Extracellular Mg2 Research Articles

Cysteine-modulated colorimetric sensing of extracellular Mg2+ in rat brain based on the strong chelation interaction between dithiothreitol and Mg2+

Simple and effective measurement of Mg(2+) in the brain of living animals is of great physiological and pathological importance. In this study, we report a facile yet highly selective colorimetric method for effective sensing of cerebral Mg(2+). The method is based on rational design of surface chemistry of gold nanoparticles (Au-NPs) with functional molecules including 1,4-dithiothreitol (DTT) and cysteine, enabling the fine tuning of the surface chemistry of Au-NPs in such a way that the addition of Mg(2+) into the Au-NPs dispersion could selectively trigger the change of the dispersion/aggregation states of Au-NPs. The strong chelation interaction between Mg(2+) and the hydroxyls in 1,4-dithiothreitol and the co-existence of cysteine on the surface of Au-NPs could, on one hand, enable the selective colorimetric detection of Mg(2+) and, on the other hand, avoid the aggregation of Au-NPs induced by DTT itself. As a result, the addition of Mg(2+) into the dispersion of the Au-NPs containing both cysteine and DTT results in the changes in both the color and the UV-vis spectra of the Au-NPs dispersion. The signal readout shows a linear relationship of Mg(2+) within the concentration range from 1 μM to 40 μM with a detection limit of 800 nM (S/N = 3). Moreover, the assay demonstrated here is free from the interference of some physiological species commonly existing in rat brain. Although Ca(2+) could interfere with the detection of Mg(2+) because of its strong chelation with DTT, it could be selectively masked by masking agent (i.e., ethyleneglcol-bis (2-aminoethylether) tetraacetic acid). By combining the microdialysis technique, the basal dialysate level of Mg(2+) is determined to be 299.2 ± 41.1 μM (n = 3) in the cerebral systems. The method essentially offers a new method for the detection of Mg(2+) in the cerebral system.

Calcium reabsorption in the distal tubule: regulation by sodium, pH, and flow

We developed a mathematical model of Ca(2+) transport along the late distal convoluted tubule (DCT2) and the connecting tubule (CNT) to investigate the mechanisms that regulate Ca(2+) reabsorption in the DCT2-CNT. The model accounts for apical Ca(2+) influx across transient receptor potential vanilloid 5 (TRPV5) channels and basolateral Ca(2+) efflux via plasma membrane Ca(2+)-ATPase pumps and type 1 Na(+)/Ca(2+) exchangers (NCX1). Model simulations reproduce experimentally observed variations in Ca(2+) uptake as a function of extracellular pH, Na(+), and Mg(2+) concentration. Our results indicate that amiloride enhances Ca(2+) reabsorption in the DCT2-CNT predominantly by increasing the driving force across NCX1, thereby stimulating Ca(2+) efflux. They also suggest that because aldosterone upregulates both apical and basolateral Na(+) transport pathways, it has a lesser impact on Ca(2+) reabsorption than amiloride. Conversely, the model predicts that full NCX1 inhibition and parathyroidectomy each augment the Ca(2+) load delivered to the collecting duct severalfold. In addition, our results suggest that regulation of TRPV5 activity by luminal pH has a small impact, per se, on transepithelial Ca(2+) fluxes; the reduction in Ca(2+) reabsorption induced by metabolic acidosis likely stems from decreases in TRPV5 expression. In contrast, elevations in luminal Ca(2+) are predicted to significantly decrease TRPV5 activity via the Ca(2+)-sensing receptor. Nevertheless, following the administration of furosemide, the calcium-sensing receptor-mediated increase in Ca(2+) reabsorption in the DCT2-CNT is calculated to be insufficient to prevent hypercalciuria. Altogether, our model predicts complex interactions between calcium and sodium reabsorption in the DCT2-CNT.

