize the specificity of ion binding to phospholipids in terms of headgroup structure, hydration and lyotropic phase behavior we studied 1-palmitoyl-2-oleoyl-phosphatidylcholine as a function of relative humidity (RH) at 25 degrees C in the presence and absence of Li+, Na+, K+, Be2+, Mg2+, Ca2+, Sr2+, Ba2+, Zn2+ and Cu2+ ions by means of infrared (IR) spectroscopy. All divalent cations and Li+ shift the gel-to-liquid crystalline phase transition towards bigger RH values indicating stabilization of the gel state. The observed shift correlates in a linearly fashion with the electrostatic solvation free energy for most of the ions in water that in turn, is inversely related to the ionic radius. This interesting result was interpreted in terms of the excess chemical potential of mixing of hydrated ions and lipids. Calcium, zinc and partially lithium, cause a positive deviation from the linear relationship. IR spectral analysis shows that the carbonyl groups become more accessible to the water in the presence of Mg2+, Ca2+, Sr2+ and Ba2+ probably because of their involvement into the hydration shell of the ions. In contrast, Be2+, Zn2+ and Cu2+ dehydrate the carbonyl groups at small and medium RH. The ability of the lipid to take up water is distinctly reduced in the presence of Zn2+ and, partially, of Cu2+ meaning that the headgroups have become less hydrophilic. The binding mode of Be2+ to lipid headgroups involves hydrolyzed water. Polarized IR spectra show that complex formation of the phosphate groups with divalent ions gives rise to conformational changes and immobilization of the headgroups. The results are discussed in terms of the lyotropic Hofmeister series and of fusogenic activity of the ionic species.     

Conformational studies of A23187 with mono-, di- and trivalent metal ions by circular dichroism spectroscopy

CD studies carried out on A23187 indicate a solvent-dependent conformation for the free acid. Alkali metal ions were found to bind to the ionophore weakly. Divalent metal ions such as Mg2+, Ca2+, Sr2+, Ba2+ and Co2+ and trivalent lanthanide metal ions like La3+ were found to form predominantly 2:1 (ionophore-metal ion) complexes at low concentrations of metal ions, but both 2:1 and 1:1 complexes were formed with increasing salt concentration. Mg2+ and Co2+ exhibit similar CD behaviour that differs from that observed for the other divalent and lanthanide metal ions. The structure of 2:1 complexes involves two ligand molecules coordinated to the metal ion through the carboxylate oxygen, benzoxazole nitrogen and keto-pyrrole oxygen from each ligand molecule along with one or more solvent molecules. Values of the binding constant were determined for 2:1 complexes of the ionophore with divalent and lanthanide metal ions.     

Concentrations of trace elements in osteoarthritic knee-joint effusions

Concentrations of the 18 elements, barium (Ba), beryllium (Be), bismuth (Bi), calcium (Ca), cadmium (Cd), cesium (Cs), copper (Cu), lanthanum (La), lithium (Li), magnesium (Mg), molybdenum (Mo), lead (Pb), rubidium (Rb), antimony (Sb), tin (Sn), strontium (Sr), thallium (Tl), and zinc (Zn), were determined in the synovial fluids of osteoarthritic knee joints and in the corresponding sera of 16 patients by inductively coupled plasma-mass spectrometry. Knee-joint effusions have lower elemental concentrations than their corresponding sera. For the essential elements Ca, Cu, Mg, and Zn and for the nonessential and toxic elements Ba, Be, Bi, La, and Sb, this difference was highly significant. Strong positive correlations between concentrations in effusions and sera for the essential elements Cu and Mg and for the nonessential elements Cs, Li, Rb, and Sr could be established. The grade of localized hyperperfusion of the knee region in the blood pool phase of ^99mTc HDP bone scan indicating inflammation did not correlate with any elemental concentration determined. Deceased.     

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.     

