Allosteric Interactions at the NMDAReceptor Channel Complex
Manolo Mugnaini
Biology Department, Psychiatry Center of Excellence for Drug Discovery,GlaxoSmithKline Medicines Research Center, Verona, Italy
INTRODUCTION
Historical Perspective
Glutamic acid is the main excitatory neurotransmitter of the mammaliancentral nervous system (CNS) and mediates neurotransmission across mostexcitatory synapses (1). Soon after the earliest description of the markedexcitatory action of L-glutamic acid on the general electrical activity of themammalian cerebral cortex (2), it became evident that a variety of aminoacids had a depolarizing effect on single neurons of the CNS (3), the mostpotent of which was the synthetic derivative of the D-form of the naturallyoccurring L-aspartic acid, N-methyl-D-aspartic acid (NMDA) (4). In thefollowing decades, a huge number of electrophysiological and pharmacologi-cal studies, together with the development of selective antagonists, led to theunequivocal distinction of NMDA receptors, within the wide variety ofglutamate receptors, as those selectively activated by NMDA (5).
Indeed, the NMDA receptor was the first class of glutamate-gatedion channels to be clearly identified, followed by a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptor classes.Overall, NMDA, AMPA, and kainate receptors, also termed ionotropic glu-tamate receptors (iGluRs), are responsible for signal transduction at the
postsynaptic level in the vast majority of fast excitatory synapses of CNS.NMDA receptors, in particular, are ubiquitously distributed in the brainand play a fundamental role in normal CNS function. Their peculiar charac-teristics, like calcium (Ca2þ) permeability (6) and voltage-dependent blockadeby magnesium (Mg2þ) ions (7), make NMDA receptors critical for importantphysiological mechanisms, like long-term potentiation (LTP) and synapticplasticity (the mechanisms underlying learning and memory), as well aspathological events like excess elevation of intracellular Ca2þ, which isthought to be the cause of cell death in many neurodegenerative diseases(8,9). Competitive agonists and antagonists, i.e., compounds that activateor block NMDA receptors through direct interaction with the neurotransmit-ter binding site, have proved to be neurotoxic or to have strong sideeffects, respectively. For this reason, positive and negative allosteric modula-tors are preferred to restore NMDA receptor activity to physiologicallevels, without causing excitotoxicity or complete blockade of normalreceptor functions.
In the 1970s and 1980s, the NMDA receptor channel complex wasextensively investigated in terms of pharmacology and electrophysiology,with the result of the discovery of many allosteric modulatory sites, the most
NMDA receptors were cloned, revealing the multiple subunit compositionof these channels and their complex heterogeneity (11).
Following a brief summary of the molecular biology of NMDA recep-tors (and some considerations about the concept of allosteric interactionsfor these proteins), the present chapter will deal with a description of thepharmacology and the structural determinants of the most important allo-steric modulatory sites of the NMDA receptor channel complex, in thecontext of its molecular diversity. Finally, the therapeutic potential of com-pounds designed for selectively targeting the different modulatory sites ofthe NMDA receptor will be discussed.
Molecular Biology of NMDA Receptors
So far, three families of NMDA receptor (NR) subunits have been identifiedby molecular cloning: the NR1, which is composed of eight isoforms gener-ated by alternative splicing of a single gene; the NR2, which contains foursubunits (NR2A, NR2B, NR2C, and NR2D) encoded by four differentgenes; and the NR3 family, which is formed by two subunits (NR3A andNR3B) encoded by two genes (12–20). NR1 variants differ according tothe presence or absence of three different amino acid cassettes: N1 (exon5), a segment of 21 amino acids in the N-terminus domain, and C1 (exon21) and C2 (exon 22), segments of 37 and 38 amino acids, respectively, inthe C-terminus domain. In the present review, NR1 splice variants will beidentified according to the nomenclature proposed by Durand et al. (21), inwhich three subscripts (one for every cassette) indicate the presence (1) or the
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important of which is the glycine binding site (Table 1) (10). In the 1990s,
absence (0) of a certain cassette, or if it is indeterminate (X). NR1011, forexample, indicates the splice variant lacking the N1 cassette but containingboth C1 and C2 cassettes, whereas NR11XX is used to indicate all splice var-iants containing the N1 cassette, independently of the presence or absence ofC1 and C2 cassettes.
Table 1 Allosteric Sites on NMDA Receptor
Localization
Allosteric site Effecta Subunit Domain
Glycine " NR1 LBDPolyamines (glycine
independent)" NR10XX/NR2B
interfaceATD (R2)
Polyamines (glycinedependent)
" NR1 Not determined
Histamine " NR10XX, NR2B Not determinedArachidonic acid " NR1, NR2A Not determinedSteroid positive
modulatory site" NR2A, NR2B SMD1
Adenosine triphosphate " NR2A, NR2B Not determinedZn2þ (very high affinity,
voltage independent)" NR10XX Not determined
Polyamines (lowglutamateconcentration)
# NR2B Not determined
Steroid negativemodulatory site
# NR2 Not determined
Zn2þ (high affinity,voltage independent)
# NR2A ATD
Phenylethanolamines # NR2B ATDFelbamate # NR2B Not determinedProton modulatory
sites, primary# NR1, NR2 M3–S2 linker,
M4–S2 linkerProton modulatory
sites, secondary# NR10XX, NR2A ATD
Redox modulatory site,primary
" # NR1 LBD
Redox modulatory site,secondary
" # NR10XX, NR2A ATD
a", The allosteric site is responsible for an increase of NMDA receptor function; #, the allosteric
site is responsible of a decrease of NMDA receptor function; " #, the allosteric site can either
NMDA receptor subunits share the same structural features of otheriGluR subunits: a large extracellular N-terminus domain; four hydrophobicdomains (M1, M2, M3, and M4); and an intracellular C-terminus domain
transmembrane spanning segments, whereas one, M2, makes a re-entrantloop within the membrane, which, together with the M2 regions of the othersubunits forming the ion channel complex, lines the pore (22). Finally,NMDA subunits possess an additional extracellular amino acid segmentlocated between M3 and M4 transmembrane spanning regions, and twointracellular domains, between M1 and M2 and between M2 and M3.
