Physiological Mg2 Research Articles

The Non-mevalonate Pathway of Isoprenoid Precursor Biosynthesis

The recently discovered non-mevalonate biosynthetic route to isoprenoid precursors is an essential metabolic pathway in plants, apicomplexan parasites, and many species of bacteria. The pathway relies on eight enzymes exploiting different cofactors and metal ions. Structural and mechanistic data now exist for most components of the pathway though there remain some gaps in our knowledge. The individual enzymes represent new, validated targets for broad spectrum antimicrobial drug and herbicide development. Detailed knowledge of the pathway may also be exploited to genetically modify microorganisms and plants to produce compounds of agricultural and medical interest.

Secondary Structure and Dynamics of the r(CGG) Repeat in the mRNA of the Fragile X Mental Retardation 1 (FMR1) Gene

The CGG triplet repeat found within the 5’UTR of the FMR1 gene is involved in the pathogenesis of both fragile X syndrome and fragile X-associated tremor/ataxia syndrome (FXTAS). The repeat has been shown to form both hairpins and tetraplexes in DNA; however, the secondary structure of CGG-repeat RNA has not been well defined. To this end, we have performed NMR spectroscopy on in vitro transcribed CGG-repeat RNAs and see clear evidence of intramolecular hairpins, with no evidence of tetraplex structures. Both C•G and G◦G base pairs form in the hairpin stem, though in a dynamic equilibrium of conformations. In addition, we investigated the effect of an AGG repeat interruption on hairpin stability; such interruptions are often interspersed within the CGG repeat element and are thought to modulate secondary structure of the RNA. While the AGG repeat lowers the Tm of the hairpin at low Mg2+ concentrations, this difference disappears at physiological Mg2+ levels.

Efficient Ligation of the Schistosoma Hammerhead Ribozyme

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by an approximately 2000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2 to 3 at physiological Mg2+ ion concentrations (0.1-1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme.

Liposome reconstitution and modulation of recombinant N-methyl-D-aspartate receptor channels by membrane stretch.

In this study, the heteromeric N-methyl-D-aspartate (NMDA) receptor channels composed of NR1a and NR2A subunits were expressed, purified, reconstituted into liposomes, and characterized by using the patch clamp technique. The protein exhibited the expected electrophysiological profile of activation by glutamate and glycine and internal Mg2+ blockade. We demonstrated that the mechanical energy transmitted to membrane-bound NMDA receptor channels can be exerted directly by tension developed in the lipid bilayer. Membrane stretch and application of arachidonic acid potentiated currents through NMDA receptor channels in the presence of intracellular Mg2+. The correlation of membrane tension induced by either mechanical or chemical stimuli with the physiological Mg2+ block of the channel suggests that the synaptic transmission can be altered if NMDA receptor complexes experience local changes in bilayer thickness caused by dynamic targeting to lipid microdomains, electrocompression, or chemical modification of the cell membranes. The ability to study gating properties of NMDA receptor channels in artificial bilayers should prove useful in further study of structure-function relationships and facilitate discoveries of new therapeutic agents for treatment of glutamate-mediated excitotoxicity or analgesic therapies.

Kinetic Analysis of Interaction of Eukaryotic Release Factor 3 with Guanine Nucleotides

Eukaryotic translation termination is mediated by two release factors: eRF1 recognizes stop codons and triggers peptidyl-tRNA hydrolysis, whereas eRF3 accelerates this process in a GTP-dependent manner. Here we report kinetic analysis of guanine nucleotide binding to eRF3 performed by fluorescence stopped-flow technique using GTP/GDP derivatives carrying the fluorescent methylanthraniloyl (mant-) group, as well as thermodynamic analysis of eRF3 binding to unlabeled guanine nucleotides. Whereas the kinetics of eRF3 binding to mant-GDP is consistent with a one-step binding model, the double-exponential transients of eRF3 binding to mant-GTP indicate a two-step binding mechanism, in which the initial eRF3.mant-GTP complex undergoes subsequent conformational change. The affinity of eRF3 for GTP (K(d), approximately 70 microM) is about 70-fold lower than for GDP (K(d), approximately 1 microM) and both nucleotides dissociate rapidly from eRF3 (k(-1)(mant-GDP) approximately 2.4 s(-1); k(-2)(mant-GTP) approximately 3.3 s(-1)). Whereas not influencing eRF3 binding to GDP, association of eRF3 with eRF1 at physiological Mg(2+) concentrations specifically changes the kinetics of eRF3/mant-GTP interaction and stabilizes eRF3.GTP binding by two orders of magnitude (K(d) approximately 0.7 microM) due to lowering of the dissociation rate constant approximately 24-fold (k(-1)(mant-GTP) approximately 0.14s(-1) approximately 0.14 s(-1)). Thus, eRF1 acts as a GTP dissociation inhibitor (TDI) for eRF3, promoting efficient ribosomal recruitment of its GTP-bound form. 80 S ribosomes did not influence guanine nucleotide binding/exchange on the eRF1 x eRF3 complex. Guanine nucleotide binding and exchange on eRF3, which therefore depends on stimulation by eRF1, is entirely different from that on prokaryotic RF3 and unusual among GTPases.

