Effects of Ca2+ and other divalent cations on K+-evoked force production of slow muscle fibers from Rana esculenta and Rana pipiens

Summary Slow muscle fibers were dissected from cruralis muscles of Rana esculenta and Rana pipiens. Isometric contractures were evoked by application of K⁺-rich Ringer's containing Ca²⁺, Ni²⁺, Co²⁺, Mn²⁺ or Mg²⁺. High (7.2 mmol/liter) external Ca²⁺ concentration raised, 0 Ca²⁺ lowered the K⁺ threshold. Replacing Ca²⁺ by Ni²⁺ or Co²⁺ had an effect similar to that of high Ca²⁺ Ringer's. In Mg²⁺ Ringer's the K⁺ concentration-response curve was flattened. These effects were observed already after short exposure times in both species of slow fibers. When Ca²⁺ was removed for long periods of time the slow fibers of R. esculenta lost their contractile response to application of high K⁺ concentrations much more quickly than those of R. pipiens, while the response to caffeine (20 mmol/liter) was maintained. Upon readmission of Ca²⁺ contractile ability was quickly restored in the slow fibers of both R. esculenta and R. pipiens, but the effects of Ni²⁺ (or Co²⁺, Mn²⁺ and Mg²⁺) were much larger in R. esculenta than in R. pipiens slow fibers. It is concluded that divalent cations have two different sites of action in slow muscle fibers. K⁺ threshold seems to be affected through binding to sites at the membrane surface; these sites bind Ni²⁺ and Co²⁺ more firmly than Ca²⁺. The second site is presumably the voltage sensor in the transverse tubular membrane, which controls force production, and where Ca²⁺ is the most effective species of the divalent cations examined.We are grateful to Mrs. S. Pelvay for technical assistance.     

The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase ofRhodospirillum rubrum: Effects of pH and divalent cations

The relation that exist between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores ofRhodospirillum rubrum was studied. The two reactions have a markedly different requirement for pH. The optimal pH for hydrolysis was 6.5 while the Pi-PPi exchange reaction was at 7.5; the pH affects mainly theK _m of Mg²⁺ or Pi for the enzyme; Mn²⁺ and Co²⁺ support the Pi-PPi exchange reaction partially (50%), but the reaction is slower than with Mg²⁺; other divalent cations like Zn²⁺ or Ca²⁺ do not support the exchange reaction. In the hydrolytic reaction, Zn²⁺, at low concentration, substitutes for Mg²⁺ as substrate, and Co²⁺ also substitutes in limited amount (50%). Other cations (Ca²⁺, Cu²⁺, Fe²⁺, etc.) do not act as substrates in complex with PPi. The Zn²⁺ at high concentrations inhibited the hydrolytic reaction, probably due to uncomplexed free Zn²⁺. In the presence of high concentration of substrate for the hydrolysis (Mg-PPi) the divalent cations are inhibitory in the following order: Zn²⁺>Mn²⁺>Ca²⁺Co²⁺>Fe²⁺>Cu²⁺>Mg²⁺. The data in this work suggest that H⁺ and divalent cations in their free form induced changes in the kinetic properties of the enzyme.     

Ca<Superscript>2+</Superscript> increases the specific coenzyme Q<Subscript>10</Subscript> content in <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Of various metal ions (Ca²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Ni²⁺ and Zn²⁺) added to the culture medium of Agrobacterium tumefaciens at 1 mM, only Ca²⁺ increased Coenzyme Q10 (CoQ₁₀) content in cells without the inhibition of cell growth. In a pH-stat fed-batch culture, supplementation with 40 mM of CaCO₃ increased the specific CoQ₁₀ content and oxidative stress by 22.4 and 48%, respectively. Also, the effect of Ca²⁺ on the increase of CoQ₁₀ content was successfully verified in a pilot-scale (300 L) fermentor. In this study, the increased oxidative stress in A. tumefaciens culture by the supplementation of Ca²⁺ is hypothesized to stimulate the increase of specific CoQ₁₀ content in order to protect the membrane against lipid peroxidation. Our results improve the understanding of Ca²⁺ effect on CoQ₁₀ biosynthesis in A. tumefaciens and should contribute to better industrial production of CoQ₁₀ by biological processes.     

