Introduction

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Dynorphin is thought to be an endogenous ligand for -opioid receptors (Chavkin et al., 1982). However, the application of exogenous dynorphin to the central nervous system produces a number of non-opioid receptor-mediated effects, including hind-limb paralysis, motor activity impairment, and loss of tail-flick response (Walker et al., 1982; Caudle and Isaac, 1987). These neurotoxic effects of dynorphin may be mediated by the N-methyl-D-aspartate receptors (NMDARs) based on the observation that they can be prevented by pretreatment with NMDAR antagonists (Bakshi and Faden, 1990; Long et al., 1994). Endogenous levels of dynorphin also have been implicated in pathological states in which NMDAR appears to play an important role. It has been observed that spinal dynorphin is significantly elevated in response to peripheral nerve injury or tonic pain (Kajander et al., 1990; Drasci et al., 1991). In a rat model of neuropathic pain, antisera to dynorphin have the same profile of actions as the NMDAR antagonist MK-801 in blocking thermal hyperalgesia and restoring the efficacy of morphine against tactile allodynia brought on by nerve-ligation injury (Nichols et al., 1997). These findings suggest that the elevated levels of spinal dynorphin may contribute to the hyperesthetic states, possibly through modulation of NMDAR function in the maintenance of neuropathic pain.

Many previous studies have attempted to elucidate the mechanisms by which dynorphin may induce or potentiate the excitatory actions of NMDAR. It has been proposed that dynorphin could contribute to excitotoxicity indirectly by enhancing the release of excitatory amino acids such as aspartate and glutamate (Faden, 1992; Long et al., 1994). Dynorphin has also been proposed to mediate its effects through direct interaction with the NMDAR based on radioligand binding analysis. In rat brain membranes, dynorphin and its related fragments attenuate the binding of [³H]glutamate (Massardier and Hunt, 1989) and [³H]MK-801 (Shukla et al., 1992) but potentiate that of [³H]CGP39,653 (Dumont and Lemaire, 1994). This binding profile suggests that dynorphin is inhibitory on NMDARs; the affinity of the peptide for NMDAR based on these analyses, however, is generally quite low (in the micromolar range). Electrophysiological studies of NMDA-mediated currents in isolated trigeminal neurons (Chen et al., 1995) or in Xenopus oocytes that expressed NMDAR subunit complexes (Brauneis et al., 1996) also indicated that dynorphin attenuates NMDAR conductance. This inhibitory action of dynorphin on NMDAR is not consistent with the apparent excitatory actions of dynorphin in vivo. An alternative hypothesis to this paradox is that dynorphin may produce its excitatory effects through a disinhibition mechanism (Chen et al., 1995) or through an inhibitory action on melanocortin receptors (Quillan and Sadee, 1997). Dynorphin has also been shown to potentiate NMDAR-mediated current under an extremely low glycine concentration in the assay buffer (Zhang et al., 1997). However, these data were obtained under nonphysiological conditions and may not reflect the action of dynorphin in vivo.

Both radioligand binding and electrophysiological analyses cited above have defined the apparent affinity and potency of dynorphin at the NMDAR in the micromolar range. However, it is important to note that indirect radioligand competition analysis may not truly reflect the affinity of dynorphin for the NMDAR if both the NMDAR ligand and dynorphin have modulatory effects on the conformation of the NMDAR complex or if the ligands do not compete at the same binding site. It is certainly true of the NMDAR, which consists of multiple binding sites for different agonists and modulators. A more reliable approach to definition of ligand affinity is through direct binding analysis. This study tested the hypothesis that a high-affinity binding site for dynorphin on the NMDAR may exist and that interaction of dynorphin may promote an active state of the receptor. To test this hypothesis, we characterized the interaction of dynorphin with the NMDAR using ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) in a direct radioligand saturation binding analysis on membranes prepared from rat brain cortex as well as that prepared from human embryonic kidney (HEK) 293 cells that have been transfected with the cDNAs for the NR1a and NR2A subunits of the NMDAR. Our data demonstrate that dynorphin binds directly to the NMDAR complex and defines a novel high-affinity site for the peptide. The modulatory effect of a number of NMDAR ligands on this high-affinity binding for dynorphin, however, suggests that dynorphin preferentially binds to the closed/desensitized state of the NMDAR.

