To evaluate the possible role of the plasma membrane Na+/Ca2+-exchanger (NCX) in regulation of N-methyl-d-aspartate (NMDA) receptors (NMDARs), we studied effects of 2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate (KB-R7943; KBR) and lithium (inhibitors of NCX) on NMDA-elicited whole-cell currents using the patch-clamp technique on rat cortical neurons and human embryonic kidney 293T cells expressing recombinant NMDARs. KBR inhibited NMDAR currents in a voltage-independent manner with similar potency for receptors of GluN1/2A and GluN1/2B subunit compositions that excludes open-channel block and GluN2B-selective inhibition. The inhibition by KBR depended on glycine (Gly) concentration. At 30 μM NMDA, the KBR IC50 values were 5.3 ± 0.1 and 41.2 ± 8.8 μM for 1 and 300 μM Gly, respectively. Simultaneous application of NMDA + KBR in the absence of Gly induced robust inward NMDAR currents that peaked and then rapidly decreased. KBR, therefore, is an agonist (EC50 is 1.18 ± 0.16 µM) of the GluN1 subunit coagonist binding sites. The decrease of NMDA-elicited currents in the presence of KBR was abolished in Ca2+-free solution and was not observed in the presence of extracellular Ca2+ on 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-loaded neurons, suggesting that Ca2+ affects NMDARs from the cytosol. In agreement, the substitution of Li+ for extracellular Na+ caused a considerable decrease of NMDAR currents, which was not observed in the absence of extracellular Ca2+. Most likely, the accumulation of intracellular Ca2+ is caused by the inhibition of Ca2+ extrusion via NCX. Thus, KBR and Li+ provoke Ca2+-dependent receptor inactivation due to the disruption of Ca2+ extrusion by the NCX. The data reveal the role of NCX in regulation of Ca2+-dependent inactivation of NMDARs.
In the central nervous system, the plasma membrane sodium-calcium exchanger (NCX) plays an important role in neuronal calcium homeostasis. In the forward transport mode, the NCX lowers cytosolic Ca2+ by extruding one Ca2+ accompanied by the entry of three extracellular Na+ ions into the cell (Blaustein and Lederer, 1999). Under normal conditions, inhibition of NCX causes an elevation of intracellular Ca2+ concentration (Mattson et al., 1989) and forces normally nonexcitotoxic glutamate concentrations to produce a lethal Ca2+ overload (Bano et al., 2005). During excitotoxic stress, the excessive Na+ and Ca2+ entry into the cytoplasm results in loss of the Na+ gradient and thus of driving force for Ca2+ extrusion, which turns the NCX into the reverse mode of exchange. The NCX in reverse mode produces additional Ca2+ entry (Kiedrowski et al., 1994; Hoyt et al., 1998) and contributes to Ca2+ deregulation (Khodorov, 2004), promoting apoptotic neuronal death in stroke and brain trauma. Inhibition of the NCX in the reverse mode is a promising approach to decrease Ca2+ deregulation and glutamate excitotoxicity. KB-R7943 2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl] isothiourea methanesulfonate (KB-R7943; Fig. 1) was the first discovered selective blocker of the NCX in reverse mode (Iwamoto et al., 1996; Watano et al., 1996). Since that time, KB-R7943 (KBR) has been a widely used inhibitor of NCX. This compound demonstrates neuroprotection in stroke, ischemia, glutamate receptor overactivation, and hypoxia (Schroder et al., 1999; Breder et al., 2000; Li et al., 2000; MacGregor et al., 2003; Luo et al., 2007). Recently, it has been demonstrated that KBR is able to inhibit both the forward and the reverse modes of NCX activity. For example, its IC50 for fish NCXs is 1.9 and 3 μM for the reverse and forward modes of action, respectively (Abramochkin et al., 2013). In cultured cortical neurons, 10 μM KBR caused an immediate rise of intracellular Ca2+, suggesting inhibition of NCX forward mode of transport (Sibarov et al., 2012).
Whereas KBR has great potential for experimental and therapeutic usage, this compound exhibits additional effects that influence neuronal survival, including inhibition of L-type voltage-gated Ca2+ channels (Ouardouz et al., 2005), suppression of store-operated Ca2+ influx in cultured neurons and astrocytes (Arakawa et al., 2000), inhibition of N-methyl-d-aspartate (NMDA) receptors (NMDARs; Sobolevsky and Khodorov, 1999; Brustovetsky et al., 2011), and inhibition of mitochondrial complex I in rat cultured hippocampal neurons (Brustovetsky et al., 2011).
