Sergei M. Antonov

Binding sites for permeant ions in the channel of NMDA receptors and their effects on channel block.

We report the presence of binding sites for permeant monovalent cations at the internal and external entrances to the channel of NMDA receptors. We measured the effects of changing internal cesium (Cs+) and external sodium (Na+) concentrations on the channel-blocking kinetics of the adamantane derivatives IEM-1754 and IEM-1857. Binding of Na+, or of Cs+ after it permeates the channel, to sites at the external channel entrance prevents blockers from entering the channel. Binding of Na+ to a blocked channel prevents blocker unbinding. Cs+ binding to a site at the internal channel entrance prevents IEM-1754 from occupying the deeper of its two sites of block. The results show the critical effects of permeant ions on the kinetics, affinity and voltage-dependence of channel blockers.

A fluorescence vital assay for the recognition and quantification of excitotoxic cell death by necrosis and apoptosis using confocal microscopy on neurons in culture.

An automated fluorescence method for the detection of neuronal cell death by necrosis and apoptosis with sequential acridine orange (AO) and ethidium bromide (EB) staining using confocal microscopy is described. Since cell nuclei during apoptosis become acidic, AO staining was utilized to distinguish live neurons from neurons undergoing apoptosis, using the AO property to shift its fluorescence from green at normal pH toward brilliant orange-red in the process of acidification. Further EB application labels nuclei of necrotic neurons in red. Sequential treatment by AO and EB can be employed as an express vitality test to count fractions of live and dead cell via apoptosis and necrosis, respectively. An algorithm of automatic quantification of cell types is based on the image correlation analysis. Our conclusion is validated by experiments with the vital dye trypan blue and the pharmacological study of receptor subtypes involved in the excitotoxicity. The approach described here, therefore, offers an express, easy, sensitive and reproducible method by which necrosis and apoptosis can be recognized and quantified in a population of living neurons. Because this assay does not require any preliminary tissue treatment, fixation or dissociation in a cell suspension its utility is likely to be extended for measuring cell viability and cytotoxicity on a variety of living preparations (tissues, brain slices and cell cultures).

Modulation by permeant ions of Mg2+ inhibition of NMDA‐activated whole‐cell currents in rat cortical neurons

Whole‐cell N‐methyl‐d‐aspartate (NMDA)‐activated currents were recorded from cultured rat cortical neurons. We report here a powerful effect of changing permeant ion concentrations on the voltage‐dependent inhibition by external Mg2+ (Mg2+ (Mgo2+) of these currents. Internal Cs+ (Csi+) affected Mgo2+ inhibition of the NMDA‐activated currents in a voltage‐dependent manner. A decrease in Csi+ concentration ([Cs+]i) from 125 to 8 mm reduced Mgo2+ IC50 by 1.4‐fold at −105 mV and by 11.5‐fold at – 15 mV. A decrease in external Na+ (Nao+ concentration ([Na+]o) also reduced Mgo2+ IC50. This effect was voltage independent. A decrease in [Na+]o from 140 to 70 mm reduced Mgo2+ IC50 by 1.4‐fold at–105 mV and by 1.6‐fold at–15 mV. Varying external Ca2+ (Cao2+) concentrations ([Ca2+]o) from 0.1 to1 mm did not affect Mgo2+ inhibition, even though changing [Ca2+]o in the same range strongly influenced the magnitude of NMDA‐activated currents in the absence of Mgo2+. However, increasing [Ca2+]o to higher concentrations (2–20 mm) greatly increased Mgo2+ IC50 at hyperpolarized voltages. These data are consistent with a model in which Nai+ and Csi+ modulate Mgo2+ inhibition of NMDA‐activated currents by occupying external permeant ion binding sites. The Mgo2+ IC50 values reported here are similar to Mgo2+KD values calculated from previous single‐channel measurements of Mgo2+ blocking kinetics. This similarity implies that Mgo2+ does not affect gating while blocking the channel.

