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. Author manuscript; available in PMC: 2009 Dec 1.
Published in final edited form as: J Neurochem. 2008 Nov 5;107(6):1566–1577. doi: 10.1111/j.1471-4159.2008.05729.x

Binding of spermine and ifenprodil to a purified, soluble regulatory domain of the N-methyl-d-aspartate receptor

Xia Han *, Hideyuki Tomitori †, Satomi Mizuno *, Kyohei Higashi *,§, Christine Füll *, Tomohide Fukiwake *, Yusuke Terui †, Pathama Leewanich *, Kazuhiro Nishimura *, Toshihiko Toida *, Keith Williams ‡, Keiko Kashiwagi †, Kazuei Igarashi *,§
PMCID: PMC2690642  NIHMSID: NIHMS106289  PMID: 19014388

Abstract

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N-methyl-d-aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1-R, NR2A-R and NR2B-R. The R domains were overexpressed in Escherichia coli and purified to near homogeneity. The Kd values for binding of [14C]spermine to NR1-R, NR2A-R and NR2B-R were 19, 140 and 33 µM, respectively. [3H]Ifenprodil bound to NR1-R (Kd, 0.18 µM) and NR2B-R (Kd, 0.21 µM), but not to NR2A-R at the concentrations tested (0.1 to 0.8 µM). These Kd values were confirmed by circular dichroism measurements. The Kd values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1-R and NR2A-R or NR1-R and NR2B-R was additive with that of each individual R domain. Binding of spermine to NR1-R and NR2B-R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1-R and NR2B-R are distinct.

Keywords: spermine, ifenprodil, aminoglycoside antibiotics, regulatory domain of N-methyl-d-aspartate receptor

Introduction

N-Methyl-d-aspartate (NMDA) receptors are activated by glutamate and glycine and modulated by a variety of ligands including spermine, protons, and Zn2+ (Dingledine et al. 1999). Spermine has several effects on NMDA receptors including “glycine-independent” stimulation (seen with saturating concentrations of glycine), which may involve relief of tonic proton inhibition (Traynelis et al. 1995; Williams 1997). At physiologic pH, spermine stimulation is seen at NR1/NR2B receptors but not at NR1/NR2A receptors. Another form of spermine stimulation, “glycine-dependent” stimulation, is seen with low concentrations of glycine and involves an increase in the affinity of the receptor for glycine in the presence of spermine (Durand et al. 1993; Williams et al. 1994).

NMDA receptor subunits include NR1, NR2A, B, C, and D and NR3A and B. The receptors are thought to be tetramers containing two NR1 and two NR2 (or two NR3) subunits, and the type of NR2 subunit included in the receptor complex affects its functional and pharmacological properties (Dingledine et al. 1999; Chatterton et al. 2002). The receptor subunits have a large extracellular amino terminal region, and part of this region (the S1 domain), together with part of the extracellular M3–M4 loop (the S2 domain) forms the agonist binding site—the glycine binding site in NR1 and the glutamate binding site in NR2 (Kuryatov et al. 1994; Laube et al. 1997). The S1–S2 domains of NMDA and other glutamate receptor subunits were proposed to have structural similarity with bacterial periplasmic binding proteins such as the glutamine binding protein (QBP) (Moriyoshi et al. 1991; Arvola and Keinänen 1996). Indeed, the structures of the agonist binding domains of several glutamate receptor subunits, including GluR0, GluR2, GluR5, NR1 and NR2A have been determined by X-ray crystallography, and these domains do indeed form bi-lobed “clamshell” structures similar to bacterial periplasmic binding proteins (Armstrong et al. 1998; Mayer et al. 2001; Furukawa and Gouaux 2003; Mayer 2005; Furukawa et al. 2005).

The region of the amino terminus preceding S1 also has sequence similarity and a predicted structure similar to bacterial periplasmic binding proteins, in particular the leucine/isoleucine/valine binding protein (LIVBP) (Masuko et al. 1999a). We have termed this region the regulatory (R) domain. Mutations at a number of residues in this region of the NR1 subunit affect spermine stimulation (Williams et al. 1995; Masuko et al. 1999a). This region has also been referred to as the “amino terminal domain” (ATD or NTD) and the “LIVBP-like domain” (Paoletti et al. 2000; Herin and Aizenman 2004; Wong et al. 2005). We have suggested that a spermine binding site is located in or near the cleft of the R domain of the NR1 subunit (Masuko et al. 1999a).

Ifenprodil is an atypical NMDA antagonist that binds outside the ion channel pore and selectively inhibits NMDA receptors containing the NR2B subunit (Williams 1993; Williams et al. 1993). Inhibition by ifenprodil is pH-dependent and, mechanistically, may involve an increase in tonic proton inhibition (Pahk and Williams 1997; Mott et al. 1998). Although it was initially suggested that ifenprodil is an antagonist at the stimulatory polyamine site, this does not seem to be the case. There is evidence that spermine and ifenprodil act at discrete sites with an allosteric interaction (Kew and Kemp 1998; Masuko et al. 1999a). Mutations at several residues in the R domain of NR1 greatly influenced ifenprodil inhibition, and we have suggested that this region may form part of the ifenprodil binding site (Masuko et al. 1999a). It has also been reported that mutations at residues in the R domain of NR2B strongly affect ifenprodil inhibition (Perin-Dureau et al. 2002), and it was proposed that the R domain of NR2B forms the ifenprodil binding site whereas the equivalent region of NR2A forms a high-affinity Zn2+ binding site, involving several amino acid residues equivalent to those found at the proposed ifenprodil site in NR2B (Paoletti et al. 2000; Perin-Dureau et al. 2002).

