Glutamate-induced increases in intracellular free Mg2+ in cultured cortical neurons

doi:10.1016/0896-6273(93)90084-5

Neuron

Volume 11, Issue 4, October 1993, Pages 751-757

https://doi.org/10.1016/0896-6273(93)90084-5 Get rights and content

Abstract

Intracellular free Mg²⁺ concentrations ([Mg²⁺]_i) in single rat brain neurons were measured with the Mg²⁺-sensitive fluorescent dye magfura-2. Addition of glutamate with glycine raised [Mg²⁺]_i from 1 to more than 11 mM compared with the resting concentration of 0.5 mM, an effect mediated by N-methyl-d-aspartate receptors. Most of the increase in [Mg²⁺]_i was independent of extracellular Mg²⁺, but was dependent on extracellular Ca²⁺. The second component of the increase induced by glutamate was independent of extracellular Ca²⁺, but required extracellular Mg²⁺ and was amplified by extracellular Na⁺ removal. These results indicate that regulation of [Mg²⁺]_i by neurotransmitters such as glutamate may be important in controlling neuronal excitability.

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Citation Excerpt :
Although this study measured Kd of fluorescent Ca2+ dyes in vitro, it should provide reasonable estimates of the respective Kd in neurons. We found that the Kd values of both OGB-6F and OGB-1 were not significantly affected by addition of ∼0.7 mM Mg2+, a concentration observed in neurons ([40–42]; Figs. 1B vs. 2B and data not shown). This suggests that these dyes have a relatively low affinity for Mg2+, most likely similar to that of fura-2 and fluo-3 (i.e Kd ≥ 5 mM; [34,43]).
Fluorescent Ca²⁺ indicators are widely used to measure the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in living cells, including neurons. By calibrating an indicator in solutions that mimic the main ionic constituents of the actual cytoplasm, [Ca²⁺]_i can be determined from the measured fluorescence intensity. However, different studies have reported considerably different Ca²⁺-binding affinities (K_d) for the same indicator, even though they used calibrating solutions with similar compositions. In this paper, we present a method to accurately determine the K_d values of non-ratiometric Ca²⁺ indicators in solutions that mimicked a standard patch-clamp internal solution. The free Ca²⁺ concentration ([Ca²⁺]) in these solutions, which was set by either EGTA or HEDTA, was measured with a Ca²⁺-selective macroelectrode. We found that such a measurement was critical for an accurate calibration of the Ca²⁺ indicators. The K_d values of OGB-1, OGB-6F, fluo-5F, and fluo-4FF were 0.26 ± 0.01, 8.7 ± 0.4, 1.00 ± 0.05, and 23.0 ± 0.7 μM, respectively. Calculating [Ca²⁺] with Maxchelator, a widely used computer program, led to a significant underestimation of the K_d values of OGB-6F, fluo-5F, and fluo-4FF. This is because the purity of EGTA was considerably less than that advertised by the manufacturer. In addition, the K_d value of HEDTA was overestimated by Maxchelator. Therefore, besides batch-to-batch variations, the fact that [Ca²⁺] in the calibrating solutions of many studies was estimated with Maxchelator is very likely a reason for the different published values of K_d of Ca²⁺ indicators. Using a reaction-diffusion model to reproduce Ca²⁺ rises in a nerve terminal, we further showed that incorrect calibration of fluorescent Ca²⁺ indicators can underlie the large variation of the endogenous Ca²⁺ binding ratio between different types of excitatory synapses.
Neural depolarization triggers Mg2+ influx in rat hippocampal neurons
2015, Neuroscience
Citation Excerpt :
However, little is known about [Mg2+]i changes under physiological conditions in healthy neurons. Previous studies reported, in neurons loaded with mag-fura-2, a Mg2+ mobilization elicited by glutamate and veratridine (Brocard et al., 1993) and neuronal depolarizer, a high concentration of KCl (Kato et al., 1998; Gotoh et al., 1999). The [Mg2+]i imaging, using a conventional Mg2+ probe, should be carefully performed in cells with the wide dynamic range of intracellular calcium ion concentration ([Ca2+]i) increase by using Mg2+ probes with low selectivity (Stout and Reynolds, 1999).
Homeostasis of magnesium ion (Mg²⁺) plays key roles in healthy neuronal functions, and deficiency of Mg²⁺ is involved in various neuronal diseases. In neurons, we have reported that excitotoxicity induced by excitatory neurotransmitter glutamate increases intracellular Mg²⁺ concentration ([Mg²⁺]_i). However, it has not been revealed whether neuronal activity under physiological condition modulates [Mg²⁺]_i. The aim of this study is to explore the direct relationship between neural activity and [Mg²⁺]_i dynamics. In rat primary-dissociated hippocampal neurons, the [Mg²⁺]_i and [Ca²⁺]_i dynamics were simultaneously visualized with a highly selective fluorescent Mg²⁺ probe, KMG-104, and a fluorescent Ca²⁺ probe, Fura Red, respectively. [Mg²⁺]_i increase concomitant with neural activity by direct current stimulation was observed in neurons plated on an indium-tin oxide (ITO) glass electrode, which enables fluorescent imaging during neural stimulation. The neural activity-dependent [Mg²⁺]_i increase was also detected in neurons whose excitability was enhanced by the treatment of a voltage-gated K⁺ channel blocker, tetraethylammonium (TEA) at the timings of spontaneous Ca²⁺ increase. Furthermore, the [Mg²⁺]_i increase was abolished in Mg²⁺-free extracellular medium, indicating [Mg²⁺]_i increase is due to Mg²⁺ influx induced by neural activity. The direct neuronal depolarization by veratridine, a Na⁺ channel opener, induced [Mg²⁺]_i increase, and this [Mg²⁺]_i increase was suppressed by the pretreatment of a non-specific Mg²⁺ channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB). Overall, activity-dependent [Mg²⁺]_i increase results from Mg²⁺ influx through 2-APB-sensitive channels in rat hippocampal neurons.
Hypoxia induces an increase in intracellular magnesium via Transient Receptor Potential Melastatin 7 (TRPM7) channels in rat hippocampal neurons in vitro
2011, Journal of Biological Chemistry
Citation Excerpt :
The different time phase of these two important cations implied there may be some kind of interaction. It has been reported that glutamate caused a large increase in [Mg2+]i through a Ca2+ influx in cerebral neurons (29), and an intracellular Mg2+ surge followed Ca2+ increase during depolarization in cultured neurons (37). According to the time window differences and the reports above, it is accepted that [Ca2+]i overload may contribute to the [Mg2+]i overload to a certain degree in OGD/CI and that needs our further investigation.
TRPM7, a divalent cation channel, plays an important role in neurons damaged from cerebral ischemia due to permitting intracellular calcium overload. This study aimed to explore whether magnesium was transported via a TRPM7 channel into the intracellular space of rat hippocampal neurons after 1 h of oxygen-glucose deprivation (OGD) and acute chemical ischemia (CI) by using methods of the Mg²⁺ fluorescent probe Mag-Fura-2 to detect intracellular magnesium concentration ([Mg²⁺]_i) and flame atomic absorption spectrometry to measure extracellular magnesium concentration ([Mg²⁺]_o). The results showed that the neuronal [Mg²⁺]_i was 1.51-fold higher after 1 h of OGD at a basal level, and the increase of neuronal [Mg²⁺]_i reached a peak after 1 h of OGD and was kept for 60 min with re-oxygenation. Meanwhile, the [Mg²⁺]_o decreased after 1 h of OGD and recovered to the pre-ischemic level within 15 min after re-oxygenation. In the case of CI, the [Mg²⁺]_i peak immediately appeared in hippocampal neurons. This increase of [Mg²⁺]_i declined by removing extracellular magnesium in OGD or CI. Furthermore, by using Gd³⁺ or 2-aminoethoxydiphenyl borate to inhibit TRPM7 channels, the [Mg²⁺]_i increase, which was induced by OGD or CI, was attenuated without altering the basal level of [Mg²⁺]_i. By silencing TRPM7 with shRNA in hippocampal neurons, it was found that not only was the increase of [Mg²⁺]_i induced by OGD or CI but also the basal levels of [Mg²⁺]_i were attenuated. In contrast, overexpression of TRPM7 in HEK293 cells exaggerated both the basal levels and increased [Mg²⁺]_i after 1 h of OGD/CI. These results suggest that anoxia induced the increase of [Mg²⁺]_i via TRPM7 channels in rat hippocampal neurons.
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^†: The first two authors contributed equally to this work.