Na+,K+-ATPase Functionally Interacts with the Plasma Membrane Na+,Ca2+ Exchanger to Prevent Ca2+ Overload and Neuronal Apoptosis in Excitotoxic Stress

Using a fluorescent viability assay, immunocytochemistry, patch-clamp recordings, and Ca2+ imaging analysis, we report that ouabain, a specific ligand of the Na+,K+-ATPase cardiac glycoside binding site, can prevent glutamate receptor agonist-induced apoptosis in cultured rat cortical neurons. In our model of excitotoxicity, a 240-min exposure to 30 μM N-methyl-d-aspartate (NMDA) or kainate caused apoptosis in ∼50% of neurons. These effects were accompanied by a significant decrease in the number of neurons that were immunopositive for the antiapoptotic peptide Bcl-2. Apoptotic injury was completely prevented when the agonists were applied together with 0.1 or 1 nM ouabain, resulting in a greater survival of neurons, and the percentage of neurons expressing Bcl-2 remained similar to those obtained without agonist treatments. In addition, subnanomolar concentrations of ouabain prevented the increase of spontaneous excitatory postsynaptic currents frequency and the intracellular Ca2+ overload induced by excitotoxic insults. Loading neurons with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or inhibition of the plasma membrane Na+,Ca2+-exchanger by 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate (KB-R7943) eliminated ouabains effects on NMDA- or kainite-evoked enhancement of spontaneous synaptic activity. Our data suggest that during excitotoxic insults ouabain accelerates Ca2+ extrusion from neurons via the Na+,Ca2+ exchanger. Because intracellular Ca2+ accumulation caused by the activation of glutamate receptors and boosted synaptic activity represents a key factor in triggering neuronal apoptosis, up-regulation of Ca2+ extrusion abolishes its development. These antiapoptotic effects are independent of Na+,K+-ATPase ion transport function and are initiated by concentrations of ouabain that are within the range of an endogenous analog, suggesting a novel functional role for Na+,K+-ATPase in neuroprotection.

The role of NMDA and mGluR5 receptors in calcium mobilization and neurotoxicity of homocysteine in trigeminal and cortical neurons and glial cells

Recent studies suggested contribution of homocysteine (HCY) to neurodegenerative disorders and migraine. However, HCY effect in the nociceptive system is essentially unknown. To explore the mechanism of HCY action, we studied short‐ and long‐term effects of this amino acid on rat peripheral and central neurons. HCY induced intracellular Ca2+ transients in cultured trigeminal neurons and satellite glial cells (SGC), which were blocked by the NMDA antagonist AP‐5 in neurons, but not in SGCs. In contrast, 3‐((2‐Methyl‐4‐thiazolyl)ethynyl)pyridine (MTEP), the metabotropic mGluR5 (metabotropic glutamate receptor 5 subtype) antagonist, preferentially inhibited Ca2+ transients in SGCs. Prolonged application of HCY induced apoptotic cell death of both kinds of trigeminal cells. The apoptosis was blocked by AP‐5 or by the mGluR5 antagonist MTEP. Likewise, in cortical neurons, HCY‐induced cell death was inhibited by AP‐5 or MTEP. Imaging with 2′,7′‐dichlorodihydrofluorescein diacetate or mitochondrial dye Rhodamine‐123 as well as thiobarbituric acid reactive substances assay did not reveal involvement of oxidative stress in the action of HCY. Thus, elevation of intracellular Ca2+ by HCY in neurons is mediated by NMDA and mGluR5 receptors while SGC are activated through the mGluR5 subtype. Long‐term neurotoxic effects in peripheral and central neurons involved both receptor types. Our data suggest glutamatergic mechanisms of HCY‐induced sensitization and apoptosis of trigeminal nociceptors.

Complex rectification of Müller cell Kir currents.

Although Kir4.1 channels are the major inwardly rectifying channels in glial cells and are widely accepted to support K+‐ and glutamate‐uptake in the nervous system, the properties of Kir4.1 channels during vital changes of K+ and polyamines remain poorly understood. Therefore, the present study examined the voltage‐dependence of K+ conductance with varying physiological and pathophysiological external [K+] and intrapipette spermine ([SP]) concentrations in Müller glial cells and in tsA201 cells expressing recombinant Kir4.1 channels. Two different types of [SP] block were characterized: “fast” and “slow.” Fast block was steeply voltage‐dependent, with only a low sensitivity to spermine and strong dependence on extracellular potassium concentration, [K+]o. Slow block had a strong voltage sensitivity that begins closer to resting membrane potential and was essentially [K+]o‐independent, but with a higher spermine‐ and [K+]i‐sensitivity. Using a modified Woodhull model and fitting i/V curves from whole cell recordings, we have calculated free [SP]in in Müller glial cells as 0.81 ± 0.24 mM. This is much higher than has been estimated previously in neurons. Biphasic block properties underlie a significantly varying extent of rectification with [K+] and [SP]. While confirming similar properties of glial Kir and recombinant Kir4.1, the results also suggest mechanisms underlying K+ buffering in glial cells: When [K+]o is rapidly increased, as would occur during neuronal excitation, “fast block” would be relieved, promoting potassium influx to glial cells. Increase in [K+]in would then lead to relief of “slow block,” further promoting K+‐influx.