	Materials and Methods

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Chemicals. MK-801, AP-5, 7-chlorokynurenic acid (CKA), ifenprodil, (+)-HA-966, and glutamic acid were obtained from Research Biochemicals Inc. (Natick, MA). Glycine, bestatin, captopril, bacitracin, phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin (BSA), and other buffering reagents were from Sigma Chemical Co. (St. Louis, MO).

Radiochemicals. (des-Tyrosyl)dynorphin A(2-17) (sequence: GGFLRRIRPKLKWDNQ; see below for synthesis) was radioiodinated according to the Bolton and Hunter procedure (1973) and purified with the use of reverse phase (RP)-HPLC on a Vydac C18 column (Custom Labeling Service; Amersham Life Sciences, Arlington Heights, IL). The ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) was supplied lyophilized with a specific activity of about 2000 Ci/mmol. When reconstituted in 1 ml of water, it contains 5% lactose, 0.25% BSA, and 0.3 TIU/ml aprotinin in 50 mM sodium phosphate buffer, pH 7.4. [³H]MK-801 and [³H]CGP39,653 were from NEN (Boston, MA).

Synthesis of (des-Tyrosyl)dynorphin A(2-17). Dynorphin A(2-17)-OH was synthesized by standard solid phase F_moc chemistry protocols on an Applied Biosystems 431A peptide synthesizer using Wang resin presubstituted with F_moc-Gln (Trt)-OH (0.68 mmol/g substitution level; Advanced Chem Tech, Louisville, KY) on a 0.25 mmol scale. Purification was effected with RP-HPLC (C18, Vydac Protein and Peptide column, 5.0 × 25.0 cm), with a gradient elution from 0 to 70% acetonitrile (0.1% aq. trifluoroacetic acid makeup solvent) over 70 min with a flow rate of 40 ml/min. The purity of the peptide was assessed as being >95% by analytical RP-HPLC (two-gradient systems) and thin-layer chromatography (four-solvent systems). The structure was determined by mass spectrometry (Department of Chemistry, Mass Spectrometry Facility, University of Arizona). Some of the peptide used in this study was also kindly provided by the National Institute on Drug Abuse (the peptide was synthesized by Multiple System Peptides Inc.: purity, 98%; protein content, 74%).

Preparation of Membranes from Rat Brain Cortex. Sprague-Dawley rats (male, 250-300 g) were anesthetized with ether and decapitated. Tissues from rat brain cortex were rapidly removed and homogenized in ice-cold 5 mM Tris-Cl, pH 8.0. The homogenate was centrifuged at 48,000g for 25 min at 4°C. The pellet was resuspended in 5 mM Tris-Cl/10 mM EDTA, pH 8.0, and incubated at 37°C for 10 min. The suspension was then centrifuged at 48,000g for 25 min at 4°C. The membrane pellet was washed with 5 mM Tris-Cl, pH 8.0, and centrifuged as above. Membrane pellets were stored at 80°C until use. Protein content was determined according to the method of Lowry et al. (1951). Aliquots of membranes were thawed and washed twice in ice-cold 5 mM Tris-Cl, pH 7.5, before each experiment.

Radioligand Binding Assays in Rat Brain Membranes. Assays using ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) were carried out in a shaking water bath at 25°C for 2 h in 0.5 ml of binding buffer (5 mM Tris-Cl buffer, pH 7.5, containing 0.2% BSA, 100 µM PMSF, 30 µM bestatin, 50 µg/ml bacitracin, and 10 µM captopril). Naloxone (10 µM) was included in the assay buffer to prevent potential binding of the peptide to opioid receptors. For saturation analysis, rat brain cortical membranes (300 µg) were incubated with 1) a low concentration range of 0.6 to 25 nM (des-tyrosyl)dynorphin A(2-17) or 2) a high concentration range of 1 to 10 µM (des-tyrosyl)dynorphin A(2-17), where ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) was added to the unlabeled peptide stock solution as a tracer before serial dilution. Nonspecific binding was defined by the binding of radioligand in the presence of 200 µM (des-tyrosyl)dynorphin A(2-17). To examine the modulation of ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) binding by NMDAR ligands, 300 µg of the brain membranes/assay tube was used in the binding assay at a final concentration of 1 nM or 1 µM (des-tyrosyl)dynorphin A(2-17), containing ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) as a tracer, in the presence of 1) AP-5, 2) CKA, 3) ifenprodil, 4) (+)-HA-966, or 5) MK-801. Binding was terminated with rapid filtration through Whatman GF/B filters (pretreated with 0.2% BSA at 4°C for at least 2 h) on a Brandel cell harvester, and the filters were rinsed six times with 3 ml of cold saline. Filters were transferred to polypropylene tubes, and the amount of radioactivity in each filter was determined by gamma counting (Gamma 5500; Beckman).

