Inhibition of Acid Sensing Ion Channel 3 Aggravates Seizures by Regulating NMDAR Function

The existing data about whether acid sensing ion channels (ASICs) are proconvulsant or anticonvulsant are controversial. Particularly, acid sensing ion channel 3 (ASIC3) is the most sensitive to extracellular pH and has the characteristic ability to generate a biphasic current, but few studies have focused on the role of ASIC3 in seizure. Here we found ASIC3 expression was increased in the hippocampus of pilocarpine induced seizure rats, as well as in hippocampal neuronal cultures undergoing epileptiform discharge elicited by Mg²⁺-free media. Furthermore, ASIC3 blockade by the selective inhibitor APETx2 shortened seizure onset latency and increased seizure severity compared with the control in the pilocarpine induced seizure model. Incubation with APETx2 enhanced the excitability of primary cultured hippocampal neurons in Mg²⁺-free media. Notably, the aggravated seizure was associated with upregulation of the N-methyl-d-aspartate subtype of glutamate receptors (NMDARs), increased NMDAR mediated excitatory neurotransmission and subsequent activation of the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cAMP-response element binding protein (CREB) signaling pathway. Moreover, co-immunoprecipitation confirmed the interaction between ASIC3 and NMDAR subunits, and NMDARs blockade prevented the aggravated seizure caused by ASIC3 inhibition. Taken together, our findings suggest that ASIC3 inhibition aggravates seizure and potentiates seizure induced hyperexcitability at least partly by the NMDAR/CaMKII/CREB signaling pathway, which implies that ASIC3 agonists may be a promising approach for seizure treatment.     

The aquaporin MePIP2;7 improves MeMGT9-mediated Mg2+ acquisition in cassava

Aquaporins are important transmembrane water transport proteins which transport water and several neutral molecules. However, how aquaporins are involved in the synergistic transport of Mg²⁺and water remains poorly understood. Here, we found that the cassava aquaporin Me PIP2;7 was involved in Mg²⁺transport through interaction with Me MGT9, a lower affinity magnesium transporter protein. Knockdown of Me PIP2;7 in cassava led to magnesium deficiency in basal mature leaves wi...     

Synaptic mechanisms of use-dependent potentiation and use-dependent inhibition of evoked burst discharges in rat hippocampal slices

The mechanisms of activity-dependent modulation of burst discharges in rat hippocampal slices have been studied. The extracellular registration of field responses (population spike, PS, and field excitatory postsynaptic potential, fEPSP) induced by repetitive electrical stimulation (1–4 Hz) of Schaffer collaterals (with 30 pulses trains separated by 5-min resting intervals) was performed in cellular and dendritic layers of CA1 area. It has been established that repetitive orthodromic stimulation exerts biphasic modulation of burst discharges in Mg²⁺-free medium: use-dependent potentiation (UDP) and use-dependent inhibition (UDI). The former was manifested as an increase in the number of PS in the burst discharge associated with a corresponding lengthening of the fEPSP. During the UDI development the number of NMDA-dependent PS in the burst was diminished despite the continuing increase in the fEPSP duration. In some cases UDI was followed by spreading depression. Both UDP and UDI were reversible. The development of UDP and UDI could be effectively suppressed either by the NMDA antagonists or by the GABAergic inhibition enhancer, diazepam. The picrotoxin (PTX)-induced burst discharges did not undergo either UDP or UDI development. However, removal of Mg²⁺ from PTX-containing solution during continuing repetitive stimulation led to the appearance of NMDA-dependent UDI. Analysis of the data obtained indicates that: (1) UDP results from a progressive decrease in GABA-mediated inhibition in the course of low-frequency (1–4 Hz) repetitive stimulation (the so-called “fatigue of synaptic inhibition”); (2) UDI is caused by excessive Ca²⁺ influx into the neurons due to overactivation of NMDA receptors. The article is published in the original.     

Induction of L-Phase Variant from Protoplast of Staphylococcus aureus

Protoplasts of Staphylococcus aureus 209P and Cowan 1 were induced by treatment with lysostaphin. These protoplasts were sensitive to detergent, a low concentration of sodium chloride and low temperature. Almost all protoplast cells spread on CLYS agar medium (casein hydrolysate, yeast extract, Na-lactate, and NaCl) formed typical L-form colonies. Horse serum (0.25%) and Mg²⁺ (109 mm) are essential factors for formation of the L-form colonies of 209P. In the case of Cowan 1, Mg²⁺ was not required. The active factor(s) in horse serum was heat-resistant and protein in nature.     

Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABAA receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on γ-aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA-and muscimol (Mus)-activated currents (I_gaba and I_Mus), respectively in the Mg²⁺-free external solution containing 1 μmol/L glycine at a holding potential (V _H ) of −40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 γmol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of I_gaba; (ii) when the neurons were incubated in a Ca²⁺-free bath or pre-loaded with a membrane-permeable Ca²⁺ chelator, BAPTA AM (10 μmol/L), the inhibitory effect of NMDA on I_GAba disappeared. Cd²⁺ (10 μmol/L) or La³⁺ (30 μmol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of I_gaba by NMDA application; (iii) the suppression of I_GAba by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca²⁺-dependent and CaMKII is involved in the process of the Ca²⁺-dependent inhibition.     

The role of calcium ions for the expression of ricin toxicity in cultured macrophages

Ricin toxin, which consists of two distinct polypeptide moieties, A and B chains, is cytotoxic to the cultured macrophage cell line, J774A.1. Ricin is a protein synthesis inhibitor, and incubating macrophages for 4 hours with ricin (1 pM to 10 nM) in standard medium containing calcium and magnesium inhibited ³H-leucine incorporation into protein (97%, at 1 nM ricin). However, in Ca²⁺-free medium, protein synthesis was inhibited only 19%. EGTA pretreatment (to deplete intracellular calcium) also partly protected cells from protein synthesis inhibition, in spite of added calcium (2 mM) in the incubation medium. Decreased toxicity in the absence of extracellular calcium resulted from decreased toxin binding. Adding or deleting Mg²⁺ did not affect protein synthesis or binding of ¹²⁵I-ricin in cultured macrophages. We conclude that calcium is required for ricin to exert its inhibitory effect on protein synthesis in cultured macrophages.     

Low magnesium induced epileptiform discharges in neocortical slices (guinea pig): Increased antiepileptic efficacy of organic calcium antagonist verapamil with elevation of extracellular K+ concentration

1. The antiepileptic effect of the organic calcium antagonist verapamil on low Mg²⁺ induced epileptiform discharges in the neocortex was tested.2. The experiments were carried out on slices of the frontal neocortex (guinea pigs). Verapamil was tested at normal (4 mmol/l) and elevated (8 mmol/l) KCl levels.3. Verapamil (40, 60 μmol/l) suppressed epileptiform activity in any case.4. With elevated K⁺ concentration the suppressive effect of verapamil was significantly accelerated.5. Epileptic activity reappeared when verapamil was omitted from the Mg²⁺-free superfusate.     

Interaction of Triton X-100 with particulate cardiac guanylate cyclase: comparison between Mg2+- and Mn2+-supported enzyme activities in particulate. detergent-solubilized and detergent-insoluble fractions