Structural modeling of calcium binding in the selectivity filter of the L-type calcium channel

Calcium channels play crucial physiological roles. In the absence of high-resolution structures of the channels, the mechanism of ion permeation is unknown. Here we used a method proposed in an accompanying paper (Cheng and Zhorov in Eur Biophys J, 2009) to predict possible chelation patterns of calcium ions in a structural model of the L-type calcium channel. We compared three models in which two or three calcium ions interact with the four selectivity filter glutamates and a conserved aspartate adjacent to the glutamate in repeat II. Monte Carlo energy minimizations yielded many complexes with calcium ions bound to at least two selectivity filter carboxylates. In these complexes calcium-carboxylate attractions are counterbalanced by calcium-calcium and carboxylate-carboxylate repulsions. Superposition of the complexes suggests a high degree of mobility of calcium ions and carboxylate groups of the glutamates. We used the predicted complexes to propose a permeation mechanism that involves single-file movement of calcium ions. The key feature of this mechanism is the presence of bridging glutamates that coordinate two calcium ions and enable their transitions between different chelating patterns involving four to six oxygen atoms from the channel protein. The conserved aspartate is proposed to coordinate a calcium ion incoming to the selectivity filter from the extracellular side. Glutamates in repeats III and IV, which are most distant from the repeat II aspartate, are proposed to coordinate the calcium ion that leaves the selectivity filter to the inner pore. Published experimental data and earlier proposed permeation models are discussed in view of our model.     

Interaction between calcium ions and surface charge as it relates to calcium currents

Summary Calcium ions affect the gating of Ca currents. Surface charge is involved but to what extent is unknown. We have examined this, using isolated nerve cell bodies ofHelix aspersa and the combined microelectrode-suction pipette method for voltage-clamp and internal perfusion. We found that Ba and Sr currents produced by substitution of these ions for extracellular Ca ions are activated at less positive potentials than Ca currents. Mg ions do not permeate the Ca channel and changes in [Mg]₀ produce shifts in the activation-potential curves that are comparable to the effects of changes in [Ba]₀ or [Sr]₀. Inactivation of Ba currents also occurs at less positive potentials. Perfusion intracellularly with EGTA reduced inactivation of Ca currents as a function of potential, but did not shift the inactivation-potential curve. Hence, Ca current-dependent inactivation which is blocked by intracellular EGTA probably does not involve a similar change of intracellular surface potential. The voltage shifts of activation and inactivation produced by extracellular divalent cations used singly or in mixtures can be described by the Gouy-Chapman theory for the diffuse double layer with binding (Gilbert & Ehrenstein, 1969; McLaughlin, Szabo & Eisenman, 1971). From the surface potential values and the Boltzman distribution, we have computed surface concentrations that predict the following experimental observations: 1) saturation of current-concentration relationships when surface potential is changing maximally; 2) the increase in peak current when Ca ions are replaced by Sr or Ba ions; and 3) the greater inhibitory effect of Mg onI _Ba thanI _Ca. Theory indicates that surface charge cannot be screened completely even at 1m [Mg]₀ and thus that Ca channel properties must be evaluated in the light of surface charge effects. For example, after correction for surface charge effects the relative permeabilities of Ca, Ba and Sr ions are equivalent. In the presence of Co ions, however, Ca ions are more permeable than Ba ions suggesting a channel binding site may be involved.     

Untersuchungen an synthetischem Hydroxylapatit im Hinblick auf den Knochenstoffwechsel von Calcium,Strontium, Barium und Radium

Zusammenfassung Das System Hydroxylapatitkristallite in einer geeigneten Lösung stellt in gewisser Hinsicht einin vitro-Analogon für den Knochenstoffwechsel der Erdalkalien dar. Es wird über Versuche berichtet, Unterschiede im Verhalten von Ca, Sr, Ba und Ra in synthetischem Hydroxylapatit festzustellen. Dazu wurden die Diskriminierungsfaktoren von Ca gegen Sr, Ba und Ra bei der Herstellung der Kristallite gemessen. Weiterhin wurde der Austausch der Erdalkalien zwischen vorgefertigten Kristalliten und einer geeigneten Lösung über eine Zeitdauer von 1000 h verfolgt. Die Diskriminierungsfaktoren nehmen mit zunehmender Ionengröße in der Reihenfolge Sr, Ba, Ra zu. Während Sr, Ca-Ionen auch im Inneren der Kristallite ersetzen kann, sind Ba und Ra fast ausschließlich auf die Oberfläche beschränkt. Für die Diskriminierung des Gesamtknochens muß angenommen werden, daß zusätzliche Prozesse eine Rolle spielen.

