Mechanism Of Mg2 Research Articles

Functional reconstitution and characterization of the Arabidopsis Mg2+ transporter AtMRS2-10 in proteoliposomes

Magnesium (Mg2+) plays critical role in many physiological processes. The mechanism of Mg2+ transport has been well documented in bacteria; however, less is known about Mg2+ transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg2+ transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg2+ transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg2+ uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg2+ without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg2+ uptake. The AtMRS2-10-mediated Mg2+ influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co2+ and Ni2+; however, it was not inhibited by Ca2+, Fe2+, or Fe3+. While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al3+. These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure–function relationships.

Mechanisms of Mg2+ inhibition of BzATP-dependent Ca2+ responses in THP-1 monocytes

We have recently reported effects of Mg2+ to confer neuroprotection against toxicity of purinergic stimulated microglia and THP-1 monocytes. To examine mechanisms underlying neuroprotection, we have studied Mg2+ modulation of transient changes in intracellular Ca2+ ([Ca2+]i) in THP-1 cells induced by P2X7R agonist 2′,3′-[benzoyl-4-benzoyl]-ATP (BzATP). Application of BzATP caused a rapid transient increase in [Ca2+]i followed by a prolonged component. The time course of the secondary slower phase was significantly reduced with Ca2+-free extracellular solution, with treatment of THP-1 cells by the P2X7R antagonist, oxATP or with exposure of cells to the store-operated channel (SOC) inhibitor, SKF96365. These results suggest that Ca2+ influx, mediated by both the P2X7R or by SOC, contribute to the slow component of [Ca2+]i. Treatment of THP-1 cells with 10mMMg2+ was highly effective in reducing the time course of BzATP-induced Ca2+ decay; unlike the other modulatory protocols, Mg2+ markedly inhibited the amplitudes of slow and rapid components. In addition, acute application of Mg2+ during BzATP-induced responses elicited in the presence of either oxATP or SKF96365 to block respective P2X7R and SOC contributions, rapidly attenuated [Ca2+]i to baseline levels. Priming of cells with the inflammatory stimulus LPS/IFN-γ markedly enhanced the slower, but not rapid, phase of BzATP-induced [Ca2+]i with application of 10mMMg2+ inhibiting both components of response. A model is proposed to account for BzATP stimulation of both ionotropic P2XR and metabotropic P2YR which provides a mechanistic basis for elevated Mg2+ anti-inflammatory and neuroprotective actions in inflamed brain.

Development and Optimization of a High-Throughput Bioassay for TRPM7 Ion Channel Inhibitors

TRPM7 is a ubiquitously expressed and constitutively active divalent cation channel essential for cell survival and proliferation because it provides a mechanism for Mg2+ entry. This makes the channel an attractive target for proliferative diseases. In keeping with its role in Mg2+ homeostasis, TRPM7 is inhibited by intracellular Mg2+ and Mg-ATP. TRPM7 has been implicated in anoxia-mediated cell death following brain ischemia. Despite its critical role in ischemic cell death and cell proliferation, there are no reports of selective inhibitors of TRPM7. The authors developed and optimized a fluorescent dye-based bioassay measuring the fluorescence quench of fura-2 by TRPM7-mediated Mn2+ influx in HEK293 cells that stably overexpress TRPM7. The following bioassay conditions were evaluated: (a) cell density, (b) dye loading conditions, (c) bioassay temperature, (d) concentration of the fura-2 quenching agent Mn2+, and (e) concentration of vehicle solvent. The bioassay was validated by measuring the effects of the known (nonselective) inhibitor 2-APB and La3+ on Mn2+ influx, and furthermore, the performance of the assay was evaluated by screening a subset of a marine bacteria-derived extract library. The quality of the bioassay window is excellent based on an established statistical parameter used to evaluate high-throughput screening window quality (Z and Z' factors > or =0.5).

Investigation on the micro-mechanisms of Al3+ interfering the reactivities of aspartic acid and its biological processes with Mg2+

Density functional theory (DFT) has been applied to study the micro-mechanisms of Al3+ interfering the reactivities of aspartic acid (H2asp) and its biological processes with Mg2+. All the 46 stable conformers of Hasp- and 3 of asp2- have been determined at the B3LYP/6-311++G** level, showing that the 7 most stable conformers of Hasp- all present a very strong and linear O-H...O H-bond between carboxyl and carboxylic acid groups with the bond energy high up to 162 kJ mol(-1). The reaction thermodynamics and micro-mechanism between Al3+ and Hasp- (or asp2-) in aqueous phase have been investigated by the combined application of supramolecular model and polarizable continuum IEFPCM solvent model, firstly revealing Al3+ interfering in the biological processes of aspartic acid. The substitution thermodynamics and mechanisms of Mg2+ by Al3+ in the biological processes between the species of aspartic acid and Mg2+ in aqueous phase were probed, revealing the facile displacement of Mg2+ by Al3+. These results may provide a reasonable mechanism of Al3+ biological toxicity at the microscopic level.