Inhibition of NMDARs by KBR can potentially contribute to neuronal survival during excitotoxic stress by limiting glutamate-induced calcium overload via NMDAR channels. Although possible KBR neuroprotective effects that are realized through inhibition of NMDARs represent a subject of great interest, the mechanisms of KBR action on NMDARs is still not clearly understood. Here, we study the effects of KBR on NMDARs in a wide range of KBR concentrations and demonstrate that KBR is an agonist of the coagonist binding site on the GluN1 subunit. Currents activated by NMDA + KBR exhibit strong Ca2+-dependent NMDAR desensitization most likely due to the impairment of intracellular local Ca2+ homeostasis determined by the inhibition of Ca2+ extrusion via NCX. In agreement, Li+, a substrate inhibitor of the NCX, induces Ca2+-dependent inactivation of NMDAR currents as well. This reveals the role of NCX in regulation of Ca2+-dependent inactivation of NMDARs.
Materials and Methods
Primary Culture of Cortical Neurons.
The procedure of culture preparation from embryos was previously described (Antonov and Johnson, 1996; Mironova et al., 2007). All procedures using animals were in accordance with recommendations of the Federation for Laboratory Animal Science Associations and approved by the local Institutional Animal Care and Use Committees. Female Wistar rats (provided by the Sechenov Institute’s animal facility, St. Petersburg, Russia) that were 16 days pregnant (14 animals overall in this study) were sacrificed by CO2 inhalation. Both male and female embryos were used as tissue donors. Fetuses were removed, and their cerebral cortices were isolated, enzymatically dissociated, and used to prepare primary neuronal cultures. Cells were used for experiments after 10–15 days in culture (Mironova et al., 2007; Han and Stevens, 2009). Cells were grown in Neurobasal culture medium supplemented with B-27 (Gibco-Invitrogen, Paisley, UK) on glass coverslips coated with poly-d-lysine.
Human Embryonic Kidney 293 Cells with Recombinant NMDAR.
Human embryonic kidney (HEK) 293T cells were maintained as previously described (Qian et al., 2005). HEK293T cells were plated onto 7-mm glass coverslips pretreated with poly-l-lysine (0.2 mg/ml) in 35-mm culture dishes at 1 × 105 cells per dish. Eighteen to 24 hours after plating, cells were transiently transfected with pcDNA1 encoding rat NMDA receptor subunits GluN2A and GluN2B and pcDNA3.1 encoding GluN1 and enhanced green fluorescent protein using FuGene HD reagent (Promega, Madison, WI). In brief, transfection was performed by adding to each dish 50 μl of serum-free medium containing 1 μg of total DNA and 2 μl FuGene. The ratio of cDNA used was 1 enhanced green fluorescent protein:1 GluN1:3 GluN2 (A or B). After incubation of cells for 6–8 hours, the transfection solution was replaced with fresh culture medium containing 200 μM DL-2-amino-5-amino-5-phosphono-valeric acid and 2 mM Mg2+ to prevent NMDA receptor–mediated excitotoxicity. Experiments were performed 24–72 hours after transfection.
Loading of BAPTA AM.
Neurons were loaded with BAPTA AM (1 μM) using conventional protocols. In brief, neuronal cultures were incubated with BAPTA AM and 0.02% Pluronic F-127 (Sigma-Aldrich, St. Louis, MO) added to the external solution for 20 minutes in the dark at 20–23°C. Then, BAPTA AM was washed out, and cells were incubated in the external solution for a further 30 minutes.
Patch Clamp Recordings.
Whole-cell patch clamp recordings of membrane currents were performed on cultured rat cortical neurons (10–15 days in vitro) and HEK293T cells expressing recombinant NMDARs of GluN1/2A or GluN1/2B composition. We used a MultiClamp 700B patch clamp amplifier with a Digidata 1440A acquisition system controlled by pClamp v10.2 software (Molecular Devices, Sunnyvale, CA). The acquisition rate was 20,000 seconds−1. The signal was 8-order low-pass Butterworth filtered at 200 Hz to remove high-frequency noise. Micropipette positioning was performed with an MP-85 micromanipulator (Sutter Instrument Company, Novato, CA) under the visual control of a Nikon Diaphot TMD microscope (Nikon, Tokyo, Japan). For fast medium exchange, we used a BPS-4 fast perfusion system (Ala Scientific Instruments, Farmingdale, NY). The tip of the multichannel manifold was placed at a distance of 0.2 mm from the patched cell, which allowed the solution exchange in 80 ms. Unless otherwise specified, the following extracellular medium was used for recording (the external bathing solution, in mM): 140 NaCl; 2.8 KCl; 1.0 CaCl2; and 10 HEPES, at pH 7.2–7.4. Patch pipette solution had the following composition (in mM): 120 CsF, 10 CsCl, 10 EGTA, and 10 HEPES. The pH was adjusted to 7.4 with CsOH. Patch pipettes (2–4 MΩ) were pulled from 1.5-mm (outer diameter) borosilicate standard wall capillaries with inner filament (Sutter Instrument Company). Experiments were performed at room temperature (23–25°C). The default membrane voltage (Vm) was set to −55 mV for neurons and −35 mV for HEK293T cells.
