I. Introduction

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Since the finding by Johnson and Ascher (1987) demonstrating that glycine enhances electrophysiological responses mediated by N-methyl-D-aspartate (NMDA)^b-sensitive glutamatergic receptors, considerable interest has been devoted to this topic (for reviews, see Dingledine et al., 1990; Thomson, 1990; Huettner, 1991; Carter, 1992; Kemp and Leeson, 1993; Leeson, 1993; Wood, 1995). Although Johnson and Ascher (1987) were the first to connect the strychnine-insensitive glycine recognition site with the NMDA receptor, the strychnine-insensitive effects of glycine had been recognized earlier, indicating the diversity of presumed glycine receptors (table 1). Kishimoto et al. (1981) reported that the saturation isotherm of Na⁺-independent [³H]glycine binding in the medulla oblongata and spinal cord (but not cerebral cortex) showed biphasic features. In this assay, D-serine was surprisingly >40 times more potent as an inhibitor of [³H]glycine binding in the cortex than was L-serine, and strychnine was inactive, indicating that these sites were not related to the inhibitory glycine receptors forming Cl channels (Young and Snyder, 1974). Later, it was reported that the distribution of [³H]glycine and [³H]strychnine binding is not colocalized, but rather complementary, being strongest in the forebrain and pons/spinal cord, respectively (Bristow et al., 1986). Johnson and Ascher (1987) then published their observations, based on patch-clamp experiments in primary cultures of mouse cortical neurons, that the magnitude of NMDA responses was dependent on the speed of perfusion of agonist solution, i.e., slower flow resulted in larger responses. Subsequently, by using conditioned medium or adding exogenous glycine, it was clarified that the enhancement of NMDA responses was the result of glycine released from cultured cells. These authors reported similar augmentation when serine or alanine was used, although the differences between isomers were not established. Soon after, at the Society for Neuroscience meeting in 1987, it was reported that glycine enhanced 3',5'-cyclic guanidine monophosphate (cGMP) levels in the cerebellum in vivo after intracisternal application (Danysz et al., 1987, 1989c). This finding was soon confirmed in the same experimental model (Wood et al., 1989), and both studies indicated that the glycine site of NMDA receptors is not always saturated in vivo (see Section III.C.7. for a more detailed discussion). At the same Neuroscience Society Meeting, glycine was also shown to potentiate a slow excitatory postsynaptic potential (EPSP) between hippocampal neurons in culture (Forsythe et al., 1987), and the first glycine_B antagonist, kynurenic acid, was introduced (Kessler et al., 1987, 1989b); this is of particular interest considering that kynurenic acid is an endogenous substance (Stone et al., 1987).

Another important milestone was the report by Kleckner and Dingledine (1988) indicating that glycine is in fact probably obligatory for the activation of NMDA receptors. In turn, the term "coagonist" was proposed. This finding has very important implications, because it indicates that maximal inhibition of the glycine site should result in total inhibition of NMDA receptor activity, which in fact has been confirmed in several studies (see Section III.A.). Therefore, this opened new avenues for drug development targeted at the modulation of NMDA receptors.

One of the concerns regarding glycine have been its dual role in the central nervous system (CNS). It activates both inhibitory strychnine-sensitive receptors and the NMDA receptor-coupled site. Therefore, a selective endogenous ligand for the latter site has been sought. Hashimoto et al. (1993c) were the first to show that D-serine is present in the brain at levels that might affect the glycine_B recognition site (for review, see Hashimoto and Oka, 1997). Since that report, at least two candidates have been proposed to be endogenous agonists for the glycine site of NMDA receptors.

One of the major problems in accepting the dynamic role of glycine or D-serine in the regulation of NMDA receptor function was the apparent lack of sufficiently robust regulatory mechanisms (uptake, release, and metabolism). Also, considering the micromolar concentrations of glycine present in the extracellular fluid (ECF) (Matsui et al., 1995), some authors strongly suggested that these glycine sites must be saturated under physiological conditions. However, using coexpression of glycine transporter (GLYT)1 and NMDA receptors in oocytes, it was recently shown that very efficient buffering of local glycine concentration exists, leading to a >100-fold concentration gradient (Supplisson and Bergman, 1997). This strongly implies that the glycine concentration might be well below its K_d value for the glycine site at NMDA receptors if this transporter is expressed sufficiently at the right location in vivo.

There is no uniformly accepted term for the "NMDA receptor-coupled, strychnine-insensitive, glycine modulatory site," which is both confusing and highly impractical. For purely pragmatic reasons, we abbreviate this as the "glycine_B site," in contrast to the inhibitory, strychnine-sensitive, "glycine_A site."

	II. N-Methyl-D-Aspartate Receptors

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NMDA-sensitive ionotropic glutamate receptors probably consist of tetrameric, heteromeric, subunit assemblies that have different physiological and pharmacological properties and are differentially distributed throughout the CNS (Seeburg, 1993; Hollmann and Heinemann, 1994; McBain and Mayer, 1994; Danysz et al., 1995a; Parsons et al., 1998b). The exact subunit stoichiometry of these subunit assemblies is still a matter of debate. Although previous data were consistent with pentameric -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptors (Wenthold et al., 1992; Brose et al., 1993, Wu and Chang, 1994; Ferrer Montiel and Montal, 1996; Sutcliffe et al., 1996; Premkumar and Auerbach, 1997), recent findings indicate that both AMPA receptors (Rosenmund et al., 1998) and NMDA receptors (Laube et al., 1998) are in fact tetrameric assemblies. However, the debate is not yet over, because two studies using similar single-channel recording techniques came to very different conclusions regarding the number of NR1 subunits, with Behe et al. (1995) claiming two copies and Premkumar and Auerbach (1997) claiming three.