Fluoxetine suppresses synaptically induced [Ca2+]i spikes and excitotoxicity in cultured rat hippocampal neurons

Fluoxetine is a widely used antidepressant with an action that is primarily attributed to the inhibition of serotonin re-uptake into the synaptic terminals of the central nervous system. Fluoxetine also has blocking effects on various ion channels, including Ca2+ channels. It remains unclear, however, how fluoxetine may affect synaptically induced [Ca2+]i spikes. We investigated the effects of fluoxetine on [Ca2+]i spikes, along with the subsequent neurotoxicity that is synaptically evoked by lowering extracellular Mg2+ in cultured rat hippocampal neurons. Fluoxetine inhibited the synaptically induced [Ca2+]i spikes in p-chloroamphetamine-treated and non-treated neurons, in a concentration-dependent manner. However, other selective serotonin reuptake inhibitors, such as paroxetine and citalopram, did not significantly affect the spikes. Pretreatment with fluoxetine for 5min inhibited [Ca2+]i increases induced by glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and N-methyl-d-aspartate. Fluoxetine also inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced currents. In addition, fluoxetine decreased the [Ca2+]i responses induced by the metabotrophic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine or the ryanodine receptor agonist caffeine. Fluoxetine inhibited [Ca2+]i responses induced by 20mM KCl. Fluoxetine decreased the release of FM1-43 induced by electric field stimulation. Furthermore, fluoxetine inhibited 0.1mM [Mg2+]o-induced cell death. Collectively, our results suggest that fluoxetine suppresses the spikes and protects neurons against excitotoxicity, particularly in cultured rat hippocampal neurons, presumably due to both direct inhibition of presynaptic glutamate release and postsynaptic glutamate receptor-mediated [Ca2+]i signaling. In addition to an indirect inhibitory effect via 5-HT levels, these data suggest a new, possibly direct inhibitory action of fluoxetine on synaptically induced [Ca2+]i spikes and neuronal cell death.

Kcnh1 Voltage-gated Potassium Channels Are Essential for Early Zebrafish Development

The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca(2+)/calmodulin and modulation of voltage-dependent gating by extracellular Mg(2+). Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning.

A new signalling pathway for parallel fibre presynaptic type 4 metabotropic glutamate receptors (mGluR4) in the rat cerebellar cortex

In the rodent cerebellum, pharmacological activation of mGluR4 acutely depresses excitatory synaptic transmission at parallel fibre–Purkinje cell synapses. This depression involves the inhibition of presynaptic calcium (Ca2+) influx that ultimately controls glutamate release. In this study, we investigate the molecular basis of mGluR4-mediated inhibition of presynaptic Ca2+ transients. Our results demonstrate that the mGluR4 effect does not depend on selective inhibition of a specific type of presynaptic voltage-gated Ca2+ channel, but rather involves modulation of all classes of Ca2+ channels present in the presynaptic terminals. In addition, this inhibitory effect does not involve the activation of G protein-activated inwardly rectifying potassium channels, TEA-sensitive potassium channels or two-pore-domain potassium channels. Furthermore, this inhibition does not require pertussis toxin-sensitive G proteins, and is independent of any effect on adenylyl cyclases, protein kinase A, mitogen-activated protein kinases or phosphoinositol-3 kinase activity. Interestingly we found that mGluR4 inhibition of presynaptic Ca2+ influx employs a newly defined signalling pathway, notably that involving the activation of phospholipase C and ultimately protein kinase C.

In vivo electrochemical recording of continuous change of magnesium in medial vestibular nucleus following vertigo induced by ice water vestibular stimulation

Vertigo is one of the most common clinical symptoms. However, the chemical processes involved in the pathological mechanism of vertigo remain to be fully understood. In this study, we investigate the dynamic changes in the magnesium (Mg2+) concentration in medial vestibular nucleus (MVN) of guinea pigs following vertigo induced by vestibular ice water stimulation with an electrochemical detection method consisting of in vivo microdialysis and on-line selective electrochemical detection. Electrochemical detection of Mg2+ was accomplished based on the current enhancement of Mg2+ towards the electrocatalytic oxidation of NADH at the electrodes modified with the polymerized film of toluidine blue O (TBO). Selectivity for the on-line electrochemical detection against Ca2+ was achieved by using ethyleneglcol-bis(2-aminoethylether) tetraacetic acid (EGTA) as the selective masking agent for Ca2+. The basal level of the extracellular Mg2+ in the MVN of guinea pigs was determined to be 759.7 ± 176.2 μM(n = 16). Upon ice water irrigation of the left external ear canal, the concentration of Mg2+ in the MVN decreases significantly, reaches 72 ± 6% (n = 8) of the basal level, and maintains for at least 1000 s. Control experiments reveal that neither warm water irrigation of the external ear canal nor ice water irrigation of the auricle induces the decrease in the concentration of Mg2+ in the MVN. These results demonstrate that the extracellular Mg2+ in the MVN decreases significantly following vertigo induced by vestibular ice water stimulation. This demonstration suggests that Mg2+ might play an important role in the pathological mechanism of vertigo.