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Studies on synthetic hydroxyapatite crystals with regard to metabolism of calcium, strontium, barium and radium, in boneI. The discrimination against calcium

Summary The system hydroxyapatite crystals in a proper solution, in a way, represents anin vitro analogue to metabolism of alkaline earths in bone. Experiments are described to find differencies in the behaviour of Ca, Sr, Ba and Ra to synthetic hydroxyapatite crystals. Synthesizing the crystals the discrimination ratios of Ca to Sr, Ba and Ra were measured. Furthermore the exchange of alkaline earths between preformed crystals and a proper solution was studied over a period of 1000 h. The discrimination ratios grow larger with increasing ionic radius in the order Sr, Ba, Ra. While Sr can replace Ca ions also in the interior of the crystals, Ba and Ra are limited to crystal surface. With respect to the discrimination of bone it must be concluded that further processes participate.

     

IR and Raman study on the interactions of the 5′-GMP and 5′-CMP phosphate groups with Mg(II), Ca(II), Sr(II), Ba(II), Cr(III), Co(II), Cu(II), Zn(II), Cd(II), Al(III) and Ga(III)

The chief motive behind this research is the interest provoked by the presence of metal ions as necessary stabilizers of the negative charges of phosphate groups in nucleic acids. The effect that the presence of different metal ions produces on the band principally assigned to the nu(s) PO(3)(2-) mode has been studied using FT-IR and FT-Raman spectroscopy. The results obtained reveal the diagnostic capacity of these techniques in determining the type of metal ion interaction with respect to the mononucleotides that form DNA and RNA, providing a tool for improving the knowledge of the stabilizing or destabilizing effects of these ions on such macromolecules. The metal complexes of the ribonucleotides 5'-CMP and 5'-GMP with Mg(II), Ca(II), Sr(II), Ba(II), Cr(III), Co(II), Cu(II), Zn(II), Cd(II), Al(III) and Ga(III) were obtained in this study. After studying and analyzing the IR and Raman spectra of all these complexes and comparing them with the spectra of the corresponding disodium salts, it was verified that, independently of the type of nucleotide involved, the presence of the metal in the vicinity of the phosphate group produces an alteration in the aforementioned nu(s) PO(3)(2-) band. This effect is related to the type of interaction that the phosphate group has with the metal. Three components are observed: (1) one near 983-975 cm(-1) (detectable in IR and Raman), associated with phosphate groups in an electrostatic type of interaction with the metal ion, separated by two or more water molecules; (2) another near 989-985 cm(-1) (only in IR), associated with phosphate groups in indirect interaction through the water molecules of the coordination sphere of the metal ions; and (3) the IR and Raman bands near 1014-1001 cm(-1), which represent phosphate groups directly bonded to the metal ion. These results are supported by the behavior of 5'-CMP in aqueous solution in the presence of Mg(II) ions.     

Interactive effects of Al, Ca and other cations on root elongation of rice cultivars under low pH

BACKGROUND AND AIMS: As with other crop species, Al tolerance in rice (Oryza sativa) is widely different among cultivars, and the mechanism for tolerance is unknown. The Ca2+-displacement hypothesis, that is, Al displaces Ca2+ from critical sites in the root apoplast, was predicted to be the essential mechanism for causing Al toxicity in rice cultivars. If displacement of Ca is an essential cause of Al toxicity in rice, Al toxicity may show the same trend as toxicities of elements such as Sr and Ba that are effective in displacing Ca. METHODS: The interactive effects of Al, Ca, Sr and Ba on root elongation of rice cultivars with different Al tolerances were evaluated in hydroponic culture. Al and Ca accumulation in root tips was also investigated. KEY RESULTS AND CONCLUSIONS: Not only Al but also Sr and Ba applications inhibited root growth of rice cultivars under low Ca conditions. As expected, rice cultivars more tolerant of Sr and Ba were also tolerant of Al (japonica > indica). Although Mg application did not affect Sr or Ba toxicity, Mg alleviated Al toxicity to the same level as Ca application. In addition, Ca application decreased the Al content in root tips without displacement. These results suggest that Ca does not have a specific, irreplaceable role in Al toxicity, unlike Sr and Ba toxicities. Alleviation of Al toxicity with increasing concentrations of Ca in rice cultivars is due to increased ionic strength, not due to decreased Al activity. The difference in Al tolerance between indica and japonica cultivars disappears under high ionic strength conditions, suggesting that different electrochemical characteristics of root-tip cells are related to the significant difference in Al tolerance under low ionic strength conditions.     