The part of the protein composed of a portion of the large extracellu-lar N-terminus domain preceeding M1 (a polypeptide segment of around150 amino acids, termed S1) and a portion of the segment between M3 andM4 (another segment of around 170 amino acids, S2) has structural simi-larities with some bacterial periplasmic binding proteins such as lysine/arginine/ornithine binding protein and glutamine binding protein (23).Similar to these proteins, this part of the NR subunits, termed ‘‘ligand bind-ing domain’’ (LBD), has a bilobate structure, with two domains (S1 and S2)that, adjusting the amino acid substrate in the cleft, pass from an open to aclosed conformation (24). The neurotransmitter glutamate binds to the LBDof the NR2 subunits (25–27), whereas glycine binds to the LBD of NR1 sub-units (28–31). At present, it is not clear if the LBD of NR3 subunits, whichhas structural homology to the LBD of both NR1 and NR2 subunits, bindsglutamate, glycine, or a yet unidentified ligand.
The first part (around 400 amino acids) of the large extracellularN-terminus domain has structural homology with bacterial leucine/isoleucine/valine binding protein (32) and polyamine binding protein (33).Similar to these proteins, this amino terminal domain (ATD) has a bilobatestructure, with two regulatory domains (R1 and R2) that facilitate theaccommodation of the ligand in a clam shell–like feature. Many allostericmodulators of the NMDA receptor bind to the ATD, which is thought totranslate binding of these agents into alterations of the LBD and/or stabi-lize specific receptor conformations, therefore changing NMDA receptorfunction (34).
Recent molecular modeling studies suggest that while in the resting(closed) state of the NMDA receptor the LBD has an open conformation,while the ATD has a closed conformation. Upon binding of the agonist (glu-tamate and glycine), the LBD closes and the receptor passes to an open state(in which both the LBD and the ATD have a closed conformation). Conver-sely, the desensitized (closed) state of the receptor is characterized by anATD in its open conformation (35).
Homomeric assembling of any of the subunits (NR1, NR2, or NR3)does not lead to the formation of functional channels in mammalian celllines. Homomeric expression of NR1 subunits in Xenopus oocytes gives
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(Fig. 1). Three of the four hydrophobic domains, M1, M3, and M4, are
Figure 1 Schematic representation of a tetrameric NMDA receptor composed oftwo NR1 subunits (only one subunit is shown), an NR2A and an NR2B subunit.The NR1 subunit lacks the amino terminal N1 cassette and contains both theC-terminal cassettes C1 and C2 and is therefore termed NR1011, according tothe nomenclature proposed by Durand et al. (21). Every subunit contains a largeextracellular N-terminus, four hydrophobic domains (M1, M2, M3, and M4), andan intracellular C-terminus tail. Three of the four hydrophobic domains, M1, M3,and M4, are transmembrane spanning segments, whereas one, M2, makes a re-entrant loop within the membrane, which, together with the M2 regions of the othersubunits, lines the pore. The extracellular portion of every subunit contains twobilobate structures: an LBD, with the two polypeptide segments S1 and S2, andan ATD, with two regulatory regions (R1 and R2). The neurotransmitter glutamatebinds to the LBD of NR2A and NR2B subunits. The coagonist/positive alloste-ric modulator glycine binds to the LBD of the NR1 subunit. The negative allostericmodulators Zn2þ and ifenprodil bind to the ATD of the NR2A and NR2B subunits,respectively. To exert its positive allosteric modulation, PS binds to the SMD(SMD1, small grid), whereas to exert its glycine-independent positive allosteric mod-ulation, spermine binds to a site located on the R2 regulatory domain of both NR1and NR2B subunits. Abbreviations: NMDA, N-methyl-D-aspartic acid; NR, NMDAreceptor; LBD, ligand binding domain; ATD, amino terminal domain; PS, pregne-nolone sulfate; SMD, steroid modulatory domain.