Restoration of Synaptic Plasticity and Learning in Young and Aged NCAM-Deficient Mice by Enhancing Neurotransmission Mediated by GluN2A-Containing NMDA Receptors

Neural cell adhesion molecule (NCAM) is the predominant carrier of the unusual glycan polysialic acid (PSA). Deficits in PSA and/or NCAM expression cause impairments in hippocampal long-term potentiation and depression (LTP and LTD) and are associated with schizophrenia and aging. In this study, we show that impaired LTP in adult NCAM-deficient (NCAM(-/-)) mice is restored by increasing the activity of the NMDA subtype of glutamate receptor (GluN) through either reducing the extracellular Mg2+ concentration or applying d-cycloserine (DCS), a partial agonist of the GluN glycine binding site. Pharmacological inhibition of the GluN2A subtype reduced LTP to the same level in NCAM(-/-) and wild-type (NCAM(+/+)) littermate mice and abolished the rescue by DCS in NCAM(-/-) mice, suggesting that the effects of DCS are mainly mediated by GluN2A. The insufficient contribution of GluN to LTD in NCAM(-/-) mice was also compensated for by DCS. Furthermore, impaired contextual and cued fear conditioning levels were restored in NCAM(-/-) mice by administration of DCS before conditioning. In 12-month-old NCAM(-/-), but not NCAM(+/+) mice, there was a decline in LTP compared with 3-month-old mice that could be rescued by DCS. In 24-month-old mice of both genotypes, there was a reduction in LTP that could be fully restored by DCS in NCAM(+/+) mice but only partially restored in NCAM(-/-) mice. Thus, several deficiencies of NCAM(-/-) mice can be ameliorated by enhancing GluN2A-mediated neurotransmission with DCS.

Detailed Examination of TRPM7 Channel Mg2+ Dependence

TRPM7, a member of transient receptor potential superfamily of ion channels, underlies the Mg2+-inhibited cation currents highly expressed in various cell types of hematopoietic lineage. TRPM7 channels are sensitive to intracellular as well as extracellular Mg2+ and other polyvalent cations. The present study was undertaken to characterize the inhibition of TRPM7 channels by intracellular Mg2+ in detail. In order to generate a dose-response curve for Mg2+i, we systematically varied the internal Mg2+ concentration and measured the corresponding maximal TRPM7 current amplitude with whole-cell patch clamp in Jurkat T lymphocytes and HEK293 cells. Mg2+-containing solutions were buffered with HEDTA. We tested free Mg2+ concentrations of 100 nM to 400 μM in n ≥ 70 cells. Unexpectedly, we find that the dose-response curve for Mg2+ is biphasic, yielding IC50 values of ∼8 μM and ∼200 μM. 300 μM free Mg2+ is sufficient for complete inhibition of channel activity. This finding suggests the existence of two inhibitor sites for TRPM7 activity. Since in whole-cell configuration the channel interior is only exposed to one solution throughout the recording, it is impossible to separate Mg2+ effect on the likelihood of channel opening from its effect on an already open channel. Moreover, reversible inhibition cannot be distinguished from time-dependent rundown of activity. In order to examine the nature of Mg2+-mediated inhibition more thoroughly, we recorded TRPM7 channels in inside-out patch configuration. Membrane patches were excised into a Mg2+-free bath solution and TRPM7 single-channel activity recorded. Mg2+ was then added to the solution facing intracellular side of the channel, using rapid application. We observed reversible inhibition by Mg2+ in the 35 - 400 micromolar concentration range and have characterized changes in single-channel characteristics responsible for the inhibition. A comparison of Mg2+ and proton sensitivities is also presented.