Functional activity of NMDARs requires binding of both glutamate and a coagonist, glycine. Unless otherwise stated, to activate NMDARs, we applied 30 μM NMDA with 30 μM glycine (Gly). The current voltage (I-V) relations of NMDA-induced conductance were determined by subtraction of the current recorded during a control voltage ramp from the current obtained in the presence of either NMDA + Gly or NMDA + Gly + KBR (10 μM). For NMDA + Gly + KBR, the control was measured in the external bathing solution + KBR. I-V relationships and membrane holding voltages were corrected for the liquid junction potential (approximately −15 mV; see Neher, 1992). Taking into account the value of pipette liquid junction potential, the actual ramp voltage range was from −60 to +50 mV.
KB-R7943 was from Tocris (Minneapolos, MO). Other compounds were from Sigma-Aldrich (St. Louis, MO). Mammalian expression vectors were supplied by Dr. J. W. Johnson (University of Pittsburgh, Pittsburgh, PA).
Quantitative data are expressed as means ± S.E.M. Analysis of variance (ANOVA) and Bonferroni multiple comparison methods as well as Student’s two-tailed t tests were used for statistical analysis. The number of experiments is indicated by n throughout. The data were considered as significantly different based on a confidence level of 0.05. Current measurements and I-V graphs were plotted using ClampFit 10.2 (Molecular Devices). The EC50 (half-maximal effective concentration for KBR as an agonist), IC50 (half-maximal concentration for KBR as an inhibitor), and Hill coefficient (h) were estimated by fitting dose-response curves with the Hill equation, I[C]/Imax = 1 / (1 + [KBR]b/C50b), where C50 represents IC50 or EC50. For IC50, the Imax is the current elicited by NMDA + Gly at 0 KBR and b = h, and for EC50, the Imax is the current elicited by NMDA + KBR at 0 Gly and b = −h. I[C] is the current, measured at different [KBR]s.
Voltage Independence of KBR Block of NMDARs.
We first studied KBR effects on the cellular conductance, because the ion exchange by NCX is electrogenic, and an inhibition of KBR could potentially interfere with measurements of NMDAR-mediated currents. In the majority of cells, the effect of KBR on transmembrane currents was rather small and did not considerably affect the resting I-V relationships. Considering the large amplitudes of NMDA-activated currents in neurons, we may conclude that the KBR block of NCX transport currents may not strongly affect amplitude measurements. Because KBR is able to inhibit voltage-sensitive Na+ and L-type Ca2+ channels (Watano et al., 1996; Török, 2007), we also tested the effects of tetrodotoxin (0.5 μM) and nifedipine (10 μM) on KBR-blocked currents. Neither compound affected the I-V relationships of NCX transport currents. We therefore ignored these KBR effects as factors that could complicate interpretation of our data in further experiments.
To get some clues into the mechanism of KBR inhibition of NMDARs, we performed experiments in which NMDA-induced currents were studied over a wide range of Vm values (from −85 to +45 mV) using application of NMDA + Gly and KBR at a single voltage and the ramp protocol. At Vm = −85 mV, 10 μM KBR inhibited the NMDA-induced currents to approximately 30% of the steady-state amplitude (Fig. 2A), which is consistent with previously published data (Sobolevsky and Khodorov, 1999; Brustovetsky et al., 2011). The voltage dependence of block may provide key information about the mechanism of compound action. Interestingly, when we studied the effect of KBR at Vm = +45 mV, the inhibition of currents transferred through NMDAR channels was found similar to that obtained at Vm = −85 mV (Fig. 2A). This observation was confirmed in experiments with ramp protocols in which currents elicited by NMDA + Gly and in NMDA + Gly + KBR were recorded in the Vm range of −60 mV to +50 mV (Fig. 2B). The fraction of blocked current at various Vm values (−85, −55, −25, and +45 mV) did not differ significantly (P > 0.5, ANOVA, Fig. 2C), suggesting that inhibition of NMDARs is voltage-independent. This suggests that the block of NMDARs by KBR is not related to the open-channel block.
KBR Effect Is Not GluN2 Subunit–Dependent.