Allosteric Interactions at the NMDA Receptor Channel Complex 97
functional receptors, but it is not clear if this is due to the association ofmammalian NR1 subunits with Xenopus glutamate receptor subunit XenU1(36). However, the contemporary coexpression of at least a member of theNR1 family and a member of the NR2 family gives functional receptorswith much bigger currents in both Xenopus oocytes and mammalian celllines (37–39). When the NR3 subunit is present in the complex, the resultis inhibition of channel function (40). Coexpression of NR1 and NR3 sub-units leads to the formation of non-NMDA, glycine excitatory receptors(19), whereas the functional assembly of NR2 and NR3 receptors has neverbeen reported. Similar to other iGluRs (41), NMDA receptors are thoughtto assemble with a preliminary step of family-specific subunit dimerization[to form, for example, (NR1)2 or (NR2A)2], followed by dimerization ofthese dimers, to form a ‘‘dimer-of-dimers’’ or tetrameric receptor [e.g.,(NR1)2(NR2A)2 or (NR1)2(NR2B)2]. Recent studies strongly support thishypothesis (42–44), confirming previous reports suggesting a tetramericstructure with two NR1 and two NR2 subunits in the same channel complex(45). A schematic representation of a tetrameric (NR1)2(NR2A) (NR2B)
The Concept of ‘‘Allosteric Interaction’’ for NMDA Receptors
The term ‘‘allosteric’’ was used for the first time by Monod et al. (46)to define, in enzymes, accessory sites topographically distinct from thesubstrate-binding site or ‘‘isosteric’’ site (the terms were derived fromthe ancient Greek words ‘‘allos’’ and ‘‘ısos,’’ which mean ‘‘other’’ and‘‘equal,’’ respectively). Binding of a ligand to the allosteric site was able toinduce a conformational change in the protein and modulate the bindingof the substrate to the isosteric site, with the result of a modification in enzy-matic activity. The term was later introduced to define any binding site, on areceptor protein, that was able to modulate the binding properties of the pri-mary ligand (e.g., a neurotransmitter or a hormone) to its binding site or‘‘orthosteric’’ site (from the Greek ‘‘orthos,’’ ‘‘straight’’), with the resultof a change in receptor activity (47,48). The ability of the allosteric site tomodulate the binding properties of the orthosteric site was termed ‘‘allo-steric interaction’’ or ‘‘allosteric modulation.’’
According to this definition, the term ‘‘allosteric’’ can be used also todescribe interactions between multiple, identical sites present on the samereceptor protein, a phenomenon better known as ‘‘cooperative interaction.’’It is the case of many ligand-gated ion channels, for which more moleculesof the same ligand bind to the same receptor and binding of one molecule toone site can affect the binding properties to the other site [as occurs for thetwo c-aminobutyric acid (GABA) molecules, which bind to the sameGABAA receptor (49), or two acetylcholine (Ach) molecules, which bindto the same Ach receptor (50)]. In the present review, to avoid confusion,
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receptor is shown in Figure 1 (only one NR1 subunit is shown).
‘‘allosteric sites’’ are defined as sites topographically and morphologicallydistinct from the primary ligand binding site(s), or ‘‘orthosteric site(s).’’ Inparallel, the term ‘‘allosteric interaction’’ will be used to identify the inter-actions between allosteric and orthosteric sites and ‘‘cooperative interac-tion’’ or ‘‘cooperativity’’ to define the interactions between binding sitestopographically distinct (they are either orthosteric or allosteric sites) butidentical in terms of ligand specificity.
The term ‘‘allosteric’’ has also been widely used in the literature todescribe the properties of proteins that exist in an equilibrium mixture ofdifferent conformational states, corresponding to different pharmacologicalactivities (51). An example of allosteric proteins are nicotinic Ach receptors,oligomers composed of five subunits that may shift, through so-called allo-steric transitions, between resting, active, quickly desensitized, and slowlydesensitized conformations (52). Every binding site of the receptor protein,irrespective of whether it is an orthosteric or an allosteric site, has differentbinding properties depending on the conformational state. A specific ligandmight have a greater affinity for one state rather than another and, there-fore, increase the proportion of the protein in that state. To avoid confusion,in a recent review, Christopoulos and Kenakin (48) suggested the use of theterm ‘‘receptor isomerization,’’ rather than ‘‘allosteric transition,’’ to de-scribe the conversion of receptors between multiple conformations. The wayin which a ligand, by binding to a preexisting major conformational state,can shift the equilibrium toward that conformation is called conformationalselection, whereas the mechanism by which a ligand modifies the conforma-tion through the binding process, as occurs during the allosteric interactionsand the cooperative interactions, is termed conformational induction.
It is reasonable to think that in NMDA receptors, being ligand-gatedion channels composed of multiple protein subunits, all these phenomena(that is, allosteric interactions, cooperative interactions, and receptor iso-merization) might coexist. Indeed, many allosteric sites have been identified
mate binding sites are present in a single NMDA receptor (53) and althoughinitial studies seemed to suggest that the occupancy of one site does notaffect the affinity at the other site (54,55), the absence of cooperative inter-actions cannot be completely excluded. Finally, the presence of multipleclosed and open states has been revealed by many authors (56–59). Furthercomplication derives from the fact that glycine, similar to glutamate, is anessential requirement for NMDA receptor activation (60) and binds to theLBD rather than other structures of the NR1 subunit, so that many authorsconsider this substance a coagonist rather than an allosteric modulator,raising the query of which site can be considered as the othosteric site(the glutamate binding site, the glycine binding site, or both?).