Kainate-induced calcium overload of cortical neurons in vitro: Dependence on expression of AMPAR GluA2-subunit and down-regulation by subnanomolar ouabain

Whereas kainate (KA)-induced neurodegeneration has been intensively investigated, the contribution of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in neuronal Ca2+ overload ([Ca2+]i) is still controversial. Using Ca2+ imaging and patch-clamp techniques, we found different types of Ca2+ entry in cultured rat cortical neurons. The presence of Ca2+ in the extracellular solution was required to generate the [Ca2+]i responses to 30 μM N-methyl-d-aspartate (NMDA) or KA. The dynamics of NMDA-induced [Ca2+]i responses were fast, while KA-induced responses developed slower reaching high [Ca2+]i. Ifenprodil, a specific inhibitor of the GluN2B subunit of NMDARs, reduced NMDA-induced [Ca2+]i responses suggesting expression of GluN1/GluN2B receptors. Using IEM-1460, a selective blocker of Ca(2+)-permeable GluA2-subunit lacking AMPARs, we found three neuronal responses to KA: (i) IEM-1460 resistant neurons which are similar to pyramidal neurons expressing Ca(2+)-impermeable GluA2-rich AMPARs; (ii) Neurons exhibiting nearly complete block of both KA-induced currents and [Ca2+]i signals by IEM-1460 may represent interneurons expressing GluA2-lacking AMPARs and (iii) neurons with moderate sensitivity to IEM-1460. Ouabain at 1 nM prevented the neuronal Ca2+ overload induced by KA. The data suggest, that cultured rat cortical neurons maintain functional phenotypes of the adult brain cortex, and demonstrate the key contribution of the Na/K-ATPase in neuroprotection against KA excitotoxicity.

Calcium alterations signal either to senescence or to autophagy induction in stem cells upon oxidative stress

Intracellular calcium ([Ca2+]i) has been reported to play an important role in autophagy, apoptosis and necrosis, however, a little is known about its impact in senescence. Here we investigated [Ca2+]i contribution to oxidative stress-induced senescence of human endometrium-derived stem cells (hMESCs). In hMESCs sublethal H2O2-treatment resulted in a rapid calcium release from intracellular stores mediated by the activation of PLC/IP3/IP3R pathway. Notably, further senescence development was accompanied by persistently elevated [Ca2+]i levels. In H2O2-treated hMESCs, [Ca2+]i chelation by BAPTA-AM (BAPTA) was sufficient to prevent the expansion of the senescence phenotype, to decrease endogenous reactive oxygen species levels, to avoid G0/G1 cell cycle arrest, and finally to retain proliferation. Particularly, loading with BAPTA attenuated phosphorylation of the main DNA damage response members, including ATM, 53BP1 and H2A.X and reduced activation of the p53/p21/Rb pathway in H2O2-stimulated cells. Next, we revealed that BAPTA induced an early onset of AMPK-dependent autophagy in H2O2-treated cells as confirmed by both the phosphorylation status of AMPK/mTORC1 pathway and the dynamics of the LC3 lipidization. Summarizing the obtained data we can assume that calcium chelation is able to trigger short-term autophagy and to prevent the premature senescence of hMESCs under oxidative stress.