Optimization of parameters for semiempirical methods IV: extension of MNDO,AM1, and PM3 to more main group elements

The NDDO semiempirical methods MNDO, AM1, and PM3 have been extended to all the remaining non-radioactive elements of the main group, excluding the noble gases. Most of the new elements are of Groups I and II. 44 sets of parameters are presented for the following methods and elements. MNDO: Na, Mg, K, Ca, Ga, As, Se, Rb, Sr, In, Sb, Te, Cs, Ba, Tl, and Bi; AM1: Li, Be, Na, Mg, K, Ca, Ga, As, Se, Rb, Sr, In, Sn, Sb, Te, Cs, Ba, Tl, Pb, and Bi; PM3: B, Na, K, Ca, Rb, Sr, Cs, and Ba. Average errors are presented for heats of formation, molecular geometries, etc.     

The activation of phospholipase C from Clostridium Welchii by quinine: an absolute requirement for calcium ions.

Quinine activates the hydrolysis of phosphatidyl choline suspensions by phospholipase C (E.C. 3.1.4.3) obtained from Clostridium welchii. Low levels of calcium are an absolute requirement for this activation: Mg2+, Ba2+, Sr2+, and Zn2+ are ineffective. The induction period, or lag phase for this enzyme is dependent upon both calcium concentration and substrate interfacial surface area. At low concentrations (less then 50 muM) calcium ions affect the induction period but not the maximal rate of hydrolysis, whereas guinine predominantly affects the rate of hydrolysis by alterations in the surface charge carried by the substrate.     

STABILIZATION OF SPIDER CRAB NERVE MEMBRANES BY ALKALINE EARTHS,AS MANIFESTED IN RESTING POTENTIAL MEASUREMENTS

1. The alkaline earths, Ba, Sr, Ca, and Mg, in isotonic solutions of their chlorides, have, in general, no effect upon the resting potential of non-medullated spider crab nerve. 2. Ba, Sr, and Ca can, however, prevent the depressing action of K upon the resting potential. The order of effectiveness of these ions in this regard is the following: Ba > Sr > Ca. 3. Ba, Sr, Ca, and Mg oppose the depressing action of veratrine sulfate upon the resting potential. The order of effectiveness is Ba > Sr > Ca > Mg. The relation between drop in potential caused by veratrine sulfate and the logarithm of the veratrine sulfate concentration is a linear one. 4. The action of various other organic ions and molecules which depress the resting potential: saponin, amyl urethane, chloral hydrate, and Na salicylate is neutralized by Ba. 5. Hypertonic sea water solutions do not affect the resting potential. Also, preliminary experiments indicate that the nerves do not shrink in hypertonic solutions although they swell in hypotonic sea water. 6. The alkaline earths depress excitability reversibly. The various organic agents which depress the resting potential also depress excitability, in most cases, reversibly, but the concentrations necessary to depress excitability are much smaller than those necessary to depress the resting potential. 7. The relation of these findings to theories put forward as possible explanations of resting potential phenomena is considered.     

Metal ion requirement of bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase from rat liver

The metal ion requirement for both enzymatic activitiesof the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosaminekinase (E.C. 5.1.3.14/ 2.7.1.60), the key enzyme of N-acetylneuraminic acidbiosynthesis in ratliver, was investigated. UDP-N-acetylglucosamine 2-epimerase was active inimida-zole/HCl buffer in the complete absence of any metal ion. 200 mM Na + , K + , Rb + and Cs +activated enzymeactivity up to five-fold, whereas lower concentrations of thesemonovalent metal ions showed only a small effect on UDP-N-acetylglucosamine 2-epimeraseactivity. In sodium phosphate buffer the enzyme activitywas increased by 0.5 mM Mg , Sr , Ba and Mn , while in the presence of 200 mM NaCl UDP-N-acetyl-glucosamine2-epimerase activity showed astronger activation by these divalent metal ions. In imidazole/HClbuffer, UDP-N-acetylglucosamine2-epimerase activity was partially inhibited by 0.5 mM Be , Mg , Ba ,Mn , Sn and Fe , and completely inhibited by 0.5 mM Zn and Cd . Divalent metal ions were essen-tialforN-acetylmannosamine kinase activity, the most effective being Mg , followed byMn and Co .The optimal concentration of these metal ions was 3 mM. Less effective were Ni and Cd , whereas Ca ,Ba , Cu , Fe and Zn showed no effect on enzyme activity.     

