The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

(1) Background: The N-methyl-D-aspartate receptors (NMDARs) mediate fast excitatory currents leading to depolarization. Postsynaptic NMDARs are ionotropic glutamate receptors that mediate excitatory glutamate or glycine signaling in the CNS and play a primary role in long-term potentiation, which is a major form of use-dependent synaptic plasticity. The overstimulation of NMDARs mediates excessive Ca2+ influx to postsynaptic neurons and facilitates more production of ROS, which induces neuronal apoptosis. (2) Methods: To confirm the induced inward currents by the coapplication of glutamate and ergotamine on NMDARs, a two-electrode voltage clamp (TEVC) was conducted. The ergotamine-mediated inhibitory effects of NR1a/NR2A subunits were explored among four different kinds of recombinant NMDA subunits. In silico docking modeling was performed to confirm the main binding site of ergotamine. (3) Results: The ergotamine-mediated inhibitory effect on the NR1a/NR2A subunits has concentration-dependent, reversible, and voltage-independent properties. The major binding sites were V169 of the NR1a subunit and N466 of the NR2A subunit. (4) Conclusion: Ergotamine effectively inhibited NR1a/NR2A subunit among the subtypes of NMDAR. This inhibition effect can prevent excessive Ca2+ influx, which prevents neuronal death.


Introduction
The N-methyl-D-aspartate (NMDA) receptor (NMDAR), which mediates fast-acting excitatory currents leading to depolarization, is a tetrameric ionotropic channel belonging to the ligand-gated glutamate receptor family. The pore blockade by Mg 2+ is decreased with increased neuronal activity, and the permeation of the NMDAR pore is stimulated to enable activation and allow the influx of Ca 2+ and Na + into neurons; however, the NMDAR is largely inactive when the cell membrane is at resting potential [1]. NMDAR activity is induced by: (1) binding of active compounds such as neurotransmitters, for example, agonists (glutamate) or other co-agonists (D-serine or glycine), and (2) agents causing membrane depolarization, which impact the channel pore blockade by Zn 2+ or Mg 2+ . NMDARs are classified into three groups based on sequence homology, namely glycine/D-serine-binding NR1, glutamate-binding NR2 subunits (NR2A, NR2B, NR2C, and NR2D), and glycine-binding NR3 (NR3A and NR3B) subunits [2][3][4]. All NMDARs have the same membrane topology: one large extracellular N-terminal domain, four transmembrane domains (M1, M2, M3, and M4; M2 encodes the re-entrant pore [approximately Figure 1. Schematic diagram of the NMDA receptor and excessive Ca 2+ ions influx induces cellular apoptosis in the event of the overstimulation of this receptor. The overstimulation of NMDAR increases the intracellular Ca 2+ influx, thus increasing ROS accumulation, damaging genetic material, and ultimately leading to apoptosis. Ergotamine derived from ergot alkaloids can modulate intracellular Ca 2+ concentration by inhibiting hyperstimulation to NMDAR.

Materials
Ergotamine (Wuchan Chem Faces Biochemical, Hubei, China) was dissolved in a dimethyl sulfoxide solvent and diluted to prepare a recording solution for the next experiment (less than 0.03% DMSO in the final recording solution). Figure 2A shows the chemical structure of ergotamine. The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA). All other compounds were supplied by Merck (Darmstadt, Germany).  2+ ions influx induces cellular apoptosis in the event of the overstimulation of this receptor. The overstimulation of NMDAR increases the intracellular Ca 2+ influx, thus increasing ROS accumulation, damaging genetic material, and ultimately leading to apoptosis. Ergotamine derived from ergot alkaloids can modulate intracellular Ca 2+ concentration by inhibiting hyperstimulation to NMDAR.

Materials
Ergotamine (Wuchan Chem Faces Biochemical, Wuhan, China) was dissolved in a dimethyl sulfoxide solvent and diluted to prepare a recording solution for the next experiment (less than 0.03% DMSO in the final recording solution). Figure 2A shows the chemical structure of ergotamine. The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA). All other compounds were supplied by Merck (Darmstadt, Germany).