Transient Expression of NR1a/NR2A Complex of NMDAR in HEK 293 Cells. HEK 293 cells (ATCC CRL 1573; American Type Culture Collection, Rockville, MD) were maintained in 5% FCS, 5% newborn calf serum, 90% Dulbecco's modified Eagle's medium, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere containing 5% CO₂ (culture reagents were from Life Technologies, Grand Island, NY). Before transfection, cells were seeded into 175-cm² flasks at approximately 3 × 10⁶ cells/flask and harvested at approximately 50% confluency (while they were still in logarithmic growth). The cells were resuspended by trypsinization and pelleted by low-speed centrifugation at room temperature. The cells were then washed once in HEPES-buffered saline (HBS) and resuspended in the same buffer at a density of 20 × 10⁶ cells/ml. For the electroporation of these cells, 0.7 ml of cell suspension was mixed with the following: 30 µg of cytomegalovirus pRc containing the cDNA for NR1a, 30 µg of pCDNA1 containing the cDNA for NR2A, and 440 µg of salmon testes DNA (Sigma Chemical Co.) to a final volume of 0.8 ml with HBS. For nontransfected control cells, 0.7 ml of cell suspension and 440 µg of salmon testes DNA were made up to 0.8 ml with HBS. Each 0.8-ml batch of cells was electroporated separately in disposable 0.4-cm² cuvettes in a Bio-Rad Gene Pulser II Unit fitted with a Capacitance Extender II Module (Hercules, CA). Electroporation was carried out under constant 200 V and 900 . The cells were left undisturbed for 10 min before plating into two 75-cm² flasks containing culture media as described above. The cell cultures were supplemented with a final concentration of 200 µM MK-801 6 h after plating to inhibit any potential excitotoxicity that might occur with overexpression of the NMDAR complexes in transfected cells. Cells were harvested 72 h after plating. Before cell harvest, the media were aspirated and cells were incubated in serum-free medium containing 500 µM glutamic acid and 500 µM glycine for 1 h. The cells were then washed in serum-free medium and resuspended in ice-cold 5 mM Tris-Cl, pH 8.0, and pelleted. Crude membranes were prepared from these cells in a similar manner as described above for brain cortical membranes. Protein content was determined according to the method of Lowry. Membranes were stored at 80°C until use.

Radioligand Binding Assays in Transfected HEK 293 Cells. For each experiment, aliquots of membranes were thawed and washed twice in ice-cold 5 mM Tris-Cl, pH 7.5. For ¹²⁵I-(des-tyrosyl) dynorphin A(2-17) binding, membranes were resuspended in the binding buffer as described above for brain membranes. Nonspecific binding was defined as that in the presence of 50 µM (des-tyrosyl) dynorphin A(2-17). For [³H]MK-801 and [³H]CGP39,653 binding, membranes were resuspended in 5 mM Tris-Cl, pH 7.5, containing 100 µM PMSF. Reactions using [³H]MK-801 were carried out in the presence of 100 µM glutamic acid, 100 µM glycine, and 50 µM spermidine. Nonspecific binding for [³H]MK-801 and [³H]CGP39,653 was defined as that in the presence of 10 µM MK-801 and 100 µM glutamate, respectively. All assays were carried out using 80 to 100 µg of membrane protein in a total volume of 0.5 ml. Incubation and filtration of the samples were as described for brain membranes. Radioactivity of ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) was determined by gamma counting; that of [³H]MK-801 and [³H]CGP39,653 was determined by liquid scintillation counting.