Stabilities of HIV-1 DIS type RNA loop-loop interactions in vitro and in vivo

RNA loop–loop interactions are a prevalent motif in the formation of tertiary structure and are well suited to trigger molecular recognition between RNA molecules. We determined the stabilities of several loop–loop interactions with a constant 6 bp core sequence and varying unpaired flanking nucleotides and found that the flanking bases have a strong influence on the stability and ion dependence of the kissing complex. In general, the stabilities determined in 1 M Na+ are equivalent to those in the presence of near physiological Mg2+ concentrations. Therefore we further tested whether the stabilities determined in vitro and within yeast cells correlate, using a recently developed yeast RNA-hybrid system. For the majority of the loop types analyzed here, the melting temperatures determined in vitro are in good agreement with the relative β-galactosidase activity in yeast cells, showing that data derived from in vitro measurements reflect in vivo properties. The most stable interactions are the naturally occurring HIV-1 DIS MAL and LAI derived loops with the motif (5′ AA/GN6A 3′), emphasizing the crucial role of stable kissing complexes in HIV genome dimerization.

Potentiation of TRPM7 Inward Currents by Protons

TRPM7 is unique in being both an ion channel and a protein kinase. It conducts a large outward current at +100 mV but a small inward current at voltages ranging from −100 to −40 mV under physiological ionic conditions. Here we show that the small inward current of TRPM7 was dramatically enhanced by a decrease in extracellular pH, with an ∼10-fold increase at pH 4.0 and 1–2-fold increase at pH 6.0. Several lines of evidence suggest that protons enhance TRPM7 inward currents by competing with Ca2+ and Mg2+ for binding sites, thereby releasing blockade of divalent cations on inward monovalent currents. First, extracellular protons significantly increased monovalent cation permeability. Second, higher proton concentrations were required to induce 50% of maximal increase in TRPM7 currents when the external Ca2+ and Mg2+ concentrations were increased. Third, the apparent affinity for Ca2+ and Mg2+ was significantly diminished at elevated external H+ concentrations. Fourth, the anomalous-mole fraction behavior of H+ permeation further suggests that protons compete with divalent cations for binding sites in the TRPM7 pore. Taken together, it appears that at physiological pH (7.4), Ca2+ and Mg2+ bind to TRPM7 and inhibit the monovalent cationic currents; whereas at high H+ concentrations, the affinity of TRPM7 for Ca2+ and Mg2+ is decreased, thereby allowing monovalent cations to pass through TRPM7. Furthermore, we showed that the endogenous TRPM7-like current, which is known as Mg2+-inhibitable cation current (MIC) or Mg nucleotide–regulated metal ion current (MagNuM) in rat basophilic leukemia (RBL) cells was also significantly potentiated by acidic pH, suggesting that MIC/MagNuM is encoded by TRPM7. The pH sensitivity represents a novel feature of TRPM7 and implies that TRPM7 may play a role under acidic pathological conditions.

Functional Hammerhead Ribozymes Naturally Encoded in the Genome of Arabidopsis thaliana

The hammerhead ribozyme (HHRz) is an autocatalytic RNA motif found in subviral plant pathogens and transcripts of repetitive DNA sequences in animals. Here, we report the discovery and characterization of unique HHRzs encoded in a plant genome. Two novel sequences were identified on chromosome IV of Arabidopsis thaliana in a database search, which took into account recently defined structural requirements. The HHRzs are expressed in several tissues and coexist in vivo as both cleaved and noncleaved species. In vitro, both sequences cleave efficiently at physiological Mg(2+) concentrations, indicative of functional loop-loop interactions. Kinetic analysis of loop nucleotide variants was used to determine a three-dimensional model of these tertiary interactions. Based on these results, on the lack of infectivity of hammerhead-carrying viroids in Arabidopsis, and on extensive sequence comparisons, we propose that the ribozyme sequences did not invade this plant by horizontal transfer but have evolved independently to perform a specific, yet unidentified, biological function.