GluN2A Subunit-Containing NMDA Receptors Are the Preferential Neuronal Targets of Homocysteine

Homocysteine (HCY) is an endogenous redox active amino acid, best known as contributor to various neurodegenerative disorders. Although it is known that HCY can activate NMDA receptors (NMDARs), the mechanisms of its action on receptors composed of different NMDA receptor subunits remains almost unknown. In this study, using imaging and patch clamp technique in cultured cortical neurons and heterologous expression in HEK293T cells we tested the agonist activity of HCY on NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors) and GluN1 and GluN2B subunits (GluN1/2B receptors). We demonstrate that the time courses of Ca2+ transients and membrane currents activated by HCY and NMDA in cortical neurons are drastically different. Application of HCY to cortical neurons induced responses, which in contrast to currents induced by NMDA (both in the presence of glycine) considerably decreased to steady state of small amplitude. In contrast to NMDA, HCY-activated currents at steady state were resistant to the selective GluN2B subunit inhibitor ifenprodil. In calcium-free external solution the decrease of NMDA evoked currents was abolished, suggesting the Ca2+-dependent NMDAR desensitization. Under these conditions HCY evoked currents still declined almost to the baseline suggesting Ca2+-independent desensitization. In HEK293T cells HCY activated NMDARs of GluN1/2A and GluN1/2B subunit compositions with EC50s of 9.7 ± 1.8 and 61.8 ± 8.9 μM, respectively. Recombinant GluN1/2A receptors, however, did not desensitize by HCY, whereas GluN1/2B receptors were almost fully desensitized by HCY. Thus, HCY is a high affinity agonist of NMDARs preferring the GluN1/2A subunit composition. Our data suggest that HCY induced native NMDAR currents in neurons are mainly mediated by the “synaptic type” GluN1/2A NMDARs. This implies that in hyperhomocysteinemia, a disorder with enlarged level of HCY in plasma, HCY may persistently contribute to post-synaptic responses mediated by GluN2A-containing NMDA receptors. On the other hand, HCY toxicity may be limited by desensitization typical for HCY-induced activation of GluN2B-containing extrasynaptic receptors. Our findings, therefore, provide an evidence for the physiological relevance of endogenous HCY, which may represent an effective endogenous modulator of the central excitatory neurotransmission.

Functional Properties of Human NMDA Receptors Associated with Epilepsy-Related Mutations of GluN2A Subunit

Genetic variants of the glutamate activated N-methyl-D-aspartate (NMDA) receptor (NMDAR) subunit GluN2A are associated with the hyperexcitable states manifested by epileptic seizures and interictal discharges in patients with disorders of the epilepsy-aphasia spectrum (EAS). The variants found in sporadic cases and families are of different types and include microdeletions encompassing the corresponding GRIN2A gene as well as nonsense, splice-site and missense GRIN2A defects. They are located at different functional domains of GluN2A and no clear genotype-phenotype correlation has emerged yet. Moreover, GluN2A variants may be associated with phenotypic pleiotropy. Deciphering the consequences of pathogenic GRIN2A variants would surely help in better understanding of the underlying mechanisms. This emphasizes the need for functional studies to unravel the basic functional properties of each specific NMDAR variant. In the present study, we have used patch-clamp recordings to evaluate kinetic changes of mutant NMDARs reconstituted after co-transfection of cultured cells with the appropriate expression vectors. Three previously identified missense variants found in patients or families with disorders of the EAS and situated in the N-terminal domain (p.Ile184Ser) or in the ligand-binding domain (p.Arg518His and p.Ala716Thr) of GluN2A were studied in both the homozygous and heterozygous conditions. Relative surface expression and current amplitude were significantly reduced for NMDARs composed of mutant p.Ile184Ser and p.Arg518His, but not p.Ala716His, as compared with wild-type (WT) NMDARs. Amplitude of whole-cell currents was still drastically decreased when WT and mutant p.Arg518His-GluN2A subunits were co-expressed, suggesting a dominant-negative mechanism. Activation times were significantly decreased in both homozygous and heterozygous conditions for the two p.Ile184Ser and p.Arg518His variants, but not for p.Ala716His. Deactivation also significantly increased for p.Ile184Ser variant in the homozygous but not the heterozygous state while it was increased for p.Arg518His in both states. Our data indicate that p.Ile184Ser and p.Arg518His GluN2A variants both impacted on NMDAR function, albeit differently, whereas p.Ala716His did not significantly influence NMDAR kinetics, hence partly questioning its direct and strong pathogenic role. This study brings new insights into the functional impact that GRIN2A variants might have on NMDAR kinetics, and provides a mechanistic explanation for the neurological manifestations seen in the corresponding human spectrum of disorders.