Kalzium Ist Nicht Alles

Elevation of cerebral Mg2+ with a novel orally delivered ionophore, magnesium threonate, enhances cognition in young and old rats over a 12-24 day treatment interval, as outlined in a paper by Slutsky et al. in this issue of Neuron. Despite both Mg2+ and Zn2+ blocking the NMDA receptor channel, sustained extracellular Mg2+ elevation mimics sustained synaptic Zn2+ concentrations by increasing hippocampal NR2B expression and bouton density.

Mg2+ Enhances Voltage Sensor/Gate Coupling in BK Channels

BK (Slo1) potassium channels are activated by millimolar intracellular Mg2+ as well as micromolar Ca2+ and membrane depolarization. Mg2+ and Ca2+ act in an approximately additive manner at different binding sites to shift the conductance–voltage (GK-V) relation, suggesting that these ligands might work through functionally similar but independent mechanisms. However, we find that the mechanism of Mg2+ action is highly dependent on voltage sensor activation and therefore differs fundamentally from that of Ca2+. Evidence that Ca2+ acts independently of voltage sensor activation includes an ability to increase open probability (PO) at extreme negative voltages where voltage sensors are in the resting state; 2 μM Ca2+ increases PO more than 15-fold at −120 mV. However 10 mM Mg2+, which has an effect on the GK-V relation similar to 2 μM Ca2+, has no detectable effect on PO when voltage sensors are in the resting state. Gating currents are only slightly altered by Mg2+ when channels are closed, indicating that Mg2+ does not act merely to promote voltage sensor activation. Indeed, channel opening is facilitated in a voltage-independent manner by Mg2+ in a mutant (R210C) whose voltage sensors are constitutively activated. Thus, 10 mM Mg2+ increases PO only when voltage sensors are activated, effectively strengthening the allosteric coupling of voltage sensor activation to channel opening. Increasing Mg2+ from 10 to 100 mM, to occupy very low affinity binding sites, has additional effects on gating that more closely resemble those of Ca2+. The effects of Mg2+ on steady-state activation and IK kinetics are discussed in terms of an allosteric gating scheme and the state-dependent interactions between Mg2+ and voltage sensor that may underlie this mechanism.

Magnesium cation initiates nucleoside triphosphate to monophosphate conversion by a radical mechanism: Quantum molecular dynamics simulation

A DFT:B3LYP (6-311G** basis set) quantum molecular dynamics simulation was used to study the mechanism of Mg2+-induced conversion of guanosine triphosphate (GTP) to guanosine monophosphate (GMP). The computations were performed at 310 K in a bath of 178 water molecules surrounding the Mg(H2O)2-GTP complex and mimicking the hydration shells. Dissociation of the Mg2+-GTP complex (process duration, 5 ps) produces two phosphate anions (Pi), a hydrated Mg2+ cation, atomic oxygen, and a highly reactive GMP radical. This radical appears as a result of the action of Mg2+, which initiates the radical mechanism of GTP cleavage. At the initial stage of the interaction with GTP, Mg2+ is reduced to Mg+, producing an ion-radical pair +Mg·-·GTP3−. In the absence of Mg2+, an inert GMP molecule forms instead of the GMP radical as a result of hydrolytic cleavage of GTP via an ionic mechanism. Presumably, formation of the GMP radical and analogous radicals with adenosine, cytidine, thymidine, and uridine is a key point in the syntheses of deoxyribonucleic and ribonucleic acids.