Guanylate cyclase activity was determined in a 1000g particulate fraction derived from rabbit heart homogenates using Mg²⁺ or Mn²⁺ as sole cation in the presence and absence of Triton X-100. With Mg²⁺, very little guanylate cyclase activity could be detected in the original particulate fraction assayed with or without Triton, or in the particulate fraction treated with varying concentrations of Triton (detergent-treated mixture) prior to enzyme assay. However, the detergent-solubilized supernatants as well as the detergent-insoluble residues (pellets) derived from detergent-treated mixtures possessed appreciable Mg²⁺-supported enzyme activity. With Mn²⁺, significant enzyme activity was detectable in the original particulate fraction assayed without Triton. Much higher activity was seen in particulate fraction assayed with Triton and in detergent-treated mixtures; the supernatants but not the pellets derived from detergent-treated mixtures possessed even greater activity. The sum of enzyme activity in pellet and supernatant fractions greatly exceeded that of the mixture. When the pellets and supernatants derived from detergenttreated mixtures were recombined, measured enzyme activities were similar to those of the original mixture. With Mg²⁺ or Mn²⁺, the specific activity of guanylate cyclase in pellet and supernatant fractions varied considerably depending on the concentration of Triton used for treatment of the particulate fraction; treatment with low concentrations of Triton (0.2–0.7 μmol/mg protein) gave supernatants showing high activity whereas treatment with relatively greater concentrations of the detergent (>0.7 μmol/mg protein) gave pellets showing high activity. The relative distribution of guanylate cyclase in pellet and supernatant fractions expressed as a function of Triton concentration during treatment (of the particulate fraction) showed that 50 to 80% of the recovered enzyme activity remained in supernatants at low detergent concentrations whereas 50 to 80% of the recovered activity resided in the pellets at higher detergent concentrations. Inclusion of excess Triton in the enzyme assay medium did not alter the specific activity profiles and the relative distribution patterns of the cyclase in pellet versus supernatant fractions. The results demonstrate the inherent potential of cardiac particulate guanylate cyclase to utilize Mg²⁺ in catalyzing the synthesis of cyclic GMP. However, it appears that some factor(s) endogenous to the cardiac particulate fraction severely impairs the expression of Mg²⁺-dependent activity; Mn²⁺-dependent activity is also affected by such factor(s) but apparently less severely. Further, the results suggest that previously reported activities of cardiac particulate guanylate cyclase, despite being assayed with Mn²⁺ and in the presence of Triton X-100, represent underestimation of what otherwise appears to be a highly active enzyme system capable of utilizing physiologically relevant divalent cation such as Mg²⁺.     

The structure of MgtE in the absence of magnesium provides new insights into channel gating

MgtE is a Mg²⁺ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg²⁺ homeostasis. The previously determined MgtE structures in the Mg²⁺-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg²⁺ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg²⁺ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg²⁺-free conditions, and the pore-opening mechanism has thus remained unclear.Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg²⁺ ions. The Mg²⁺-free MgtE TM domain structure and its comparison with the Mg²⁺-bound, closed-state structure, together with functional analyses, showed the Mg²⁺-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

MgtE is a magnesium-selective ion channel whose gating is regulated by cytoplasmic magnesium concentration; this cryo-EM study reveals how MgtE undergoes magnesium-dependent structural changes to open the pore on the cytoplasmic side.     

N-Methyl-d-Aspartic Acid/Glycine Interactions on the Control of 5-Hydroxytryptamine Release in Raphe Primary Cultures

Abstract: Glutamic acid and glycine were quantified in cells and medium of cultured rostral rhombencephalic neurons derived from fetal rats. In the presence of 1 mM Mg²⁺, NMDA (50 μM) significantly stimulated (by 69%) release of newly synthesized 5-[³H]hydroxytryptamine ([³H]5-HT). d -2-Amino-5-phosphonopentanoate (AP-5; 50 μM) blocked the stimulatory effect of NMDA. AP-5 by itself inhibited [³H]5-HT release (by 25%), suggesting a tonic control of 5-HT by glutamate. In the absence of Mg²⁺, basal [³H]5-HT release was 60% higher as compared with release with Mg²⁺. AP-5 blocked the increased [³H]5-HT release observed without Mg²⁺, suggesting that this effect was due to the stimulation of NMDA receptors by endogenous glutamate. Glycine (100 μM) inhibited [³H]5-HT release in the absence of Mg²⁺. Strychnine (50 μM) blocked the inhibitory effect of glycine, indicating an action through strychnine-sensitive inhibitory glycine receptors. The [³H]5-HT release stimulated by NMDA was unaffected by glycine. In contrast, when tested in the presence of strychnine, glycine increased NMDA-evoked [³H]5-HT release (by 22%), and this effect was prevented by a selective antagonist of the NMDA-associated glycine receptor, 7-chlorokynurenate (100 μM). 7-Chlorokynuren-ate by itself induced a drastic decrease in [³H]5-HT release, indicating that under basal conditions these sites were stimulated by endogenous glycine. These results indicate that NMDA stimulated [³H]5-HT release in both the presence or absence of Mg²⁺. Use of selective antagonists allowed differentiation of a strychnine-sensitive glycine response (inhibition of [³H]5-HT release) from a 7-chlorokynurenate-sensitive response (potentiation of NMDA-evoked [³H]5-HT release).     