In this paper, we have studied binding of radiolabeled spermine and ifenprodil to purified soluble R domains of NR1, NR2A, and NR2B, to determine whether these ligands can bind to the isolated R domains and to determine the relationship between binding to the R domains and the effects of these ligands at intact, functional receptors. We found that spermine binds to all three domains, but with the highest affinity at NR1-R. Ifenprodil binds with high affinity to NR1-R and NR2B-R but not to NR2A-R.

Materials and methods

Plasmids

For production of COOH-terminal histidine-tagged R domain proteins, DNA fragments encoding NR1(19-380), NR2A(23-389) and NR2B(27-390) (numbering is from the initiator methionine, thus these constructs lack the signal peptide) were amplified by polymerase chain reaction (PCR) using primer pairs of (5’-TATACATATGCGCGCCGCCTGCCGACCC-3’ and 5’-GGTGCTCGAGGATGATCTTCCTGTCAT-3’) for NR1, (5’-TATACATATGCA-GAACGCGGCGGCG-3’ and 5’-GGTGCT-CGAGGATGATCT-TCCTGTCAT-3’) for NR2A and (5’-TAT-ACATATG-CGTTCCCAAAAGAGC-3’ and 5’-CTCGAGTGC-GGCCG-CCA-CATA-ATACTTCATCTGCAG-3’) for NR2B. The rat NR1A clone, which lacks the exon-5 insert (Moriyoshi et al. 1991), and rat NR2A and 2B clones (Monyer et al. 1992) were used as templates. The DNA fragments obtained by PCR were digested with NdeI and XhoI for NR1 and NR2A, and with NdeI and NotI for NR2B, and inserted into the same restriction sites of pET29b (Novagen) to construct plasmids pET29b-NR1(19-380), pET29b-NR2A(23-389) and pET29b-NR2B(27-390). The PCR fragments were sequenced over the entire length of each fragment, and the sequences correspond to those in the NR subunit cDNAs.

Purification of R domain proteins

Escherichia coli BL21(DE3) (Novagen) cells carrying pET29b-NR1(19-380), pET29b-NR1A(19-380, E181Q), pET29b-NR2A(23-389) or pET29b-NR2B(27-390) were grown in 2 L of modified Luria-Bertani medium (10 g tryptone, 10 g yeast extract and 5 g NaCl per liter) containing 50 µg/ml kanamycin at 37 °C until A600 reached 0.3. Protein production was induced by 1 mM isopropyl-β-d-thiogalactopyranoside for 4 h. Inclusion bodies were prepared using methods similar to those described by Chen and Gouaux (1997). Cells were collected by centrifugation, washed once with 10 mM Tris-HCl, pH 8.0, containing 1 mM MgCl2, and suspended in 100 ml of 20% sucrose containing 30 mM Tris-HCl, pH 7.5, 10 mM EDTA and 0.1 mg/ml lysozyme. Cells were kept on ice for 20 min, and spheroplasts were collected by centrifugation at 30,000 g for 30 min. Spheroplasts were resuspended in 200 ml of 50 mM Tris-HCl, pH 7.5, 0.01% Triton X-100, 1 mM dithiothreitol (DTT) and 100 mM KCl, and sonicated for 2 min using an Ultrasonic Disruptor UD201 (TOMY, Tokyo). After treatment of the cell lysate with 20 µg/ml DNase I plus 10 mM MgCl2 (30 min on ice), inclusion bodies were collected by centrifugation at 30,000 g for 15 min. Inclusion bodies were resuspended in 50 ml of buffer (20 mM Tris-HCl, pH 7.5, 6 M guanidine-HCl, 500 mM NaCl, and 5 mM imidazole), kept on ice for 1 h, and centrifuged at 30,000 g for 15 min. The supernatant contained about 70 mg protein, half of which (about 35 mg protein) was applied to each of two 5 ml of Ni-NTA columns (Qiagen). After washing with buffer (20 mM Tris-HCl, pH 7.5, 6 M guanidine-HCl, 500 mM NaCl, and 20 mM imidazole), R domain proteins were eluted with an elution buffer (20 mM Tris-HCl, pH 7.5, 6 M guanidine-HCl, 500 mM NaCl, and 100 mM imidazole).