Spectral control of metal admixtures in tetracycline]

Analysis of circular dichroism spectra made it possible to offer a method for estimation of tetracycline solutions contamination with metal ions. By its sensitivity the method is much superior to the spectrophotometric one used at present for determination of the antibiotic purity. In the latter method formation of complexes with metals is traced by batochromic displacement of the absorption spectra. The new method is rapid, relatively selective and requires comparatively small quantities of the substance for the analysis, which provides its use under both laboratory and manufacture conditions. The method is based on identification of the circular dichroism spectra of tetracycline complexes with metals in the long wavelength region. The presence of the circular dichroism concervative bands with strictly defined extremums in the spectra of tetracycline low acid solutions contaminated by multiply charged metal ions allowed vs. the circular dichroism spectra of pure tetracycline sample to conclude that the solution contained admixtures and to suggest their nature. It was shown that the charge, ion radius and tetracycline:metal relation were the factors defining the mark and location of the dichroism band extremums. At lambda(extr)-410-415 nm the tetracycline complexes with light metal ions such as Mg2+, Al3+ and Ca2+ were detected by the circular dichroism negative band in the spectra, while the complexes with heavy metal ions such as Sc3+, Sr3+, Cu3+, Cd3+, Ba2+, Y3+ and the cerium subgroup lanthanides were detected by the circular dichroism positive band. The tetracycline complexes with the lanthanides of the second half of the yttrium subgroup (Ho(3+)-Lu3+) were characterized by the presence of the circular dichroism minimum at lambda(min)-425 nm. When the tetracycline concentration was above 1.5 x 10(-3) M, multiligand complexes with circular dichroism negative extremum at lambda(min)-400 nm formed.     

Synthesis, spectroscopic characterization and structural studies of mixed ligand complexes of Sr(II) and Ba(II) with 2-hydroxybenzophenone and salicylaldehyde, hydroxyaromatic ketones or β-diketones: Crystal structure of 2-HOC6H4C(O)C6H5

Reactions of Sr(II) and Ba(II) chlorides with 2-hydroxybenzophenone and salicylaldehyde, hydroxyaromatic ketones or β-diketones in 1:1:1 molar ratios have resulted in the formation of mixed ligand complexes of the type [MLL′(H₂O)₂] (M = Sr(II) or Ba(II); HL = 2-hydroxybenzophenone and HL′ = salicylaldehyde, 2-hydroxyacetophenone, 2-hydroxypropiophenone, pentane-2,4-dione, 1-phenylbutane-1,3-dione or 1,3-diphenylpropane-1,3-dione). These complexes have been characterized by elemental analyses, TLC, IR and ¹H NMR spectroscopy.     

A study of strontium binding to albumins, by a chromatographic method involving atomic emission spectrometric detection

A chromatographic method involving ICP-AES (inductively coupled plasma atomic emission spectrometry) detection has been successfully applied for the study of strontium-protein complexes. The chromatographic step involves the use of gel filtration-a large-zone Hummel and Dreyer method-which allows to dissociate the bound metallic ions and the free ones. This step is followed by an ICP-AES analysis of fractions collected throughout the chromatographic experiment: the concentration of ionic metallic species in solution can therefore be calculated. Two proteins have been tested: bovine serum albumin, which showed only weak interactions with Sr2+ ions, and bovine alpha-lactalbumin: this protein, well-known for its calcium binding capacity, proved to interact strongly with strontium. The influence of various parameters on the formation of strontium-lactalbumin complexes were determined, namely temperature, pH. Competition experiments between Sr2+ ions and, respectively Na+ and Ca2+ ions were also performed, by varying ionic strength of the medium, and by using both apo and native forms of bovine alpha-lactalbumin.     