Mg2+ and Ca2+ Differentially Regulate DNA Binding and Dimerization of DREAM

DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca(2+) binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca(2+) binding to the various mutants revealed that, in the absence of Mg(2+), Ca(2+) binds independently and sequentially to EF-3 (DeltaH = -2.4 kcal/mol), EF-4 (DeltaH = +5.2 kcal/mol), and EF-2 (DeltaH = +1 kcal/mol). By contrast, only two Ca(2+) bind to DREAM in the presence of physiological levels of Mg(2+) for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg(2+). ITC measurements demonstrate that one Mg(2+) binds enthalpically with high affinity (K(d) = 13 mum and DeltaH = -0.79 kcal/mol) and two or more Mg(2+) bind entropically in the millimolar range. Size-exclusion chromatography studies revealed that Mg(2+) stabilizes DREAM as a monomer, whereas Ca(2+) induces protein dimerization. Electrophoretic mobility shift assays indicated that Mg(2+) is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca(2+) levels, suggesting that binding of a single Ca(2+) at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca(2+) and Mg(2+) induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg(2+) binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca(2+)-induced protein dimerization disrupts DNA binding.

Evaluation of magnesium fluxes in rat erythrocytes using a stable isotope of magnesium

The mechanisms that maintain intracellular Mg concentration at physiologic levels are not fully understood. In this work, we described for the first time, a new method using 25Mg stable isotopes, that allows simultaneous determination of Mg2+ efflux and Mg2+ influx in non-loaded cells at physiological levels of extracellular Mg. Erythrocytes from rats were suspended as a 10% suspension in NaCl medium or choline medium. The erythrocyte suspension was incubated at 37C, and aliquots of the cell suspension were centrifuged at the beginning of the incubation and after 60 and 120 min. The quantification of 24Mg, 25Mg and 26Mg in supernatants and in erythrocytes were determined by ICP/MS. Simultaneous Mg2+ efflux and Mg2+ influx were calculated from the intra-extracellular distribution of the three isotopes. By this new approach we characterized Mg2+ influx and Mg2+ efflux at 0.4 mM extracellular Mg in both NaCl and choline Cl medium. Mg2+ efflux and Mg2+ influx were largely inhibited by amiloride in NaCl medium and by cinchonine in choline Cl medium. Apparent velocity and LineWeaver-Burk kinetics showed that Mg2+ influx is different from Mg2+ efflux suggesting the involvement of two distinct transport mechanisms. Moreover, modifying extracellular Mg concentrations, to mimic hypo- or hyper-magnesaemia, we showed that Mg2+ efflux and Mg2+ influx increased with extracellular Mg up to 0.8 mM, the physiologic concentration of total extracellular Mg. Our data demonstrate that Mg2+ fluxes are directly related to the levels of extracellular Mg and that in physiological conditions, Na-dependent and Na-independent Mg2+ efflux counterbalance Mg influx to maintain constant intracellular Mg level.

Evidence for simultaneous 1Na+:1Mg2+ and ping pong 2Na+:1Mg2+ exchangers in rat thymocytes

Rat thymocytes showed two Na+/Mg2+ exchangers with high- and low- affinities for external Na+ (Na+o) at physiological internal Mg2+content. The total internal Mg2+ content (Mg2+it) was enhanced by loading with MgCl2 and the ionophore A-23187. Under these conditions, Na+/Mg2+ exchangers were dramatically stimulated by the Mg2+it increase. Na+-induced Mg2+ effluxes were independent of Cl-o or H+. The Na+/Mg2+ exchangers, which we named HANao (high affinity for Na+o) and LANao (low affinity for Na+o), were dissected in Mg2+-loaded thymocytes according to their kinetics and stoichiometries. HANao, which showed an apparent dissociation constant for Na+o (KNa H) = 9.2 +/- 1.6 mmol l(-1) Na+o and a maximal Na+ influx rate (VNa(Na H)max) = 30.5 +/- 6.1 mmol (l cells)(-1) h(-1), was a 1Na+:1Mg2+ simultaneous antiporter insensitive to external magnesium (Mg2+o) whereas that LANao, with KNa L = 65.1 +/- 8.6 mmol l(-1) Na+ and a VNa(Na L)max = 79.5 +/- 14.3 mmol (l cells)(-1) Na+ h(-1), was a 2Na+:1Mg2+ "ping-pong" antiporter which was strongly inhibited by Mg2+o. At physiological concentration of Mg2+o (1 mM), the Na+/Mg2+ exchange through the LANao was inhibited by approximately 50%. Amiloride (10(-4) M) inhibited at similar extent both Na+ and Mg2+ fluxes at high and at low Na+o.