Proteins (100 µg/ml) were refolded through gradual decrease in the concentration of guanidine-HCl by stepwise dialysis against Buffer 1 [25 mM Hepes-KOH, pH 7.5, 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 10 µM E-64-C, a thiol protease inhibitor, 10 µM FUT-175, a serine protease inhibitor, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 10% glycerol and 0.01% Brij-35] containing 1 M guanidine-HCl, Buffer 1 containing 0.5 M guanidine-HCl, Buffer 1 containing 0.2 M guanidine-HCl, and Buffer 1 at 4 °C for at least 4 h each. R domain proteins were concentrated to approximately 1 mg/ml by ultrafiltration using PM10 filter (Amicon), and precipitates were removed by centrifugation. Protein concentration was determined by the method of Lowry et al. (1951). About 20 mg of R domain protein was obtained from a 2 liter culture. Monomers of the three R domain proteins (41.6 kDa of NR1-R, 42.3 kDa of NR2A-R and 42.7 kDa of NR2B-R) were isolated by gel filtration using TSK gel G3000SW (2.15 × 67.5 cm) (TOSOH) and Buffer 2 containing 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1 mM dithiothreitol, and 0.01% Brij-35. Monomers of R domains were eluted at an elution volume of 132 to 152 ml. The protein was concentrated to approximately 2 mg/ml by ultrafiltration using PM10 filter (Amicon), and used for the experiments. The percentage of monomer and dimer of NR1-R, NR2A-R and NR2B-R during the assay was confirmed by gel filtration using TSK gel G3000SW (0.75 × 30 cm) (TOSOH) and Buffer 2. As a molecular marker, phosphorylase B (94 kDa), ovalbumin (45 kDa) and lysozyme (14.5 kDa) were used.

Binding of spermine and ifenprodil to R domain proteins

Binding assays were carried out as described previously (Kashiwagi et al. 1996). The reaction mixture (0.5 ml) containing 50 to 100 µg R-domain proteins, 10 mM Tris-HCl, pH 7.5, and 50 mM KCl was preincubated at 30 °C for 10 min, and [14C]spermine (463 MBq/mmol, Amersham) or [3H]ifenprodil (15 GBq/mmol, PerkinElmer) was added at 5 to 250 µM for spermine and 0.05 to 0.8 µM for ifenprodil. After incubation at 30 °C for 20 min, the reaction was stopped by addition of 2 ml cold Buffer (10 mM Tris-HCl, pH 7.5, and 50 mM KCl) and the protein was collected on HAWP02400 filter (0.45 µm, Millipore). After the filter was washed 2 times with 2 ml of cold Buffer, radioactivity on the filter was measured by a liquid scintillation spectrometer. Non-specific binding was defined by filtration assay of a reaction mixture kept at 0 °C and was subtracted from total radioactivity to obtain specific binding. The level of non-specific binding obtained using this procedure was very similar to non-specific binding obtained by filtration assay of a reaction mixture containing all assay components except the R domain protein. The dissociation constant (Kd) and maximum binding capacity (Bmax) were calculated from Scatchard plots as described previously (Kashiwagi et al. 1993). The association (k1) and dissociation (k−1) rate constants were calculated with GraphPad Prism V. 4 (GraphPad software).

Determination of the Kd values of spermine and ifenprodil for NR1-R, NR2A-R and NR2B-R by CD (circular dichroism) measurements

The CD measurements were performed at 25 °C using a J-820 spectropolarimeter (Jasco International Co) equipped with a 1.0-mm path-length cuvette. The samples were scanned from 190 to 250 nm and accumulated 10 times at a resolution of 0.1 nm with a scanning speed of 50 nm/min and sensitivity of 200 mdeg. All of the CD data are expressed as molar ellipticity. The concentration of NR1-R, NR2A-R and NR2B-R used was 0.3-, 0.5- and 0.5 mg/ml, respectively. The buffer used was the same as that used for binding assay of spermine and ifenprodil. Increasing amounts of spermine or ifenprodil were added in small aliquots from stock solutions to 300 µl of protein solution and were allowed to bind for 5 min at room temperature. The CD spectra of the protein in the absence and presence of increasing concentrations of spermine (5 – 360 µM) or ifenprodil (0.05 – 2 µM) were recorded. In this experiment, ifenprodil hemitartrate was used instead of ifenprodil to avoid the precipitation of proteins. The final change of assay volume was < 3%. The Kd values were determined according to the double-reciprocal equation plot shown in the figure.

Preparation of oocytes and voltage clamp recording

Adult female Xenopus laevis (Saitama Experimental Animals Supply Co. Ltd., Saitama, Japan) were chilled on ice, and the preparation and maintenance of oocytes were carried out using the methods similar to those described previously (Williams et al. 1993). Capped cRNAs were prepared from linearized cDNA templates using mMessage mMachine kits (Ambion). Oocytes were injected with NR1 and NR2 cRNAs in a ratio of 1:5 (0.2 – 4 ng of NR1 plus 1 – 20 ng of NR2). Macroscopic currents were recorded with a two-electrode voltage-clamp using Dual Electrode Voltage Clamp Amplifier CEZ-1250 (Nihon Koden, Tokyo). Electrodes were filled with 3 M KCl. Oocytes were continuously superfused (ca. 5 ml/min) with a Mg2+-free saline solution (96 mM NaCl, 2 mM KCl, 1.8 mM BaCl2, 10 mM HEPES, pH 7.5). This solution contained BaCl2 rather than CaCl2, and in most experiments oocytes were injected with K+-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (100 nl; 40 mM, pH 7.0) on the day of recording to eliminate Ca2+-activated Cl− currents (Williams 1993). Oocytes were voltage-clamped at a holding potential of −20 mV. Data were recorded by using a Digidata 1322A interface with software pCLAMP8 for Windows (Axon Instruments, USA).