Preparation of Xenopus Oocytes and Microinjection
According to the Chonnam National University guidelines for animal care (CNU IACUC-YB-2016-07), X. laevis frogs were cared for and handled in adherence to the Korea Xenopus Resource Center for Research (KXRCR000001) manual. Surgery was performe (C-F) Glutamate induced inward currents with or without ergotamine (30 µM and 10 µM). For each subunit of NMDARs, the responses after treating with either glutamate (100 µM) alone or together with ergotamine (30 and 10 µM). Voltage clamp recording was conducted at a holding potential of −80 mV. The coapplication of ergotamine with glutamate resulted in modulation of the recombinant receptors (C) NR1a/NR2A, (D) NR1a/NR2B, (E) NR1a/NR2C, and (F) NR1a/NR2D, which in turn reduced glutamate-evoked inward current in a reversible manner (n = 6-8 oocytes from four different frogs).

Preparation of Xenopus Oocytes and Microinjection
According to the Chonnam National University guidelines for animal care (CNU IACUC-YB-2016-07), X. laevis frogs were cared for and handled in adherence to the Korean Xenopus Resource Center for Research (KXRCR000001) manual. Surgery was performed to manually collect the X. laevis oocytes. The selected oocytes were isolated by shaking incubation in Ringer solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 5 mM HEPES; pH 7.4) supplemented with 0.5 µg/µL of collagenase within 2 h and then maintained in ND96 incubation solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, 2.5 mM sodium pyruvate, 50 mg/mL gentamicin solution; pH 7.4) at 16 • C. All the incubation solutions were changed daily. Aliquots of 40 nL mRNA solutions were prepared, and the mRNAs were pulled with a glass capillary tubing (15-20 µm in diameter) by using a 10 nL nanoinjector (VWR Scientific, Seattle, WA, USA). The electrophysiological experiments were performed within 3-5 days of the oocyte isolation.

NR1a/NR2A Receptor Mutation and In Vitro Transcription of cDNAs
MAX QuikChange mutagenesis kits (Stratagene, CA, USA) were used for mutating NMDAR subunits prior to amplification by PCR. The success of PCR was evaluated using DNA sequencing analysis by Cosmo Gentech Inc. (Seoul, Seongdong-gu, Korea) after the PCR product was transfected into XL1-Blue supercompetent cells, followed by screening. The identified DNA was linearized using the restriction enzyme NotI, and then transcribed into RNA using T7 in vitro transcription kits (Ambion, Austin, TX, USA). The final RNA products were resuspended in diethyl pyrocarbonate and stored as aliquots at a final concentration of 1 µg/µL at −80 • C for the next experiment.

Molecular Docking Study with Three-Dimensional (3D) Modeling
For the molecular docking study of the NMDAR and ergotamine interaction, the protein structure was obtained from the Protein Data Bank (PDB); the PDB ID of the selected protein structure was 7EOS. The 3D structure of ergotamine was referenced in PubChem (CID code: 8223). The docking study was performed in a basic setting using AutoDock Tools (Scripps Research Institute [version 4.2.6], La Jolla, CA, USA). The performance state of the protein was enhanced by removing water molecules from the macromolecule, adding polarity and hydrogen ions, and computing the Gasteiger charges. The models were selected on the basis of intermolecular energy, inhibition constant, binding structures, and binding energy. The complex of ergotamine and NMDAR (NR1a/NR2A subunits) was analyzed using LIGPLOT, which calculated the binding activity between ergotamine and NMDAR. The distance between the NMDAR and ergotamine molecule interaction site was measured using PyMol.