Polymerization of ADP-Ribose Pyrophosphatase: conversion mechanism of Mg2+-dependent ADP-Ribose Pyrophosphatase into Mg2+-independent form

ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose (ADPR) into AMP and ribose-5'-phosphate. It is classified into two groups, Mg(2+)-dependent and Mg(2+)-independent ADPRase, depending on its Mg2+ requirement. Here, we purified Mg(2+)-dependent ADPRase from rabbit liver and examined what factors affect Mg2+ requirement. The purified enzyme showed a single band with the molecular weight of 34 kDa on SDS-PAGE both in the presence and absence of 2-mercaptoethanol. The molecular weight of the native enzyme calculated by gel filtration was 68 kDa, indicating that ADPRase is a dimer made up of two identical subunits. Mg(2+)-dependent ADPRase with the highest ADPR affinity had a Km of 160 +/- 10 microM and a pH optimum of around pH 9.5. Treatment of the purified ADPRase with heated cytosol fractions at 37 degrees C for 3 h caused some changes in the chemical properties of the enzyme, including an increase in molecular weight, a decrease in solubility, and a loss of Mg(2+)-dependency. The molecular weight of the cytosol-treated ADPRase measured by gel filtration was over 420 kDa, suggesting, for the first time, that ADPRase could be polymerized by undefined cytoplasmic factors, and that polymerization is accompanied by changes in the solubility and metal ion dependency of the enzyme.

Action of hexaamminecobalt on the activity of Serratia marcescens nuclease.

Using CD spectroscopic and kinetic analysis, a refined mechanism of Co(NH3)6(3+) action on activity of Serratia marcescens nuclease was elucidated. The mechanism was identical with previously found mechanisms of Mg2+ and C7H5O2Hg+. Similarly to Mg2+ and C7H5O2Hg+, Co(NH3)6(3+) binding to the DNA substrate induced changes in the secondary structure which resulted in changes of the enzymatic activity of the S. marcescens nuclease. Upon binding of 0.03 Co(NH3)6(3+) per DNA phosphate, highly polymerized DNA displayed A-form characteristics. The DNA transition from B-form to A-form intermediate was followed by a decrease of the nuclease activity. The diminishing nuclease activity was consistent with diminishing values of Km and Kcat. Co(NH3)6(3+) binding to the highly polymerized DNA caused a 1.7-2.8-fold decrease in Km, and 13.3-19.9 decrease in Vmax compared with Mg-DNA complex. A vast excess of Co(NH3)6(3+) did not affect the activity of S. marcescens nuclease if the DNA in the assay mixture remained in its B-form conformation. Preincubation of S. marcescens nuclease with Co(NH3)6(3+) did not influence the tertiary structure of the enzyme.

Mg2+ transport in the kidney.

Magnesium is abundant in biological systems and an important divalent cation in the human body. Mg2+ helps mediate cellular energy metabolism, ribosomal and membrane integrity. Additionally Mg2+ modulates the activity of several membrane transport and signal transduction systems. Despite its importance however, little is known about the molecular mechanisms of Mg2+ transport and homeostasis in mammals. In mammals the amount of Mg2+ absorption is about the same as the amount of Mg2+ excretion in urine. Additionally, when total Mg2+ intake is deficient, the kidney is capable of reabsorbing all filtered Mg2+. This balance between intake and excretion indicates that the kidney plays a principal role in maintenance of total body Mg2+ homeostasis. Within the kidney, Mg2+ filtered by the glomerulus is handled in different ways along the nephron. About 10-20% of Mg2+ is reabsorbed by the proximal tubule. the bulk of Mg2+ (about 50-70%) is reabsorbed by the cortical thick ascending limb of the loop of Henle. In this region, Mg2+ moves across the epithelium through the paracellular pathway, driven by the positive lumenal transepithelial voltage. A recently cloned human gene, paracellin-1 was shown to encode a protein localized to the tight junctions of the cortical thick ascending limb and is thought to mediate Mg2+ transport via the paracellular space of this epithelium. The distal convoluted tubule reabsorbs the remaining 5-10% of filtered Mg2+. This segment seems to play an important role in determining final urinary excretion, since there is no evidence for significant Mg2+ absorption beyond the distal tubule. Although many renal Mg2+ transport activities have been characterized, no Mg2+ transporter cDNAs have been cloned from mammalian tissues. Recent research has certainly expanded our knowledge of Mg2+ transport in kidney; but details of the transport processes and the mechanisms by which they control Mg2+ excretion must await cloning of renal Mg2+ transporters and/or channels. Such information would provide new concepts in our understanding of renal Mg2+ handling.