For the purpose of this review, considering that most NMDA recep-tors are contained in pathways in which glutamate has the role of the
Allosteric Interactions at the NMDA Receptor Channel Complex 99
on the NMDA receptor channel complex (Table 1). Moreover, two gluta-
neurotransmitter (being released from the presynaptic terminals in anactivity-dependent manner, while glycine is present in the extracellular fluidat more constant levels), the glutamate (or NMDA) binding site will be con-sidered as the orthosteric site and the glycine binding site as the allostericsite. In addition, the effect of all compounds modulating NMDA receptorfunction in a noncompetitive manner will be described. This can be broadlyclassified as (i) compounds binding to an allosteric site and changingNMDA receptor activity through a mechanism of conformational induction(i.e., proper allosteric interaction); (ii) compounds binding to an allostericsite and changing NMDA receptor activity with a mechanism of conforma-tional selection; (iii) compounds changing NMDA receptor activity in anindirect way (i.e., affecting the levels or the activity of other biological fac-tors, which in turn modulate NMDA receptor function); and (iv) com-pounds that change NMDA receptor function through yet unidentifiedroutes. Substances like (þ)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine (MK801), which, rather than modulating NMDAreceptor function, completely block the receptor activity through bindingto a site deep within the channel, will not be considered in this review.Similarly, this chapter will not deal with the mechanisms of the voltage-dependent block of NMDA receptors by Mg2þ, block by high concentra-tions of Ca2þ, glycine-independent desensitization, and Ca2þ-dependentinactivation. Also, modulation of NMDA receptor function by mechanismsof phosphorylation, dephosphorylation, and interaction with intracellularregulatory proteins will not be covered in this review.
ALLOSTERIC SITES OF NMDA RECEPTORS
The Glycine Binding Site
The exciting discovery that the classical inhibitory neurotransmitter, glycine,markedly increased the action of glutamate at the NMDA receptor was firstmade by Johnson and Ascher in 1987 (61). In the following years, severalfunctional studies [reviewed by Thomson (62)] demonstrated that glycineinteracted with a distinct recognition site, later found to reside on NR1 sub-units. The positive allosteric nature of this interaction was further supportedby some receptor binding studies in which it was shown, by means of satura-tion and displacement experiments performed at equilibrium conditions,that glycine increased the affinity of glutamate for its binding site onNMDA receptors (63,64) and that this effect was reciprocal, with glutamateincreasing the affinity of glycine (65,66).
It became even more interesting when glycine–glutamate interactionswere investigated by means of fast perfusion systems, which made feasi-ble the direct measurement of activation and desensitization kinetics ofthe NMDA receptor channel complex. These techniques made possible the
discovery of the so-called glycine-dependent desensitization, a time-dependent loss of NMDA receptor function (in the continuous presenceof glutamate or NMDA) that could be almost completely overcome byincreasing glycine concentration (67). Glycine potentiation of NMDA-evoked current was detected both during the initial (peak) response and atequilibrium conditions (i.e., during the steady-state response recorded afterthe onset of desensitization), therefore confirming both previous functionalstudies reporting a glycine-induced increase in NMDA response at equilib-rium conditions (60,61) and binding studies reporting an increase of NMDAor glutamate affinity at equilibrium conditions (63,64). Glycine potentiationof the steady state was much larger than that of the peak response, with theresult that, by increasing glycine concentration, the difference between thesteady state and the peak response (in other words, desensitization) progres-sively diminished. This effect was found to depend on the ability of glycineto dramatically speed up the rate of the recovery from desensitization (67).Considering that the affinity of glycine during the peak response was higherthan during the steady state (68,69), these data suggest that the affinity ofglycine for the resting and open states of the NMDA receptor was higherthan its affinity for the desensitized state of the NMDA receptor and thatglycine favored the nondesensitized states of the NMDA receptor for amechanism of conformational selection.
To explain glycine reduction of desensitization, some authors havehypothesized the existence of further NMDA receptor states: a glutamate-(or NMDA-) unbound, closed conformation, with high affinity for glycine,and a glutamate- (or NMDA)-bound, closed conformation with low affinityfor glycine, but in rapid equilibrium with an open (active) conformation(57). This theory was later supported by results from electrophysiologicalexperiments on recombinant NR1/NR2A receptors, showing that glycineaffinity was higher in the absence of glutamate (70). The model describedthe loss of glycine affinity upon glutamate binding in terms of negativeallosteric interaction (or negative cooperativity, following the authors’ ter-minology) between the NMDA and the glycine binding sites, which mightseem somewhat misleading in the light of the more common reports ofpositive allosteric interaction or coagonism between these two sites. A modelconsidering the coexistence of major discrete quaternary transitions (i.e.,receptor isomerization) between clear conformational states (e.g., restingstate, open state, desensitized state) and, within each state, local reorganiza-tion at the subunit level more directly linked to fractional glycine andNMDA (or glutamate) binding (in other words, conformational induction)might help in interpreting these high-resolution electrophysiological dataand reconciling them to more classical studies performed at equilibriumconditions. Unfortunately, how fractional binding of glycine and glutamatecan affect quaternary organization at each conformational state is still amatter of debate.