Characterization of Mg²⁺-regulated TRPM7-like current in human atrial myocytes.

BackgroundTRPM7 (Transient Receptor Potential of the Melastatin subfamily) proteins are highly expressed in the heart, however, electrophysiological studies, demonstrating and characterizing these channels in human cardiomyocytes, are missing.MethodsWe have used the patch clamp technique to characterize the biophysical properties of TRPM7 channel in human myocytes isolated from right atria small chunks obtained from 116 patients in sinus rhythm during coronary artery and valvular surgery. Under whole-cell voltage-clamp, with Ca2+ and K+ channels blocked, currents were generated by symmetrical voltage ramp commands to potentials between -120 and +80 mV, from a holding potential of -80 mV.ResultsWe demonstrate that activated native current has dual control by intracellular Mg2+ (free-Mg2+ or ATP-bound form), and shows up- or down-regulation by its low or high levels, respectively, displaying outward rectification in physiological extracellular medium. High extracellular Mg2+ and Ca2+ block the outward current, while Gd3+, SpM4+, 2-APB, and carvacrol inhibit both (inward and outward) currents. Besides, divalents also permeate the channel, and the efficacy sequence, at 20 mM, was Mg2+>Ni2+>Ca2+>Ba2+>Cd2+ for decreasing outward and Ni2+>Mg2+>Ba2+≥Ca2+>Cd2+ for increasing inward currents. The defined current bears many characteristics of heterologously expressed or native TRPM7 current, and allowed us to propose that current under study is TRPM7-like. However, the time of beginning and time to peak as well steady state magnitude (range from 1.21 to 11.63 pA/pF, ncells/patients = 136/77) of induced TRPM7-like current in atrial myocytes from different patients showed a large variability, while from the same sample of human atria all these parameters were very homogenous. We present new information that TRPM7-like current in human myocytes is less sensitive to Mg2+. In addition, in some myocytes (from 24 out of 77 patients) that current was already up-regulated at membrane rupture.ConclusionsThis study provides the first electrophysiological description of TRPM7-like current in native human atrial myocytes. Less sensitivity to intracellular Mg2+ suggests for channel operation under physiological conditions. The TRPM7-like current up-regulation indicates the pathophysiological evidence of that current in human heart.

Syndrome de Sweet révélant une micropolyangéite : première description

The tight junction of epithelial cells excludes macromolecules but allows permeation of ions. However, it is not clear whether this ion-conducting property is mediated by aqueous pores or by ion channels. To investigate the permeability properties of the tight junction, we have developed paracellular ion flux assays for four major extracellular ions, Na+, Cl−, Ca2+, and Mg2+. We found that the tight junction shares biophysical properties with conventional ion channels, including size and charge selectivity, dependency of permeability on ion concentration, competition between permeant molecules, anomalous mole-fraction effects, and sensitivity to pH. Our results support the hypothesis that discrete ion channels are present at the tight junction. Unlike conventional ion channels, which mediate ion transport across lipid bilayers, the tight junction channels must orient parallel to the plane of the plasma membranes to support paracellular ion movements. This new class of paracellular-tight junction channels (PTJC) facilitates the transport of ions between separate extracellular compartments.

SLC41A1 Mg2+ transport is regulated via Mg2+-dependent endosomal recycling through its N-terminal cytoplasmic domain

SLC41A1 (solute carrier family 41, member A1) is a recently described vertebrate member of the MgtE family of Mg(2+) transporters. Although MgtE transporters are found in both prokaryotic and eukaryotic organisms, and are highly conserved, little is known about the regulation of their Mg(2+) transport function. In the present study, we have shown that endogenous SLC41A1 transporter expression is post-transcriptionally regulated by extracellular Mg(2+) in TRPM7 (transient receptor potential cation channel, subfamily M, member 7)-deficient cells, suggesting that SLC41A1 transporters underlie a novel plasma membrane Mg(2+) transport function. Consistent with this conclusion, structure-function analyses of heterologous SLC41A1 transporter expression demonstrate that SLC41A1 transporters exhibit the same plasma membrane orientation as homologous bacterial MgtE proteins, are capable of complementing growth of TRPM7-deficient cells only when the Mg(2+) transporting pore is intact, and require an N-terminal cytoplasmic domain for Mg(2+)-dependent regulation of lysosomal degradation and surface expression. Taken together, our results indicate that SLC41A1 proteins are a central component of vertebrate Mg(2+) transport systems, and that their Mg(2+) transport function is regulated primarily through an endosomal recycling mechanism involving the SLC41A1 N-terminal cytoplasmic domain.