Magnesium Therapy for Subarachnoid Hemorrhage Induced Cerebral Vasospasm as Assessed by In Vitro Studies on the Porcine Carotid Artery

82 Background: Mg 2+ has been used therapeutically in the treatment of eclampsia and it has recently become a novel avenue in the treatment of stroke because it can relax vascular smooth muscle. Currently there is a clinical trial, on going in England, involving Mg 2+ administration to stroke patients. But relatively little is known regarding the function or mechanism of Mg2+ treatment following subarachnoid hemorrhage (SAH). We therefore set out to determine if Mg 2+ could be used to prevent or reverse the in vitro vasospasm caused by the CSF from vasospastic (SAH) patients. Methods: Oxygen consumption and isometric force measurements of the isolated porcine carotid artery were used to assess the functional and metabolic responses of the vascular smooth muscle following stimulation by CSF from SAH patients with cerebral vasospasm. We used a method of exposing the vascular smooth muscle to 12 mM MgCl 2 for 1 hour to increase intracellular magnesium. This method has been shown to increase intracellular Mg 2+ to levels reached with two to three days of Mg 2+ therapy. Results: CSF from SAH patients with vasospasm will cause vasospasm when applied to vessels in vitro . Following this in vitro vasospasm, Mg 2+ caused a dose dependant decrease in tension (relaxation) following exposure to CSF from vasospasm patients. Exposure of vessels to 12 mM Mg 2+ normalized the rate of relaxation in the presence of CSF from SAH patients was normalized (2.70±0.71 Nm -2 s -1 in the presence of CSF; 15.8±4.20 Nm -2 s -1 with CSF and Mg 2+ ; and 16.1±4.85 Nm -2 s -1 without CSF). Mg 2. significantly (P≤0.05) decreased the rate of oxygen consumption observed when stimulated by CSF (0.70±0.03 μmol O 2 min -1 g -1 with CSF alone and 0.46±0.08 μmol O 2 min -1 g 1 with CSF and Mg 2+ ). Conclusion: These results suggest that Mg 2+ is a potent vasodilator that helps to normalize contractile behavior and metabolism of the porcine carotid artery exposed to the CSF of SAH patients with vasospasm. Therefore, Mg 2+ therapy is a possible avenue for treating patients with cerebral vasospasm by causing a relaxation of vascular smooth muscle.

Effects of short chain fatty acids and carbon dioxide on magnesium transport across sheep rumen epithelium.

Short chain fatty acids (SCFAs) and CO2 have been shown to stimulate net Mg2+ efflux from the isolated reticulorumen in vivo. To investigate the underlying mechanisms of Mg2+ transport we performed Ussing chamber and microelectrode experiments and measured 28Mg2+ fluxes across sheep rumen epithelium in vitro. In the presence of SCFAs mucosal-to-serosal Mg2+ flux (Jm-sMg) amounted to 82.3 +/- 7.8 nmol cm-2 h-1 and serosal-to-mucosal Mg2+ flux (Js-mMg) to 3.2 +/- 0.7 nmol cm-2 h-1. Replacing SCFAs with gluconate caused a 50% reduction of Jm-sMg, whereas Js-mMg was not affected. Among the SCFAs, n-butyrate was more effective in stimulating Jm-sMg than acetate, propionate or iso-butyrate. Eliminating HCO3(-)-CO2 from SCFA-containing solutions did not affect Mg2+ fluxes, whereas the same replacement in SCFA-free solutions led to a further reduction in Jm-sMg. Jm-sMg decreased after the addition of ethoxyzolamide to SCFA-free, bicarbonate buffered solutions. Decreasing mucosal pH from 6.4 to 5.4 increased Jm-sMg in SCFA-free, bicarbonate buffered solutions. SCFAs had no effect on the apical membrane potential of rumen epithelial cells. The experiments show that both SCFAs and CO2 stimulate Mg2+ transport through an increase in Jm-sMg, most probably via stimulation of a Mg(2+)-2H+ exchange mechanism. SCFAs may have additional metabolic effects on Mg2+ transport.

Regulation of the cardiac delayed rectifier K current by neurotransmitters and magnesium.

The delayed rectifier K current plays an important role in cardiac electrophysiology: It is involved in the repolarization of the action potential and in frequency-dependent changes in action potential duration and waveform. The delayed rectifier current IK is regulated by the autonomic nervous system: Beta-adrenergic agonists increase IK. This increase is due to an increase in the maximally activatable current as well as a shift of the activation curve to more negative potentials. Thus, in response to sympathetic nerve stimulation, the action potential would be expected to repolarize more rapidly as a result of activation of more IK current and its activation at more negative potentials. Single-channel analysis suggests that the increase in IK is due to an increase in the availability of IK channels to respond to depolarization. IK is also regulated by internal free Mg2+. When the internal solution contains high [Mg2+], IK decreases, whereas low [Mg2+] results in an increase in current. The effect of Mg2+ is not detectably voltage dependent, suggesting that the mechanism of Mg2+ action involves an allosteric or enzymatic effect. Mg2+ also affects the rate of washout of the response to beta-adrenergic agonists, suggesting that Mg2+ may be affecting the activity of a protein phosphatase.