Allosteric Interactions at the NMDA Receptor Channel Complex 101
The fact that NMDA receptors are hetero-oligomers of four subunitsand that the glutamate and the glycine binding sites reside on different sub-units raises the question of the possible existence of distinct NMDA recep-tors with different glycine or glutamate affinities, and/or glycine–glutamateallosteric interactions. Monaghan et al. (71) were the first to hypothesize thepresence of NMDA receptor subtypes of this kind in rat brain. Theseauthors noticed the different regional distribution, in rat brain sections, ofradiolabeled NMDA site agonists and antagonists and initially explainedthis difference with the possible existence of ‘‘agonist-’’ and ‘‘antagonist-preferring’’ NMDA receptor subtypes, i.e., NMDA receptors with a rela-tively higher affinity for NMDA site agonists and antagonists, respectively.In the same study, the authors also noticed that glycine potentiation of[3H]glutamate binding was regionally different in rat brain sections (withstriatum, for example, less sensitive than the cerebral cortex) and thatglycine, while increasing the binding of NMDA site agonists, had theopposite effect on the binding of NMDA site antagonists. Following theseobservations, these authors (71) theorized the possibility that agonist- andantagonist-preferring NMDA receptor subtypes might have, in additionto (or as an alternative to) intrinsic differences in agonist and antagonistaffinities, also different glycine–glutamate allosteric interactions and there-fore be differently regulated (in terms of binding at the NMDA site) bythe endogenous glycine, which is difficult to be properly washed away inbrain sections. This fact could explain why, in regions like the septum andstriatum, for the predominance of NMDA receptors with a more efficientglycine allosteric modulation, there was relatively more NMDA-sensitive[3H]glutamate binding (because of a greater glycine-induced potentiationof binding) and relatively less binding of [3H]-3-((�)-2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP), an NMDA site antagonist (becauseof a greater glycine-induced inhibition of [3H]-CPP binding). On the con-trary, in other regions like the thalamus and the cerebral cortex, possiblyfor a less efficient glycine–glutamate allosteric interaction, there was arelatively lower [3H]glutamate binding (corresponding to a smaller glycine-induced potentiation) and relatively higher [3H]-CPP binding (correspond-ing to a smaller glycine-induced inhibition). These differences could notbe explained in terms of regionally different concentrations of endogenousglycine, as revealed by microdialysis studies (72,73).
The theory that the differentially distributed receptor subtypesrevealed by Monaghan et al. (71) differed in the efficiency of the glycine–glutamate interaction was not demonstrated until the discovery of [3H]-D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP39653) (74).The high affinity and selectivity of this NMDA site antagonist, as well asits interaction with a single binding site, made [3H]CGP39653 the radioli-gand of choice for detecting NMDA receptors in studies of receptor bindingand autoradiography (64,75,76). It was therefore possible to determine that
(i) glycine, while increasing the affinity of glutamate, was able to decreasethe affinity of [3H]CGP39653; (ii) this inhibition was of allosteric nature,being reversed in a competitive fashion by selective glycine site antagonists,such as 7-chlorokinurenic acid (7-CKA) and 3-[2-(phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt (GV150526A)(77,78); (iii) glycine inhibition of [3H]CGP39653 binding (similar to glycineenhancement of [3H]glutamate binding) was regionally different in rat brainsections (with striatum, for example, less sensitive than the cerebral cortex),suggesting the presence of regionally different NMDA receptor subtypeswith different glycine–glutamate allosteric interactions; (iv) also, reversalof glycine inhibition by 7-CKA or GV150526A was regionally different inrat brain sections, but in a complementary fashion (with striatum, for exam-ple, more sensitive than the cerebral cortex), revealing the role of endogen-ous glycine in determining the different distribution patterns of NMDA siteagonists and antagonists; (v) in brain membranes (in which the levels ofendogenous glycine are much lower in comparison to brain sections) striataland cerebral cortical NMDA receptors did not present significant differ-ences in terms of glycine or glutamate affinity, or the affinity of any otherligand used to demonstrate the existence of different allosteric interactions(Table 2); and (vi) the potency of glycine allosteric inhibition of[3H]CGP39653 binding was greater in the striatum than in the cerebral cor-tex (apparent pKi ¼ 7.48 and 6.98, respectively).
In other words, the existence of the regionally distinct NMDAreceptor subtypes reported previously was confirmed (71) and it was proven
Table 2 Affinity of Glycine and NMDA Site Ligands for Their Respective BindingSites and Potency of Glycine in Glycine–Glutamate Allosteric Interaction, in theStriatal and Cerebral Cortical Membranes
that the difference between these receptor subtypes (at least for the striatumand the cerebral cortex) resided in the potency of glycine–glutamate allo-steric interaction rather than in significant differences in glycine or gluta-mate affinity for their respective binding sites.