Characteristic changes of the S2/S1 difference FTIR spectrum induced by Ca2+ depletion and metal cation substitution in the photosynthetic oxygen-evolving complex

Effects of Ca2+ depletion and substitution with other metal cations on the structure of the protein matrices of the oxygen-evolving complex (OEC) and their corresponding changes upon the S1 to S2 transition were examined using Fourier transform infrared (FTIR) spectroscopy. Ca2+ depletion and further supplementation with Li+, Na+, Mg2+, Ca2+, or Sr2+ did not significantly affect the typical vibrational features in the double difference S2/S1 spectrum, including the symmetric [1365(+)/1404(-) cm(-1)] and the asymmetric [1587(+)/1566(-) cm(-1)] stretching modes of the carboxylate ligand and the amide I and II modes of the backbone polypeptides. On the other hand, supplementation with K+, Rb+, Cs+, or Ba2+ significantly modified the S2/S1 spectrum, in which the carboxylate modes disappeared and the amide I and II modes were modified. Results indicate that the binding of metal cations that have ionic radii larger than that of Ca2+ to the Ca2+ site induces perturbations in the protein matrices in the vicinity of the Mn cluster to interrupt the characteristic structural and/or conformational changes upon the oxidation of the Mn cluster accompanied with the S1 to S2 transition. The spectrum was also altered by the supplementation of Cd2+, which has an ionic radius comparable to that of Ca2+. A single-pulse-induced S2/S1 difference spectrum revealed that bands that have been assigned to the vibrational modes for the Y(Z) tyrosine and the histidine ligand for the Mn cluster were not induced in the K+-supplemented membranes, although the histidine band is likely to be preserved in the Ca2+-depleted membranes. The Y(Z) band was considerably small in the double difference S2/S1 spectrum in the Ca2+-depleted and the cation-substituted membranes but distinctively present in the Sr2+- or Ca2+-replenished membranes. Furthermore, cation supplementation induced several new bands that disappeared following the Ca2+ replenishment. These results suggest that the proper organization of the hydrogen bond network within OEC for the water oxidation chemistry requires the Ca2+ ion and indicate that the role of Ca2+ is not purely structurally defined by the physical properties of the ion, such as valence and ionic radius. On the basis of these and other findings, we propose that Ca2+ is necessary for the formation of the hydrogen bond network that is involved in the reaction step of water oxidation.     

Divalent cation conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum

The conduction properties of the alkaline earth divalent cations were determined in the purified sheep cardiac sarcoplasmic reticulum ryanodine receptor channel after reconstitution into planar phospholipid bilayers. Under bi-ionic conditions there was little difference in permeability among Ba2+, Ca2+, Sr2+, and Mg2+. However, there was a significant difference between the divalent cations and K+, with the divalent cations between 5.8- and 6.7-fold more permeant. Single-channel conductances were determined under symmetrical ionic conditions with 210 mM Ba2+ and Sr2+ and from the single-channel current-voltage relationship under bi-ionic conditions with 210 mM divalent cations and 210 mM K+. Single-channel conductance ranged from 202 pS for Ba2+ to 89 pS for Mg2+ and fell in the sequence Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. Near-maximal single-channel conductance is observed at concentrations as low as 2 mM Ba2+. Single-channel conductance and current measurements in mixtures of Ba(2+)-Mg2+ and Ba(2+)-Ca2+ reveal no anomalous behavior as the mole fraction of the ions is varied. The Ca(2+)-K+ reversal potential determined under bi-ionic conditions was independent of the absolute value of the ion concentrations. The data are compatible with the ryanodine receptor channel acting as a high conductance channel displaying moderate discrimination between divalent and monovalent cations. The channel behaves as though ion translocation occurs in single file with at most one ion able to occupy the conduction pathway at a time.     

Model for calcium binding to gamma-carboxyglutamic acid residues of proteins: crystal structure of calcium alpha-ethylmalonate

The crystal structure of a Ca2+ salt of alpha-ethylmalonic acid was determined from three-dimensional X-ray diffraction data. The dicarboxylate anion represents the functional side chain of gamma-carboxyglutamic acid (Gla) residues, which are implicated as essential calcium-binding ligands in a variety of proteins. The alpha-ethylmalonate ion chelates the Ca2+ ion in a bidentate manner that involves an O atom from each of the two malonate carboxylate groups. This type of binding arises from the constrained arrangement of carboxylate ligands in the malonate group and may be of significance to the calcium-binding properties of Gla-containing sites in proteins. The Ca2+-malonate chelation forms a six-membered ring, which is stabilized by interactions that are consistent with the preferred stereochemistries of both calcium-carboxylate and metal-malonate complexes. No other interactions are observed between Ca2+ ions and alpha-ethylmalonate ions that depend upon the malonate juxtaposition of two carboxylate groups. The potential for this type of binding distinguishes Gla residues from the monocarboxylate residues, aspartate and glutamate, and confers a novel calcium-chelation ability upon Gla-containing sites in proteins.