Activation of Soluble Guanylyl Cyclase at the Leading Edge duringDictyosteliumChemotaxis

Dictyostelium contains two guanylyl cyclases, GCA, a 12-transmembrane enzyme, and sGC, a homologue of mammalian soluble adenylyl cyclase. sGC provides nearly all chemoattractant-stimulated cGMP formation and is essential for efficient chemotaxis toward cAMP. We show that in resting cells the major fraction of the sGC-GFP fusion protein localizes to the cytosol, and a small fraction is associated to the cell cortex. With the artificial substrate Mn2+/GTP, sGC activity and protein exhibit a similar distribution between soluble and particulate fraction of cell lysates. However, with the physiological substrate Mg2+/GTP, sGC in the cytosol is nearly inactive, whereas the particulate enzyme shows high enzyme activity. Reconstitution experiments reveal that inactive cytosolic sGC acquires catalytic activity with Mg2+/GTP upon association to the membrane. Stimulation of cells with cAMP results in a twofold increase of membrane-localized sGC-GFP, which is accompanied by an increase of the membrane-associated guanylyl cyclase activity. In a cAMP gradient, sGC-GFP localizes to the anterior cell cortex, suggesting that in chemotacting cells, sGC is activated at the leading edge of the cell.

Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity.

The hammerhead ribozyme (HHRz) is a small, naturally occurring ribozyme that site-specifically cleaves RNA and has long been considered a potentially useful tool for gene silencing. The minimal conserved HHRz motif derived from natural sequences consists of three helices that intersect at a highly conserved catalytic core of 11 nucleotides. The presence of this motif is sufficient to support cleavage at high Mg2+ concentrations, but not at the low Mg2+ concentrations characteristic of intracellular environments. Here we demonstrate that natural HHRzs require the presence of additional nonconserved sequence elements outside of the conserved catalytic core to enable intracellular activity. These elements may stabilize the HHRz in a catalytically active conformation via tertiary interactions. HHRzs stabilized by these interactions cleave efficiently at physiological Mg2+ concentrations and are functional in vivo. The proposed role of these tertiary interacting motifs is supported by mutational, functional, structural and molecular modeling analysis of natural HHRzs.

Activity of PP2Cβ is Increased by Divalent Cations and Lipophilic Compounds Depending on the Substrate

The chapter discusses activity of protein phosphatase type-2Cβ (PP2Cβ) that increases by divalent cations and lipophilic compounds depending on the substrate. The α- and β-isozymes of serine/threonine protein phosphatase type-2C (PP2C) belong to the phosphatases of the first generation, described side by side with PP1, PP2A and PP2B. The requirement of Mg2+- or Mn2+-ions for PP2C activity still holds true. No other mono- or divalent cation was found capable of supporting PP2C activity. It was discovered that PP2C activity is highly active at physiological Mg2+ concentrations provided certain unsaturated lipophilic compounds were present. The concentration of the lipids necessary, so far, exceeds the concentration of free fatty acids under physiological conditions. However, those assays were performed using casein as a substrate, and casein itself is known to bind free fatty acids, thus reducing the effective concentration to a much lower value. The chapter discusses determination of PP2C-activity including [32P] Casein as a substrate, BAD phosphorylated at Ser-155 as a substrate, and phosphopeptides as substrates. Differences in the dependence of divalent cations for activity and in the effect of lipophilic oxidizable compounds are also discussed.

Functional characterisation of mycobacterial DNA gyrase: an efficient decatenase.

A rapid single step immunoaffinity purification procedure is described for Mycobacterium smegmatis DNA gyrase. The mycobacterial enzyme is a 340 kDa heterotetrameric protein comprising two subunits each of GyrA and GyrB, exhibiting subtle differences and similarities to the well-characterised Escherichia coli gyrase. In contrast to E.coli gyrase, the M.smegmatis enzyme exhibits strong decatenase activity at physiological Mg2+ concentrations. Further, the enzymes exhibited marked differences in ATPase activity, DNA binding characteristics and susceptibility to fluoroquinolones. The holoenzyme showed very low intrinsic ATPase activity and was stimulated 20-fold in the presence of DNA. The DNA-stimulated ATPase kinetics revealed apparent K0.5 and kcat of 0.68 mM and 0.39 s(-1), respectively. The dissociation constant for DNA was found to be 9.2 nM, which is 20 times weaker than that of E.coli DNA gyrase. The differences between the enzymes were further substantiated as they exhibited varied sensitivity to moxifloxacin and ciprofloxacin. In spite of these differences, mycobacterial DNA gyrase is a functionally and mechanistically conserved enzyme and the variations in activity seem to reflect functional optimisation for its physiological role during mycobacterial genome replication.