Regulation of divalent cations of the membrane-bound pyrophosphatase of Rhodospirillum rubrum,as shown by the hydrolysis of tripositive-pyrophosphate complexes

Tripositive-pyrophosphate [M(III)-PPi] complexes were used to investigate the role of free divalent cations on the membrane-bound pyrophosphatase. Divalent cations remain free and the M(III)-PPi complexes were employed as substrates. Formation of a La-PPi complex was studied by fluorescence, and the fact that Zn²⁺ and Mg²⁺ remain free in the solution was validated. Hydrolysis of La-PPi is stimulated by the presence of fixed concentrations of free Mg²⁺ or Zn²⁺ and this stimulation depends on the concentration of the cations when the La-PPi complex is fixed. The divalent cation stimulation order is Zn²⁺ > Co²⁺ > Mg²⁺ > Mn²⁺ > Ca²⁺ (at 0.5 mm of free cation). With different M(III)-PPi complexes, Zn²⁺ produces the same K _m, for all the complexes and Mg²⁺ stimulates with a different K _m. The results suggest that both Mg²⁺ and Zn²⁺ activate the membrane-bound pyrophosphatase but through different mechanisms.     

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K⁺, Na⁺, Ca²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Co²⁺, Cd²⁺, Mn²⁺, Ba²⁺, Ni²⁺, Zn²⁺, and Li⁺) were analyzed. AtCCX5 expression was found to affect the response to K⁺ and Na⁺ in yeast. The AtCCX5 transformant also showed a little better growth to Zn²⁺. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K⁺ (0.5 mM), and also suppressed its Na⁺ sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K⁺ uptake and was also involved in Na⁺ transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K⁺ uptake and Na⁺ transport in yeast.     

Regulatory effect of regucalcin on (Ca2+−Mg2+)-ATPase in rat liver plasma membranes: comparison with the activation by Mn2+ and Co2+

The effect of various metals and regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes of rat liver was investigated. Of various metals (Zn²⁺, Cu²⁺, Ni²⁺, Mn²⁺, Co²⁺ and Al³⁺; 100 M as a final concentration), Mn²⁺ and Co²⁺ increased markedly (Ca²⁺–Mg²⁺)-ATPase activity, while other metals had no effect. When Ca²⁺ was not added into enzyme reaction mixture, Mn²⁺ and Co²⁺ (25–100 M) did not significantly increase the enzyme activity, indicating that heavy metals act on Ca²⁺-stimulated phosphorylation of the enzyme. Meanwhile, regucalcin (0.25–1.0 M) caused a remarkable elevation of (Ca²⁺–Mg²⁺)-ATPase activity. This increase was not inhibited by the presence of 100 M vanadate, although the effects of Mn²⁺ and Co²⁺ (100 M) were inhibited by vanadate. Also, the inhibition of the Mn²⁺ and Co²⁺ effects by vanadate was not seen in the presence of regucalcin. Moreover, regucalcin (0.5 M) increased significantly the enzyme activity in the absence of Ca²⁺. This effect of regulcalcin was not altered by increasing concentrations of Ca²⁺ added, indicating that the regucalcin effect does not depend on Ca²⁺. The present results suggest that regucalcin activates directly (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes, and that the activation is not involved in the Ca²⁺-dependent phosphorylation of the enzyme.     