Many compounds (for review, see Traynelis et al., 2010) reveal voltage-independent noncompetitive antagonism of NMDARs. Some of them, including ifenprodil (Williams, 1993; Whittemore et al., 1997) and Co 101244 (Zhou et al., 1999), are selective for NMDARs that contain the GluN2B subunit. To study whether KBR has selectivity for NMDARs containing GluN2A or GluN2B subunits, we performed experiments on neurons utilizing a pharmacological approach and on transfected HEK293T cells expressing recombinant NMDARs of GluN1/2A or GluN1/2B subunit compositions. Figure 3, A and B demonstrates the effects of KBR and ifenprodil on currents evoked by activation of native NMDARs in neurons. Whereas ifenprodil at 1 μM selectively inhibits GluN2B subunit–containing NMDARs, in different neurons it induces a variable degree of current inhibition from very weak (Fig. 3A) to complete block (Fig. 3B). KBR at 10 μM revealed similar inhibitions (Fig. 3, A and B) in all experiments. Since the experiments on native NMDARs do not provide conclusive evidence, we therefore undertook experiments on recombinant NMDARs consisting of GluN1/2A or GluN1/2B subunits expressed in HEK293T cells (Fig. 3, C and D). KBR at 10 μM effectively inhibited currents elicited by activation of both GluN1/2A (Fig. 3C) and GluN1/2B (Fig. 3D) receptors by 85 ± 5% (n = 3) and 73 ± 9% (n = 3), respectively. Data obtained on native (neurons, n = 5) and recombinant (HEK293T cells, n = 3) NMDARs are summarized in Fig. 3E and did not differ significantly between conditions under study (P > 0.2, ANOVA, Fig. 3E). This may suggest that KBR does not distinguish between GluN2A and GluN2B subunits and does not bind to the ifenprodil binding sites. Therefore, KBR binding requires a site that would have to be similar on GluN2A and GluN2B subunits or KBR may interfere with NMDAR function via an interaction with the GluN1 subunit.
GluN1 Subunit as a Target for KBR Action.
We further studied possible competitive antagonism of KBR at glycine binding sites, which are located on GluN1 subunits. We investigated the KBR effects within the concentration range of 0.5–200 μM on 30 μM NMDA–activated whole-cell currents in the presence of 1, 30, or 300 μM Gly (Fig. 4). Notably, the concentrations of KBR required for similar inhibition of currents at 300 μM glycine were much larger than in the presence of 1 μM (Fig. 4A). Average data from 12 experiments are summarized in the concentration-inhibition curves for KBR (Fig. 4B). These curves were fitted using the Hill equation with the following parameters: for 1 μM Gly, KBR IC50 = 5.3 ± 0.1 μM and Hill coefficient (h) = 2.7 ± 0.3 (n = 4); for 30 μM Gly, IC50 = 15.0 ± 1.2 μM and h = 2.0 ± 0.1 (n = 4); and for 300 μM Gly, IC50 = 41.2 ± 8.8 μM and h = 1.0 ± 0.1 (n = 4). The IC50 values are significantly different for all Gly concentrations under study (P < 0.01, ANOVA, post-hoc Bonferroni test). Therefore, the IC50 of KBR inhibition of NMDA-induced currents increases with the increase of Gly concentration, suggesting that KBR competes with Gly for the Gly binding sites on GluN1 subunits of NMDARs. The observed tendency of a decrease of the Hill coefficient values with rising Gly concentration, however, suggests a more complex mechanism of current inhibition by KBR, rather than simple competitive antagonism.
In addition, we tested possible antagonism of KBR at the glutamate binding sites on GluN2. Currents elicited by 10, 30, and 100 μM NMDA in the presence of 30 μM Gly were blocked by KBR with similar IC50 values (Fig. 4C). KBR IC50 values were 12.9 ± 1.5 μM (h = 1.8 ± 0.2, n = 4), 15.0 ± 1.2 μM (h = 1.8 ± 0.2, n = 4), and 15.6 ± 1.2 μM (h = 1.9 ± 0.4, n = 4) for 10, 30, and 100 μM NMDA, respectively, and did not differ significantly (P > 0.4, ANOVA). This observation suggests the lack of KBR interaction with glutamate recognition sites.
KBR as an Agonist of NMDAR Coagonist Binding Sites.
To obtain more evidence in favor of competition between Gly and KBR, we undertook experiments in which NMDAR currents were activated by various Gly concentrations when 30 μM NMDA and 10 μM KBR were already present in the external bathing solution. We expected that, at concentrations of NMDA and KBR used in this series of experiments, the recognition of glutamate binding sites and the KBR binding sites that are responsible for the NMDAR inhibition will be predominantly occupied, and the channels will be closed. Then, in case of competition between Gly and KBR, it is predicted that the amplitude of current response caused by Gly application should depend on [Gly]. In Fig. 5A, 30 μM NMDA + 30 μM Gly can be seen to induce typical NMDA-activated current. In the presence of 30 μM NMDA + 10 μM KBR, applications of 1, 30, and 300 μM Gly caused NMDAR currents which were substantially smaller than the current caused by NMDA + Gly in the absence of KBR, presumably because of KBR inhibition. The amplitude of current evoked by 1 μM Gly was much smaller than the amplitude of current evoked by 30 μM Gly, and the current caused by the application of 30 μM Gly was much smaller than the current activated by 300 μM Gly. This observation demonstrates that the degree of NMDAR inhibition by 10 μM KBR depends on [Gly] and is consistent with competition of Gly and KBR for the NMDAR coagonist binding sites. In the experiments illustrated in Fig. 5B, NMDAR currents were triggered by 30 and 100 μM Gly in the presence of 30 μM NMDA + 10 μM KBR. The current activated by 100 μM Gly is much larger than that activated by 30 μM Gly. However, the amplitude of current caused by 100 μM Gly when the KBR concentration was increased to 33 μM to keep the same [KBR]/[Gly] as for 30 μM Gly was similar to the amplitude of current activated by 30 μM Gly in the presence of 10 μM KBR. This suggests that there is a reciprocal relationship between [KBR] and [Gly] in terms of NMDAR inhibition, which is also consistent with competitions between these two ligands for the same binding sites in NMDARs.