It is difficult to explain the features of these allosterically differentNMDA receptor subtypes in terms of possible differences in subunit compo-sition. In functional studies, the sensitivity of recombinant NR1/NR2Areceptors to glycine is around 10-fold lower than that of NR1/NR2B,NR1/NR2C, and NR2D receptors (79–82). To some extent, binding studiesalso show similar results, with the affinity of glycine for NR1/NR2A recep-tors being two to three times lower than that for NR1/NR2B receptors(83,84). Native NMDA receptors from the cerebral cortex and striatum,however, do not present significant differences in terms of glycine affinity,despite their relatively greater abundance in NR2A and NR2B subunits,
Following the finding that NMDA site antagonists and agonists havea slightly higher affinity for NR1/NR2A and NR1/NR2B recombinantreceptors, respectively (79,80), some authors have suggested that these sub-unit combinations might represent the antagonist- and agonist-preferringNMDA receptor subtypes previously found with receptor binding studiesin native tissues (84,85). This result, however, was not confirmed by otherauthors, who found a higher affinity of both agonists and antagonists forthe NR1/NR2A rather than the NR1/NR2B combination (25). In addition,receptor binding studies suggest that regions containing antagonist- andagonist-preferring NMDA receptor subtypes (namely, the cerebral cortexand the striatum) do not present significant differences in terms ofagonist and antagonist affinities at the NMDA binding site (Table 2). More-over, the relatively higher abundance of the NR2B mRNA in the striatumthan the cerebral cortex (with respect to the NR2A) might suggest that thissubunit confers agonist-preferring characteristics on NMDA receptors, butthis is not true for the thalamus, which also contains relatively more NR2Bthan NR2A mRNA (14,86). Finally, the affinity of [3H]glutamate was onlyslightly increased by glycine in the NR1/NR2B combination, when com-pared with the NR1/NR2A combination (84), which agrees with the smallerglycine-induced increase of [3H]glutamate binding and smaller glycine-induced decrease of [3H]CGP39653 binding in regions containing agonist-preferring receptors, like the striatum. This finding, however, does notexplain why the same region presented the greatest enhancement of[3H]CGP39653 binding by 7-CKA or GV150526A.
Interestingly, NR1/NR2A recombinant receptors appeared to formmore efficiently than other combinations and looked very similar to nativereceptors in terms of binding properties (i.e., NMDA site agonist andantagonist affinity) and allosteric interactions, with glycine increasing theaffinity of [3H]glutamate and inhibiting [3H]CGP39653 binding (25,84,87).
Given the widespread distribution of the NR2A subunit mRNA in rat brain(which overlaps that of NR2B, NR2C, and NR2D subunits mRNA), it isreasonable to imagine that NR1/NR2A receptors may provide a core towhich the other subunits can attach and influence the properties of thewhole receptor. Indeed, the coexistence of more than one member ofthe NR2 family in the same receptor has been revealed by several authors(88–90). However, there are no reports of studies describing glycine–glutamate allosteric interactions in such complex combinations of NMDAreceptor subunits (such as NR1/NR2A/NR2B, NR1/NR2A/NR2C, orNR1/NR2A/NR2D), so that a comparison with the NR1/NR2A combi-nation and native receptors is not possible. In addition, different splicevariants of the NR1 subunit family might account for the different glycine–glutamate allosteric interactions observed in native receptors.
Glycine affinity in native cerebellar NMDA receptors is lower thanthat of the cerebral cortex, especially at cerebellar granule cells (91). In addi-tion, glycine did not potentiate NMDA-induced currents in cerebellarmRNA-injected oocytes, while, as expected, it potentiated cerebral mRNA-injected oocytes (92). These data might suggest that native NMDA receptorscontaining the NR2C subunit (which is highly expressed in the granularlayer of cerebellum) have a lower affinity for glycine and/or lower potencyin the glycine–glutamate interactions. Functional and binding studies onheteromeric NR1/NR2 receptors, however, do not support this hypothesis,with glycine affinity for NR1/NR2C receptors being similar or higher thanthat for NR1/NR2A and NR1/NR2B receptors (14,70,80,84). Glycineaffinity in recombinant NMDA receptors in a tri-heteromeric combinationcontaining the NR2C subunit (e.g., NR1/NR2A/NR2C), however, hasnever been determined.
The Zinc (Zn21) Binding Site
Many experimental results have demonstrated that zinc ions (Zn2þ) inhibitNMDA receptor function by interaction with a specific site, distinct fromthe Mg2þ binding site and located on the extracellular surface of the recep-tor (93–96). In cultured neurons, Zn2þ inhibits NMDA receptor function ina voltage-independent manner, at concentrations as low as 1–10 mM, andin a voltage-dependent manner, at concentrations of 10–100 mM (97,98).These two effects of Zn2þ on NMDA receptor function have been termedas high-affinity, voltage-independent inhibition and low-affinity, voltage-dependent inhibition, respectively.
Among heteromeric NR1/NR2 receptors, the highest sensitivity forthe high-affinity, voltage-independent Zn2þ inhibition is given by those con-taining the NR2A subunit, suggesting the presence of a specific Zn2þ nega-tive modulatory site responsible for this activity on this subunit (99–102).Indeed, more recent studies have revealed that Zn2þ resides in the cleft of
Allosteric Interactions at the NMDA Receptor Channel Complex 105
the bilobate structure of the NR2A subunit (103–105) and that inhibition ofZn2þ is involved in the fast desensitization of NR1/NR2A receptors (106).Recent molecular modeling studies suggest that Zn2þ stabilizes the openstate of the ATD, which corresponds to a desensitized form of the NMDAreceptor (35). The sensitivity for the high-affinity, voltage-independent Zn2þ
inhibition of NR1/NR2A receptors is higher in the presence of protons (lowpH), not only for the activation of the proton sensor located between the
per se increase the rate of formation of the desensitized state (35,104).The sensitivity of heteromeric NMDA receptors to voltage-independent
Zn2þ inhibition is determined also by the presence of the N1 insert in the NR1subunits, which, in fact, decreases both voltage-independent Zn2þ inhibitionand proton (Hþ) inhibition of the NR1/NR2A and NR1/NR2B combina-tions, suggesting that Zn2þ and Hþmay share similar structural determinants(101,102). In line with this finding, glycine-independent spermine potentiationof NMDA receptor function relieves both voltage-independent Zn2þ and Hþ
inhibition (102,108).The low-affinity, voltage-dependent inhibition by Zn2þ is likely to occur
within the ion channel pore and seems to involve the same residue on the M2segment of the NR1 subunit implicated in Mg2þ block and Ca2þ permeabilityof the receptor channel (109–111).
In addition to its well-known inhibitory effect at high concentrations,Zn2þ potentiates agonist-induced currents at submicromolar concentra-tions (EC50¼ 0.5 mM) in a voltage-independent manner (112). This effect,however, is seen only in homomeric receptors made of subunits lackingthe N1 insert (NR10XX) and is absent in all kinds of NR1/NR2 hetero-meric receptors, independently of the N1 insert in the NR1 subunit. Thepresence of native functional homomeric NMDA receptors, however, hasnever been demonstrated and the physiological relevance of the very highaffinity, voltage-independent stimulation of NMDA receptors is still tobe resolved.
The Phenylethanolamines Binding Site
Ifenprodil, a phenylethanolamine derivative, is a noncompetitive NMDAsite antagonist, initially thought to have antagonistic effect at the polyaminepositive modulatory sites (113–115). In line with the glycine-dependent stim-ulatory effect of polyamines, which is present only with NR1/NR2Brecombinant receptors, ifenprodil selectively inhibited NMDA receptors com-posed of the NR2B subunit, with a 400-fold higher affinity for the NR1/NR2B than for the NR1/NR2A combination (116,117). Later reports, how-ever, suggested that ifenprodil acts on a site distinct from the polyaminebinding site (118–120). In fact, residues on the NR2B subunit important
106 Mugnaini
LBD and the transmembrane region (see section ‘‘The Proton ModulatorySite’’), but also for the protonation of histidine residues in the ATD, which
for polyamine stimulation are not required for ifenprodil inhibition andvice versa. In addition, ifenprodil’s effect is independent of the presence ofthe N1 cassette in NR1 splice variants, unlike polyamine stimulation.Rather, polyamine and ifenprodil binding sites on the NR2B subunits seemto be allosterically linked in a negative manner (33,121,122).
More recently, several mutagenesis studies have proved that the bind-ing site of ifenprodil is located deep in the cleft of the ATD of the NR2Bsubunit (123–125). Functional studies suggest that ifenprodil (andstructurally related compounds, like Ro 8–4304) antagonizes NMDA recep-tor function with an activity-dependent mechanism (126,127) and byincreasing fast desensitization (106). Similar to the action of Zn2þ at theNR2A subunit, ifenprodil is thought to bind to the open state of theATD domain of the NR2B subunit and stabilize the desensitized form ofthe NMDA receptor (35). As with Zn2þ inhibition, ifenprodil inhibitionof NMDA receptor function is also potentiated by protons (128,129). How-ever, mutation of several residues in the ATD of the NR1 subunit alsoaffects sensitivity to ifenprodil, suggesting that some molecular determinantsof the ifenprodil binding site may also be located on the NR1 subunits, orthat these residues are important for the stabilization of the binding siteor subunit–subunit interactions (124).
Interestingly, neonatal rat brain NMDA receptors have a uniformlyhigh affinity for ifenprodil, which decreases during development becauseof the appearance of a lower affinity component (117). This finding corre-lates well with the fact that NR2B subunits are expressed much earlier indevelopment than NR2A subunits (130).
The Polyamine Binding Sites
Modulation of NMDA receptor activation by polyamines was first reportedby Ransom and Stec (131), who showed that spermine and spermidineincreased the binding of [3H](þ)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine (MK801) to rat brain membranes, an index ofNMDA receptor channel activation (132–134). In the following years,multiple, often opposing, effects of polyamines (i.e., stimulation and inhibi-tion of NMDA receptor function) have been described and more than one
(135–137). Nevertheless, the most relevant effect of spermine as an endoge-nous modulator remains its facilitatory influence on NMDA receptor–mediated neurotransmission (138), especially under pathological conditionssuch as brain ischemia, where its production is dramatically enhanced (139).The effect of polyamine on the affinity of the primary neurotransmitter glu-tamate on native receptors is still uncertain, with studies reporting a smalldecrease of glutamate affinity by 100 mM spermine (140) and higherconcentrations of spermine (up to 1 mM) increasing the binding of
Allosteric Interactions at the NMDA Receptor Channel Complex 107
interaction site has been hypothesized for this class of compounds (Table 1)
[3H]glutamate (135). In contrast, receptor binding studies on native recep-tors clearly support the evidence that polyamines increase glycine affinityto its binding site (65,141).
Electrophysiological studies have proved the existence of two differentmechanisms of positive modulation of NMDA receptor function by poly-amines: so-called glycine-dependent and glycine-independent stimulation.Glycine-dependent stimulation of NMDA receptor function is directly linkedto the spermine-induced increase of the affinity for glycine at its modulatorysite (see earlier paragraphs). Glycine-independent stimulation involves anincrease of channel opening frequency and a decrease of the onset rate ofglycine-independent desensitization (136,142–147). This effect, which can berevealed only at saturating concentrations of glycine, probably correspondsto the increase of [3H]MK801 binding observed in initial studies and islikely to be the most relevant effect of spermine on NMDA receptorfunction in physiological conditions.