Identification of heterodimer formation of R domain by immunoprecipitation and Western blot analysis

The reaction mixture (0.3 ml) containing either 45 µg NR1-R, 30 µg NR2A-R, 30 µg NR2B-R, 45 µg NR1-R plus 30 µg NR2A-R or 45 µg NR1-R plus 30 µg NR2B-R, 10 mM Tris-HCl, pH 7.5, 50 mM KCl, and 2 µg of anti-NR1 (sc-9058, Santa Cruz) was kept at 4 °C for 2 h. Then, antibody-coupled protein was precipitated by addition of 2 µl PANSORBIN® Cells (CALBIOCHEM) and incubation at 4 °C for 2 h. The precipitate was collected by centrifugation at 30,000 g for 5 min, washed 3 times with Buffer 3 [0.5 M Na-phosphate buffer, pH 7.5, 100 mM NaCl, 1% Triton-X100 and 0.1% sodium dodecyl sulfate (SDS)], and boiled for 5 min in SDS-PAGE sample buffer. Western blotting was performed by the method of Nielsen et al. (1982) using 3 µg protein and ECL Western blotting reagents (GE Healthcare). NR1-R, NR2A-R and NR2B-R were detected with anti-NR1, anti-NR2A (sc-9056, Santa Cruz), and anti-NR2B (sc-9057, Santa Cruz). These commercially available antibodies were raised against epitopes in the first 70 to 300 amino acids of the amino terminus (i.e., the R domain) of each subunit.

Results

Purification of R domain proteins

The regulatory (R) domains of native NMDA receptor subunits are in the extracellular environment, as is the S1–S2 agonist-binding domain (Fig. 1a), and we reasoned that the R domains could be expressed and purified as soluble proteins, as has been reported for S1–S2 fusion proteins from several glutamate receptors (Arvola and Keinänen 1996; Chen and Gouaux 1997; Armstrong et al. 1998; Mayer et al. 2001; Furukawa and Gouaux 2003; Mayer 2005). The R domains of NR1, NR2A, and NR2B, comprising the first 360 to 370 amino acids after the signal peptide (Fig. 1b) were subcloned into pET29b with a histidine tag at their COOH-terminus. These R domain proteins were expressed separately in E. coli, purified from inclusion bodies using Ni-NTA column chromatography, and refolded by dialysis. As shown in Fig. 1c, the soluble NR1-R, NR2A-R and NR2B-R proteins were purified to near homogeneity. The molecular mass of these proteins on SDS-PAGE is similar to that predicted based on their amino acid sequences (41.6 kDa, NR1-R; 42.3 kDa, NR2A-R; 42.7 kDa, NR2B-R), although the mobility of NR2A-R was slightly greater than that of NR1-R and NR2B-R. Fig. 1d shows the molecular size of these purified R domains in solution, as determined by gel-filtration chromatography. NR2A-R and NR2B-R mainly consisted of monomer and dimer forms, but NR1-R contained a polymerized form together with monomers and dimers. The percentages of the monomer forms of NR1-R, NR2A-R and NR2B-R were 37, 73 and 71%, respectively. The elution profile of NR1-R, NR2A-R and NR2B-R was not influenced by adding 0.1 mM spermine to the elution buffer, suggesting that spermine is not involved in polymerization or dimerization of each regulatory domain (data not shown). These preparations were used for subsequent binding assays.

Fig. 1.

Fig. 1

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Structure and purity of R domain proteins. (a) Schematic of an NMDA receptor subunit showing the extracellular R and S1–S2 domains, three membrane-spanning domains (M1, M3, M4), a re-entrant loop (M2) and intracellular C terminus. (b) Schematics of NR1, NR2A, and NR2B subunits showing the positions of the signal peptides (sp), the R domains used in this study, the S1 and S2 domains, and the membrane spanning and re-entrant loops (M1–M4). Amino acids are numbered (above each schematic) from the initiator methionine. (c) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified R domains (NR1-R, NR2A-R and NR2B-R). Each lane of the 12% acrylamide gel contained 2 µg protein, and the gel was stained with Coomassie Brilliant Blue R250. Numbers on the left represent molecular mass in kDa. (d) Gel filtration of purified R domain proteins used for radioligand binding assays. The pattern indicates the extent of dimerization and polymerization after the isolation and concentration of the monomer fraction of R domain proteins. Peaks corresponding to monomers are indicated by an arrow in each panel.

Binding of spermine and ifenprodil to R domain proteins

Binding of [14C]spermine to the soluble R domain proteins was determined. The binding of spermine to NR1-R, NR2A-R and NR2B-R was saturable (Fig. 2, upper panels), and the Kd values for spermine at these regulatory domains were 19, 140 and 33 µM, respectively (Fig. 2, lower panels). Thus, although spermine can bind to all three R domains, it binds with highest affinity to NR1-R. Bmax values, which indicate the proportion of active protein, for NR1-R, NR2A-R, and NR2B-R were 6.3, 6.9 and 7.3 nmol/mg R domain protein, which were equivalent to 0.24, 0.29 and 0.32 mol/mol R domain, respectively. Because of the rapid rate of dissociation of [14C]spermine (see below), it is possible that some [14C]spermine bound to the R domain protein is lost during the washing step in the binding assay. In that case, Bmax values may in fact be higher than reported here. Judging from the percentage of monomer and dimer (Fig. 1d) and Bmax values for spermine binding (Fig. 2b), spermine may bind to both the monomer and dimer forms of NR1-R, NR2A-R and NR2B-R. The Hill coefficient for spermine binding to these three R domains was close to unity, suggesting that spermine binding to each R domain is independent, even though dimers can be formed. As a control, binding of spermine was studied using an unrelated protein, ovalbumin, and a mutant of NR1-R, E181Q, which greatly reduces spermine stimulation as studied in functional assays (Masuko et al. 1999a). There was little binding of spermine to ovalbumin, and binding to the E181Q mutant was reduced compared to the wild-type NR1-R domain (Fig. 2). The Kd value of spermine at the E181Q mutant was 74 µM compared with 19 µM for wild type NR1-R. This is consistent with the finding that spermine stimulation is reduced at receptors containing NR1(E181Q) as measured in functional assays, and with the proposal that residue E181 forms part of the spermine binding site on NR1 (Masuko et al. 1999a).