Magnesium deprivation inhibits a MEK–ERK cascade and cell proliferation in renal epithelial Madin-Darby canine kidney cells

AimsLoss of magnesium (Mg²⁺) inhibits cell proliferation and augments nephrotoxicant-induced renal injury, but the role of Mg²⁺ has not been clarified in detail. We examined the effect of extracellular Mg²⁺ deprivation on a MEK–ERK cascade and cell proliferation using a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells.Main methodsMDCK cells were cultured in Mg²⁺-containing or Mg²⁺-free media. A HA-tagged constitutively active (CA)-MEK1 and a dominant negative (DN)-MEK1 were transfected into MDCK cells. The level of protein was examined by Western blotting. The intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was measured using a fluorescent dye, mag-fura 2. Cell proliferation was determined by WST-1 assay. Dead cells were identified by staining with annexin V-FITC and propidium iodide.Key findingsIn the presence of fetal calf serum (FCS), Mg²⁺ deprivation decreased phosphorylated-ERK1/2 (p-ERK1/2) levels and [Mg²⁺]_i. Re-addition of Mg²⁺ increased p-ERK1/2 levels, which were inhibited by U0126, a specific inhibitor of a MEK–ERK cascade. Glutathione-S-transferase pull-down and coimmunoprecipitation assays showed that CA-MEK1 and DN-MEK1 binds with ERK1/2 in the presence of Mg²⁺. In contrast, neither CA-MEK1 nor DN-MEK1 bound to ERK1/2 in the absence of Mg²⁺. These results indicate that the MEK–ERK cascade is regulated by [Mg²⁺]_i. Cell proliferation was increased by the treatment with FCS or the expression of CA-MEK1 in the presence of Mg²⁺, but was inhibited by Mg²⁺ deprivation. Mg²⁺ deprivation did not increase the number of dead cells.SignificanceMg²⁺ is involved in the regulation of the MEK–ERK cascade and cell proliferation in MDCK cells.     

Purification and characterization of a species-specific aggregation factor in sponges

The events during re-aggregation of sponge cells, dissociated in Ca²⁺- and Mg²⁺-free artificial sea water, containing trypsin, can be subdivided into three phases. The first event is the formation of primary aggregates with a diameter of 82 μm. It is Ca²⁺- and Mg²⁺-dependent and insensitive towards trypsin and puromycin even at high concentrations. The formation of secondary aggregates with a diameter greater than 1 000 μm, is initiated by an aggregation factor. This factor can be separated from the formed elements with calcium- and magnesium-free artificial sea water. Its action is Ca²⁺- and Mg²⁺-dependent, temperature-independent and insensitive to puromycin. The last event is the reconstitution of functional aquiferous systems in the aggregates.The aggregation factor which was found to be species-specific could be purified 158-fold. Its functional group seems to be a protein probably with polar amino acids in critical positions. Secondary aggregates generated with the aggregation factor show high viability. Some evidence is presented that the aggregation factor may be an annular particle with a circular contour length of 3 500 Å with about 25 filaments attached to it.     

Divalent cations activate TRPV1 through promoting conformational change of the extracellular region

Divalent cations Mg²⁺ and Ba²⁺ selectively and directly potentiate transient receptor potential vanilloid type 1 heat activation by lowering the activation threshold into the room temperature range. We found that Mg²⁺ potentiates channel activation only from the extracellular side; on the intracellular side, Mg²⁺ inhibits channel current. By dividing the extracellularly accessible region of the channel protein into small segments and perturbing the structure of each segment with sequence replacement mutations, we observed that the S1–S2 linker, the S3–S4 linker, and the pore turret are all required for Mg²⁺ potentiation. Sequence replacements at these regions substantially reduced or eliminated Mg²⁺-induced activation at room temperature while sparing capsaicin activation. Heat activation was affected by many, but not all, of these structural alternations. These observations indicate that extracellular linkers and the turret may interact with each other. Site-directed fluorescence resonance energy transfer measurements further revealed that, like heat, Mg²⁺ also induces structural changes in the pore turret. Interestingly, turret movement induced by Mg²⁺ precedes channel activation, suggesting that Mg²⁺-induced conformational change in the extracellular region most likely serves as the cause of channel activation instead of a coincidental or accommodating structural adjustment.     