These receptors are positively modulated by glycine, which, as mentioned, binds to a specific, strychnine-insensitive, glycine_B site (see Section II.D.), by polyamines (spermine and spermidine), by histamine, and, under some conditions, by cations (fig. 1). NMDA receptors are coupled to high conductance cationic channels permeable to K⁺, Na⁺, and Ca²⁺ (McBain and Mayer, 1994).

View larger version (62K): [in this window] [in a new window]   Fig. 1.   Model showing that NMDA receptors are probably heteromeric assemblies of four subunits. Each subunit has four hydrophobic regions, although only three form membrane-spanning domains (TM1, TM2, and TM4). TM2 makes a hairpin bend within the membrane and forms the channel pore; the "TM" terminology is therefore inappropriate. Functional NMDA receptor complexes are formed by combinations of NR1 and NR2 subunits, which contain the glycine and glutamate recognition sites, respectively. Alternative splicing at three exons, one in the amino-terminal domain (N1) and two in the carboxyl-terminal domain (C1 and C2), generates eight isoforms for the NR1 subfamily. The NR2 subfamily consists of four individual subunits, NR2A to NR2D. Competitive antagonists such as APV probably bind to one site, which may be distinct from the agonist recognition sites but isosterically coupled in such a way as to allow competitive interactions. Glycine is a coagonist at the glycineB site and prevents Ca2+-independent receptor desensitization. MRZ 2/576 is an example of a glycineB antagonist. Polyamines such as spermine and spermidine are positive modulators but also block the channel at higher concentrations. All heteromeric and homomeric NMDA receptor subtype complexes are permeant to Ca2+, Na+, and K+. The open NMDA channel is blocked by Mg2+ and uncompetitive NMDA receptor antagonists, such as memantine and (+)MK-801, in a voltage-dependent manner, although the speed and voltage dependence of this effect depend on the antagonist affinity and the subunit composition. Ifenprodil is a selective antagonist for NR2B-containing receptors. Zn2+ is a potent, voltage-independent antagonist at NR2A-containing receptors. In addition, most NMDA receptors are influenced by Zn2+ ions in a voltage-dependent manner, as well as by oxidation/reduction and pH.

The NMDA channel is blocked in a use- and voltage-dependent manner by Mg²⁺ (fig. 1). This means that NMDA receptors are activated only after depolarization of the postsynaptic membrane by, for example, AMPA receptor activation, which relieves the voltagedependent blockade by Mg²⁺. This biophysical property and their high Ca²⁺ permeability render NMDA receptors inherently suitable for their role in mediating synaptic plasticity, such as that underlying learning processes and development (Collingridge and Singer, 1990; Danysz et al., 1995b). Similar to Mg²⁺, uncompetitive NMDA receptor antagonists such as ketamine, dextromethorphan, memantine, phencyclidine (PCP), and (+)MK-801 [(+)5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate] block the NMDA channel in the open state, although the blocking kinetics and voltage dependence of this effect depend on the antagonist (Rogawski, 1993; Parsons et al., 1995).

To date, two major subunit families, designated NR1 and NR2, have been cloned. It is generally accepted that functional receptors in the mammalian CNS are only formed by combination of NR1 and NR2 subunits, which express the glycine and glutamate recognition sites, respectively (Kuryatov et al., 1994; Grimwood et al., 1995b; Wafford et al., 1995; Hirai et al., 1996; Williams et al., 1996; Laube et al., 1997; Anson et al., 1998) (figs. 1 and 2).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Schematic presentation of the NMDA receptor subunit family. The tree indicates the degree of homology. For clarity, a NR1 receptor subunit splice variant having all alternatively spliced exons, i.e., N1 (exon 5, 21 amino acids), C1 (exon 21, 37 amino acids), and C2 (exon 22, 38 amino acids), is shown (Zukin and Bennett, 1995). Therefore, the TM and point mutation positions are numbered taking into account the presence of the N1 exon, i.e., positions are shifted by 21 amino acids, compared with original publications. Amino acid residues crucial for the binding of glutamate at NR2A or NR2B subunits and that of glycine at NR1 subunits are shown. The marks (closed and open circles) indicate residues at which point mutations resulted in at least 100-fold decreases in the affinity of glycine or glutamate. Glutamate binding sites also exist in the NR2C and NR2D subunits, but their exact locations have not been defined and are therefore not indicated in the scheme.

The NR2 subfamily consists of four individual subunits, i.e., NR2A to NR2D (Nakanishi et al., 1990, 1992; Kutsuwada et al., 1992; Monyer et al., 1992; Hollmann and Heinemann, 1994; McBain and Mayer, 1994; Danysz et al., 1995a; Parsons et al., 1998b). Various heteromeric NMDA receptor channels formed by combinations of NR1 and NR2 subunits are known to differ in gating properties, magnesium sensitivity, and pharmacological profile (Sucher et al., 1996; Parsons et al., 1998b). The heteromeric assembly of NR1 and NR2C subunits, for instance, has much lower sensitivity to Mg²⁺ but increased sensitivity to glycine and very restricted distribution in the brain. In situ hybridization has revealed overlapping but different expression profiles for NR2 messenger ribonucleic acid (mRNA). For example, NR2A mRNA is distributed ubiquitously (like NR1), with the highest densities occuring in hippocampal regions, and NR2B is expressed predominantly in forebrain but not in cerebellum, where NR2C predominates; NR2D is localized mainly in the brainstem (Moriyoshi et al., 1991; Monyer et al., 1992; Nakanishi, 1992; McBain and Mayer, 1994). NMDA receptors cloned from murine CNS have a different terminology, compared with those from rats (Kutsuwada et al., 1992); 1 remains the designation for the mouse equivalent of NR1, and 1 to 4 represent NR2A to -2D subunits, respectively.