We noticed that NMDA and KBR applied simultaneously (without Gly) induce a peak inward current followed by a steady-state current (Fig. 5, A and B). This could be interpreted as KBR being a partial agonist or agonist of the NMDAR coagonist binding sites. In further experiments, we therefore tested this possibility. Either KBR or NMDA applied alone did not induce considerable inward currents (Fig. 6A). Whereas 30 µM NMDA with 30 µM Gly induced a typical large inward current that reached the peak and then declined a little to the steady state, 30 µM NMDA with 10 µM KBR induced a robust inward peak current that declined quickly to a steady state of small current amplitude (Fig. 6A). The amplitude at the steady state was approximately 1/7 of the peak amplitude. The previously described observation suggests that KBR is an agonist of the NMDAR coagonist binding sites that causes fast desensitization of NMDARs. Based on this hypothesis, KBR inhibition of NMDA-induced currents may represent NMDAR desensitization induced by KBR binding.
Ca Dependence of KBR Inhibition of NMDARs.
To study whether Ca2+-dependent inactivation of NMDARs contributed to the inhibition of NMDA-induced currents by KBR, we measured currents in neurons activated by combined applications of 30 µM NMDA and 10 µM KBR in the presence and absence of Ca2+ in the external bathing solution. Whereas currents evoked by NMDA + KBR in the presence of 1 mM Ca2+ increased to a peak and then declined to a steady state of small amplitude (Fig. 6, A and B), when Ca2+ was removed from the extracellular bathing solution, the amplitudes of currents at the steady state largely increased (Fig. 6B). In many experiments, amplitudes of currents in Ca2+-free solution elicited by NMDA + Gly and NMDA + KBR were similar (Fig. 6C). Apparently, Ca2+-dependent inactivation of NMDARs contributes to the decrease of the current amplitude when KBR is used as a coagonist of NMDARs. In agreement, in the absence of extracellular Ca2+, we did not find any inhibition of NMDA + Gly–elicited current by 10 µM KBR (Fig. 6C) in the experimental protocol used to study KBR inhibition of NMDARs (Figs. 2–4), whereas kynurenate (50 µM), a competitive antagonist of Gly binding sites, inhibited currents activated by NMDA + KBR. On average, the amplitude of currents evoked by NMDA + KBR at the steady state was 239 ± 21 pA (n = 4) and decreased in the presence of 50 µM kynurenate to 19 ± 6 pA (n = 4, P < 0.0001, Student’s two-tailed t test), further supporting our conclusion that KBR and Gly bind to the same binding sites (Fig. 6D). These observations may suggest that KBR inhibition of NMDARs results predominantly from enhancement of Ca2+-dependent inactivation.
We directly addressed this question by experiments in which NMDA + KBR–elicited currents in the presence and absence of Ca2+ in the external bathing solution were recorded in paired applications on the same neurons. In the presence of Ca2+, NMDA + KBR applications caused currents which reached the peak and then declined to a steady state of small amplitude (Fig. 6E), which is typical for these conditions (Fig. 6, A and B). When NMDA + KBR were applied to the same neuron in Ca2+-free media, the elicited currents had a similar amplitude as the peak current, recorded in the presence of Ca2+, but did not reveal the amplitude decrease as long as 1 mM Ca2+ was not added. Application of Ca2+ induced a substantial decrease of the amplitude, and its washout caused the recovery of the current amplitude (Fig. 6E). Average data (Fig. 6F) depict that current amplitudes measured at the steady stated in the presence of Ca2+ (n = 7) and in the Ca2+-free media when Ca2+ was briefly applied (n = 3) do not differ significantly, whereas they are significantly smaller than the amplitudes of NMDAR-mediated currents (n = 7) measured in the Ca2+-free solution (P < 0.0001, ANOVA, post-hoc Bonferroni test). Based on the previously described experiments, we may conclude that the decrease of NMDAR-mediated currents activated by NMDA + KBR in the presence of extracellular Ca2+ results predominantly from the enhancement of Ca2+-dependent inactivation that requires an interaction of Ca2+ with intracellular domains of NMDARs. Our experiments on neurons loaded with BAPTA support this conclusion, since in the Ca2+-free media, currents activated by NMDA + KBR and NMDA + Gly in neurons loaded with BAPTA were similar and did not demonstrate the amplitude decrease in the presence of Ca2+ in the extracellular bathing solution (Fig. 6G). Average data (Fig. 6H) depict that current amplitudes measured at the steady stated in the presence of Ca2+ (n = 6) and in the Ca-free media (n = 6) do not differ significantly (P > 0.1, ANOVA).