Modulation by Ionotropic Excitatory Amino Acids and Potassium of (±)-1-Aminocyclopentane-trans-1,3-Dicarboxylic Acid-Stimulated Phosphoinositide Hydrolysis in Mouse Cerebellar Granule Cells

Abstract: The effect of ionotropic excitatory amino acids and potassium on the formation of inositol phosphates elicited by the metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) was studied in mouse cerebellar granule cells. In Mg²⁺-containing buffers, NMDA (50–100 µM), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 10–1,000 µM), and high potassium (10–30 mM) enhanced synergistically the response to a maximally effective concentration of 500 µMtrans-ACPD. Potentiation of the trans-ACPD response was blocked by higher concentrations of NMDA (>500 µM) and potassium (>35 mM) but not by AMPA (up to 1 mM). The potentiation by NMDA of the trans-ACPD-stimulated phosphoinositide hydrolysis was blocked by d,l -2-amino-5-phosphonopentanoic acid (APV), a competitive NMDA-receptor antagonist. Under Mg²⁺-free conditions, the accumulation of inositol phosphates in the presence of trans-ACPD alone was equal to that attained by trans-ACPD in Mg²⁺-containing buffers when costimulated with maximally enhancing concentrations of NMDA (50 µM). trans-ACPD potentiated synergistically the NMDA-evoked increases in cytosolic free-Ca²⁺ levels in Mg²⁺-containing but not in Mg²⁺-free solutions, and moreover did not enhance the AMPA-evoked increases in cytosolic free-Ca²⁺ levels. The calcium ionophore A23187 caused a dose-dependent increase in inositol phosphate accumulation but did not enhance the response stimulated by trans-ACPD alone. These results demonstrate the existence of cross talk between metabotropic and ionotropic glutamate receptors in cerebellar granule cells. The exact mechanism remains unclear but appears to involve interplay of G protein-coupled phospholipase C activation and regulated elevation of cytosolic free-Ca²⁺ levels. This study may provide a framework for future investigations at the cellular and molecular level that clarify the functional relevance and molecular mechanisms that are described.     

Differential effects of ca2+ and Mg2+ on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

In autodigestion assays, endonucleaw activity in non-apoptotic HL-60 promydocytic leukemia cell nuclei cleaved the chromatin of he autologous cells to an oligonucleosomal length pattern. Both EGTA and EDTA inhibited the activation of endonuclease activity in isolated HL-60 cell nuclei. The inhibition by EDTA could be reversed by exogenous Ca²⁺. but not by exogenous Mg²⁺. In Ca²⁺/Mg²⁺-free nuclei digation buffer, addition of Ca^2→ (1-10 mmol/L) induced endonuclease activity in the isolated nuclei, while addition of Mg²⁺ had no effect. In the presence of Ca²⁺(0.1 mmol/L), endonuclease activity was enhanced by exogenous Mg²⁺ (0.1-10mmol/L). These results suggest that the endonuclease responsible for internucleosomal DNA fragmentation in HL-60 cells during apoptosis is activated by Ca²⁺ and further modulated by Mg²⁺ in the presence of ca²⁺.     

Effects of Divalent Cations,Trypsin, and Phospholipases on the Passive Permeability to Sodium of Inside-Out Vesicles From Human Red Cells

Inside-out vesicles (IOV) were prepared from human red blood cells. Steady-state uptake of ²²Na was observed to generally follow an exponential time course with a rate constant of 1.57 ± 0.09 h^?1 (SE). One week of cold storage (0–4°C) increased the rate constant to 2.50 ± 0.12 h ^?1 (SE). Mg²⁺, Ca²⁺, or Sr²⁺ decreased the rate of ²²Na uptake with no observable differences between the three divalent cations when tested at concentrations of 50 μM. Mg²⁺ was shown to decrease the rate of ²²Na uptake at concentrations as low as 5 μM with maximal effect at 50 to 100 μM. The decrease in rate of ²²Na uptake induced by Mg²⁺ could be enhanced by exposure of IOV to Mg²⁺ for longer periods of time. Trypsin treatment of IOV increased the rate of uptake of ²²Na and was dependent on the concentration of trypsin added between 5 to 25 μg/ml (treated for 5 min at 25°C). The ability of Mg²⁺ (50 μM) to decrease the rate of ²²Na uptake was still observed after maximal trypsin treatment. Phospholipase A₂ or phospholipase C treatment of IOV increased the rate of ²²Na uptake and was dependent on the amount of phospholipase A₂ (0.1 to 1.0 units/ml) or phospholipase C (0.25 to 2.5 units/ml) added (treated for 5 min at 25°C). After phospholipase A₂ treatment, the observed decrease in the rate of ²²Na uptake induced by Mg²⁺ (50 μM) was generally greater than controls. After phospholipase C treatment, the observed decrease in rate of ²²Na uptake induced by Mg²⁺ (50 μM) was less or absent when compared with controls. Phospholipase C treatment was less effective in preventing the Mg²⁺ effect the longer IOV were exposed to Mg²⁺. The results suggest that Mg²⁺ binds to phospholipid head-groups to reduce Na permeability perhaps by inducing a change in bilayer structure or phospholipid association.     