In addition to NR1 and NR2, the NR3A subunit has recently been discovered. This receptor subunit (previously termed chi-1, or NMDAR-L) is a relatively recently identified member of a new class in the ionotropic glutamate receptor family that attenuates NMDA receptor currents when coexpressed with NR1/NR2 subunits in Xenopus oocytes but has no effect when tested with non-NMDA receptors or when expressed alone (Ciabarra et al., 1995; Sucher et al., 1995; Das et al., 1998). This subunit has an open reading frame coding for a predicted polypeptide of 1115 amino acids (with a predicted mass of 110 kDa) and shares 23% homology with other NMDA subunits and 27% homology with non-NMDA subunits. Highest levels are present in the spinal cord, brainstem, hypothalamus, thalamus, CA1 field of the hippocampus, and amygdala, and this distribution remains the same throughout life. However, the absolute levels show a strong peak between postnatal days 7 and 14, which then declines to adulthood. This subunit is expressed as a glycosylated protein subunit (135 kDa) with a distribution that parallels that observed for its mRNA, as determined by in situ hybridization (Ciabarra and Sevarino, 1997). Genetic knockout of NR3A in mice results in enhanced NMDA responses and increased dendritic spines in early postnatal cortical neurons, suggesting that NR3A is involved in the development of synaptic elements by modulating NMDA receptor activity (Das et al., 1998).

Before the molecular biological features of NMDA receptors were resolved, it was reported (Sekiguchi et al., 1990) that NMDA receptors expressed from mRNA from guinea pig cerebellum (as opposed to other brain regions) do not show sensitivity to glycine. However, strychnine-insensitive glycine binding is present in the cerebellum (Bristow et al., 1986; Danysz et al., 1989c), and glycine potentiates electrophysiological responses to NMDA in the cerebellum in situ (Netzeband et al., 1990). It should be stressed that in the cerebellum there are mainly NMDA receptors containing NR2C subunits (with NR1), which show strong sensitivity to glycine (Matsui et al., 1995), and the failure of Sekiguchi et al. (1990) to observe further facilitation may have been the result of saturation of the glycine_B site by background levels of glycine.

Because of the location of the glycine recognition site on the NR1 subunit, this review focuses more on the molecular biological features and ontogeny of this subunit. For more information on the molecular biological characteristics of NR2 subunits, readers are referred to previous overviews (Seeburg, 1993; Hollmann and Heinemann, 1994; McBain and Mayer, 1994).

Alternative splicing generates eight isoforms for the NR1 subfamily (Nakanishi et al., 1992; Durand et al., 1993; Zukin and Bennett, 1995). The variants arise from splicing at three exons; one encodes a 21-amino acid insert in the amino-terminal domain (N1, exon 5), and two encode adjacent sequences of 37 and 38 amino acids in the carboxyl-terminal domain (C1, exon 21, and C2, exon 22, respectively) (figs. 1 and 2). NR1 variants are sometimes denoted by the presence or absence of these three alternatively spliced exons (N1, C1, and C2); NR1₁₁₁ has all three exons, NR1₀₀₀ has none, and NR1₁₀₀ has only the amino-terminal exon (Durand et al., 1993; Zukin and Bennett, 1995). The variants from NR1₀₀₀ to NR1₁₁₁ are alternatively denoted as NR1E, -C, -D, -A, -G, -F, -"H", and -B or NMDAR1-4a, -2a, -3a, -1a, -4b, -2b, -3b, and -1b, respectively, but the more frequently used terminology uses noncapitalized suffixes for the most common splice variants, i.e., NR1a (NR1₀₁₁, NMDAR1-1a, or NR1A) and NR1b (NR1₁₀₀, NMDAR1-4b, or NR1G). This terminology is very confusing, and in our opinion the most logical and useful is that proposed by Zukin and Bennett (1995), i.e., NR1_xxx. The human NR1 gene is composed of 21 exons distributed over a total length of approximately 31 kilobases. Exons 4, 20, and 21 are identical in their amino acid sequences to N1, C1, and C2, respectively, in rats, suggesting that all eight NR1 isoforms found in rats would also be expressed in the human brain (Lebourdelles et al., 1994; Zimmer et al., 1995). In contrast, others reported that the human NR1 sequence diverges from the rodent and murine homologues near the carboxyl terminus (Planellscases et al., 1993). Studies on the function of these splice variants in homomeric receptors expressed in Xenopus oocytes must be viewed with caution, because homomeric NR1 receptors (Durand et al., 1993; Planellscases et al., 1993; Rodriguez Paz et al., 1995) are probably only functionally expressed because of the presence of an endogenous NR2-like protein (XenU1) in these cells (Barnard, 1997; Soloviev and Barnard, 1997).

The highest levels of NR1 mRNA in the adult rat and mouse CNS are in the olfactory bulb, and the lowest levels are expressed in the spinal cord. Intermediate levels were found in frontal cortex, hippocampus, cerebellum, and whole brain (Franklin et al., 1993; Akazawa et al., 1994). Similar findings have been reported with antibodies to NR1 subunits (Petralia et al., 1994; Benke et al., 1995). mRNA for double-splice variants in the C1/C2 regions, such as NR1₀₁₁ (NR1a), show an almost complementary pattern with respect to those lacking both of these inserts, such as NR1₁₀₀ (NR1b); the former are more concentrated in rostral structures such as cortex, caudate, and hippocampus, whereas the latter are principally found in more caudal regions such as thalamus, colliculi, locus coeruleus, and cerebellum (Laurie and Seeburg, 1994b; Luque et al., 1995a; Paupard et al., 1997). Others reported that the predominant splice variants in cortex and hippocampus were those without the N1 insert (also NR1a), whereas in the cerebellum the major variant was NR1₁₀₀ (NR1b), containing N1 (Zhong et al., 1995). In other words, cell-specific patterns for NR1 mRNA lacking N1 inserts parallel those for mRNA containing C1/C2 inserts (NR1₀₁₁, NR1a) and vice versa (NR1₁₀₀, NR1b). In the hippocampus, NR1a mRNA shows high levels in all regions, whereas NR1₁₀₀ is expressed more intensely in CA3 pyramidal neurons (Paupard et al., 1997). mRNA for NR1₀₀₁ and NR1₁₀₁ splice forms is found nearly homogeneously throughout the adult CNS, whereas mRNA with alternative splicing at C1 but not C2 (NR1₀₁₀ and NR1₁₁₀) is scarce, being detected only at very low levels in postnatal cortex and hippocampus (Laurie and Seeburg, 1994b; Paupard et al., 1997). Important from a methodological perspective is the finding that the predominant splice variants in cultured cortical neurons are also those lacking the N1 insert, such as NR1₀₁₁ (NR1a) (Zhong et al., 1994).

In developing rats, NR1 mRNA levels in cortex and hippocampus increased nearly three-fold from postnatal day 3 to day 15 and approximately doubled from day 15 to day 67 (Franklin et al., 1993; Riva et al., 1994; Nowicka and Kaczmarek, 1996). In contrast, cerebellum and brainstem showed no change in NMDAR1 mRNA levels between postnatal days 3 and 15 but levels also doubled from day 15 to day 67 (Franklin et al., 1993). Similar results were reported by a different group, although levels in the hippocampus peaked at postnatal day 10 and declined thereafter (Pujic et al., 1993). In the hippocampus, NR1 mRNAs lacking the N1 insert (such as NR1₀₁₁) dominate at birth and exhibit mature patterns of labeling, with high levels of expression in the CA1 and CA3 regions and the dentate gyrus. In contrast, mRNAs containing this insert (such as NR1₁₀₀) are initially expressed at lower uniform levels but levels increase more in the CA3 region than in the CA1 region or the dentate gyrus in the second and third postnatal weeks (Paupard et al., 1997).

Antisera against the carboxyl- and amino-terminal domains of NR1 receptors revealed similar distributions, which increased strongly in most brain regions until postnatal day 21; the exception was that carboxyl-terminal domain staining decreased in the thalamus, tectum, and brainstem, possibly because of the emergence of carboxyl-terminal splice variants not recognized by the antiserum (Benke et al., 1995; Luo et al., 1996). Interestingly, the amino acid sequences contained within the seven-amino acid, carboxyl-terminal domain of C1 NR1 splice variants and all NR2 subunits may serve to localize NMDA receptors to synaptic domains by interactions with postsynaptic density protein-95 (Ehlers et al., 1995; Kornau et al., 1995). An important finding is that the absolute density of NR1 receptors detected with antisera is close to that found using [³H]MK-801 binding, suggesting that most of the NR1 subunits expressed in the brain exist in an active form (Luo et al., 1996).

NR1 subunit immunostaining in the rat visual cortex is associated with the plasma membrane at early stages of development, before innervation by axons, whereas clustering of receptors at junctions may be promoted by axonal contact (Aoki et al., 1994; Aoki, 1997). At all ages, the prevalence of NR1-immunoreactive profiles was lamina 1 > laminae 4/5 > laminae 6/6B (Aoki et al., 1994). In contrast, others reported that cat and ferret cortical neurons initially show high levels of immunostaining for NR1, which then decline gradually during development, with the notable exception of cortical layers 2/3, where levels of NMDAR1 immunostaining remain high into adulthood (Catalano et al., 1997).

In human embryonic kidney (HEK) 293 cells expressing homomeric NMDAR1 receptors, significant levels of specific binding of the glycine_B antagonists ³H-labeled L-689,560 [4-trans-2-carboxy-5,7-dichloro-4-phenylamino-carbonylamino-1,2,3,4-tetrahydroquinoline] (fig. 3) and MDL-105,519 [(E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1H-indole-2-carboxylic acid] (fig. 4) but not of glutamate antagonists or [³H]MK-801 were seen (Grimwood et al., 1995b; Siegel et al., 1996). Similarly, Lynch et al. (1994) reported, in HEK 293 cells transfected with the NR1 subunit, significant binding to the glycine_B site [³H-labeled 5,7-dichlorokynurenic acid (5,7-diCl-KYN)] (fig. 5) but not to the NMDA site [³H-labeled CGP-37849 [DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid]] or the PCP site ([³H]MK-801).

View larger version (9K): [in this window] [in a new window]   Fig. 3.   2-Carboxytetrahydroquinoline antagonists of the glycineB site.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   2-Carboxyindole (CI) antagonists of the glycineB site.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Selected kynurenic acid derivatives that are antagonists of the glycineB site.

It should also be noted that NR2 subunits contribute to the affinity of glycine. With homomeric NR1 receptors the glycine K_i is in the range of 1 to 5 µM, but with some heteromeric receptors it is in the nanomolar range (Grimwood et al., 1995b). Interestingly, glycine affinity with wild-type receptors is usually higher, i.e., 100 to 300 nM (Kishimoto et al., 1981; Danysz et al., 1990; Grimwood et al., 1992).

Site-directed mutagenesis of the NR1 subunit at residues corresponding to positions forming the binding site of homologous, bacterial, amino acid-binding proteins indicates conservation of a common amino acid-binding fold from prokaryotic periplasmic proteins to glutamate receptors in the mammalian brain (Kuryatov et al., 1994). Glutamine substitutions at position 387 in NR1 subunits reduce glycine affinity (Kuryatov et al., 1994). Similarly, replacement of cysteines 402 and 418 by alanine largely abolishes the potentiation of glutamate currents by glycine (Laube et al., 1993) (fig. 2). These residues in the amino-terminal domain of NR1 subunits are extracellular before transmembrane domain (TM)1. It should be noted that these mutations in NR1 subunits had little effect on glutamate binding or the affinity of the glycine_B antagonist 7-chlorokynurenic acid (7Cl-KYN) (fig. 5). However, site-directed mutagenesis of the NR1 subunit expressed in Xenopus oocytes revealed that aromatic residues at positions 390, 392, and 466 are crucial determinants of both agonist and antagonist binding, as observed in patch-clamp experiments (Kuryatov et al., 1994). Glutamate efficacy was little affected by these mutations, but inhibition by 7-Cl-KYN was also greatly reduced (Kuryatov et al., 1994). Very similar findings were reported for human NR1 receptors, expressed in Xenopus oocytes, with mutation of residues 481 and 483 (Wafford et al., 1995).

Valine and serine substitutions in NR1 subunits at positions 666 and 669, respectively (in the loop between TM1 and TM4) (figs. 1 and 2), were also found to reduce glycine efficacy (Kuryatov et al., 1994). Others found that alternative mutation of D669 in NR1a to asparagine, alanine, or glutamate had little effect on the potency of glycine but abolished the "glycine-independent" form of spermine stimulation, indicating the importance of this residue for the binding of polyamines and/or the formation of part of the proton sensor (Kashiwagi et al., 1996). Mutation of alanine at position 714 also greatly reduced the apparent affinity for glycine (Wood et al., 1997), as did substitutions of the phenylalanine residues at positions 735 or 736 (Hirai et al., 1996). Interestingly, these mutations did not alter the affinity of 7-Cl-KYN, indicating that this part of the extracellular domain contributes to glycine binding but not antagonist binding. Mutation of D732 to glutamate (D732E), asparagine (D732N), alanine (D732A), or glycine (D732G) not only dramatically reduced the potency of glycine but also changed the sensitivity to other glycine site agonists (and, in some cases, their efficacy) (Williams et al., 1996) (fig. 2). For example, D-serine was a full coagonist at receptors containing NR1(D732N) and NR1(D732A), a partial agonist at receptors containing NR1(D732G), and a competitive antagonist at receptors containing NR1(D732E) (Williams et al., 1996). All of these residues are found in the extracellular M3-M4 loop (figs. 1 and 2).

Homology-based molecular modeling of the glutamate and glycine binding domains indicates that the NR2 and NR1 subunits use similar residues to form their respective agonist binding sites. Therefore, similar mutations of residues within the amino-terminal domain (E387A and K459E) and the loop region between segments TM3 and TM4 (S664G) of NR2B subunits reduced the potency of glutamate >100-fold but had no effect on glycine affinity (Laube et al., 1997). Mutations in NR2A subunits (T671A) dramatically reduced glutamate potency and produced faster deactivation kinetics, without changing channel gating or the affinity for glycine (Anson et al., 1998). Similarly, NR2 subunits containing mutations at NR2A position D731 and NR2B position D732, which correspond to NR1 position D732, did not produce functional receptors when coexpressed with NR1 (Williams et al., 1996). Taken together, these results indicate that the extracellular region before TM1 and the extracellular TM3-TM4 loop form a ligand-binding pocket for glutamate and glycine in NR2 and NR1 subunits, respectively; these findings provide the basis for a refined model for agonist and coagonist binding sites of the NMDA receptor (Hirai et al., 1996).

The absolute stoichiometry of NMDA receptor subunits is not clear. It is widely accepted that NMDA receptors are activated only after the binding of glutamate to at least two NR2 subunits for each receptor (Mayer et al., 1989b; Javitt et al., 1990; Patneau and Mayer, 1990; Curras and Dingledine, 1992; Sather et al., 1992; Wafford et al., 1993; Hirai et al., 1996; Laube et al., 1997), and the same is true for glycine as a coagonist at NR1 subunits (Thedinga et al., 1989; Benveniste et al., 1990a,b; Benveniste and Mayer, 1991; Clements and Westbrook, 1991; Siegel et al., 1996; Williams et al., 1996). This assumption is normally made on the basis of Hill coefficients for steady-state responses, which are considerably greater than unity. Similarly, sigmoidal activation kinetics of NMDA channels in outside-out patches from cultured hippocampal neurons were best fitted by a kinetic model with two glutamate binding sites and two glycine sites, with agonist and coagonist binding being better described by an independent, rather than a sequential, model (Clements and Westbrook, 1991). Taken together, these data led to the hypothesis that the NMDA receptor is at least a tetramer containing four ligand-binding subunits, with a single binding site in each subunit (Clements and Westbrook, 1991). In contrast, others have reported Hill coefficients of less than unity for glycine (Lerma et al., 1990), and detailed analysis of glycine concentration-response curves and kinetics indicated Hill coefficients for glycine of 1.1, with a corrected affinity of 130 nM, consistent with the idea that there is only one glycine binding site (Johnson and Ascher, 1992).

	III. Physiological Role of Glycine

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Kleckner and Dingledine (1988) were the first to report that glycine is essential for activation of the NMDA receptors. Using Xenopus oocytes injected with whole-brain mRNA, those authors observed that the response to NMDA vanished when the contamination with glycine was reduced to negligible levels. A similar conclusion regarding the necessity of glycine for the activation of NMDA receptors was later drawn on the basis of patch-clamp studies in neuronal cultures (Mayer et al., 1989a; Henderson et al., 1990; Huettner, 1990; Vornov and Coyle, 1991; Aoshima et al., 1992; Chen et al., 1997). Further evidence that the activation of the glycine_B site is a prerequisite for NMDA receptor activation in vitro or in vivo was obtained by showing that selective antagonists of the glycine_B site completely block the effects of NMDA receptor stimulation (see Section IV.C.). This suggests that residual responses in the nominal absence of glycine are the result of contamination with low background levels of glycine (Benveniste et al., 1990a; Lerma et al., 1990; Kemp and Priestley, 1991; Parsons et al., 1993, 1997; Molnar and Erdo, 1996). Indeed, extrapolation of the lower linear part of glycine concentration-response curves indicates that approximately 20 to 40 nM glycine is found in the extracellular solution, a value that agrees very well with that measured by high pressure liquid chromatography (Benveniste et al., 1990). This provides support for the notion that glycine is an essential coagonist at the NMDA receptor and that responses to NMDA cannot be obtained in the complete absence of glycine (Mayer et al., 1989a,b; Vornov and Coyle, 1991; Aoshima et al., 1992). However, it remains a remote possibility that blockade of the residual peak in the nominal absence of glycine by glycine_B site full antagonists is the result of inverse agonistic effects and that a component of the peak response would still be present in the complete absence of glycine (Kemp et al., 1988b; Mayer et al., 1989a).

The binding of use-dependent NMDA receptor channel blockers, such as [³H]MK-801, under nonequilibrium conditions can be used to investigate certain aspects of receptor function; levels of bound ligand are proportional to the degree of activation (Foster and Wong, 1987; Wong et al., 1988). Using this method, in most cases it was only possible to demonstrate the essential role of glycine by blocking this site with an antagonist, probably because of widespread contamination with glycine. However, in extensively washed membranes it was shown that glutamate and glycine failed to enhance [³H]MK-801 binding when used separately but did produce enhancement when applied together (Ratti et al., 1990). On the other hand, spermine enhanced functional [³H]MK-801 binding in the presence of the glycine_B antagonist 7-Cl-KYN but not when the NMDA site was blocked by 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP) (Marvizon and Baudry, 1993). It was suggested by the authors of the latter study that glycine is not an absolute requirement for NMDA receptor activation. However, binding is probably not the optimal method to explore such questions, because, for example, [³H]MK-801 has been reported to access the NMDA channel in a closed state via the so-called "lipophilic pathway" (Javitt and Zukin, 1989).

It is also noteworthy that, although glycine concentrations up to 3 mM do not activate NMDA receptors in vitro without NMDA agonists, there are indications that at concentrations above these levels glycine can actually damage neurons (Kleckner and Dingledine, 1988; McNamara and Dingledine, 1990; Wallis et al., 1994; Newell et al., 1997) and induce inward currents in cultured hippocampal neurons (Pace-Asciak et al., 1992), via activation of both glycine_B and NMDA sites. It has also been suggested that, under certain conditions, glycine might achieve such high levels and contribute to neurotoxicity in vivo (Newell et al., 1997).

In vivo activation of glycinergic inhibitory interneurons in the spinal cord by stimulation of 1b afferents elicited a classical, short-latency, glycinergic inhibitory postsynaptic potential followed by an NMDA receptor-mediated EPSP. The EPSP was blocked by ketamine and R(+)HA-966 [R(+)-3-amino-1-hydroxypyrrolidin-2-one] and showed classical voltage dependence. The authors proposed that glycine released at inhibitory interneurons spills over to activate nonsaturated glycine_B sites in vivo. This interpretation is controversial, because 1b afferents also activate excitatory interneurons and NMDA EPSPs are always delayed because of slower activation kinetics (Fern et al., 1996).

Glycine greatly potentiates NMDA receptor-mediated responses by reducing desensitization both in native mammalian neurons and in Xenopus oocytes or HEK 293 cells expressing NMDA receptors (Mayer et al., 1989a; Vornov and Coyle, 1991; Aoshima et al., 1992; Chen et al., 1997) (fig. 6). Glycine-sensitive desensitization is accompanied by a five- to seven-fold decrease in the affinity of the glycine_B site in the presence of agonists for the NMDA site (Lerma et al., 1990; Parsons et al., 1993). With higher concentrations of glycine, the magnitude of this desensitization is decreased but the rate becomes faster. Furthermore, recovery from desensitization after step increases in the concentration of glycine or lower affinity glycine agonists in the continuous presence of NMDA reflects the association kinetics of the agonist concentrations used (Lerma et al., 1990; Parsons et al., 1993). Therefore, desensitization probably occurs rapidly upon binding of both glutamate and glycine, and the apparent rate reflects the balance between slow dissociation from and concentrationdependent reassociation of glycine with the altered receptor. In other words, at higher concentrations of glycine, the forward rate constant for rebinding greatly exceeds the rate of dissociation and the time course of desensitization appears to be faster. The affinities of agonist to induce desensitization are five-fold higher than their respective affinities as agonists at the peak of the response (Chizhmakov et al., 1992).

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Patch-clamp experiments with four different cultured hippocampal neurons, showing that glycineB antagonists have different effects on glycine-sensitive desensitization. NMDA (200 µM) was applied with a rapid concentration clamp (exchange times, 20 msec) for 2.5 sec every 30 sec, in the continuous presence of glycine (0.03 to 1 µM), with low Ca2+ levels (0.2 mM) and a constant membrane potential of 70 mV. Responses at equilibrium have been superimposed for each concentration of glycine or glycineB antagonist tested (continuously present for at least 2 min). Reduction of glycine concentrations reveals glycine-sensitive desensitization. 5,7-diCl-KYN (5,7-DCKA) (0.3 to 3 µM) concentration-dependently reveals glycine-sensitive desensitization in the continuous presence of glycine (1 µM). The higher affinity glycineB antagonist MDL-105,519 (0.1 to 1 µM) reveals less pronounced glycine-sensitive desensitization in the continuous presence of glycine (1 µM). The low intrinsic activity, glycineB site partial agonist R(+)HA-966 (10 to 300 µM) does not reveal glycine-sensitive desensitization in the continuous presence of glycine (1 µM). Note that the current scale bars are different for each antagonist tested.

The opposite has been reported in binding experiments, namely a mutually positive allosteric interaction, with glutamate increasing glycine affinity and glycine increasing glutamate affinity (table 2). Moreover, other electrophysiological studies concluded that desensitization involves structural changes in the channel-lining section of the protein, rather than the glycine or NMDA binding sites, because the induction of desensitization was dependent on channel opening (Zilberter et al., 1991). This seems unlikely, because single-channel recordings and fluctuation analysis show an increase in opening frequency with no change in mean open time or conductance in the presence of glycine and the opposite in the presence of glycine_B antagonists, suggesting that glycine regulates transitions to states that are intermediate between the binding of NMDA receptor agonists and ion-channel gating (Mayer et al., 1989a; Vornov and Coyle, 1991; Parsons et al., 1993). It has also been claimed that aspartate induces desensitization in the absence of glycine, but this interpretation is again complicated by the presence of background levels of glycine in all experiments (Chizhmakov et al., 1992).

A similar form of desensitization is seen in the presence of some glycine_B site full antagonists (Kemp and Priestley, 1991; Parsons et al., 1993), but these vary in their ability to induce glycine-sensitive desensitization. L-689,560, L-701,324 [7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1H)-quinolinone], L-695,902 [7-chloro-4-hydroxy-3-methoxycarbonyl-2(1H)-quinolinone] (fig. 7), and RPR-104,632 [2-(3-bromobenzyl)-6,8-dichloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-1,1-dioxide-3-carboxylic acid] (fig. 5) reveal little or no desensitization but other compounds, such as 7-Cl-KYN and 5,7-diCl-KYN, show 10-fold higher potencies against plateau responses (Molnar and Erdo, 1996; Karcz-Kubicha et al., 1997). Other compounds, such as ACEA 1021 (5-nitro-6,7-dichloro-1,4-dihydro-2,3-quinoxalinedione) (fig. 8), MRZ 2/571 [8-fluoro-4-hydroxy-1-oxo-1,2-dihydropyridazinol[4,5-b]quinoline-5-oxide (choline salt)], and MRZ 2/576 [8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazinol[4,5-b]quinoline-5-oxide (choline salt)] (fig. 9), induce moderate desensitization. The glycine_B site partial agonist R(+)HA-966 (fig. 10) does not induce desensitization but produces a three-fold allosteric reduction in the affinity of agonists for the glutamate recognition site, which is reflected by slowing of the response onset rise time and acceleration of offset kinetics (Kemp and Priestley, 1991). Taken alone, this result could be considered indicative of a negative reciprocal interaction between the glycine and glutamate recognition sites. However, D-cycloserine and 1-aminocyclopropanecarboxylic acid (ACPC), which are partial agonists with higher intrinsic activity (fig. 10), are also unable to induce desensitization (Karcz-Kubicha et al., 1997) but have intermediate effects on glutamate deactivation kinetics, in line with their relative intrinsic activities (Priestley and Kemp, 1994). Furthermore, the full antagonists L-701,324 and L-695,902 (fig. 7) were recently reported to decrease glutamate affinity in a manner similar to that of R(+)HA-966 (Priestley et al., 1996). The same group reported a reciprocal three-fold interaction between partial agonists at the NMDA recognition site and glycine affinity, with the off-rate of glycine being fastest in the presence of a saturating concentration of the competitive NMDA receptor antagonist cis-2,3-piperidinedicarboxylic acid and progressively slower in the presence of quinolinate, NMDA, and L-glutamate (Priestley and Kemp, 1994). Taken together, these data were interpreted as being more in agreement with binding studies showing reciprocal positive interactions between full agonists at the NMDA and glycine recognition sites, with the extent of the modulation of one site by the other being related to the intrinsic activity of the agonist used, rather than its affinity.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   4-Hydroxyquinolone antagonists of the glycineB site. HNQ, 4-hydroxy-3-nitroquinolin-2(1H)-one.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Quinoxaline-diones and related compounds that are antagonists of the glycineB site. CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; HBAD, 3-hydroxy-1H-1-benzazepine-2,5-dione.

View larger version (14K): [in this window] [in a new window]   Fig. 9.   Tricyclic antagonists of the glycineB site.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   Selected agonists and partial agonists of the glycineB site. MSD low effic PA, Merck Sharp and Dohme low efficiency partial agonist.

However, it should be stressed that the comparisons in offset kinetics were all made at steady state and did not address the relative changes in apparent affinities at the peak and plateau of the response. The same is true for binding studies, which all assessed affinities at steady state under highly nonphysiological conditions. Alternatively, the fact that R(+)HA-966 and D-cycloserine do not exhibit glycine-sensitive desensitization (fig. 6) could be the result of both their low potency and allosteric slowing in onset kinetics. It may be that an agonist-induced change in the affinity of the glycine site still occurs and would be seen as very fast desensitization, reflecting the rapid unbinding of this low affinity partial agonist, if it were not masked by the slower rise time of the response. This idea is supported by the fact that desensitization is seen with intermediate concentrations of the higher affinity partial agonist ACPC in the absence of glycine (Karcz-Kubicha et al., 1997). A similar argument can be applied to the apparent lack of desensitization seen with some glycine_B site full antagonists. We previously showed a trend for less desensitization with higher potency full antagonists (Parsons et al., 1997) (fig. 6). In this case, the association of low concentrations of antagonist after agonist-induced dissociation of glycine is much slower than the forward rate constant for reassociation of glycine. Therefore, glycine-sensitive desensitization would be predicted to be revealed with a much slower time course.