Data Recording
An oocyte clamp (OC-725C; Warner Instruments, Hamden, CT, USA) with a perfusion chamber was used for the two-electrode voltage-clamp recordings at room temperature. A recording solution (ND96 bath solution) was prepared as described previously [23] and applied with ergotamine and glutamate during recording, according to the experiment design. Oocytes were placed into the chamber with the ND96 bath solution, flowing at a rate of 1 mL/min. Two electrodes filled with 3M KCl (electrolyte solution, 0.2-0.7 MΩ resistance) were stabbed at a random position in every oocyte. Experiments were set with a −80 mV holding potential for the current recording and −100 to +60 mV within 300 ms for ramping the voltage relationship of NMDARs. All the data were collected and analyzed using Digidata 1320 (Molecular Devices, Sunnyvale, CA, USA) and pCLAMP 9 software (Axon Instruments, Union City, CA, USA).

Data Analysis
To investigate the concentration-response curves of the role of ergotamine on glutamate-stimulated inward current (I Glu ), various concentrations of ergotamine were examined to measure I Glu by voltage-clamp recording in the presence of glutamate. Origin Pro 8.0 (Origin, Northampton, MA, USA) was used to describe the relationship between the concentration of ergotamine and percent inhibition of I Glu by ergotamine, based on the Hill equation described as: where y is the peak current at a given concentration of ergotamine, V max is the maximal peak value, IC 50 is the half-maximal inhibitory concentration of ergotamine on I Glu , [x] is the concentration of ergotamine and glutamate, and n is the interaction coefficient. All the values are presented as the standard error of the mean. The differences between the means of the control and application values were determined using one-way ANOVA with a Tukey test. p <0.01 was considered statistically significant.

Inhibitory Effect of Ergotamine on Various N-Methyl-D-Aspartate Receptors
To assess the impact of ergotamine on various NMDAR subunits, 100 µM glutamate was supplemented into a bath solution to stimulate a large inward current in oocytes that were injected with NMDAR subunit (NR1a/NR2A, NR1a/NR2B, NR1a/NR2C, and NR1a/NR2D) mRNAs. Figure 2B shows that H 2 O-injected oocytes were not affected by the application of glutamate (100 µM). The effects of ergotamine (30 and 10 µM) on glutamate (100 µM)-evoked current are shown in Figure 2C-F. The inhibition response of I Glu is caused by the application of (30 µM) ergotamine in the presence of glutamate (100 µM) on each type of NMDAR subunit (NR1a/2A, NR1a/2B, NR1a/2C, and NR1a/2D) was recorded at 65.2 ± 6.5%, 45.5 ± 8.5%, 10.2 ± 4.3%, and 18.6 ± 5.3%, respectively. The coapplication of ergotamine with glutamate (100 µM) had nearly no impact on I Glu on NR1a/2C and NR1a/2D subunits. On the NR1a/2B subunits, the coapplication of ergotamine with glutamate could only slightly reduce the intensity of the currents elicited by glutamate compared to treatment with glutamate only; however, it markedly inhibited I Glu on the NR1a/2A subunit receptor in the same condition. These results implied that the coapplication of ergotamine on inward currents of NR1a/2A subunits has an inhibitory manner, and the inhibition was reversible.