Effects of Metal Ions on the Conformation and Activity of Acutolysin D from Agkistrodon Acutus Venom

Acutolysin D, isolated from the venom of Agkistrodon acutus, possesses marked haemorrhagic and proteolytic activities. The molecular weight and the absorption coefficients (A ^1% ₂₈₀) of acutolyisn D have been determined to be 47,850 ± 8 amu and 9.3 by mass spectrometer and UV spectrum, respectively. The effects of metal ions on the conformation and activity of acutolysin D have been studied by following fluorescence, circular dichroism and biological activity measurements. Acutolysin D contains two Ca²⁺-binding sites and two Zn²⁺-binding sites determined by atomic absorption spectrophotometer. Zn²⁺ is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn²⁺ significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)₂ precipitate formation. Ca²⁺ is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca²⁺ markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu²⁺, Co²⁺, Mn²⁺ or Tb³⁺ and inhibited completely by Cd²⁺, is enhanced by Mg²⁺. The fluorescence intensity of the protein decreases in the presence of Cu²⁺, Co²⁺, Cd²⁺ or Mn²⁺, but neither for Ca²⁺, Mg²⁺ nor for Tb³⁺. Zn²⁺, Ca²⁺, Mg²⁺, Cu²⁺, Mn²⁺, Co²⁺ and Tb³⁺ have slight effects on its secondary structure contents. In addition, Cd²⁺ causes a marked increase of antiparallel β-sheet content from 45.5% to 60.2%.     

Purification and characteristics of Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases from spermatozoa of the sea urchin Strongylocentrotus intermedius

Ca²⁺,Mg²⁺- and Ca²⁺,Mn²⁺-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca²⁺,Mg²⁺-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mg²⁺) > Mn²⁺ = (Ca²⁺ + Mn²⁺) > (Mg²⁺ + EGTA) > Ca²⁺. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn²⁺ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca²⁺,Mn²⁺-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mn²⁺) > (Ca²⁺ + Mg²⁺) > Mn²⁺ > (Mg²⁺ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed.     

Purification and characterization of a Ca2+/Mg2+ ecto-ATPase from rat heart sarcolemma

The Ca²⁺/Mg²⁺ ATPase of the rat heart sarcolemmal particles was solublized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12 – 0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca²⁺ (Ka = 0.4 mM) and Mg²⁺ (Ka = 0.2 mM) as well as by other cations in the order Ca^2– > Mg²⁺ > Mn²⁺ > Sr²⁺ > Ba²⁺ > Ni²⁺ > Cu²⁺. The ATPase activity in the presence of Ca²⁺ was markedly inhibited by Mg²⁺, Mn²⁺, Ni²⁺ and Cu²⁺ whereas the monovalent cations such as Na⁺ and K⁺ were without effect. The enzyme did not exhibit Ca²⁺ stimulated Mg²⁺ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca²⁺ or Mg²⁺ ATPase activity was 8.5 – 9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca²⁺ metabolism in the myocardium is discussed.     

Kinetic evidence for a dual cation role for muscle pyruvate kinase

The activation of muscle pyruvate kinase by divalent cations was studied by steady-state kinetics. Under experimental conditions the enzyme exhibits activation by Mg²⁺, Co²⁺, Mn²⁺, Ni²⁺, and Zn²⁺ in descending order of maximal velocity. Combinations of cations were also studied. A synergistic activation was observed with a fixed concentration of Mg²⁺ and varying concentrations of Mn²⁺ or of Co²⁺. This synergism indicates at least two roles for the cations for enzymatic activation and a differential specificity among the cations for the separate functions. Synergistic activation was also observed with fixed Co²⁺ and varying Mn²⁺. These results are consistent with a cation specifically required to activate the enzyme and a cation which serves as a cation-nucleotide complex which is a substrate for the reaction. The response observed suggests that Mn²⁺ is a better activator of the enzyme than is Mg²⁺, however, MgADP is a better substrate than is MnADP. The lack of a synergistic effect by Ni²⁺ or Zn²⁺ with Mg²⁺ suggests that Ni²⁺ and Zn²⁺ are poor activators either because they serve one catalytic function poorly but bind to that site tightly or they serve both catalytic functions poorly in contrast to Mg²⁺. These studies yield the first simple kinetic evidence that muscle pyruvate kinase, under catalytic conditions of the overall reaction, has a dual divalent cation requirement for activity.     