Lithium Enhances Ca-Dependent NMDAR Inactivation.
Li+ is widely used in therapy of mood disorders, including bipolar disorders and depression as well as suicidal behaviors (Can et al., 2014). Since Li+ is a substrate inhibitor of Na+-dependent neurotransmitter transporters (Antonov and Magazanik, 1988; Can et al., 2014) and exchangers (for review, see Török, 2007), at least a portion of its therapeutic effects is determined by this type of pharmacological action. Accordingly, the substitution of Li+ for Na+ in the external bathing solution should considerably decrease the efficacy of Ca2+ extrusion via NCX. The effects of such substitution on NMDAR kinetics and conductance is expected to be negligible, because NMDARs have similar Li+ and Na+ channel permeabilities (Karkanias and Papke, 1999). In agreement, currents activated by NMDA + Gly in our experiments on neurons in Ca2+-free Na+ and Li+ bathing solutions had similar time courses (Fig. 7, A and B, left overlay), and their steady-state amplitudes did not differ significantly (Fig. 7C, P > 0.5, n = 5, Student’s two-tailed t tests). In the presence of Ca2+ in the external bathing solutions, currents activated by NMDA + Gly revealed the amplitude decrease in both Na+ and Li+ (Fig. 7, A and B) because of the Ca2+-dependent NMDAR inactivation. The extent of Ca2+-dependent inactivation achieved in the Li+ external bathing solution was greater than in the Na+ one (Fig. 7B, right overlay), since the current amplitudes measured at the steady state under these conditions differed significantly (Fig. 7C, P < 0.01, n = 5, Student’s two-tailed t tests). This observation is consistent with the assumption that Ca2+ extrusion via NCX might be involved in the regulation of Ca2+ inactivation of NMDARs.
EC50 of KBR as an NMDAR Coagonist.
In further experiments, we measured KBR EC50 for the NMDA-elicited currents. To raise the amplitude resolution of currents at the steady state, these experiments were performed in the Ca2+-free external bathing solution. At [NMDA] = 30 µM, increasing [KBR] caused an increase of NMDAR currents measured at the steady state (Fig. 8A). The dependence of the steady-state current amplitude on KBR concentration is shown in Fig. 8B. Fits of the Hill equation to the data reveal the following parameters of KBR activation: EC50 = 1.18 ± 0.16 µM (n = 5) and h = 1.5 ± 0.4 (n = 5). It should be noted that the KBR EC50 value is very similar to the EC50 for glycine and d-serine (for review, see Traynelis et al., 2010). KBR and Gly have similar efficacy as coagonists, since currents elicited by 100 µM NMDA in the presence of saturating concentrations of KBR (10 µM) or Gly (10 µM) recorded in the paired applications on the same neurons had similar amplitudes (Fig. 8C) that do not differ significantly (P > 0.7, two-tailed Student’s t test, n = 7, Fig. 8D).
In addition to the KBR effects described earlier, we found that the increase of [KBR] to a rather high concentration (above 10 µM), even in the Ca2+-free external bathing solution, causes a decrease of current amplitudes (Fig. 8E). This effect may presumably be explained by weakly voltage-dependent channel block of NMDARs by KBR described previously (Sobolevsky and Khodorov, 1999) and an accumulation of Ca2+ released from the intracellular Ca2+ stores due to KBR inhibition of mitochondrial metabolism (Brustovetsky et al., 2011).
To clarify the mechanism of KBR inhibition of NMDARs, we examined the voltage dependence of the KBR effects because such information could be crucial with respect to the mechanism. For instance, open-channel blockers of NMDARs reveal the voltage dependence of inhibition of NMDA-induced currents that is determined by the location of their binding sites in the ion pore within the membrane electric field (Woodhull, 1973) and interaction of the channel-blocking molecule or cation with permeant ions (Antonov et al., 1998; Antonov and Johnson, 1999). In our experiments, the KBR inhibition of NMDA-induced currents was not voltage-dependent (Fig. 2). We therefore may exclude channel block as a main mechanism of KBR action and suggest that KBR most likely interacts with functionally important NMDAR structures other than the ion pore. This conclusion to some extent differs from a previous study in which a portion of KBR-induced NMDAR inhibition was slightly voltage-dependent (Sobolevsky and Khodorov, 1999).