Determination of [Mg(2+)]i - an update on the use of Mg(2+)-selective electrodes.

Since their invention, ion-selective microelectrodes have become an indispensable tool for investigations of intracellular ion regulation and transport. While highly selective sensors for all major intracellular monovalent ions have been available for decades, the development of sensors for divalent cations seems to have presented more difficulties. As ion-selective microelectrodes typically have time-constants in the range of 0.5 to several seconds they turned out to be inapt for the investigation of intracellular Ca2+. The development of sensors for Mg2+-selective electrodes has made its most striking progress only over the past few years. While the first Mg2+ sensor, ETH 1117, was barely able to detect physiological Mg2+ concentrations in the presence of other mono- and divalent cations, the newest sensors allow measurements in the micromolar range. When used in macroelectrodes, the most recent developments, ETH 5506 and ETH 5504, have even been reported to do so in the presence of millimolar Ca2+ concentrations. Although there is still room for improvement to make these sensors applicable in microelectrodes, some preliminary data look extremely promising and indicate that a new era for Mg2+-selective microelectrodes is about to start.

A collapsed non-native RNA folding state.

At physiological Mg2+ concentrations, the catalytic core of the bI5 group I intron does not fold into its native structure. In contrast, as judged by the global size, this RNA undergoes structural collapse at Mg 2+ concentrations much lower than required to drive folding of the RNA completely to the native state. The bI5 RNA therefore exists in equilibrium between expanded and collapsed non-native states. The activation energy of RNA folding from the collapsed state to the native state is negligible and the reaction is not accelerated by the addition of urea. This collapsed state is thus distinct from the kinetic traps observed during folding of other large RNAs. The collapsed non-native state forms readily in the case of bI5 RNA and may exist generically prior to assembly of other ribonucleoprotein holoenzymes, such as the ribosome.

Exon 5 and spermine regulate deactivation of NMDA receptor subtypes.

Deactivation of N-methyl-D-aspartate (NMDA) channels after brief agonist exposure determines the duration of their synaptic activation during excitatory neurotransmission. We performed patch-clamp recordings of L-glutamate responses from human embryonic kidney tumoral cells (HEK293) expressing NR1 subunit variants lacking exon 5 together with the NR2B subunit. These responses had deactivation components that lasted several seconds. The presence of exon 5 or spermine greatly accelerated deactivation of L-glutamate responses through alterations in desensitization. These effects were also observed at positive holding potentials and in the presence of physiological Mg(2+). Thus NR1 splicing and polyamines may have profound effects on the kinetics of NMDA receptor-mediated synaptic transmission.

Ca2+ influx in mouse neocortex via Glu-R ξ 1/ε 2 subunit complex at developmental stage under physiological Mg2+ concentration

Ca2+ influx in mouse neocortex via Glu-R ξ 1/ε 2 subunit complex at developmental stage under physiological Mg2+ concentration

Studies of Adhesion-Dependent Platelet Activation

The molecular differences between native-type collagen type I fibrils (NC) and their pepsinated monomers (PC) were used to uncover receptors involved in platelet-collagen interaction along the adhesion-activation axis. The platelet-depositing capacity of NC and PC under blood flow and their adhesive properties and respective morphologies, aggregation, procoagulant capacity, and tyrosine phosphorylation were compared under different cationic milieus, including or excluding the glycoprotein (GP) Ia/IIa. NC was consistently a more preferable and activating substrate than PC during flow (5 minutes) and in platelet aggregation. In PPACK-treated blood, both NC (3.3-fold) and PC (2.7-fold) increased platelet attachment on elevation of the shear rate from 500 to 1640 s(-1), whereas in citrated blood, adhesion and thrombus growth on PC were negligible under the high shear rate, unlike on NC (1.9-fold increase). The complete lack of platelet deposition on PC in citrated blood could be overcome by restoring physiological Mg(2+) concentration, and in contrast to NC, platelets interacting with PC were highly dependent on Mg(2+) during adhesion, aggregation, and procoagulant response. Monoclonal antibody (mAb 131.7) against GP IV inhibited platelet deposition to NC in citrated blood (2 minutes) by 49%, which was not further increased by coincubation with mAb against GP Ia (6F1). These results stress the importance of GP Ia/IIa in shear-resistant platelet deposition on collagen monomers. In native fibers, however, the preserved quaternary structure with telopeptides activates additional platelet receptors capable of substituting GP Ia/IIa and GP IV.