Specificity and Actions of an Arylaspartate Inhibitor of Glutamate Transport at the Schaffer Collateral-CA1 Pyramidal Cell Synapse

In this study we characterized the pharmacological selectivity and physiological actions of a new arylaspartate glutamate transporter blocker, L-threo-ß-benzylaspartate (L-TBA). At concentrations up to 100 µM, L-TBA did not act as an AMPA receptor (AMPAR) or NMDA receptor (NMDAR) agonist or antagonist when applied to outside-out patches from mouse hippocampal CA1 pyramidal neurons. L-TBA had no effect on the amplitude of field excitatory postsynaptic potentials (fEPSPs) recorded at the Schaffer collateral-CA1 pyramidal cell synapse. Excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons were unaffected by L-TBA in the presence of physiological extracellular Mg2+ concentrations, but in Mg2+-free solution, EPSCs were significantly prolonged as a consequence of increased NMDAR activity. Although L-TBA exhibited approximately four-fold selectivity for neuronal EAAT3 over glial EAAT1/EAAT2 transporter subtypes expressed in Xenopus oocytes, the L-TBA concentration-dependence of the EPSC charge transfer increase in the absence of Mg2+ was the same in hippocampal slices from EAAT3 +/+ and EAAT3 −/− mice, suggesting that TBA effects were primarily due to block of glial transporters. Consistent with this, L-TBA blocked synaptically evoked transporter currents in CA1 astrocytes with a potency in accord with its block of heterologously expressed glial transporters. Extracellular recording in the presence of physiological Mg2+ revealed that L-TBA prolonged fEPSPs in a frequency-dependent manner by selectively increasing the NMDAR-mediated component of the fEPSP during short bursts of activity. The data indicate that glial glutamate transporters play a dominant role in limiting extrasynaptic transmitter diffusion and binding to NMDARs. Furthermore, NMDAR signaling is primarily limited by voltage-dependent Mg2+ block during low-frequency activity, while the relative contribution of transport increases during short bursts of higher frequency signaling.

Distinct pharmacological and functional properties of NMDA receptors in mouse cortical astrocytes

Astrocytes of the mouse neocortex express functional NMDA receptors, which are not blocked by Mg(2+) ions. However, the pharmacological profile of glial NMDA receptors and their subunit composition is far from complete. We tested the sensitivity of NMDA receptor-mediated currents to the novel GluN2C/D subunit-selective antagonist UBP141 in mouse cortical astrocytes and neurons. We also examined the effect of memantine, an antagonist that has substantially different affinities for GluN2A/B and GluN2C/d-containing receptors in physiological concentrations of extracellular Mg(2+). UBP141 had a strong inhibitory action on NMDA receptor-mediated transmembrane currents in the cortical layer II/III astrocytes with an IC(50) of 2.29 µM and a modest inhibitory action on NMDA-responses in the pyramidal neurons with IC(50) of 19.8 µM. Astroglial and neuronal NMDA receptors exhibited different sensitivities to memantine with IC(50) values of 2.19 and 10.8 µM, respectively. Consistent with pharmacological differences between astroglial and neuronal NMDA receptors, NMDA receptors in astrocytes showed lower Ca(2+) permeability than neuronal receptors with P(Ca) /P(Na) ratio of 3.4. The biophysical and pharmacological properties of the astrocytic NMDA receptors strongly suggest that they have a tri-heteromeric structure composed of GluN1, GluN2C/D and GluN3 subunits. The substantial difference between astroglial and neuronal NMDA receptors in their sensitivity to UBP141 and memantine may enable selective modulation of astrocytic signalling that could be very helpful for elucidating the mechanisms of neuron-glia communications. Our results may also provide the basis for the development of novel therapeutic agents specifically targeting glial signalling.