Ergotamine Concentration-Dependent Inhibition of I Glu
Previous data showed the inhibitory effect of ergotamine on the application of NMDA subunit (NR1a/2A and NR1a/2B) receptors. Different concentrations of ergotamine, ranging from 1-100 µM, and different potencies for I Glu were analyzed. The results demonstrated that ergotamine-mediated inhibition of glutamate-induced inward currents for NR1a/2A and NR1a/2B subunits was concentration-dependent. As shown in Table 1, the IC 50 values for each recombinant NR1a/NR2A-D were 15.3 ± 6.1, 15.7 ± 4.8, 6.5 ± 3.6, and 13.8 ± 6.4 µM, respectively; the values of the Hill coefficient for recombinant NR1a/NR2A-D were 2.1 ± 1.5, 2.1 ± 1.0, 0.8 ± 0.5, and 1.2 ± 0.6, respectively, corroborating previous analysis results. In summary, the potent inhibitory effect of ergotamine on glutamateevoked currents on the NR1a/NR2A subunits was highest compared to that on other NMDAR subunits. A further study on the NR1a/NR2A subunits focused on clarifying the role of the inhibition of ergotamine in NMDARs is required. The homology models of wild-type NR1a/NR2A subunits and mutant channels were built to predict the possible ligand-binding site of ergotamine on the NR1a/NR2A subunits. The in silico docking of ergotamine to wild-type and mutant models of the subunit was analyzed. Using the docking tool AutoDock, we predicted the binding affinity of ergotamine to NR1a/NR2A subunits based on the binding free energy of each site. The values of all the binding free energies are shown in Table 2. The best-fit docking was noted as NR1a W167, NR1a H168, NR1a V169, NR2A P435, and NR2A N466 (lowest energy pose) at −7.45 kcal/mol with intermolecular energy at −9.54 kcal/mol. Other complexes with a docking score of −6.45 kcal/mol showed a strong interaction with intermolecular energy that was higher than that for the above-predicted complexes. The values of the rootmean-square deviation (RMSD) were insignificantly dissimilar between the two predicted groups (complex #1 and #2) and lower than 0.25 nm, indicating the binding state stability of the protein. The lowest docking score included binding energy, intermolecular energy, and RMSD at 0.21 (complex #1), indicating that the change of predicting conformation is favorable and a near-native pose. The following residues were constructed as a binding site (with active radium 2.1 Å): W167, H168, and V169 residues in NR1a subunit; and P435 and N466 residues in NR2A subunit. Virtual screening and computational molecular conformation of ergotamine docked to the NR1a/NR2A channels were performed. The pose was generated and then evaluated, as shown in Figures 3 and 4, respectively. Thus, ergotamine could not only interact with three residues (W167, H168, and V169) on the NR1a subunit but also bind to the two residues (P435 and N466) on the NR2A subunit. According to these results, the in silico docking model suggests that these residues, which mediate the inhibition of I Glu , may allow ergotamine to interact with specific amino acids of NR1a/NR2A subunits via hydrogen bond formation. * RMSD (root-mean-square deviation of atomic positions). Binding energy, intermolecular energy, and internal energy (kcal/mol).

Effect of Ergotamine in Various NR1a/NR2A Subunit Mutations
To validate the best near-native pose for the selected residues from the NR1a/NR2A subunits with ergotamine, each residue was mutated into alanine to elucidate its functional role. Thus, the inhibitory effect of ergotamine on the I Glu current on oocytes expressing these mutants was further analyzed to determine whether ergotamine interacted with the channel on these residues as the binding site or not. The values of the inhibitory response of ergotamine on I Glu for each mutant type are shown in Table 3. According to the results of the predicted mutants, the representative inward current traces of various mutant types with or without ergotamine are shown in Figure 5. A single mutation in each subunit receptor seems to exert effects on the inhibition of ergotamine (63.7% ± 3.5% to 32.7% ± 6.8%).