Data Analysis. Data from saturation binding were analyzed through nonlinear regression analysis and Rosenthal transformation. Statistical analysis was carried out using the Student's t test. Statistical significance is defined at the 95% confidence level. Both binding and statistical analyses were made with the program Prism (GraphPAD, San Diego, CA).

	Results

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Saturation Binding of ¹²⁵I-(des-Tyrosyl)dynorphin A(2-17) in Rat Brain Cortical Membranes. Initial experiments showed that ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) rapidly associated with rat brain cortical membranes. The binding reached an equilibrium after 30 min at 25°C and remained stable for at least 4 h (data not shown). Hence, all binding assays were carried out for 2 h at 25°C. Under these conditions, ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) exhibited a saturable, specific binding in the concentration range of 0.6 and 25 nM (Fig. 1). The specific binding typically constituted approximately 25 to 30% of the total binding. Rosenthal transformation of the binding data yielded a K_d value of 9.4 ± 1.6 nM and a B_max value of 2.4 ± 0.6 pmol/mg protein for this high-affinity site. At high concentrations of ¹²⁵I-(des-tyrosyl) dynorphin A(2-17) (ranging from 1 to 10 µM), the specific binding increased further in a dose-dependent manner, but this increase was not saturable (data not shown), suggesting that cortical brain membranes contain a high capacity of low-affinity binding for (des-tyrosyl)dynorphin A(2-17). Thus, only the high-affinity site could be defined for (des-tyrosyl) dynorphin A(2-17).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Saturation and Rosenthal (inset) analysis of 125I-(des-tyrosyl) dynorphin A(2-17) binding to rat brain cortical membranes. An experiment for each ligand concentration was conducted in duplicate. The data are representative of three independent experiments. Kd = 9.4 ± 1.6 nM (mean ± S.E., n = 3); Bmax = 2.4 ± 0.6 pmol/mg (mean ± S.E., n = 3).

Modulation of ¹²⁵I-(des-Tyrosyl)dynorphin A(2-17) Binding to Rat Brain Cortical Membranes by Ligands Specific for NMDAR. To test whether the observed specific binding of ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) in the brain membranes was associated with the NMDAR, the binding of 1 nM ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) to brain cortical membranes was carried out in the presence of 100 µM concentration of a number of ligands specific for the NMDAR (Fig. 2A). AP-5, an antagonist at the glutamate binding site of the NMDAR; CKA and (+)-HA-966, an antagonist and a partial agonist, respectively, at the glycine binding site; and ifenprodil, an antagonist at the spermidine binding site of NMDAR (Reynolds and Miller, 1989), significantly potentiated the specific binding of 1 nM ¹²⁵I-(des-tyrosyl)dynorphin A(2-17). In contrast, MK-801, an NMDAR-specific open channel blocker, significantly attenuated the specific binding of 1 nM ¹²⁵I-(des-tyrosyl)dynorphin A(2-17). Specific binding of ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) at high concentration of the peptide (1 µM), presumably also occupying the low-affinity binding sites for the peptide, was not modulated by any of the NMDAR ligands tested (Fig. 2B). Further analysis of the dose-effect of CKA and MK-801 on the specific binding of 1 nM ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) showed that the potentiating effect of CKA had a half-maximal concentration of 18 µM and a maximal 2-fold potentiation, whereas the inhibitory effect of MK-801 was significant only at high concentrations of the ligand (100 µM), showing a maximal inhibition of 50% (Fig. 3).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of NMDAR ligands on the binding of 125I-(des-tyrosyl) dynorphin A(2-17) to brain cortical membranes. The changes are expressed as percentage of specific binding of 125I-(des-tyrosyl)dynorphin A(2-17) in the absence of NMDAR ligands and represent the mean ± S.E. of at least three experiments conducted in triplicate. Statistical significance was determined by 95% confidence interval (*P < .05 compared with 0). A, with 1 nM 125I-(des-tyrosyl)dynorphin A(2-17). B, with 1 µM 125I-(des-tyrosyl)dynorphin A(2-17).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Dose-effect of CKA (top) and MK-801 (bottom) on the binding of 1 nM 125I-(des-tyrosyl)dynorphin A(2-17) to rat brain cortical membranes. The results represent mean ± S.E. values of three experiments conducted in triplicates. *P < .05 compared with 0.