It is well known that cortical neurons express GluN1, GluN2A, and GluN2B subunits of NMDARs (Zhong et al., 1994; Mizuta et al., 1998; Abushik et al., 2013). Neurons vary substantially with respect to the GluN2A/GluN2B expression ratio (Tovar and Westbrook, 1999; Traynelis et al., 2010). The NMDAR-mediated currents in both neuronal types (ifenprodil-sensitive and -resistant) were effectively inhibited by 10 μM KBR (Fig. 3, A and B). In addition, KBR effectively inhibited currents elicited by NMDA + Gly activation of recombinant NMDARs consisting of GluN1/2A (Fig. 3C) or GluN1/2B (Fig. 3D) subunits expressed in HEK293T cells. These observations provide evidence that KBR is not selective with respect to GluN1/2A and GluN1/2B receptors and does not interact with sites on the GluN2B subunit at which subunit-specific inhibitors bind.
Based on the phenomenology of NMDAR inhibition by KBR (Figs. 2–4), one cannot exclude that this compound competes with agonists for glutamate recognition sites and with glycine (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988) or d-serine (Schell et al., 1995; Wolosker et al., 1999), which are endogenous ligands at the coagonist binding sites on NMDARs. In our experiments, the values of KBR IC50 were similar when measured on currents elicited by different NMDA concentrations (10, 30, or 100 μM; Fig. 4C). This suggests a lack of competition between KBR and NMDA for the glutamate recognition sites and is consistent with the conclusion that, during inhibition, KBR does not interact with the glutamate site of GluN2 subunits of NMDARs. In contrast, increasing the Gly concentration in the range of 1–300 μM while keeping the receptor in a rather high NMDA concentration (30 μM) resulted in an 8-fold increase of the IC50 value of KBR for inhibition of NMDA-activated whole-cell currents (Fig. 4B). Moreover, the KBR inhibition decreased when NMDAR currents were successively triggered by 1, 30, and 300 µM Gly in the presence of 10 µM KBR + 30 µM NMDA (Fig. 5A). Both observations may favor competition between KBR and Gly for the coagonist binding sites on GluN1 subunits of NMDARs. It should be mentioned, however, that a 300-fold increase of [Gly] induces an 8-fold increase of the KBR IC50, and h (Hill coefficient) of KBR dose-inhibition curves falls from a value of 2 at 1 and 30 µM Gly to a value of 1 at 300 µM Gly (Fig. 4B). These peculiarities of KBR-induced NMDAR inhibition are not consistent with simple competitive inhibition at the Gly binding sites.
Indeed, in later experiments, it appeared that KBR itself is an agonist at the NMDAR coagonist binding sites. The KBR EC50 for NMDAR activation is about 1 µM (Fig. 8B), which is similar to glycine and d-serine (for review, see Traynelis et al., 2010). In Ca2+-free solution, amplitudes of currents elicited by NMDA + KBR and NMDA + Gly coincide well (Figs. 6C and 8C), suggesting that Gly and KBR have similar efficacy as coagonists of NMDARs. Simultaneous application of NMDA and KBR induced robust inward NMDAR currents, which briefly reached a peak and then demonstrated fast decrease to a steady state of about 1/7 of the peak amplitude (Fig. 6, A, B, and E). The decrease of NMDA + KBR–elicited currents was abolished (Fig. 6, B and C) by removal of Ca2+ from the external bathing solution, whereas episodic applications of extracellular Ca2+ in the course of current activated by NMDA + KBR caused the inhibition (Fig. 6E). Taken together, these data suggest that the presence of KBR in the external bathing solution somehow enforces Ca2+-dependent receptor inactivation of NMDARs, which requires an accumulation of intracellular Ca2+ in close proximity to the intracellular NMDAR structures which are responsible for Ca2+ binding (Tong and Jahr, 1994). In agreement with this assumption, loading neurons with BAPTA abolished these effects of Ca2+ on currents activated by NMDA + KBR (Fig. 6, G and H). This allows us to suggest that the impairment of intracellular local Ca2+ homeostasis is caused by the NCX inhibition that occurs within the same concentration range where KBR has an effect on NMDARs. We therefore propose the role of NCX that would restrict Ca2+ diffusion and thereby regulate local intracellular Ca2+ concentration in close proximity of intracellular domains of NMDARs. This “Ca2+ compartmentalization” may depend on the fast Ca2+ entry through NMDAR channels and on the fast membranous extrusion via the NCX. Similar interaction of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and NCX-forming calcium microdomains was demonstrated for aspiny dendrites of GABAergic interneurons (Goldberg et al., 2003). Results of our experiments with Li+ external bathing solutions are consistent with this explanation. Whereas Li+ inhibits Ca2+ extrusion from neurons via NCX (Török, 2007), it does not affect currents mediated by NMDARs (Karkanias and Papke, 1999). In agreement, NMDAR-mediated currents in Ca2+-free Na+ and Li+ external bathing solutions had similar amplitudes (Fig. 7, A and B). Addition of Ca2+ (1 mM) in the external bathing solutions caused Ca2+-dependent NMDAR inactivation, which was much more pronounced in the Li+ external bathing solution than in the Na+ one (Fig. 7), suggesting a role of NCX in regulation of Ca2+ inactivation of NMDARs.