Sodium-dependent magnesium transport across in situ perfused rat placenta

Placentas of anesthetized rats were perfused in situ on the fetal side to study mechanisms of Mg2+ transport. The perfusate was a Mg(2+)-free Krebs-Ringer, and the unidirectional transfer of Mg2+ from maternal plasma to this Ringer was compared with that of 45Ca and 51Cr-EDTA, the latter being employed as a paracellular diffusional marker. Placental perfusion with amiloride (0.5 mM) or ouabain (1 mM) both rapidly (4 min) reduced maternal-fetal clearance (Kmf) for Mg2+ but had no effect on Kmf for 45Ca. In contrast, perfusion of the carbonic anhydrase inhibitor acetazolamide (1 mM) did not affect Kmf for Mg2+ or 45Ca. Placental perfusion with a Na+-free Ringer reduced Kmf for both Mg2+ and 45Ca, although the latter response was delayed. Kmf for 51Cr-EDTA was increased by amiloride and was unaffected by perfusion of ouabain, acetazolamide, or Na+-free Ringer, indicating that the effects of these treatments on Kmf of Mg2+ do not reflect nonspecific effects on placental permeability. These data suggest that maternal-fetal transfer of Mg2+ across the perfused rat placenta is Na+ dependent.

Bovine kidney alkaline phosphatase. Catalytic properties, subunit interactions in the catalytic process, and mechanism of Mg2+ stimulation.

Kidney alkaline phosphatase is an enzyme which requires two types of metals for maximal activity: zinc, which is essential, and magnesium, which is stimulatory. The main features of the Mg2+ stimulation have been analyzed. The stimulation is pH-dependent and is observed mainly between pH 7.5 and 10.5. Mg2+ binding to native alkaline phosphatase is characterized by a dissociation constant of 50 muM at pH 8.5,25 degrees. Binding of Zn2+ is an athermic process. Both the rate constants of association, ka, and of dissociation, kd, have low values. Typical values are 7 M(-1) at pH 8.0, 25 degrees, for ka and 4.10(-4) S(-1) at pH 8.0, 25 degrees, for kd. The on and off processes have high activation energies of 29 kcal mol (-1). Mg2+ can be replaced at its specific site by Mn2+, Co2+, Ni2+, and Zn2+. Zinc binding to the Mg2+ site inhibits the native alkaline phosphatase. Mn2+, Co2+, and Ni2+ also bind to the Mg2+ site with a stimulatory effect which is nearly identic-al with that of Mg2+, Mn2+ is the stimulatory cation which binds most tightly to the Mg2+ site; the dissociation constant of the Mn2+ kidney phosphatase complex is 2 muM at pH 8.5. The stoichiometry of Mn2+ binding has been found to be 1 eq of Mn2+ per mol of dimeric kidney phosphatase. The native enzyme displays absolute half-site reactivity for Mn2+ binding. Mg2+ binding site and the substrate binding sites are distinct sites. The Mg2+ stimulation corresponds to an allosteric effect. Mg2+ binding to its specific sites does not affect substrate recognition, it selectively affects Vmax values. Quenching of the phosphoenzyme formed under steady state conditions with [32P]AMP as a substrate as well as stopped flow analysis of the catalyzed hydrolysis of 2,4-dinitrophenyl phosphate or p-nitrophenyl phosphate have shown that the two active sites of the native and of the Mg2+-stimulated enzyme are not equivalent. Stopped flow analysis indicated that one of the two active sites was phosphorylated very rapidly whereas the other one was phosphorylated much more slowly at pH 4.2. Half of the sites were shown to be reactive at pH 8.0. Quenching experiments have shown that only one of the two sites is phosphorylated at any instant; this result was confirmed by the stopped flow observation of a burst of only 1 mol of nitrophenol per mol of dimeric phosphatase in the pre-steady state hydrolysis of p-nitrophenyl phosphate. The half-of-the-sites reactivity observed for the native and for the Mg2+-stimulated enzyme indicates that the same type of complex, the monophosphorylated complex, accumulates under steady state conditions with both types of enzymes. Mg2+ binding to the native enzyme at pH 8.0 increases considerably the dephosphorylation rate of this monophosphorylated intermediate. A possible mechanism of Mg2+ stimulation is discussed.