Fig. 2.

Fig. 2

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Binding of spermine to R domain proteins. Saturation isotherms (upper panels) and Scatchard plots (lower panels) of the binding of [14C]spermine to NR1-R (a), NR2A-R (b), and NR2B-R (c). The Hill coefficient was calculated from the plot of the logarithmic form of a Hill equation. Binding of [14C]spermine to NR1-R (E181Q) and that to ovalbumin was also shown in upper figures of (a) and (b), respectively. Values are means ± S. D. from three experiments. SPM, spermine.

The Kd value for binding of spermine to the NR2B R domain was also calculated by determining the association (k1) and dissociation (k−1) rate constants from studies of the time course of association and dissociation of [14C]spermine (Fig. 3). Values of k1 were obtained using spermine concentrations of 12.5 to 100 µM, and values of k−1 were obtained from dissociation plots after addition of nonlabeled 3 mM spermine. Under these conditions, the association rate was not fully concentration dependent. The Kd value was calculated as the average using values of k1 and k−1 at concentrations of 12.5, 25 and 50 µM [14C]spermine. The values for k1 and k−1 were 8,500 M−1·min−1 and 0.22 min−1, respectively. Accordingly, the Kd was determined to be as 25.9 µM, which is similar to the Kd measured in the stauration binding analysis shown in Fig. 2.

Fig. 3.

Fig. 3

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Time course of association and dissociation of [14C]spermine binding to NR2B-R. The binding assay was performed using 12.5 (◆), 25 (○), 50 (▲) and 100 (□) µM [14C]spermine as substrate. To study dissociation, nonlableled 3 mM spermine was added to the reaction mixture at 16 min after the onset of incubation. [14C]spermine bound to NR2B-R was measured as described in Materials and methods. Values are means ± S. D. from three experiments.

We measured the effects of spermine on recombinant NMDA receptors expressed in oocytes to compare the concentration of spermine required to potentiate intact, functional NMDA receptors with the effects of spermine seen at isolated R domains. Since spermine stimulation is seen at both NR1/NR2A and NR1/NR2B receptors in the presence of 10 µM glutamate and 1 µM glycine at pH 7.5 (Williams et al. 1994), the EC50 values for spermine were measured under these conditions. To minimize voltage-dependent block by spermine, oocytes were voltage-clamped at −20 mV. As shown in Fig. 4, the EC50 values for spermine stimulation at NR1/N2A and NR1/NR2B receptors were 278 µM and 78 µM, respectively. The ratio of EC50 values at NR2A versus NR2B correlates well with the Kd values for spermine binding, although EC50 values are higher than Kd values.

Fig. 4.

Fig. 4

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Effects of spermine at intact, functional NMDA receptors. The effects of spermine on currents induced by glutamate and glycine at NR1/NR2A (○) and NR1/NR2B (●) were measured in oocytes expressing recombinant receptors and voltage clamped at −20 mV. Values are mean ± S. D. from five experiments. The Hill coefficients for spermine at NR1/NR2A and NR1/NR2B were 1.17 and 1.05, respectively.

Binding of [3H]ifenprodil to the purified R domain proteins was also studied. Ifenprodil bound to NR1-R and NR2B-R, but there was no specific binding of 0.1 to 0.8 µM [3H]ifenprodil to NR2A-R. As shown in Fig. 5, binding of ifenprodil to NR1-R and NR2B-R was saturable, with Kd values of 0.18 µM and 0.21 µM, respectively. These values were similar to the IC50 (0.34 µM) of ifenprodil at NR1/NR2B (Williams 1993). Bmax values for both NR1-R and NR2B-R were 5.6 and 5.2 nmol/mg R domain protein, equivalent to 0.24 and 0.21 mol/mol R domain protein, respectively. These values were similar to the binding capacity for spermine at these proteins. The Hill coefficients for binding of ifenprodil to NR1-R and NR2B-R were close to 1.0, suggesting that ifenprodil binding to each R domain is independent, even though homodimers can be formed, and that there is likely one binding site per R domain.

Fig. 5.

Fig. 5

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Binding of ifenprodil to NR1-R and NR2-R. Saturation isotherms (upper panels) and Scatchard plots (lower panels) of the binding of [3H]ifenprodil to NR1-R (a) and NR2B-R (b). The Hill coefficient was calculated from the plot of the logarithmic form of a Hill equation. Binding of [3H]ifenprodil to NR2A-R is also shown in upper figure of (b). Values are means ± S. D. from three experiments.