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Neuron

Abstract

References (21)

Calcium clamp in single nerve cells

Cell Calcium

Regulation of free and bound magnesium in rat hepatocytes and isolated mitochondria

J. Biol. Chem.

Cytosolic free magnesium, ATP and blebbing during chemical hypoxia in cultured rat hepatocytes

Biochem. Biophys. Res. Commun.

Voltage dependent block by intracellular Mg²⁺ of N-methyl-d-aspartate activated channels

Biophys. J.

Regulation of cell magnesium

Arch. Biochem. Biophys.

Magnesium ions in cardiac function: regulator of ion channels and second messengers

Biochem. Pharmacol.

Intracellular calcium buffering capacity in isolated squid axons

J. Gen. Physiol.

Mechanisms of magnesium transport

Ann. Rev. Physiol.

Calcium buffering and free Ca²⁺ rat brain synaptosomes

J. Neurochem.

Extracellular magnesium-dependent sodium efflux in squid giant axons

Am. J. Physiol.

Cited by (133)

An improved measurement of the Ca<sup>2+</sup>-binding affinity of fluorescent Ca<sup>2+</sup> indicators

Neural depolarization triggers Mg<sup>2+</sup> influx in rat hippocampal neurons

Hypoxia induces an increase in intracellular magnesium via Transient Receptor Potential Melastatin 7 (TRPM7) channels in rat hippocampal neurons in vitro

cNTnC and fYTnC2, Genetically Encoded Green Calcium Indicators Based on Troponin C from Fast Animals

Tyrosine phosphorylation tunes chemical and thermal sensitivity of TRPV2 ion channel

Tyrosine phosphorylation tunes chemical and thermal sensitivity of TRPV2 ion channel

Neuron

Glutamate-induced increases in intracellular free Mg2+ in cultured cortical neurons

Abstract

Cell Calcium

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biophys. J.

Arch. Biochem. Biophys.

Biochem. Pharmacol.

Intracellular calcium buffering capacity in isolated squid axons

J. Gen. Physiol.

Mechanisms of magnesium transport

Ann. Rev. Physiol.

Calcium buffering and free Ca2+ rat brain synaptosomes

J. Neurochem.

Extracellular magnesium-dependent sodium efflux in squid giant axons

Am. J. Physiol.

Glutamate-induced increases in intracellular free Mg²⁺ in cultured cortical neurons

Calcium buffering and free Ca²⁺ rat brain synaptosomes