Calcium signaling mediates the response to cadmium toxicity in Saccharomyces cerevisiae cells

The involvement of Ca²⁺ in the response to high Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Hg²⁺ was investigated in Saccharomyces cerevisiae. The yeast cells responded through a sharp increase in cytosolic Ca²⁺ when exposed to Cd²⁺, and to a lesser extent to Cu²⁺, but not to Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, or Hg²⁺. The response to high Cd²⁺ depended mainly on external Ca²⁺ (transported through the Cch1p/Mid1p channel) but also on vacuolar Ca²⁺ (released into the cytosol through the Yvc1p channel). The adaptation to high Cd²⁺ was influenced by perturbations in Ca²⁺ homeostasis. Thus, the tolerance to Cd²⁺ often correlated with sharp Cd²⁺-induced cytosolic Ca²⁺ pulses, while the Cd²⁺ sensitivity was accompanied by the incapacity to rapidly restore the low cytosolic Ca²⁺.     

Divalent Metal Cations upon Coordination to the N7 of Purines Differentially Stabilize the PyPuPu DNA Triplex due to Unequal Hoogsteen-type Hydrogen Bond Enhancement

Abstract

The PyPuPu triplexes consisting of CG*G triads are stabilized by alkaline earth cations (Ca²⁺, Mg²⁺) and transition metal cations (Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺), while similar triplexes including TA*A triads are stabilized only by transition metal cations. We hypothesize that such a differential triplex stabilization by divalent metal cations can be the consequence of their coordination to the N7 of the third strand purines with concomitant polarization effects on the bases resulting in unequal Hoogsteen-type hydrogen bond enhancement.     

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2

In order to address the mechanism whereby Ca²⁺ wad crucial for the manifestation of the enzymatic activity of phospholipase A₂ (PLA₂), four divalent cations were used to assess their influences on the catalytic activity and the fine structures ofNaja naja atra PLA₂. It was found that substitution of Mg²⁺ or Sr²⁺ for Ca²⁺ in the substrate solution caused a decrease in the PLA₂ activity to 77.5% or 54.5%, respectively, of that in the presence of Ca²⁺. However, no PLA₂ activity was observed with the addition of Ba²⁺. With the exception of Mg²⁺, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA₂ markedly increased with the binding of cations to PLA₂. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate andp-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca²⁺, Sr²⁺, and Ba²⁺, but not by Mg²⁺. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba²⁺> Sr²⁺> Ca²⁺> Mg²⁺, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA₂ and that Tyr-3, Lys-6, and Tyr-63 of PLA₂ are involved in the binding with the substrate, suggest that the binding of Ca²⁺ to PLA₂ induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg²⁺ or the bigger atomic radii with Sr²⁺ and Ba²⁺ might render the conformation improperly rearranged after their binding to PLA₂ molecule.     

Chick brain glutamine synthetase and Mn2+−Mg2+ interactions

Glutamine synthetase (GS) from the chick brain was purified to apparent homogeneity by ammonium sulfate fractionation followed by affinity chromatography, electrofocusing and Sephadex G-150 chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate analysis in polyacrylamide gel. By sedimentation equilibrium analysis and gel electrophoresis analysis, it was shown that the enzyme has a subunit molecular weight of 45,000 and a native molecular weight of 364,000, which is consistent with an octameric structure. Sedimentation analysis in the presence of Mg²⁺ revealed three different forms of macromolecules corresponding respectively to a monomer, a tetramer and an octamer. Among eight cations tested (Ca²⁺, Co²⁺, Fe²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, Zn²⁺) only Co²⁺, Mg²⁺ and Mn²⁺ supported GS activity; the order of activatory ability was Mg²⁺>Co²⁺>Mn²⁺. The maximum activating effect of Mn²⁺ occurs only within a very narrow range of concentration: with an excess of cation causing strong inhibition of GS activity. For each cation, maximal GS activity occurs at a defined cation/ATP ratio. A regulatory system in which Mn²⁺, modulates the Mg²⁺ dependent GS activity, is proposed; such cation interactions may be of significance in the intracellular control of glutamine synthesis.     