In the radioligand binding assays, particularly with [14C]spermine, some radioactivity may be lost during the washing step, leading to an under-estimate of the stoichiometry of binding. Thus, the binding of spermine and ifenprodil to R domain proteins was also measured by CD. The CD spectra of R domain proteins were analyzed from 190 to 250 nm with increasing concentrations of spermine or ifenprodil, and the shifts of magnitude at wavelength 208.6 nm reflecting α-helical structure were plotted. As shown in Fig. 6 and Fig. 7, the Kd values for spermine at NR1-R, NR2A-R and NR2B-R were 20, 133 and 35 µM, respectively, and those for ifenprodil at NR1-R and NR2B-R were 0.14 and 0.17 µM, respectively. There was no change in the CD spectra of NR2A-R by 2 µM ifenprodil. The results are similar to those seen in studies with radiolabeled spermine and ifenprodil and confirm that their binding to NR1-R, NR2A-R and NR2B-R is saturable and specific.

Fig. 6.

Fig. 6

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Measurement of the Kd values for spermine at NR1-R, NR2A-R and NR2B-R by CD. CD measurements were performed as described in Materials and methods. Concentration-dependent effects of spermine on shifts of magnitude at 208.6 nm are shown in the upper panels, and the Kd values of spermine were determined from double-reciprocal plots as shown in the lower panels. Values are mean ± S. D. from three experiments.

Fig. 7.

Fig. 7

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Measurement of the Kd values for ifenprodil at NR1-R, and NR2B-R by CD. CD measurements were performed as described in Materials and methods. Concentration-dependent effects of ifenprodil on shifts of magnitude at 208.6 nm are shown in the upper panels, and the Kd values of ifenprodil were determined from double-reciprocal plots as shown in the lower panels. Values are mean ± S. D. from three experiments.

To determine whether the binding sites for spermine and ifenprodil on individual NR1 and NR2 R domains are altered through an interaction with the R domains of the other subunits, the binding of spermine and ifenprodil was compared using the R domains from NR1, NR2A, and NR2B alone, and using a mixture of the NR1 and NR2 R domains. These experiments were carried out using 20 µM spermine and 0.2 µM ifenprodil. As shown in Fig. 8, the binding of spermine and ifenprodil was quantitative, being dependent on the amount of R domain protein (50 to 100 µg). Furthermore, binding of either ligand to NR1-R and NR2A-R or NR2B-R was simply additive. We also carried out experiments to confirm that heterodimers of NR1-R + NR2A-R and NR1-R + NR2B-R do indeed form in solution under these conditions. This was done by immunoprecipitation with an anti-NR1-R antibody followed by Western blotting with anti-NR1 or anti-NR2 antibodies (Fig. 9). Although NR2A-R and NR2B-R alone were not immunoprecipitated by anti-NR1, these two R domains were coimmunoprecipitated with NR1-R by anti-NR1, indicating that formation of NR1-R/NR2A-R and NR1-R/NR2B-R heterodimers does indeed occur after the individual NR1 and NR2 R domains are added together in solution. The percentage of heterodimer formation was estimated to be 20 to 40%, which was similar to the percentage of homodimer formation (see Fig. 1). The results suggest that spermine and ifenprodil bind independently to each regulatory domain, even when dimer formation occurs. The results are in accordance with data showing Hill coefficients of unity for binding of spermine and ifenprodil to NR1-R and NR2-R (Fig. 2 and Fig. 5).

Fig. 8.

Fig. 8

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Binding of spermine and ifenprodil to various combinations of R domain proteins. R domains were expressed and purified individually, then studied alone or in combination. Binding was measured using 50 to 100 µg of NR1-R NR2A-R or NR2B-R, alone or in combination. The concentrations of [14C]spermine and [3H]ifenprodil were 20 µM and 0.2 µM, respectively. Values are means ± S. D. from three experiments.

Fig. 9.

Fig. 9

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Identification of heteromer formation of R domains by immunoprecipitation followed by Western blot analysis. The R domains were immunoprecipitated by anti-NR1 as described in Materials and methods. Immunoprecipitated protein was detected by Western blot analysis using anti-NR1, anti-NR2A and anti-NR2B antibodies. Experiments were repeated twice, and essentially the same results were obtained.

Distinct binding sites for spermine and ifenprodil on NR1-R and NR2B-R

Experiments were carried out to determine if there are interactions between spermine and ifenprodil on the purified NR1-R protein. As shown in Table 1, binding of spermine to NR1-R was inhibited by nonlabeled spermine and spermidine, but not by tribenzylspermidine (TB-34), a polyamine derivative that is a potent NMDA channel blocker but does not have effects at the stimulatory polyamine site (Igarashi et al. 1997). Binding was also unaffected by agonists (glycine and glutamate) that bind in the S1–S2 domains, and by ifenprodil. The binding of ifenprodil to NR1-R was inhibited by nonlabeled ifenprodil, but not by agonists or by spermine and spermidine (Table 1). Similarly, the binding of spermine to NR2B-R was inhibited by nonlabeled spermine and spermidine, but not by agonists and ifenprodil, and binding of ifenprodil was inhibited by nonlabeled ifenprodil, but not by agonists or by spermine and spermidine (Table 2). The results indicate that spermine and ifenprodil bind to distinct sites on NR1-R and NR2B-R.