Transient Expression of NMDAR1a/R2A Complexes in HEK 293 Cells. HEK 293 cells that were transfected with the cDNA for the NR1a and NR2A subunits expressed a saturable, specific binding of [³H]MK-801 with a K_d value of 18 nM (Fig. 4). Transient expression produced on average 202 ± 20.3 fmol/mg protein (n = 8) of specific binding of [³H]MK-801 (at 40 nM) compared with 54.6 ± 21.4 fmol/mg protein (n = 8) in nontransfected cells. The net saturable binding of [³H]MK-801 in transfected cells was 148 ± 17.5 fmol/mg protein. Specific binding of [³H]CGP39,653 (at 50 nM) was 362 ± 61 fmol/mg protein (n = 3) in transfected cells and undetectable in nontransfected cells. On the other hand, a significant level of specific binding was observed in nontransfected cells for ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) at a concentration of 10 nM (1.1 pmol/mg protein, n = 2). Parallel assays in transfected cells showed that the specific binding of this peptide was 2.6 pmol/mg protein; thus, a net specific binding of 1.45 pmol/mg may be attributed to the expression of the NR1a/NR2A complexes in the transfected cells (Table 1).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Saturation analysis of [3H]MK-801 binding in HEK 293 cells that had been transfected with the cDNAs for the NR1a and NR2A subunits. Kd = 17.8 nM and Bmax = 436 fmol/mg. Data are representative of two independent determinations.

	Discussion

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Using radioiodinated dynorphin A(2-17), which does not interact with opioid receptors, we are able to directly determine the affinity of this peptide to NMDAR in the absence of NMDAR ligands in brain cortical membranes. This is based on the observed saturable binding for dynorphin A in the nanomolar peptide concentration range and that this high-affinity binding for dynorphin A can be modulated by several antagonists for the NMDAR, suggesting that it is associated with the NMDAR complex. Incidentally, the density of this binding site for dynorphin A is similar to that of NMDAR in rat brain defined by [³H]MK-801 binding (Wong et al., 1988). The dissociation constant of dynorphin A for this binding site is also significantly lower than that previously defined for dynorphin binding to NMDAR based on indirect binding analysis as cited above. This high-affinity binding for dynorphin A associated with the NMDAR complex may thus represent a novel site for the peptide. Alternatively, the difference in the affinity for dynorphin A on the NMDAR defined by direct and indirect binding analyses may be due to the modulatory interactions between dynorphin A and NMDAR ligands. For example, the binding of [³H]MK-801 (enhanced by glycine, glutamate, and spermidine that are usually included in the binding assay) to NMDAR may promote a conformation of receptor that has lower affinity for dynorphin A, and this may explain why the IC₅₀ value of dynorphin against [³H]MK-801 is in the micromolar range in ligand competition analysis.

The high concentrations of NMDAR ligands required to modulate the high-affinity ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) binding is consistent with distinct ligand binding sites on the NMDAR for various ligands. In this regard, the agonist glutamate, coagonist glycine, spermidine, and the antagonist MK-801 occupy distinct binding sites on the receptor complex. When the binding of one ligand influences that of the other through allosteric interaction, it is often observed under concentrations higher than the affinity of the ligand for its own specific binding site. This has been previously observed in the modulation of [³H]MK-801 binding by glutamate, glycine, and polyamines such as spermine and spermidine. These compounds, alone or in combination, increased the affinity of [³H]MK-801 binding to the NMDAR, and the concentrations used were all in the micromolar range (Ransom and Stec, 1988). Dumont and Lemaire (1994) have also shown previously that dynorphin A(1-13) potentiates the binding of [³H]CGP39,653, a competitive antagonist at the glutamate binding site. This effect was observed in the micromolar range of dynorphin A. Such an effect of dynorphin A could be partially blocked by the addition of 1 µM glycine or 10 µM HA-966. Our findings indicate that in a reciprocal manner, modulation of ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) binding also requires micromolar concentrations of ligands such as CKA. Thus, the modulatory effects of the NMDAR ligands suggest that dynorphin A may occupy a site distinct from that for the other well-defined NMDAR ligands. It is certainly possible that high concentrations of the NMDAR ligands may interact with non-NMDAR sites that may also bind dynorphin A. On the other hand, it seems unlikely that all the tested NMDAR compounds interact with the same low-affinity binding site that happens to bind dynorphin A with high affinity.