Biochemical characterization of glutamine synthetase from the diazotrophic cyanobacterium,Anabaena doliolum

In cyanobacteria, the glutamine synthetase-L-glutamine-2-oxoglutarate aminotransferase (GS-GOGAT) pathway is the major ammonia-assimilating route. The GS ofAnabaena doliolum was synthesized more under N₂-fixing conditions, followed by ammonium, nitrate, and nitrite as nitrogen sources. The activities of both the glutamine synthetase, Mg²⁺-dependent biosynthetic and Mn²⁺-dependent -glutamyl transferase were optimum at pH 7. The active site of the enzyme bears sulfhydryl (-SH) groups; this was confirmed with the-SH group inhibitors, para-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM). The biosynthetic and -glutamyl transferase activities showed specificity for the divalent cations, Mg²⁺ and Mn²⁺, respectively. The other divalent cations Co²⁺, Cu²⁺, and Ni²⁺ were poor substitutes. This enzyme also required these divalent cations to stabilize its structure and function under extreme conditions such as high and low temperatures and urea denaturation. The glutamate analogl-methionine-d,l-sulfoximine, inactivated the enzyme, whereas the GOGAT inhibitor, azaserine, had no effect on the enzyme activity. The GS enzyme required de novo protein synthesis.     

Nickel resistance in fission yeast associated with the magnesium transport system

We isolated and characterized a nickel (Ni²⁺)-resistant mutant (GA1) of Schizosaccharomyces pombe. This mutant strain displayed resistance to both Ni²⁺ and Zn²⁺, but not to Cd²⁺, Co²⁺, and Cu²⁺. The growth rate of GA1 increased proportionally with increasing Mg²⁺ concentrations until 50 mM Mg²⁺. The GA1 mutation phenotype suggests a defect in Mg²⁺ uptake. Sequence analysis of the GA1 open reading frame (ORF) O13779, which is homologous to the prokaryotic and eukaryotic CorA Mg²⁺ transport systems, revealed a point mutation at codon 153 (ccc to acc) resulting in a Pro 153Thr substitution in the N-terminus of the CorA domain. Our results provide novel genetic information about Ni²⁺ resistance in fission yeast. Specifically, that reducing Mg²⁺ influx through the CorA Mg²⁺ transport membrane protein confers Ni²⁺ resistance in S. pombe. We also report that Ni²⁺ ion detoxification of the fission yeast is related to histidine metabolism and pH.     

Effects of extracellular calcium and protons on osteoclast potassium currents

During resorption of mineralized tissues, osteoclasts are exposed to marked changes in the concentration of extracellular Ca²⁺ and H⁺. We examined the effects of these cations on two types of K⁺ currents previously described in these cells. Whole-cell patch clamp recordings of membrane currents were made from osteoclasts freshly isolated from neonatal rats. In control saline (1 mm Ca²⁺, pH 7.4), the voltage-gated, outwardly rectifying K⁺ current activates at approximately 45 mV and the conductance is half-maximally activated at –29 mV (V _0.5). Increasing [Ca²⁺]_out rapidly and reversibly shifted the current-voltage (I–V) relation to more positive potentials. Current at –29 mV decreased to 28 and 9% of control current at 5 and 10 mm [Ca²⁺]_out, respectively. This effect of elevating [Ca²⁺]_out was due to a positive shift of the K⁺ channel voltage activation range. Zn²⁺ or Ni²⁺ (5 to 500 m) also shifted the I–V relation to more positive potentials and had additional effects consistent with blockade of the K⁺ channel. Based on the extent to which these divalent cations affected the voltage activation range of the outwardly rectifying K⁺ current, the potency sequence was Zn²⁺ > Ni²⁺ > Ca²⁺. Lowering or raising extracellular pH also caused shifts of the voltage activation range to more positive or negative potentials, respectively. In contrast to their effects on the outwardly rectifying K⁺ current, changes in the concentration of extracellular H⁺ or Ca²⁺ did not shift the voltage activation range of the inwardly rectifying K⁺ current. These findings are consistent with Ca²⁺ and other cations affecting voltage-dependent gating of the osteoclast outwardly rectifying K⁺ channel through changes in surface charge.This work was supported by The Arthritis Society and the Medical Research Council of Canada. S.M.S. is supported by a Scientist Award and S.J.D. by a Development Grant from the Medical Research Council.     