MRNA binding protein staufen 1-dependent regulation of pyramidal cell spine morphology via NMDA receptor-mediated synaptic plasticity

Staufens (Stau) are RNA-binding proteins involved in mRNA transport, localization, decay and translational control. The Staufen 1 (Stau1) isoform was recently identified as necessary for the protein synthesis-dependent late phase long-term potentiation (late-LTP) and for the maintenance of mature dendritic spines and synaptic activity in hippocampal CA1 pyramidal cells, strongly suggesting a role of mRNA regulation by Stau1 in these processes. However, the causal relationship between these impairments in synaptic function (spine shape and basal synaptic activity) and plasticity (late-LTP) remains unclear. Here, we determine that the effects of Stau1 knockdown on spine shape and size are mimicked by blocking NMDA receptors (or elevating extracellular Mg2+) and that Stau1 knockdown in the presence of NMDA receptor blockade (or high Mg2+) has no further effect on spine shape and size. Moreover, the effect of Stau1 knockdown on late-LTP cannot be explained by these effects, since when tested in normal medium, slice cultures that had been treated with high Mg2+ (to impair NMDA receptor function) in combination with a control siRNA still exhibited late-LTP, while siRNA to Stau1 was still effective in blocking late-LTP. Our results indicate that Stau1 involvement in spine morphogenesis is dependent on ongoing NMDA receptor-mediated plasticity, but its effects on late-LTP are independent of these changes. These findings clarify the role of Stau1-dependent mRNA regulation in physiological and morphological changes underlying long-term synaptic plasticity in pyramidal cells.

The Mg2+ transporter MagT1 partially rescues cell growth and Mg2+ uptake in cells lacking the channel-kinase TRPM7

Magnesium (Mg2+) transport across membranes plays an essential role in cellular growth and survival. TRPM7 is the unique fusion of a Mg2+ permeable pore with an active cytosolic kinase domain, and is considered a master regulator of cellular Mg2+ homeostasis. We previously found that the genetic deletion of TRPM7 in DT40 B cells results in Mg2+ deficiency and severe growth impairment, which can be rescued by supplementation with excess extracellular Mg2+. Here, we show that gene expression of the Mg2+ selective transporter MagT1 is upregulated in TRPM7−/− cells. Furthermore, overexpression of MagT1 in TRPM7−/− cells augments their capacity to uptake Mg2+, and improves their growth behavior in the absence of excess Mg2+.

Modulatory role of magnesium on the contractile response of rat aorta to several agonists in normal and calcium-free medium.

Acute withdrawal of external Mg2+ increased basal tone of rat isolated aorta incubated in the presence of Ca2+. Above normal levels of Mg2+ (1-4 mM) inhibited basal tone while much higher levels of the divalent cation (64-256 nM) evoked contractile responses regardless of the presence of Ca2+. Contractile responses to noradrenaline (1 microM) and KCl (80 mM) were inhibited by addition of cumulative concentrations of Mg2+. Acetylcholine-induced contractions in the presence of physiological concentrations of Mg2+ (1 mM) decreased gradually to the basal tone, but a sustained contraction was observed in the absence of this ion. In Ca(2+)-free medium, acetylcholine-induced phasic responses indicate the existence of an acetylcholine-sensitive Ca2+ store. KCl induced contraction only in Krebs solution, although a small residual contraction could be observed in Ca(2+)-free medium in some experiments. Mg(2+)-depletion in the extracellular medium increased contractile responses induced by acetylcholine and KCl in Ca(2+)-free medium. These results suggest that extracellular Mg2+ modulates basal tone, Ca2+ channels and responsiveness to various agents in the absence of Ca2+.