In our experiments, all tested antagonists enhance the high-affinity binding of ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) except for MK-801, which significantly attenuates the binding of ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) only at very high concentrations. One explanation is that dynorphin A has high affinity for the closed or desensitized state of NMDAR, which is promoted by NMDAR antagonists such as AP-5, CKA, and ifenprodil. The attenuation of the binding of ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) by MK-801 only at very high concentration is consistent with this postulate because MK-801 prefers the open state of the NMDAR and thus would have low affinity for that which binds dynorphin A. The direct binding analysis using high concentrations of dynorphin A(2-17) also detected nonsaturable, specific binding for the radioligand, indicating the presence of multiple low-affinity sites for dynorphin A. However, these sites are not associated with the NMDAR complex because the specific binding of dynorphin A to these sites is not sensitive to NMDAR ligands. The nature of these sites is not known.

The coexpression of specific binding for [³H]MK-801, [³H]CGP39,653, and ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) in HEK 293 cells that have been transfected with the NR1a and NR2A subunits provides further support for the existence of a dynorphin A binding site on the NMDAR. Based on the saturation analysis of [³H]MK-801, these transiently transfected cells express an appreciable density of NMDAR complexes that exhibit a high affinity for the antagonist, suggesting that the in vitro expression system using the NR1a and NR2A subunits gives rise to functional NMDAR complexes. This is further substantiated by the coexpression of [³H]CGP-39,653 in the transfected cells. For ¹²⁵I-(des-tyrosyl)dynorphin A(2-17), nontransfected HEK 293 cells exhibit a substantial capacity of specific binding sites for the peptide at 10 nM. Specific binding for ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) in transfected cell membranes under the same conditions is consistently higher than that in the nontransfected cells. Thus, this net increase in the specific binding of ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) in transfected cells represents binding sites associated with the expression of the NMDAR. The density of dynorphin A binding estimated from this net specific binding in transfected cells, however, is higher than that for MK-801 and CGP39,653 binding. This disparity is not likely to be due to differences in the affinity of the ligands for NMDAR, because the binding analysis used a saturating concentration of both [³H]CGP39,653 and [³H]MK-801, whereas the concentration of ¹²⁵I-(des-tyrosyl)dynorphin A(2-17) approximates the K_d value of dynorphin A for the NMDAR in cortical brain membranes. These findings suggest that each NMDAR complex may possibly interact with more than one molecule of dynorphin. Alternatively, because the in vitro expression of NMDAR subunits may generate a heterogeneous configuration of NMDAR complexes in the same and/or different cells, it is also possible that although MK-801 and CGP39,653 may selectively bind to the "functional" NMDAR complexes, the binding of dynorphin A may not be as conformation specific. Nevertheless, dynorphin A binding is directly correlated with the in vitro expression of NR1a/NR2A complexes.

In conclusion, the present study describes a high-affinity binding site for dynorphin A on the NMDAR. Expression of the NR1a and NR2A subunits is sufficient to confer the binding of dynorphin A to the receptor complex. The potentiation of this binding by various NMDAR antagonists suggests that this peptide may preferentially binds to the closed and/or desensitized state of the NMDAR. The possible physiological or pathophysiological importance of the direct dynorphin interaction with the NMDAR remains unclear. In view of the excitotoxicity of dynorphin in vivo, it is possible that this high-affinity binding site for dynorphin A on the NMDAR might be excitatory under some conditions or that some nonopioid, non-NMDAR site of action of dynorphin may be responsible for inducing NMDAR excitotoxicity.