Effect of Ergotamine in Various NR1a/NR2A Subunit Mutations
To validate the best near-native pose for the selected residues from the NR1a/NR2A subunits with ergotamine, each residue was mutated into alanine to elucidate its functional role. Thus, the inhibitory effect of ergotamine on the IGlu current on oocytes expressing these mutants was further analyzed to determine whether ergotamine interacted with the channel on these residues as the binding site or not. The values of the inhibitory response of ergotamine on IGlu for each mutant type are shown in Table 3. According to the results of the predicted mutants, the representative inward current traces of various mutant types with or without ergotamine are shown in Figure 5. A single mutation in each subunit receptor seems to exert effects on the inhibition of ergotamine (63.7% ± 3.5% to 32.7% ± 6.8%).  Moreover, the mutation in mutant 1 ( Figure 5A) (NR1a W167A + NR2A wild-type) showed almost no difference in the response of glutamate-stimulated current compared with the response to the wild-type NR1a/NR2A subunits, indicating that the W167A residue has no functional role in the inhibition of ergotamine. Notably, mutant 6 ( Figure 5F) (NR1a V169A and NR2A N466A) strongly eliminated the inhibitory ability of ergotamine at approximately 13.9% ± 0.7%. According to the ergotamine-induced inhibition curves in wildtype ( Figure 6A), the I max values for the NMDAR subunits NR1a/NR2A, NR1a/NR2B, NR1a/NR2D, and NR1a/NR2C were recorded at 75.2% ± 9.7%, 53.7% ± 5.8%, 24.0% ± 5.1%, and 13.9% ± 3.0%, respectively. To compare these results with mutant-type, the inhibition percentage of ergotamine applied to each mutation type was expressed as a sigmoid curve using the Hill equation, and the results are shown in Figure 6B. The inhibitory responses of ergotamine on the oocytes expressing the double-mutant type were recorded with an I max value of 13.9% ± 0.7% and IC 50 of 7.8 ± 1.1 µM. The Hill coefficient (n H ) of 1.3 ± 0.2 are shown in Table 3. Thus, ergotamine could interact with both the residues, namely NR1a V169 and NR2A N466 of the NR1a/NR2A subunits.  Moreover, the mutation in mutant 1 ( Figure 5A) (NR1a W167A + NR2A wild-type) showed almost no difference in the response of glutamate-stimulated current compared with the response to the wild-type NR1a/NR2A subunits, indicating that the W167A residue has no functional role in the inhibition of ergotamine. Notably, mutant 6 ( Figure  5F) (NR1a V169A and NR2A N466A) strongly eliminated the inhibitory ability of ergotamine at approximately 13.9% ± 0.7%. According to the ergotamine-induced mutant-type, the inhibition percentage of ergotamine applied to each mutation type was expressed as a sigmoid curve using the Hill equation, and the results are shown in Figure  6B. The inhibitory responses of ergotamine on the oocytes expressing the double-mutant type were recorded with an Imax value of 13.9% ± 0.7% and IC50 of 7.8 ± 1.1 µM. The Hill coefficient (nH) of 1.3 ± 0.2 are shown in Table 3. Thus, ergotamine could interact with both the residues, namely NR1a V169 and NR2A N466 of the NR1a/NR2A subunits. Concentration-response curves for the inhibition of ergotamine on glutamate-induced inward current of NR1a/NR2A mutant types. NR1a (W167A, H168A, and V169A) and NR2A mutants (P435A and N466A) were cross-combined between wild-type/mutants of each subunit. Ergotamine reduced IGlu in a concentration-dependent manner in the wild-type. Experiments were performed at a holding potential of −80 mV. The value of IC50, Imax, and nH (Hill coefficient) are shown in Table  3. Each point represents the mean ±S.E.M. (n = 6-8 oocytes from four different frogs).

Discussion
Various neuropathological diseases begin with the neuronal loss [24]. Neuronal death is caused by (1) beta-amyloid, (2) glutamate, (3) FeSO4, (4) haloperidol, (5) H2O2, and (6) ischemic damage. Several factors are involved in neuronal loss; however, neuronal loss can also be caused by ROS [25,26]. ROS are byproducts generated initially in cellular physiology and are involved in host defense, redox signaling, hormone biosynthesis, mitogenesis, and oxygen sensing as a result of neuronal signal transduction [27]. Excessive intracellular Ca 2+ accumulation results in the overproduction of ROS owing to a partial reduction of oxygen by mitochondrial electron transport chains, cytoplasmic lipoxygenase, and peroxisome flavoprotein oxidases. The balance of detoxification and generation of ROS induces them to play their original role. The transient generation of localized ROS plays an important role in receptor-mediated cellular signaling [16,28]. However, when the redox system is disrupted, Ca 2+ regulation is affected, leading to oxidative stress caused by excess free radicals, which is associated with various diseases [29]. Therefore, maintenance of intracellular Ca 2+ at an optimal concentration is essential.
Neuropathological disorders caused by ROS overproduction include Huntington's disease, Parkinson's disease, and AD. The pathogenesis for AD was hypothesized as follows: brain cell death due to cytotoxicity is caused by the accumulation of betaamyloid. Aggregates of beta-amyloid are found in AD patients in the form of peptides ranging from 39 to 43 amino acids in length [30]. These aggregates directly induce  Table 3. Each point represents the mean ± S.E.M. (n = 6-8 oocytes from four different frogs).