Table 1.

Spermine and ifenprodil binding to purified NR1-R

Addition [14C]Spermine bound [3H]Ifenprodil bound
nmol/mg protein % nmol/mg protein %
None 3.20 ± 0.23 100 2.98 ± 0.17 100
Spermine (300 µM) 0.08 ± 0.01 3 2.63 ± 0.14 88
Spermidine (1 mM) 0.09 ± 0.02 3 2.71 ± 0.17 91
Tribenzylspermidine (100 µM) 3.28 ± 0.21 103 n.d.
Ifenprodil (10 µM) 2.83 ± 0.25 88 0.27 ± 0.01 9
Glycine (100 µM) 3.23 ± 0.24 101 3.15 ± 0.16 106
Glutamate (100 µM) 3.45 ± 0.27 108 2.88 ± 0.19 97
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The concentration of [14C]spermine and [3H]ifenprodil used for the binding assay was 20 µM and 0.2 µM, respectively. Where indicated, nonlabeled spermine (300 µM), spermidine (1 mM), tribenzylspermidine (100 µM), ifenprodil (10 µM), glycine (100 µM) or glutamate (100 µM) was added to the reaction mixture. Data are shown as mean ± S. E. from three experiments. n.d., not determined

Table 2.

Spermine and ifenprodil binding to purified NR2B-R

Addition [14C]Spermine bound [3H]Ifenprodil bound
nmol/mg protein % nmol/mg protein %
None 3.85 ± 0.38 100 3.21 ± 0.22 100
Spermine (300 µM) 0.28 ± 0.05 7 3.17 ± 0.40 99
Spermidine (1 mM) 0.25 ± 0.04 6 3.15 ± 0.31 98
Ifenprodil (10 µM) 4.14 ± 0.36 108 0.48 ± 0.09 15
Glycine (200 µM) 3.89 ± 0.41 101 3.27 ± 0.32 102
Glutamate (200 µM) 3.93 ± 0.40 102 3.31 ± 0.34 103
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The concentration of [14C]spermine and [3H]ifenprodil used for the binding assay was 40 µM and 0.4 µM, respectively. Where indicated, nonlabeled spermine (300 µM), spermidine (1 mM), ifenprodil (10 µM) glycine (200 µM) or glutamate (200 µM) was added to the reaction mixture. Data are shown as mean ± S. E. from three experiments.

Effect of aminoglycosides on spermine binding to R domain proteins

We have previously shown that a number of aminoglycosides potentiate macroscopic currents at heteromeric NR1/NR2B receptors, probably through binding to a stimulatory polyamine site on the receptor (Masuko et al. 1999b). As shown in Fig. 10, binding of spermine to NR1-R, NR2A-R and NR2B-R was inhibited by neomycin B, paromomycin, kanamycin A and streptomycin, consistent with the idea that aminoglycosides act at the stimulatory polyamine binding sites on the R domains of NR1, NR2A and NR2B. The magnitude of inhibition of spermine binding to R domain proteins by aminoglycosides paralleled potentiation of intact NR1/NR2B receptors by aminoglycosides measured in functional studies, and occurred at similar concentrations of aminoglycosides (Masuko et al. 1999b).

Fig. 10.

Fig. 10

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Effect of aminoglycosides on spermine binding to R domain proteins. Binding of [14C]spermine was measured in the presence and absence of aminoglycosides (75 to 300 µM) as described in Materials and methods. [14C]Spermine used was 20 µM. Values are means ± S. D. from three experiments.

Discussion

Several studies have reported the purification and properties of soluble S1–S2 domains of AMPA and kainate receptor proteins (Arvola and Keinänen 1996; Chen and Gouaux 1997). The same approach has been applied to NMDA receptor subunits. The characteristics of the glycine binding site on the NR1 subunit have been reported from studies using a purified soluble S1–S2 domain (Ivanovic et al. 1998) and a membrane-bound fusion protein containing the entire extracellular portion (R domain plus S1–S2) of NR1 (Miyazaki et al. 1999). In more recent studies, some characteristics of an ifenprodil binding site on NR2B have been described using a purified soluble NR2B R domain (Perin-Dureau et al. 2002; Wong et al. 2005). We have used a similar approach to study the binding sites for spermine and ifenprodil on the R domains of NR1 and NR2 subunits. These studies indicate that spermine and ifenprodil can differentially bind to these domains. The experiments led to several interesting findings as discussed below.

We found that spermine bound to the R domains of all three NMDA receptor subunits. The affinity for spermine was NR1-R > NR2B-R > NR2A-R, and the Kd values for spermine were 19, 33 and 140 µM, respectively. The results suggest that spermine stimulation of NR1/NR2A and NR1/NR2B may be due to spermine binding to the R domain of NR2A and NR2B together with binding to the R domain of NR1. It is notable that the potency of spermine as determined in functional assays was 3- to 4-fold lower at NR1/NR2A receptors (EC50, 278 µM) than at NR1/NR2B receptors (EC50, 78 µM), which correlates with the 4-fold lower affinity for spermine of the NR2A R domain (Kd, 140 µM) compared to the NR2B R domain (Kd, 33 µM). In any event, the results indicate that there are discrete spermine binding sites on the three different R domain proteins, and the data are consistent with our previous mutagenesis studies that identified residues important for spermine stimulation in the R domains of NR1 and NR2B (Williams et al. 1995; Masuko et al. 1999a).