The interpretation of our data is illustrated in Fig. 9. In the presence of extracellular Ca2+, currents activated by NMDA + Gly demonstrate typical shape (Fig. 9A, right) exhibiting moderate receptor desensitization. The capacity of Ca2+ extrusion by the NCX is sufficient to restrict lateral diffusion along the internal surface of the plasma membrane of Ca2+ that enters the cytosol through the NMDAR channels (Fig. 9A). Currents activated by NMDA + KBR exhibit strong desensitization (Fig. 9B, right), because the inhibition of Ca2+ extrusion by NCX with KBR causes local Ca2+ accumulation and Ca2+ inactivation of NMDARs (Fig. 9B). Currents activated by NMDA + Gly (Fig. 9C) or NMDA + KBR (Fig. 9D) in the Ca2+-free external solution do not reveal receptor desensitization (Fig. 9, C and D, right) because Ca2+ does not enter the cytoplasm (Fig. 9, C and D). In the presence of extracellular Ca2+, currents activated by NMDA + KBR in neurons loaded with BAPTA do not exhibit Ca2+-dependent inactivation (Fig. 9E, right) since BAPTA binds Ca2+ entered the cytosol through NMDAR channels (Fig. 9E). Thus, functional interaction of the NMDARs with the NCXs provides a plausible explanation of the phenomenology obtained in this study. Most likely, Ca2+-dependent NMDAR inactivation provides feedback between Ca2+ extrusion via NCX and Ca2+ entry through NMDAR channels: the decrease of NCX transport induces the decrease of Ca2+ entry. Within the framework of this mechanism, the dependence of the KBR inhibition of currents activated by NMDA + Gly on Gly concentration (Figs. 4 and 5) could be determined by the relief from NMDAR Ca2+-dependent desensitization by the increase of Gly concentration (Lerma et al., 1990; Danysz and Parsons, 1998). In addition, the electrochemical gradient for Ca2+ between the extracellular media (1 mM Ca2+) and the cytoplasm (about 50 nM free Ca2+), resulting in the reversal potential for Ca2+ of about +130 mV, maintains the Ca2+ entry through NMDAR channels regardless of the direction (inward or outward) of integral NMDAR ion flow. This may explain the Vm independence of the KBR inhibition of NMDAR-mediated currents within the Vm range studied here. Whether those functional interactions between the NMDARs and the NCXs include direct molecular interplay remains to be elucidated.
It is generally thought that KBR has the clinical potential to prevent calcium overload of neurons (Amran et al., 2003). Here, we demonstrated that KBR and Li+, which inhibit the NCX by different mechanisms, both have similar effects on NMDARs and decrease amplitudes of NMDA-activated currents, enhancing their Ca2+-dependent inactivation. Since Li+ has a broad therapeutic utility, one may not exclude that this mechanism is involved in the therapy. Therefore, inhibitors of the NCX could potentially be considered as modulators of NMDARs. In addition, KBR is an effective agonist of the glycine binding sites of NMDARs. Taking into account both effects, it seems obvious that KBR should inhibit calcium entry via NMDARs, a process that can also contribute to neuroprotection in excitotoxic stress. Glycine site antagonists and partial agonists are thought to have better therapeutic indices than glutamate site antagonists because of their lesser side effects (Chen et al., 1993; Danysz and Parsons, 1998; Wood at al., 2008). Whereas the complex effects of KBR can be beneficial to its possible clinical use in stroke treatment, it is currently hard to estimate the possible therapeutic potential of the KBR effects on NMDARs.
The authors thank Dr. J. W. Johnson for careful reading and critical suggestions on the manuscript and for providing NMDAR plasmids.
Participated in research design: Antonov, Sibarov.
Conducted experiments: Abushik, Poguzhelskaya, Sibarov.
Contributed new reagents or analytic tools: Bolshakov.
Performed data analysis: Antonov, Sibarov.
Wrote or contributed to the writing of the manuscript: Antonov, Sibarov.
- Received June 23, 2015.
- Accepted September 18, 2015.
This work was supported by the Russian Foundation for Basic Research [Grants 14-04-00227 (to S.M.A.), 15-04-08283 (to D.A.S.), and 14-04-31707 (to P.A.A.)] and by the program grant (Section of Physiology and Fundamental Medicine) of Russian Academy of Sciences (to S.M.A.).
- analysis of variance
- BAPTA AM
- 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)
- Co 101244
- 1-[2-(4-Hydroxyphenoxy)ethyl]-4-[(4-methylphenyl)methyl]-4-piperidinol hydrochloride
- human embryonic kidney
- KB-R7943 (2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate)
- plasma membrane sodium-calcium exchanger
- N-methyl-d-aspartate receptor
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics