11.1. INTRODUCTION

A number of natural and synthetic molecules including polyamines, protons, Zn²⁺, steroids, redox reagents, ifenprodil, and ethanol are modulators of NMDA receptors (NMDARs), acting at extracellular sites on the receptors to increase or decrease macroscopic currents and Ca²⁺ flux through NMDA channels. Some of these modulators, for example Zn²⁺, polyamines, and protons, are endogenous molecules that may exert important regulatory effects on NMDARs under physiological and/or pathological conditions. Others, for example ifenprodil, are synthetic molecules that serve as experimental tools to study the properties of NMDARs and receptor subtypes and may provide lead compounds for the development of therapeutically useful NMDAR antagonists.

This chapter provides an overview of some of these modulators, with a focus on polyamines, ifenprodil, and Zn²⁺ and on recent studies detailing their mechanisms of action, interactions, and proposed sites of action on NMDARs. Much of the early work in this area was reviewed previously [1–11] and is not dealt with in detail in this chapter. Similarly, the list of citations at the end of this chapter is not exhaustive; rather, it draws on recent and older publications to present an overview of how the field has developed and where it stands at present (mid 2007).

11.2. POLYAMINES

Effects of the endogenous polyamines, spermidine and spermine (Figure 11.1) on NMDARs were first observed in ligand binding assays in which the polyamines were found to increase binding of the use-dependent open-channel blockers [³H]MK-801 and [³H]TCP [12–15]. It was proposed that polyamines bind to unique sites on NMDARs distinct from the agonist binding sites to potentiate receptor activity and thus potentiate binding of [³H]MK-801 [12,13]. Subsequent work demonstrated effects of polyamines on the function of NMDARs studied electrophysiologically using both native and recombinant receptors [16–27]. In studies of macroscopic NMDA currents recorded from neurons and oocytes or mammalian cells expressing recombinant receptors, four effects of spermine were described and the effects could be studied in relative isolation by adjusting experimental conditions such as the membrane potential and agonist concentration.

FIGURE 11.1

Structures of the endogenous polyamines, spermidine and spermine (A), and of ifenprodil and related antagonists that selectively inhibit NR1/NR2B receptors (B).

One effect of spermine is to increase the size of whole-cell currents evoked by saturating concentrations of glutamate and glycine, so-called glycine-independent stimulation—somewhat of a misnomer since it requires glycine. The key point is that stimulation is seen in the presence of saturating concentrations of glycine (mechanism 1, Figure 11.2) [17–20,22,24–27]. At least part of this effect involves a decrease in desensitization of NMDARs in the presence of spermine [19]. Spermine can also alter deactivation of NMDARs [28].

FIGURE 11.2

Model of the NMDAR to illustrate some of the effects and interactions of the various binding and modulatory sites. The receptor is gated by coagonists glutamate (Glu) and glycine (Gly), and currents are potentiated or inhibited by spermine, ifenprodil, (more...)

Protons inhibit NMDARs, with a tonic inhibition of 40 to 50% at physiologic pH (mechanism 2, Figure 11.2), and there is evidence that spermine stimulation involves a relief of tonic proton inhibition (mechanism 3, Figure 11.2) [29,30]. This stimulation is subunit-dependent and is seen at NR1/NR2B receptors but not at binary NR1/NR2 receptors containing NR2A, NR2C, or NR2D [26,27,31]. Furthermore, the effects of spermine and protons are both reduced in NR1/NR2B receptors containing splice variants of the NR1 subunit that include the exon-5 insert [18,24,30]. This 21-amino acid insert is located in the extracellular amino terminal domain (ATD) or regulatory (R) domain of the NR1 subunit (Figure 11.3A). The exon-5 insert, containing six basic (Lys or Arg) residues [32,33], may act as a spermine-like moiety, changing the conformation of the R domain and thus its interactions with other domains, or the insert may interfere with the binding of spermine or the modulatory effects of protons (mechanism 4, Figure 11.2).

FIGURE 11.3

Domain-based structures of NMDAR subunits. A: Schematic of an NMDAR subunit with two large extracellular domains—the regulatory R domain (comprised of R1 and R2) and the S1/S2 agonist binding domain—and a membrane-spanning/pore-forming (more...)

If spermine stimulation involves a relief of tonic proton inhibition (mechanism 3, Figure 11.2), there is a conundrum because spermine can alter deactivation and desensitization of NMDARs including recombinant NR1/NR2B receptors [19,29] whereas protons do not affect deactivation or desensitization of these receptors [34]. Relief of proton inhibition may represent only part of the mechanism of spermine stimulation at these receptors and changes in deactivation and desensitization involving proton-insensitive gating mechanisms may also be involved.

A second effect of spermine is so-called glycine-dependent stimulation seen in the presence of subsaturating concentrations of glycine (mechanism 5, Figure 11.2) [16,22,26]. This effect appears to be due to an increase in the affinity of the receptor for glycine (mechanism 5, Figure 11.2) and is distinct from glycine-independent stimulation (mechanism 1) because it is seen at both NR1/NR2A and NR1/NR2B receptors (as opposed to only NR1/NR2B), it is not affected by the presence of the exon-5 insert in NR1, and it is not sensitive to extracellular pH [7]. Thus, it may involve a second, distinct spermine binding site (Figure 11.2).

A third effect of spermine is to reduce the sensitivity to glutamate (or other glutamate site agonists) at NR1/NR2B receptors (not illustrated), presumably by reducing the affinity of the receptor for glutamate [25]. The result is that spermine stimulation is smaller at subsaturating concentrations of glutamate than at saturating concentrations. This effect is seen at NR1/NR2B receptors but not at NR1/NR2A, NR1/NR2C, or NR1/NR2D receptors and is likely mediated via the spermine binding site responsible for glycine-independent stimulation (mechanism 1) [25,31].

The fourth effect of spermine is voltage-dependent channel block due to binding of spermine within the ion channel pore at a site near to or overlapping the binding sites for extracellular Mg²⁺ (Figure 11.2) [21,22,35–37]. Voltage-dependent block by spermine and by related natural and synthetic polyamines has been reported for different types of cation channels including some subtypes of AMPA receptors, kainate receptors, and inward rectifier K⁺ channels that are blocked by intracellular spermine [7,38–46]. Spermine is a weak blocker of NMDA channels compared to the other types of cation channels.

Whether endogenous polyamines are involved in modulation of NMDARs in vivo is still unknown. The effects of intracellular polyamines on AMPA channels and inward rectifier K⁺ channels where polyamines are responsible for rectification, are well-established [7,38,47,48]. The role, if any, of extracellular polyamines in the nervous system is unclear. Selective polyamine transport systems have been identified in neurons and glia [49] and depolarization-evoked release of polyamines has been described [1,7,49]. Since NR1/NR2B receptors are most sensitive to polyamine stimulation and NR2B is the predominant subunit expressed in embryonic and neonatal forebrain [50–52], polyamines have pronounced effects during the development of the nervous system—a time when polyamine levels in the brain are higher than in the adult nervous system—and may conceivably play a role in NMDA-dependent plasticity during development.

11.3. STRUCTURES OF NMDA RECEPTOR SUBUNITS AND RELEVANCE TO THE POLYAMINE SITE

Mutations at a number of residues in the R domain of the NR1 subunit can reduce spermine stimulation and reduce sensitivity to protons; these residues may form part of a spermine binding site in the R domain of NR1 [53]. The R domains of glutamate receptor subunits are proposed to be bilobar, similar in structure to the S1/S2 domains, based on homology with bacterial amino acid–binding proteins such as the leucine–isoleucine–valine binding protein (LIVBP) as well as weak homology with the amino terminal domain of the metabotropic glutamate receptor-1 [53–57].

X-ray crystallographic studies show that the S1/S2 domains of several glutamate receptors including NMDARs do indeed have such bilobed or clamshell structures [58–65]. The S1/S2 domains (and presumably entire subunits) of NR1/NR2A receptors assemble as dimers of heterodimers, i.e., as two sets of NR1/NR2A dimers [62] (Figure 11.3B). Although no high resolution structural data are available for a glutamate receptor R domain, evidence from single-particle electron microscopy supports the idea that the R domains can exist as dimers (presumably corresponding to the S1/S2 dimers to which they are attached) and, interestingly, that their positions or orientations can change during receptor activation or desensitization [66].

Evidence from biochemical studies also suggests that R domains can assemble as dimers but, at least in the case of NR1/NR2A receptors, as homodimers (i.e., NR1–NR1 and NR2A–NR2A) or that entire subunits can assemble as homodimers with the R domain being important for assembly [67–69]. At first glance, this appears to contradict data from x-ray crystallographic studies of NR1/NR2A receptors in which the isolated S1/S2 domains are assembled as heterodimers (i.e., NR1–NR2A; Figure 11.3B) [62]. It is possible that homodimers of NR subunits are formed during the initial assembly and trafficking of the subunits, followed by later formation of heterodimers and assembly of the final, intact four-subunit receptor, or that the formations of some types of dimers are artifacts of the experimental conditions. Another possibility is that heterodimers are formed initially involving contacts between S1/S2 domains of native NR1 and NR2 subunits and that homodimerization of the R domains is involved in subsequent assembly of tetrameric NMDARs. This would differ from the assembly of AMPAr channels in which the R domains are important for the formation of initial heterodimers and contacts between the S1/S2 domains (together with other determinants) are important for the subsequent assembly of heterodimers into tetramers [57,70]. In this context, it is notable that the fundamental gating mechanisms of NMDARs and AMPA receptors are different. NMDARs undergo concerted channel opening, requiring the simultaneous binding of two molecules of glutamate and two molecules of glycine. AMPARs undergo subunit-specific gating in which binding of glutamate to one subunit causes partial opening, binding to two subunits causes further opening, and so on, with full opening requiring binding of glutamate to all four subunits [71–75]. Based on the fundamental differences in gating mechanisms, it is not unreasonable to suppose that differences also exist in the rules that guide subunit assembly and quaternary structures of NMDARs and AMPA receptors.

Based on homology modeling of the structure of the R domain, the putative spermine binding site appears to lie outside the central cleft of the NR1 R domain [53]. The identity of the endogenous ligand (if any) that binds within the cleft is unknown (see Figure 11.4). Evidence also indicates a discrete spermine binding site on the NR2 subunit, at least with regard to NR2A and NR2B [53]. This is shown in Figure 11.4.

FIGURE 11.4

Putative locations of spermine, ifenprodil, and Zn²⁺ binding sites and domain-based organization of NR1 and NR2 subunits. In NR1/NR2A and NR1/NR2B receptors, spermine may bind to the R domain of NR1 and to a second site in the R domain of NR2. Ifenprodil (more...)

It is possible that the spermine binding site on NR1 is responsible for glycine-independent stimulation and a second site on NR2A or NR2B underlies glycine-dependent stimulation, but no direct evidence supports this hypothesis. Nonetheless, it would be consistent with the effects of the exon-5 insert (mechanism 4, Figure 11.2) present in the R domains of some slice variants of NR1 (Figure 11.3A) that reduces glycine-independent spermine stimulation but not glycine-dependent stimulation at NR1/NR2B receptors. Similarly, mutations in the R domain of NR1 that affect glycine-dependent stimulation do not affect glycine-independent stimulation by spermine [76].

11.4. IFENPRODIL

Ifenprodil (Figure 11.1) was originally developed as a vasodilator, based on its activity as an α1 adrenergic antagonist [77,78]. Studies in the 1980s and early 1990s demonstrated that it was also a noncompetitive NMDAR antagonist with a novel profile and mechanism of action [79–82]. Widespread interest in the properties of ifenprodil as a tool to study NMDARs and as a potential lead compound for novel therapeutic agents followed the discovery that it is a highly selective antagonist for receptors containing the NR2B subunit [83,84]. Ifenprodil was considered an atypical antagonist [83] because it was noncompetitive and subtype-selective but did not act as an open-channel blocker like MK-801 and phencyclidine. Ifenprodil is sometimes called an ‘allosteric antagonist’ (just as spermine and other modulators are referred to as allosteric modulators), but in the absence of a detailed mechanistic understanding of how ifenprodil binds and what it does after it binds to NMDARs, the allosteric designation is probably best avoided.

Other compounds with structures similar to ifenprodil, including haloperidol and nylidrin (Figure 11.1), were subsequently found to be selective for NR1/NR2B receptors [85,86] and additional NR2B-selective antagonists were developed based on the structure of ifenprodil. These compounds include CP-101,606, Ro 8-4304, and Ro 25-6981 (Figure 11.1) and are presumed to share the same binding site as ifenprodil on NMDARs [87–89]. In addition to their subtype selectivity, ifenprodil and related antagonists exhibit a novel form of use-dependency that may contribute to a favorable in vivo profile if these compounds are eventually used in clinical settings [89,90].

11.5. STRUCTURES OF NMDA RECEPTOR SUBUNITS AND RELEVANCE TO THE IFENPRODIL SITE

Ifenprodil and related compounds such as Ro 25-6981 are several hundred- to several thousand-fold more potent at NR1/NR2B receptors than at NR1/NR2 receptors containing NR2A, NR2C, or NR2D. For example, the IC₅₀ of ifenprodil was reported to be 0.3 μM at NR1/NR2B receptors and 146 μM at NR1/NR2A receptors—a selectivity of about 500-fold [83]. Several explanations may account for the selectivity of ifenprodil for heteromeric NR1/NR2B receptors. The ifenprodil binding site may be located on the NR2B subunit (Figure 11.5A) and be absent or have a much lower affinity on NR2A, NR2C, and NR2D. Another possibility is that the ifenprodil binding site is located on the NR1 subunit and its properties or transduction mechanisms are influenced by NR2 subunits, requiring NR2B for high affinity inhibition (Figure 11.5B). A third possibility is that the binding site involves regions in both NR1 and NR2B (Figure 11.5C). The weight of the available evidence suggests that ifenprodil binds to the NR2B subunit as illustrated in Figure 11.5A, possibly within the cleft of the bilobed R domain (analogous to binding of glutamate or glycine in the S1/S2 domains). The evidence comes largely from site-directed mutagenesis studies in which mutations in the R domain of NR2B reduced ifenprodil inhibition of recombinant NR1/NR2B receptors studied electrophysiologically [55]. The same mutations also reduced the ability of ifenprodil to protect against proteolytic degradation of an isolated, purified soluble NR2B R domain [55]. In other studies, an isolated soluble NR2B R domain was found to bind ifenprodil with high affinity (Kd, 0.13 μM) based on shifts in circular dichroism [91].

FIGURE 11.5

Where is the ifenprodil binding site on NR1/NR2B receptors? The schematics show the domain-based organization of the NR1 and NR2B subunits. Mutations within the R domains of NR1 and NR2B (small circles) alter sensitivity to ifenprodil, which could bind (more...)

Interestingly, residues at which mutations in NR2B produced the most pronounced effects on sensitivity to ifenprodil or ifenprodil-like ligands are in positions analogous to residues in NR2A that appear to form the high affinity Zn²⁺ binding site [54–56,92]. Surprisingly, many of these residues are actually identical in NR2A and NR2B, despite the marked differences in the chemical natures of ifenprodil and Zn²⁺. Equally surprising, in light of the large degree of selectivity of ifenprodil for NR1/NR2B over NR1/NR2A, is that many residues in the proposed ifenprodil binding site of NR2B are identical to their corresponding residues in NR2A, at least on the basis of alignment of linear amino acid sequences [55]. This may suggest that only one or two key residues influence selectivity for ifenprodil or that the overall structure or folding of the R domain in NR2B is somewhat different from that of the R domain of NR2A despite their close sequence similarity in regions that affect sensitivity to ifenprodil. By homology modeling, these residues were proposed to lie within the cleft between the two lobes of the R domain [54,55].

Although the evidence favors a high affinity ifenprodil binding site on the NR2B subunit (Figure 11.5A), it has also been shown that mutations in the R domain of NR1 can exert profound and selective effects on ifenprodil inhibition at NR1/NR2B receptors [53]. Based on homology modeling, these mutations are in a region of the NR1 R domain that is likely outside the cleft (Figure 11.5). It is possible that at least part of the ifenprodil binding site in intact NR1/NR2B receptors is formed by regions in the R domain of NR1 (Figure 11.5C). Alternatively, some mutations in NR1-R may disrupt the properties of the NR2B R domain, particularly if these mutations are at an interface between the NR1 and NR2B subunits, perhaps at an interface between adjacent R domains. It is also notable that ifenprodil inhibits apparent homomeric NR1 receptors expressed in Xenopus oocytes with a potency similar to that at NR1/NR2B receptors [84] consistent with the idea that a high affinity ifenprodil binding site can be formed by the NR1 subunit alone, although the question whether these recombinant NR1 receptors are truly homomeric or involve the inclusion of endogenous Xenopus NR2-like subunits is still unresolved [93].

Like AMPA receptors, NMDARs are thought to be tetramers [71,94,95], likely assembled as dimers of dimers [62,96], although there is also evidence consistent with a pentameric rather than a tetrameric subunit structure for NMDARs [97,98]. Assuming a tetrameric structure (a similar argument holds if the receptor were a pentamer), then each receptor must have two identical and presumably equivalent ifenprodil binding sites just as it has two glutamate binding sites and two glycine binding sites. In the case of glutamate and glycine, the agonist concentration–response curves are steep, with Hill coefficients close to 2.0, suggesting cooperativity of binding of the agonists. In the case of ifenprodil, the Hill coefficient for inhibition of NR1/NR2B receptors is close to 1.0 [55,99], suggesting, perhaps surprisingly, that there is no positive or negative cooperativity between the two ifenprodil binding sites.

In triheteromeric receptors engineered to contain one NR2A subunit and one NR2B subunit together with two NR1 subunits, ifenprodil was found to inhibit responses with high affinity (via the NR2B subunit) but the maximum degree of inhibition was greatly reduced [92]. A similar profile was seen for high affinity inhibition of these receptors by Zn²⁺; high affinity inhibition mediated via the NR2A subunit (see below) was still present, but the degree of inhibition was greatly reduced. This may suggest that the R domains can influence channel gating in an independent rather than a concerted manner [92]—in contrast to the concerted gating of the channel by agonist binding to the S1/S2 domains [73–75].

Initial studies suggested that ifenprodil acts as an antagonist at the stimulatory polyamine site [100], but subsequent work has shown this to not be the case [81,83], and ifenprodil is thought to bind to a distinct site on the NMDAR (Figures 11.2 and 11.4). This is consistent with results from mutagenesis studies in which different residues were found to influence sensitivity to ifenprodil and spermine [53].

Ifenprodil inhibits currents activated by glutamate and glycine (mechanism 6, Figure 11.2), and this effect is not voltage-dependent but is dependent on agonist concentration. Nonetheless, there are documented interactions between spermine and ifenprodil. Spermine can reduce the affinity of the receptor for ifenprodil and, conversely, ifenprodil can reduce the affinity for spermine [101]. Spermine can affect proton inhibition (mechanism 3, Figure 11.2) and protons can, in turn, affect ifenprodil inhibition (mechanism 7, Figure 11.2) and vice versa (mechanism 8, Figure 11.2). Thus, spermine may indirectly alter sensitivity to ifenprodil by changing proton sensitivity of the receptor.

The use-dependent properties of ifenprodil arise because the affinity for ifenprodil is increased by glutamate binding and vice versa (mechanism 9, Figure 11.2) [89,90]. In addition, ifenprodil reduces the affinity for glycine at NR1/NR2B receptors (mechanism 10, Figure 11.2), which presumably contributes to its inhibitory effects at these receptors [82,83,102].

In addition to its interactions with spermine, inhibition by ifenprodil is also dependent on extracellular pH, and it was proposed that the mechanism of action of ifenprodil is to potentiate tonic proton inhibition [103]. Inhibition of NR1/NR2B receptors by ifenprodil is increased at acidic pH and reduced at alkaline pH, i.e., protons increase the apparent affinity for ifenprodil (mechanism 7, Figure 11.2) [99]. At the same time, ifenprodil (and similar NR2B-selective antagonists) can enhance the inhibitory effects of protons at NR1/NR2B receptors, and this may be the major mechanism that underlies ifenprodil inhibition (mechanism 8, Figure 11.2) [103]. The presence or absence of the exon-5 insert or the addition of spermine produces only modest effects on sensitivity to ifenprodil and related compounds. These effects are likely indirect due to changes in proton sensitivity (mechanisms 3, 4, 7, and 8, Figure 11.2) [30,99,103].

11.6. ZINC IONS

Zinc ions are present in high concentrations in some areas of the nervous system and may modulate synaptic transmission [104,105]. Their effects on NMDARs were first described in studies of native receptors on isolated neurons in which Zn²⁺ was found to be a potent inhibitor of NMDA currents [106–108]. That work indicated that Zn²⁺ likely had two effects at native NMDARs (at that time receptor subunits had not yet been cloned and NMDAR subtypes were uncharacterized): a relatively high affinity inhibition that was not voltage-dependent, presumed to be mediated at a unique extracellular site on the receptor, and a low affinity, voltage-dependent block, possibly mediated at the Mg²⁺ binding site or at a nearby site within the channel pore [106–108]. Subsequent studies characterized effects of Zn²⁺ on recombinant NMDARs. Pronounced differences in subunit-dependent sensitivity to Zn²⁺ were reported, and recent studies have identified potential Zn²⁺ binding sites in the R domains of NR2 subunits and shed some light on the mechanism of action of Zn²⁺.

Initial studies of recombinant NMDARs found marked differences in the sensitivity to Zn²⁺ of NR1/NR2A and NR1/NR2B receptors. At NR1/NR2B receptors, Zn²⁺ inhibition was not voltage-dependent and was monophasic with an IC₅₀ of 0.5 to 9 μM [109–111]. In contrast, inhibition at NR1/NR2A receptors was biphasic, with a high affinity component (IC₅₀ 5 to 80 nM) that was not voltage-dependent and a low affinity component (IC₅₀ 26 to 79 μM) that was voltage-dependent [109–111]. This low affinity, voltage-dependent component likely represents a weak open-channel block by high concentrations of Zn²⁺. Another discovery was that residual or contaminating traces of Zn²⁺ in experimental solutions can produce marked inhibitory effects at NR1/NR2A receptors, and that solutions should be buffered with a Zn²⁺ chelator such as tricine (certainly for low concentrations of Zn²⁺) to accurately determine the concentration-dependence of Zn²⁺ inhibition at these receptors [111,112]. At NR1/NR2C and NR1/NR2D receptors, Zn²⁺ was subsequently found to have an even lower affinity than at NR1/NR2B receptors [113].

Putting aside the low affinity, voltage-dependent block of NMDA channels by Zn²⁺, the difference uncovered in early studies of recombinant receptors was a very high affinity inhibition at NR1/NR2A receptors versus a lower affinity inhibition at NR1/NR2B receptors. Another difference was that inhibition at NR1/NR2B receptors was monophasic and complete (Zn²⁺ produced a complete inhibition of NMDA currents), whereas the high affinity inhibition at NR1/NR2A receptors was incomplete. Zn²⁺ inhibited responses by only 40 to 70% [109,112,114].

This is reminiscent of the effects of ifenprodil at NR1/NR2B receptors, where the maximum inhibition is about 80 to 90% at physiologic pH [83]. Interactions occur between high affinity Zn²⁺ inhibition of NR1/NR2A receptors and proton inhibition at these same receptors, and it was proposed that the mechanism of Zn²⁺ inhibition involves an increase in tonic proton inhibition at NR1/NR2A receptors, analogous to the proposed mechanism of ifenprodil inhibition at NR1/NR2B receptors (equivalent to mechanism 8, Figure 11.2) [112,115,116]. In the proposed model of Zn²⁺ inhibition of NR1/NR2A receptors, binding of glutamate to the S1/S2 domain led to increased affinity for Zn²⁺ in the R domain and binding of Zn²⁺ led to a conformational change that enhanced binding of protons to proton-sensitive gating elements leading, in turn, to a reduction in channel open probability [114,116].

11.7. STRUCTURES OF NMDA RECEPTOR SUBUNITS AND RELEVANCE TO THE ZN²⁺ SITE

Swapping the R domains between NR2A and NR2B led to reciprocal changes in sensitivity to Zn²⁺ and ifenprodil [54,55]. Thus, NR2B subunits containing R domains of NR2A showed very high sensitivity to Zn²⁺ (similar to the native NR2A subunit) whereas NR2A subunits containing R domains of NR2B showed high sensitivity to ifenprodil (similar to the native NR2B subunit) and a reduced sensitivity to Zn²⁺ [54,55]. Mutations at a number of positions in the R domain of the NR2A subunit produced marked and in some cases specific effects on inhibition by Zn²⁺ [54,112, 115,117].

Using homology modeling based on LIVBP and related proteins, the residues that affect Zn²⁺ sensitivity were proposed to lie within the cleft of the R domain of NR2A [54] (Figure 11.6). Some of these residues are conserved between NR2A and NR2B, and may form part of the high affinity Zn²⁺ binding site in NR2A and the ifenprodil binding site in NR2B [54,55]. However, there are a few other residues that are identical or similar in NR2A and NR2B at which mutations affect Zn²⁺ sensitivity in NR2A but not ifenprodil sensitivity in NR2B and vice versa [54,55].

FIGURE 11.6

Putative locations of Zn²⁺ binding sites in NR1/NR2A and NR1/NR2B receptors and domain-based organization of these receptors, with a high affinity Zn²⁺ binding site in the cleft of the R domain of NR2A and a lower affinity site in the homologous region (more...)

As with most mutagenesis studies, the results may mean that the residues (their side chains or peptide bond backbones) (1) interact directly with Zn²⁺ and ifenprodil, (2) are key components of the backbone of a binding pocket, or (3) interact with water molecules within the binding site that in turn make contacts with Zn²⁺ and ifenprodil. Structural studies will be required to determine whether Zn²⁺ and ifenprodil really bind within the R domain clefts and what role particular residues play in those domains.

As with ifenprodil, if the Zn²⁺ binding site lies within the cleft of the R domain in the NR2 subunit, then each tetrameric receptor must have two presumably identical and equivalent Zn²⁺ binding sites. Again, as with ifenprodil acting at NR1/NR2B receptors, the Hill coefficient for inhibition of NR1/NR2A receptors by Zn²⁺ is close to unity [109,113,114,117], suggesting a lack of cooperativity between the two Zn²⁺ binding sites. As discussed above with regard to ifenprodil, triheteromeric NR1/NR2A/NR2B receptors still had a very high affinity for Zn²⁺ acting at the NR2A subunit, but the degree of inhibition by Zn²⁺ was greatly reduced [92].

Although there is good evidence for localization of the high affinity Zn²⁺ binding site on the NR2A subunit (Figure 11.6), the NR1 subunit can also influence Zn²⁺ sensitivity of NR1/NR2A receptors. Thus, the exon-5 insert in the R domain of the NR1 subunit (Figure 11.3A) has been shown to alter inhibition by Zn²⁺ [118], as have mutations at residues in the extracellular region preceding M1 and in the M3-M4 loop region, both of which contribute to the S1/S2 domain in the NR1 subunit [118,119]. However, the effects of these mutations on sensitivity to Zn²⁺ may be indirect and arise from changes in pH sensitivity and/or changes in the redox state of the receptor protein, both of which can alter Zn²⁺ inhibition in NR1/NR2A receptors [118,119].

Results of studies using truncated NR2 subunits in which the R domain was removed and the remainder of the subunit was intact, and studies of point mutations in the R domain of NR2B suggest that the low affinity Zn²⁺ binding site in NR2B is located in the R domain and is structurally homologous to the high affinity Zn²⁺ binding site in NR2A (Figure 11.6), and that this site in NR2B shares many structural determinants with the ifenprodil binding site in NR2B and the high affinity Zn²⁺ binding site in NR2A [113]. Absent an R domain in the NR2 subunit, NR1/NR2A and NR1/NR2B receptors are still inhibited by Zn²⁺, albeit with very low affinity similar to NR1/NR2C and NR1/NR2D receptors [113]. This suggests the presence of yet another, very low affinity Zn²⁺ binding site that must be located somewhere other than the R domain of the NR2 subunit.

One possibility is that the very low affinity site is located on the R domain of the NR1 subunit (Figure 11.6). It is notable that some splice variants of NR1, when expressed as homomeric NR1 receptors, are inhibited by Zn²⁺ whereas others are potentiated by Zn²⁺. The presence or absence of the exon-5 insert located in the R domain of NR1 influences sensitivity to Zn²⁺ [120]. However, this interpretation is complicated by the interactions between Zn²⁺ inhibition and proton inhibition, and by the fact that proton inhibition is, itself, influenced by the absence or presence of the exon-5 insert [30,118].

11.8. PROTONS AND EXTRACELLULAR pH

Protons and thus extracellular pH exert profound effects on NMDARs over the normal physiologic pH range. The effects of protons were discussed in the preceding sections because of their interactions with modulation by polyamines, ifenprodil, and Zn²⁺. An investigation and understanding of proton modulation of NMDARs developed concurrent with studies of those other modulators, and proton inhibition may represent a common denominator linking the effects of the other modulators and a common end point through which those modulators exert their effects on activation of NMDA channels (mechanism 2, Figure 11.2). Thus, spermine stimulation may at least in part involve a relief of tonic proton inhibition (mechanism 3, Figure 11.2) whereas inhibition by ifenprodil and Zn²⁺ may involve an increase in tonic proton inhibition (mechanism 8, Figure 11.2).

As with many other modulatory effects characterized at NMDARs, effects of extracellular pH were first documented in studies of native NMDARs expressed on isolated neurons [121–124]. Protons inhibited NMDA responses with an IC₅₀ around pH 7.0 to 7.3, indicating that the receptors were tonically inhibited by about 50% at physiologic pH. Proton inhibition is not voltage-dependent and presumably does not involve block of the ion channel pore [121–124]. The effects of protons involve a decrease in channel opening frequency but not changes in unitary conductance or dwell time [34,121,123,124]. At recombinant NR1/NR2B receptors, proton inhibition can occur independent of agonist binding, and it was suggested that protonated receptors are shifted into a state from which they cannot open [34].

It was assumed that the effects of protons were due to protonation of one or more ionizable residues on the extracellular surface of the NMDAR [121–124]. Studies of recombinant receptors have begun to uncover the molecular bases for proton inhibition, but the site and mechanism of proton inhibition remain unclear and do not appear to involve only one (or even several) ionizable residues on the NR1 or NR2 subunit.

Proton sensitivity of NMDARs is influenced by the exon-5 insert in the NR1 subunit. The presence of the insert reduces proton inhibition [30]. The mechanism underlying this effect is not understood, but the insert may function similar to spermine to alter the conformation of the R domain that influences proton-sensitive gating of the intact receptor. Proton sensitivity is also influenced by NR2 subunits; receptors containing NR2C are much less sensitive to protons than receptors containing NR2A or NR2B [125]. However, most mechanistic studies of pH sensitivity focused on NR1/NR2A and NR1/NR2B receptors.

Mutations in various regions of the NR1 subunit were reported to affect proton sensitivity in NR1/NR2 receptors. These include mutations in the R domain [53,76,115,118], the S2 portion of the S1/S2 domain formed by the loop between M3 and M4 [29,125,126], the linker between M3 and S2 [125], and at the critical Asn residue in the M2 loop that controls Mg²⁺ block and Ca²⁺ permeability of the channel [36]. Some of these residues, particularly those in the R domain and within the channel pore, likely have indirect effects on pH sensitivity rather than being ionizable residues that directly form part of a ‘proton sensor’ on the NMDAR. Residues in NR1 at which mutations have the largest effects on proton sensitivity are clustered in several regions that may be important for channel gating—at the top of M3, in the M3–S2 linker, in S2 near that linker, and in S2 near the S2–M4 linker (Figure 11.4) [125].

Results of other studies showed that the M3 and M3–S2 linker regions are associated with gating; movement of the M3 domain may be critical for channel gating [74,127,130]. Residues that appear to be important for proton sensitivity have been identified in similar regions of the NR2A subunit—in the M3 domain and in the S2–M4 linker (Figure 11.4) [125]. Structural differences in the S2–M4 linker regions of NR2C versus NR2A or NR2B appear to account for the reduced proton sensitivity of NR1/NR2C receptors compared to NR1/NR2A or NR1/NR2B receptors [125]. Whether any of these residues form part of a proton sensor per se, or whether the mutations have indirect effects on proton sensitivity are still unknown.

11.9. WHITHER NEXT?

In the early 1990s, the cloning of cDNAs encoding NMDAR subunits and the subsequent widespread availability of these clones ushered in the era of molecular studies of NMDARs [32,33,131–136]. This activity followed molecular studies of AMPA receptors and kainate receptors begun in 1989 with the cloning of the GluR1 AMPA receptor subunit and the subsequent cloning of cDNAs encoding a family of related GluR subunits [137,138].

Subunits of NMDARs, AMPARs, and kainate receptors were recognized as members of the same superfamily of genes, likely with common ancestors and similar tertiary and quaternary structures. Subsequent work from many laboratories focused on the effects for agonists, antagonists, and intra- and extracellular modulators of NMDARs at the molecular level, often combined with studies of the properties and proposed structures of the receptor subunits and their interactions with other intracellular or membrane-bound proteins. Some of that work is reviewed in this chapter and elsewhere in this book. However, even when coupled with homology modeling and powerful algorithms for structure prediction, the results of mutagenesis and similar studies do not provide definitive information about the locations of binding sites for modulators or their mechanisms of action and interactions. These studies provide important clues and allow the formulation of testable hypotheses and descriptive models such as those illustrated in Figures 11.3 through 11.6.

Another major step forward in molecular studies of glutamate receptors came in 1998 with the determination of a high-resolution x-ray crystallographic structure of the S1/S2 domain of a GluR2 AMPA receptor subunit [59], following the demonstration three years earlier, that it was possible to isolate and purify a GluR subunit S1/S2 fusion protein that retained the glutamate binding site with appropriate pharmacological characteristics [139]. The 1998 work [59] confirmed that the S1/S2 domain is composed of two major lobes; that it is structurally related to bacterial amino acid binding proteins such as the glutamine binding protein (QBP); that glutamate (or the agonist kainate) binds within the cleft between the two lobes; and that glutamate receptor subunits can thus be considered to have modular architectures, perhaps arising minimally during evolution from a combination of two or three genes encoding an ion channel and one or more amino acid–binding proteins. The structures of a number of other GluR subunit S1/S2 domains have also been reported, in some cases with an agonist, partial agonist, or antagonist bound within the domain cleft, leading to detailed models of the binding and activation of these receptors [58,59,64,65,140–144]. Recently, structures for the S1/S2 domains of the NMDAR NR1 and NR2A subunits have been reported, providing crucial new insights into activation, regulation, and desensitization of NMDARs [60,62,63,75].

Three extracellular modulators discussed in this chapter—spermine, ifenprodil, and Zn²⁺—are thought to bind to the R domains of NMDAR subunits. High resolution structural studies of the R domains, both in isolation and together with the S1/S2 domains and ultimately together with the pore-forming regions, will be required to elucidate definitively the sites and mechanisms of action of these modulators. For example, if ifenprodil really does bind within the cleft of the R domain in NR2B, does it promote closure of the cleft, analogous to glutamate binding within the cleft of the S1/S2 domain of GluR1 and glycine binding within the cleft of the S1/S2 domain of NR1? [58,60] If so, how does that structural change in the R domain ultimately translate into a reduction of current through the channel and/or to a change in proton inhibition mediated elsewhere in the receptor protein? Or does ifenprodil stabilize an open conformation of the R domain in NR2B? If so, how does that affect receptor activity? Is there an interface between the R domains of the NR1 and NR2 subunits and, if so, are there interactions between these domains? The exact same questions can be asked of Zn²⁺ at the NR2A and NR2B subunits if it binds within the clefts of the R domains on the subunits (Figure 11.6). Similarly, if spermine binds elsewhere on the R domain (Figure 11.4), what is its site and mechanisms of action?

What is the spatial relationship of the R domain and the S1/S2 domain, and does this change during receptor activation or desensitization? A powerful experimental approach, single particle electron microscopy, has begun to provide tentative and fascinating information about the structure, position, and role of R domains in AMPA receptor GluR subunits [66]. Clearly, the models and hypotheses outlined in Figures 11.3 through 11.6 beg many questions for experimentation when techniques such as x-ray crystallography and single particle electron microscopy can be applied to isolated R domains and larger structural components of NMDAR subunits (e.g., R–S1/S2; NR1-R–NR1-R; NR1-R–NR2-R; etc.) and to fully assembled NMDARs together with the biochemical and functional approaches already being applied to the study of these receptors.

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