The results demonstrate that ifenprodil binds to NR1-R and NR2B-R, but not to NR2A-R. It has been reported that mutations at several residues in NR2B-R greatly reduced sensitivity to ifenprodil (Perin-Dureau et al. 2002). These residues were in positions analogous to residues in the NR2A subunit that affect sensitivity to Zn2+ and may form a Zn2+ binding site on NR2A (Paoletti et al. 2000). In the same report, ifenprodil was shown to retard protease digestion of a soluble NR2B-R domain (residues 28 to 375) but not of an NR2A-R domain (Perin-Dureau et al. 2002). It was concluded that ifenprodil binds within the cleft of the bi-lobed R domain of NR2B, analogous to binding of Zn2+ in the cleft of the NR2A R domain (Perin-Dureau et al. 2002). In this context, it should be noted that NR2A-R moves more rapidly on an SDS polyacrylamide gel compared with NR1-R and NR2B-R (see Fig. 1), even though the molecular masses of three regulatory domains were almost identical. This suggests that the overall structure of NR2A-R may be different from that of NR1-R and NR2B-R, accounting for differences in the binding sites for Zn2+ and ifenprodil even though these two apparent binding sites share many homologous residues in NR2A and NR2B (Paoletti et al. 2000; Perin-Dureau et al. 2002).

In the present study, we observed binding of [3H]ifenprodil to NR1-R and NR2B-R but not to NR2A-R. The Kd values for binding of ifenprodil to NR1-R and NR2B-R (0.18 µM and 0.21 µM, respectively) were similar to the IC50 for ifenprodil inhibition at intact, functional NR1/NR2B receptors (0.34 µM at pH 7.6) (Williams 1993). It has also been reported that the binding constant for ifenprodil at NR2B-R was 0.13 to 0.17 µM when measured by circular dichroism (Wong et al. 2005), a finding that we also replicated in the current study. The finding that [3H]ifenprodil binds to NR1-R is consistent with our previous finding that mutations in this region of the NR1 subunit affect sensitivity to ifenprodil (Masuko et al. 1999a). Although other reports have localized an ifenprodil binding site to NR2B-R (Perin-Dureau et al. 2002), our results suggest that the R domain of NR1 is also capable of forming an ifenprodil binding site with a high affinity, similar to NR2B-R. The finding that ifenprodil can bind to the R domain of NR1 is consistent with the high affinity inhibition by ifenprodil of apparent “homomeric” NR1 receptors expressed in oocytes (Williams et al. 1993). It has been proposed that these receptors are not truly NR1 homomers and that an endogenous “NR2-like” Xenopus subunit can combine with NR1 to form these receptors (Soloviev and Barnard, 1997). However, another study (Green et al., 2002) has convincingly shown that this endogenous subunit does not contribute to the formation of “homomeric” NR1 receptors in Xenopus oocytes, and the true nature of such receptors (NR1 homomers or NR1/NRX heteromers) remains unresolved. There is no evidence for expression of functional homomeric NR1 receptors in vivo, and ifenprodil appears to produce high affinity inhibition (Kd less than 1 µM) only at heteromeric NR1/NR2 receptors containing NR2B. Thus, even though the NR1 R domain is capable of forming a high affinity ifenprodil binding site, the affinity for ifenprodil or the coupling of binding to channel activity may be reduced in heteromeric receptors containing NR2A, NR2C, and NR2D, but retained in receptors containing NR2B.

The results from this study are consistent with the idea that spermine and ifenprodil act at distinct binding sites on the NMDA receptor (Kew and Kemp 1998; Masuko et al. 1999a). Amino acid residues involved in ifenprodil inhibition in NR1-R and NR2B-R were mainly located between residues 80 and 200 (Masuko et al. 1999a; Perin-Dureau et al. 2002). On the other hand, amino acid residues that influence spermine stimulation in NR1 and NR2B were mainly located between residues 170 and 350 (Williams et al. 1995; Masuko et al. 1999a). It may be that spermine binds within the cleft of the R domain similar to polyamine binding to the bacterial protein PotD (Sugiyama et al. 1996; Masuko et al. 1999a). In that case, ifenprodil may bind to another site within the cleft (Perin-Dureau et al. 2002) or elsewhere on the R domain. Even though spermine and ifenprodil have separate binding sites, there are interactions (presumably allosteric) between these two ligands at intact receptors (Kew and Kemp 1998).

Acknowledgments

We thank Drs. S. Nakanishi and P. H. Seeburg for the NR1, NR2A and NR2B clones. Thanks are also due to Torii Pharmaceutical Co. and Taisho Pharmaceutical Co. for providing FUT-175 and E-64-C, respectively. This work was supported by the National Institutes of Health (NS-35047), a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Science and Technology, Japan, and the Futaba Electronics Memorial Foundation, Chiba, Japan.

Abbreviations used

NMDA

N-methyl-d-aspartate

NR1-R

regulatory domain of NMDA receptor NR1 subunit

NR2A-R

regulatory domain of NMDA receptor NR2A subunit

NR2B-R

regulatory domain of NMDA receptor NR2B subunit

AMPA

alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate

CD

circular dichroism

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