Discussion
Various neuropathological diseases begin with the neuronal loss [24]. Neuronal death is caused by (1) beta-amyloid, (2) glutamate, (3) FeSO 4 , (4) haloperidol, (5) H 2 O 2 , and (6) ischemic damage. Several factors are involved in neuronal loss; however, neuronal loss can also be caused by ROS [25,26]. ROS are byproducts generated initially in cellular physiology and are involved in host defense, redox signaling, hormone biosynthesis, mitogenesis, and oxygen sensing as a result of neuronal signal transduction [27]. Excessive intracellular Ca 2+ accumulation results in the overproduction of ROS owing to a partial reduction of oxygen by mitochondrial electron transport chains, cytoplasmic lipoxygenase, and peroxisome flavoprotein oxidases. The balance of detoxification and generation of ROS induces them to play their original role. The transient generation of localized ROS plays an important role in receptor-mediated cellular signaling [16,28]. However, when the redox system is disrupted, Ca 2+ regulation is affected, leading to oxidative stress caused by excess free radicals, which is associated with various diseases [29]. Therefore, maintenance of intracellular Ca 2+ at an optimal concentration is essential.
Neuropathological disorders caused by ROS overproduction include Huntington's disease, Parkinson's disease, and AD. The pathogenesis for AD was hypothesized as follows: brain cell death due to cytotoxicity is caused by the accumulation of beta-amyloid. Aggregates of beta-amyloid are found in AD patients in the form of peptides ranging from 39 to 43 amino acids in length [30]. These aggregates directly induce oxidative stress in the cell by interacting with the nerve cell membrane and stimulating ROS accumulation, thereby inducing neurotoxicity. The accumulation of beta-amyloid also makes (1) the cells more susceptible to glutamate, (2) interferes with intracellular Ca 2+ regulation, and (3) interacts with various neurotransmitter receptors [25].
Recently, it has been shown that neuronal cell dysfunction and oxidative cell death are related to AD-associated beta-amyloid protein accumulation. Therefore, the importance of intracellular redox regulation is emphasized. Neuronal depolarization is induced when the activity of the respiratory enzyme deteriorates owing to aging, the ROS scavenging ability decreases, and the energy metabolism process is impaired. Neuronal depolarization induces activation of NMDARs in the brain, and excessive Ca 2+ accumulation in the cell increases ROS generation [29]. These hypotheses can be linked, suggesting that the accumulation of beta-amyloid (cause of AD) induces neurotoxicity, making it more susceptible to toxins such as glutamate. In addition, beta-amyloid accumulation disrupts the regulation of intracellular Ca 2+ . As a result, beta-amyloid aggregates interact with nerve cell membranes and induce the accumulation of ROS in the cells [25]. Therefore, although the beta-amyloid hypothesis is representative of AD, the free radical theory due to aging is also accepted. Nerve cells are extremely sensitive to oxidation, and antioxidant drugs are emerging as a new alternative for the prevention and treatment of neurodegenerative diseases. However, available drugs are very limited, and although treatment using antioxidants is an emerging alternative, studies supporting this research are lacking.
NMDARs are involved in many diseases, including neurodegenerative and psychiatric disorders. NMDAR subunits exhibit different functions depending on their combination. The subunit NR1 can assemble in pairwise combinations with one of four possible NR2 subunits (di-heteromeric NMDARs) or can assemble as a complex of NR2 and NR3 (triheteromeric NMDARs) [31][32][33][34]. This fact explains why various NMDARs that comprise one of the four different NR2 subunits were reported as having separate pharmacological and functional characteristics in the brain. The decrease in expression of various NM-DAR subunits during senescence strongly implies they play a crucial role in memory, cognition, synaptic transmission, and, particularly, spatial information [35][36][37]. Several neuropathology reports have revealed that glutamate excitotoxicity and Ca 2+ overload are associated and lead to neuronal death and synaptic dysfunction. Moreover, these reviews reported the role of NMDARs in aggravating chronic diseases, especially AD and Parkinson's disease [38,39]. It was confirmed that extra-synaptic NMDARs with glutamatergic transmission are associated with other neurological diseases such as schizophrenia, Huntington's disease, major depressive disorder, ischemia/reperfusion injury, amyotrophic lateral sclerosis, and stroke.
The expression of NR2A and NR2B subunits, found mostly in the cerebral cortex and hippocampus, was reported to be involved in the impairment of several functions of the brain, including cognitive performance and synaptic function [40][41][42][43]. The impact of neuronal damage on cerebral ischemia by inducing neuronal death and survival has been confirmed to involve NR2A-containing receptors [44]. In particular, NR2A-containing receptors were reported to regulate neurodegeneration in rats with hyperhomocysteinemia. Experiments on NR2A-containing receptor-knockout mice proved that NR2A mediated its pharmacological action by inhibiting ischemia-induced functional deficits [10]. In addition, the modulation of drug self-administration memory in the infralimbic medial prefrontal cortex by the NR2A-containing receptor suggests its potential as a therapeutic to reduce relapse possibility [45].
Consequently, the development of NMDAR-selective antagonists proves its immense therapeutic potential. Although ergotamine is an ergot alkaloid known to exhibit high efficacy in cardiovascular disease and migraine treatment [21], its neuroprotective effect owing to the interaction with NMDARs is unknown. Ergotamine is a drug that has been used in the past; however, its ability to cross the blood-brain barrier remains unclear. Additionally, its beneficial effect on headaches was inferred, not proved. Through in vitro porcine brain capillary endothelial cell experiments, it was confirmed that an effective concentration of ergotamine could penetrate the blood-brain barrier, owing to its exceptional transport properties, and could effectively activate nerve cells [46]; the toxicity caused by ergotamine association must also be considered. Furthermore, it was confirmed that ergotamine can regulate intracellular Ca 2+ concentration by inhibiting NR1a/NR2A subunits in vitro, and this result suggests that ergotamine may regulate redox status and may be a potential treatment for several neurological diseases.

Conclusions
The ergotamine-mediated inhibitory effects of NR1a/NR2A subunits were explored among four different kinds of recombinant NMDA subunits. The ergotamine-mediated inhibitory effect on the current evoked by glutamate on oocytes expressing NR1a/NR2A subunits was concentration-dependent and reversible and worked as voltage-independent. The pharmacological mechanism of ergotamine-mediated suppression of I Glu in cells expressing NR1a/NR2A subunits was clarified. The results of double mutations of V169 of the NR1a subunit and N466 of the NR2A subunit showed that both residues are important binding sites for ergotamine. The potential mechanisms by which ergotamine blocks the channel were visualized by performing homology modeling and ligand docking to the transmembrane domain of the NR1a/NR2A subunits. Excessive activity of NMDARs induces excessive Ca 2+ influx into cells and may lead to neuronal death. These results suggest that the inhibition of NMDAR by ergotamine may have a neuroprotective function. Through in vitro molecular study and in silico docking modeling, ergotamine effectively inhibited NR1a/NR2A, a subtype of NMDAR, and it was found that it could be a drug that can potentially modulate redox status by regulating Ca 2+ influx into the neurons.