Interaction of metal ions with polynucleotides and related compounds. XXII. Effect of divalent metal ions on the conformational changes of polyribonucleotides

Changes in the conformation of poly(G), poly(C), poly(U), and poly(I) in the presence of divalent metal ions Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Cd²⁺, and Zn²⁺ have been measured by means of ORD and u.v. spectra. Mg²⁺ and Ca²⁺ ions stabilize helical structures of all the polynucleotides very effectively at concentrations several orders of magnitude lower than the effective concentration of Na⁺ion. Cu²⁺ and Cd²⁺ destabilize the helical structure of polynucleotides to form random coils. Zn²⁺, Ni²⁺, Co²⁺, and Mn²⁺ions do not behave in such a clear-cut manner: they selectively stabilize some ordered structures, while destabilizing others, depending on the ligand strength of the nucleotide base as well as the preferred conformation of that polynucleotide.     

Bidirectional Modulation of GABA-Gated Chloride Channels by Divalent Cations: Inhibition by Ca2+ and Enhancement by Mg2+

Abstract: The effects of the divalent cations Ca²⁺, Sr²⁺, Ba²⁺, Mg²⁺, Mn²⁺, and Cd²⁺ were studied on γ-aminobutyric acid_A (GABA_A) responses in rat cerebral cortical synaptoneurosomes. The divalent cations produced bidirectional modulation of muscimol-induced ³⁶Cl^? uptake consistent with their ability to permeate and block Ca²⁺channels. The order of potency for inhibition of muscimol responses was Ca²⁺ > Sr²⁺ > Ba²⁺, similar to the order for permeation of Ca²⁺ channels in neurons. The order of potency for enhancement of muscimol responses was Cd²⁺> Mn²⁺ > Mg²⁺, similar to the order for blockade of Ca²⁺channels in neurons. Neither Ca²⁺ nor Mg²⁺ caused accumulation of GABA in the extravesicular space due to increased GABA release or decreased reuptake of GABA by the synaptoneurosomes. The inhibition of muscimol responses by Ca²⁺ was most likely via an intracellular site of action because additional inhibition could be obtained in the presence of the Ca²⁺ ionophore, A23187. This confirms electrophysiologic findings in cultured neurons from several species. In contrast, the effects of Cd²⁺, Mn²⁺, and Mg²⁺ may be mediated via blockade of Ca²⁺ channels or by intracellular sites, although the results of these studies do not distinguish between the two loci. The effects of Zn²⁺ were also studied, because this divalent cation is reported to have widely divergent effects on GABA_A responses. In contrast to other studies, we demonstrate that Zn²⁺ inhibits GABA_A responses in an adult neuronal preparation. Zn²⁺ produced a concentration-dependent inhibition (limited to 40%) of muscimol responses with an EC₅₀ of 60 μM. The inhibition of muscimol-induced ³⁸Cl^? uptake by Zn²⁺ was noncompetitive. The effect of Zn²⁺was reduced in the presence of Mg²⁺ in a competitive or allosteric manner. The portion of GABA_A receptors sensitive to Zn²⁺ may reflect a specific subunit composition in cerebral cortex as previously observed for recombinant GABA_A receptors in several expression systems. The modulation of GABA_A receptor function by Ca²⁺ and other divalent cations may play an important role in the development and/or attenuation of neuronal excitability associated with pathologic conditions such as seizure activity and cerebral ischemia.