Extracellular Mg regulates the intracellular Na concentration in rat sublingual acini

The intracellular free Na⁺ concentration ([Na⁺]_i) increases during muscarinic stimulation in salivary acinar cells. The present study examined in rat sublingual acini the role of extracellular Mg²⁺ in the regulation of the stimulated [Na⁺]_i increase using the fluorescent sodium indicator benzofuran isophthalate (SBFI). The muscarinic induced rise in [Na⁺]_i was approximately 4-fold greater in the absence of extracellular Mg²⁺. When Na⁺ efflux was blocked by the Na⁺,K⁺-ATPase inhibitor ouabain, the stimulated [Na⁺]_i increase was comparable to that seen in an Mg²⁺-free medium. Moreover, ouabain did not add further to the stimulated [Na⁺]_i increase in an Mg²⁺-free medium suggesting that removal of extracellular Mg²⁺ may inhibit the Na⁺ pump. In agreement with this assumption, ouabain-sensitive Na⁺ efflux and rubidium uptake were reduced by extracellular Mg²⁺ depletion. Our results suggest that extracellular Mg²⁺ may regulate [Na⁺]_i in sublingual salivary acinar cells by modulating Na⁺ pump activity.     

Effect of magnesium on calcium influx activated by glutamate and its agonists in cultured cerebellar granule cells

The effects of Mg²⁺ on the glutamate-, kainate-, N-methyl-d-aspartate- and quisqualate-induced influx of⁴⁵Ca²⁺ were studied in cultured cerebellar granule cells. The N-methyl-d-aspartate- and quisqualate-evoked influx was totally and the kainate- and glutamate-evoked influx partially blocked in 1.3 mM extracellular Mg²⁺. The increase in influx induced by kainate, quisqualate and glutamate was maximal at 0.1 mM Mg²⁺, whereas N-methyl-d-aspartate was most effective in totally Mg²⁺-free media.d-2-Amino-5-phosphonovalerate blocked partially and phencyclidine completely the enhancement of Ca²⁺ influx by 1 mM quisqualate in 0.1-mM Mg²⁺ medium. The effect of 10 M quisqualate was also significantly inhibited by antagonists specific for different glutamate receptor subtypes, including N-methyl-d-aspartate, (RS)-amino-3-hydroxy-5-methyl-4-isozazolepropionate and metabotropic recptors. This evidences a heterogeneous action of quisqualate, mediated by different glutamate receptor subtypes in 0.1 mM Mg²⁺ medium. The efficacy of quisqualate in inducing influx of Ca⁺ and the selectivity of antagonists for different receptors are also modified by extracellular Mg²⁺.     

Spatial distribution of cytoplasmic domains of the Mg-transporter MgtE,in a solution lacking Mg,revealed by paramagnetic relaxation enhancement

MgtE is a prokaryotic Mg²⁺ transporter that controls cellular Mg²⁺ concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg²⁺-free and Mg²⁺-bound forms. The Mg²⁺-binding sites lay at the interface of the 2 domains, making the Mg²⁺-bound form compact and globular. In the Mg²⁺-free structure, however, the domains are far apart, and the Mg²⁺-binding sites are destroyed. Therefore, it is unclear how Mg²⁺-free MgtE changes its conformation to accommodate Mg²⁺ ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg²⁺ in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg²⁺-bound crystal structure, facilitating efficient capture of Mg²⁺ with increased intracellular Mg²⁺ concentration, which is necessary to close the gate.