Several studies found that the kinetics of NMDA receptor currents in outside-out patches are dominated by a pronounced glycine-insensitive form of desensitization (Sather et al., 1991, 1992; Lester et al., 1993; Tong and Jahr, 1994). This is particularly evident after longer recording periods, indicating the importance of dialysis of intracellular factors in mediating this effect. However, the glycine and glutamate recognition sites are still allosterically coupled, as evidenced by changes in the offset kinetics of glycine in the presence of glutamate (Lester et al., 1993). The appearance of glycine-insensitive desensitization is reduced by intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (EDTA) or preincubation of neurons with the Ca²⁺ release inhibitor dantrolene, suggesting that this form of desensitization is triggered by a transient increase in intracellular Ca²⁺ levels. The extent of glycine-insensitive desensitization is also reduced by intracellular application of adenosine-5'-O-(3-thio)-triphosphate, the phosphatase inhibitor microcystin, or a peptide inhibitor of calcineurin, implying regulation by the phosphorylation state of the receptor (Tong and Jahr, 1994). Moreover, this form of desensitization seems to be dependent on activation of G proteins (Turecek et al., 1995). This may explain the relatively robust glycinesensitive desensitization seen in the study of Parsons et al. (1993), because single-channel recordings were first made after 20 to 30 min of whole-cell recording with low extracellular Ca²⁺ levels and high intracellular ethylene glycol bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, adenosine-5'-triphosphate, and 3',5'-cyclic adenosine monophosphate levels, i.e., under conditions chosen to selectively investigate glycine-sensitive desensitization.

The improved therapeutic profiles of some systemically active, glycine_B site full antagonists could be the result of their abilities to reveal glycine-sensitive desensitization (Parsons et al., 1993). Receptor desensitization may represent a physiological process serving as an endogenous control mechanism to prevent long-term neurotoxic activation of glutamate receptors but allow their transient physiological activation. Interestingly, ischemia increases not only the concentration of extracellular glutamate but also that of glycine and, although this latter effect is less pronounced, it persists for much longer (Globus et al., 1991b). Prolonged repetitive activation of NMDA receptors during ischemia would be effectively reduced at concentrations of glycine_B site full antagonists having less effect on more transient activation during EPSPs, because the time course for glycine-sensitive desensitization (300 to 500 msec) (Mayer et al., 1989a) is somewhat longer than that for NMDA receptor-mediated synaptic events (typically 100 to 200 msec) (Clements et al., 1992). This property may also allow such compounds to differentiate between various forms of NMDA receptor-mediated synaptic plasticity, e.g., to block drug tolerance and dependence and chronic pain states at concentrations having less effect on learning and memory.

Glycine has been traditionally regarded as an inhibitory transmitter in lower CNS regions, where it activates receptors forming chloride channels (Leu et al., 1987). Only during the past 10 years has its function as a positive modulator acting at NMDA receptors been recognized (see Section I.). Meanwhile, high free D-serine (fig. 10) levels, i.e., concentrations of 2 to 300 µM, depending on the brain structure and experimental conditions, have been detected in the mammalian (including human) CNS (for review, see Hashimoto and Oka, 1997). In turn, D-serine has been suggested as a possible endogenous ligand for the glycine_B site. Here the features crucial for dynamic regulation of the concentration of both ligands are discussed in parallel, according to the criteria given in table 3.

It is noteworthy that dynorphin(1-13) was also recently proposed as an endogenous agonist at the glycine_B site, as evidenced by very pronounced increases in the amplitude of NMDA-activated currents in Xenopus oocytes in the presence of low extracellular glycine concentrations (Zhang et al., 1997). Such an effect may exacerbate the well documented spinal toxicity seen with dynorphin peptides after intrathecal administration (Shukla and Lemaire, 1994), although the vasoconstrictive actions of this peptide are probably also important.

To determine the physiological importance of either glycine or D-serine, their extracellular CNS levels should be compared with their affinities. Affinity is often difficult to assess accurately, because of variable levels of contamination with glycine, which have been estimated to be between 20 and 130 nM in binding and patch-clamp studies (Benveniste et al., 1990; Lerma et al., 1990; Johnson and Ascher, 1992; Parsons et al., 1993; Berger, 1995). In the majority of binding studies in brain homogenates, the affinity of glycine is slightly (two- to three-fold) higher than that of D-serine (Kishimoto et al., 1981; Danysz et al., 1990) (table 4). On the other hand, some authors report that, in experiments with recombinant NMDA receptors, D-serine seems to be slightly more potent as an agonist than glycine (Matsui et al., 1995) (but see table 5).

1. Distribution within the central nervous system. In brain homogenates, glycine has been reported to be present at concentrations of 6 to 10 nmol/mg of protein in mice (Saransaari and Oja, 1994), 1 nmol/mg of wet tissue in rats (Katsura et al., 1992), and 27 nmol/mg of protein in humans (Waziri et al., 1993). In the same preparation in rats, D-serine concentrations range from 10 µM in the cerebellum to 400 µM in the cerebral cortex (Nagata et al., 1994b). In adult human brain homogenates, D-serine is present at 66 to 130 µM (Hashimoto et al., 1993c; Nagata et al., 1994b; Hashimoto and Oka, 1997) or 2 to 15 nmol/mg of protein (Chouinard et al., 1993; Waziri et al., 1993). It should be kept in mind that homogenate concentrations indicate very little regarding free extracellular levels, because accumulation in intracellular compartments is likely (for example, see Danysz et al., 1997). In human cerebrospinal fluid (CSF), free glycine levels of 7 to 10 µM have been detected (Ferraro and Hare, 1985).

2. Uptake. Early studies demonstrated the presence, in the cerebral cortex (Hagan et al., 1988), cerebellum (Wilson et al., 1976; Wilkin et al., 1981), and hippocampus (Toth and Lajtha, 1986), of a sodium-dependent glycine uptake system that did not seem to transport D-serine. In the hippocampus, both low affinity (minor Na⁺ dependence) and high affinity (strong Na⁺ dependence) carriers were detected (Fedele and Foster, 1992). Based on lesion studies, it was concluded that this carrier is localized on both neurons and glia (mainly astroglia) (Magnuson et al., 1988). A similar conclusion was derived from studies on cultured cerebellar granule cells and astrocytes (Eberhard and Holtz, 1988) and autoradiographic experiments with [³H]glycine in the cerebellum (Wilkin et al., 1981).