Sphingolipids regulate [Mg2+]o uptake and [Mg2+]i content in vascular smooth muscle cells: potential mechanisms and importance to membrane transport of Mg2+

Sphingolipids have a variety of important signaling roles in mammalian cells. We tested the hypothesis that certain sphingolipids and neutral sphingomyelinase (N-SMase) can regulate intracellular free magnesium ions ([Mg2+]i) in vascular smooth muscle (VSM) cells. Herein, we show that several sphingolipids, including C2-ceramide, C8-ceramide, C16-ceramide, and sphingosine, as well as N-SMase, have potent and direct effects on content and mobilization of [Mg2+]i in primary cultured rat aortic smooth muscle cells. All of these sphingolipid molecules increase, rapidly, [Mg2+]i in these vascular cells in a concentration-dependent manner. The increments of [Mg2+]i, induced by these agents, are derived from influx of extracellular Mg2+ and are extracellular Ca2+ concentration-dependent. Phospholipase C and Ca2+/calmodulin/Ca2+-ATPase activity appear to be important in the sphingolipid-induced rises of [Mg2+]i. Activation of certain PKC isozymes may also be required for sphingolipid-induced rises in [Mg2+]i. These novel results suggest that sphingolipids may be homeostatic regulators of extracellular Mg2+ concentration influx (and transport) and [Mg2+]i content in vascular muscle cells.

Bidirectional Plasticity Gated by Hyperpolarization Controls the Gain of Postsynaptic Firing Responses at Central Vestibular Nerve Synapses

Linking synaptic plasticity with behavioral learning requires understanding how synaptic efficacy influences postsynaptic firing in neurons whose role in behavior is understood. Here, we examine plasticity at a candidate site of motor learning: vestibular nerve synapses onto neurons that mediate reflexive movements. Pairing nerve activity with changes in postsynaptic voltage induced bidirectional synaptic plasticity in vestibular nucleus projection neurons: long-term potentiation relied on calcium-permeable AMPA receptors and postsynaptic hyperpolarization, whereas long-term depression relied on NMDA receptors and postsynaptic depolarization. Remarkably, both forms of plasticity uniformly scaled synaptic currents evoked by pulse trains, and these changes in synaptic efficacy were translated into linear increases or decreases in postsynaptic firing responses. Synapses onto local inhibitory neurons were also plastic but expressed only long-term depression. Bidirectional, linear gain control of vestibular nerve synapses onto projection neurons provides a plausible mechanism for motor learning underlying adaptation of vestibular reflexes.

Effect of Low Mg2+ and Bicuculline on Cell Survival in Hippocampal Slice Cultures

ABSTRACTA reliable model system of epileptiform insult would facilitate investigation into the underlying biological mechanisms. Epileptiform insult was induced in hippocampal slice cultures by lowering extracellular Mg2+, (+)-bicuculline, or (−)-bicuculline methochloride, a stable salt form of bicuculline (both forms block GABAA receptors). Cell death was assessed by propidium iodide uptake. Low Mg2+ or (+)-bicuculline did not produce cell death regardless of dose or incubation period. Exposure to 100 μM (−)-bicuculline methochloride for 48 hr resulted in prominent CA1 cell death. These findings demonstrate that not all pro-epileptic drugs/ion changes used routinely for electrophysiological recording of seizure activity lead to cell death in hippocampal slice cultures and that treatment with bicuculline methochloride can be used as a reliable model for epileptiform insult.

Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes

N-methyl-D-aspartate (NMDA) receptors mediate excitatory neurotransmission in the mammalian brain. Two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits each form highly Ca²(+)-permeable cation channels which are blocked by extracellular Mg²(+) in a voltage-dependent manner. Either GRIN2B or GRIN2A, encoding the NMDA receptor subunits NR2B and NR2A, was found to be disrupted by chromosome translocation breakpoints in individuals with mental retardation and/or epilepsy. Sequencing of GRIN2B in 468 individuals with mental retardation revealed four de novo mutations: a frameshift, a missense and two splice-site mutations. In another cohort of 127 individuals with idiopathic epilepsy and/or mental retardation, we discovered a GRIN2A nonsense mutation in a three-generation family. In a girl with early-onset epileptic encephalopathy, we identified the de novo GRIN2A mutation c.1845C>A predicting the amino acid substitution p.N615K. Analysis of NR1-NR2A(N615K) (NR2A subunit with the p.N615K alteration) receptor currents revealed a loss of the Mg²(+) block and a decrease in Ca²(+) permeability. Our findings suggest that disturbances in the neuronal electrophysiological balance during development result in variable neurological phenotypes depending on which NR2 subunit of NMDA receptors is affected.