Interestingly, both glycine-dependent and glycine-independent stimu-lation are highly dependent on the type of NR1 splice variant and NR2 sub-unit (21). Glycine-independent stimulation by polyamines involves a relief oftonic Hþ and Zn2þ inhibition of NMDA receptor function (108) and isabsent in NR1 splice variants carrying the N1 insert. This suggests that thissegment, for its structural similarities to polyamines, might relieve NMDAreceptors from tonic Hþ and Zn2þ inhibition by binding to the same site ofpolyamines. More recently, it has been shown that two residues critical forspermine potentiation are in the ATD of the NR1 subunit, very close to thesite of the N1 insert, suggesting that this spermine binding site resides inthe ATD of the NR1 subunit and that N1 insert constitutively occupies thisbinding site in NR1XX subunits. However, only receptors containing theNR2B subunit present glycine-independent stimulation by spermine, sug-gesting that this subunit also carries part of the binding site specific for theallosteric effect of polyamines (119,148,149). Recently, it has been proposedthat spermine binds to residues between the R2 lobes of NR1 and NR2B sub-units, stabilizing the dimer interface and the closed conformation of the ATDof these subunits, therefore favoring the open and resting states of the NMDAreceptor and diminishing the number of receptors in the desensitized state (35).Another hypothetical site of spermine binding is the first steroid modulatorydomain (SMD1), another segment that may be involved in dimer formations
that glycine-independent spermine stimulation of NMDA receptor function isreduced by 80% if the SMD1 of the NR2B subunit is substituted with thecorresponding sequence of the NR2D subunit, suggesting that SMD1 alsoplays a key role in this specific allosteric effect of polyamines. Finally, it isworthwhile to mention that Mg2þ ions, which block NMDA receptor channelsin a voltage-dependent manner, at very high, millimolar concentrations, canpotentiate NMDA receptor channels in a fashion similar to polyamines
108 Mugnaini
(see section ‘‘The Steroid Modulatory Sites;’’ 150). In fact, it has been shown
(i.e., in a voltage-independent and glycine-independent manner, only withNR1 splice variants lacking the N1 cassette and only with the NR1/NR2Bcombination), suggesting that Mg2þ may be the physiological agonist actingat the NR2B subunit–specific spermine site (151).
Glycine-dependent stimulation of NMDA receptor function is seenonly for homomeric NR1 channels and NMDA receptors carrying theNR2A and/or the NR2B subunits (21,33). Overall, these data are in linewith the finding that the binding of [3H]MK801 to NMDA receptors inthe cerebellum (which should contain a predominance of NMDA receptorscarrying the NR2C subunits) appears to be less sensitive to the action ofpolyamines than is the case with forebrain receptors in which the NR2Aand NR2B subunits are highly expressed (152,153).
Electrophysiological studies on recombinant receptors have also con-firmed the existence of mechanisms of negative allosteric modulation byspermine, previously shown with receptor binding studies (see earlier para-graphs). Interestingly, in the presence of saturating concentrations of glycine,the magnitude of spermine stimulation was dependent on the concentrationof glutamate (or NMDA) in the case of NR1/NR2B (but not NR1/NR2A)receptors: at low NMDA or glutamate concentration, spermine induced asmall decrease in NMDA, and glutamate affinity, which counteracted thestimulatory effect of spermine, resulting in little net effect of spermine(148). These results suggest that endogenous polyamines might act as abidirectional gain control at some native NMDA receptors, by dampeningthe response at low concentrations of glutamate and enhancing the responseat high concentrations of glutamate. Less relevant under physiological con-ditions is a mechanism of voltage-dependent inhibition of NMDA receptorfunction, which occurs only at hyperpolarized potentials and in the absenceof extracellular Mg2þ, possibly representing a direct block of the ion channelby spermine (142,144,146,154). This effect was present at NR1/NR2A andNR1/NR2B receptors but was absent on NR1/NR2C (81).
The Histamine Binding Site
The finding that histamine can modulate currents gated by the NMDAreceptor dates back to 1984 (155). Later, it was found that the potentenhancement by histamine of NMDA receptor-mediated currents in pyra-midal cells was the result of a direct interaction with NMDA receptors(156). The stimulatory effect of histamine is similar, in some respects, tothe glycine-independent stimulation by spermine: the ability of histamineto positively modulate NMDA receptor function is present only with NR1subunits lacking the N1 cassette and in the presence of the NR2B subunit(148,157). This evidence and certain similarities in structure between hista-mine and spermine may suggest that these substances act on NMDA recep-tors through interaction with the same allosteric binding site. Nevertheless,
Allosteric Interactions at the NMDA Receptor Channel Complex 109
the stimulatory effect of histamine, unlike that of spermine, is characterizedby a rapidly developing increase in magnitude followed by marked and slowdesensitization to a steady-state level, indicating that histamine acts at anovel recognition site distinct from that of spermine (157).
At high concentrations (1 mM), histamine also causes a voltage-dependentinhibition of NMDA currents, also at receptors that are not sensitive tostimulation by histamine. The nature of this inhibition is still unknown.
The Arachidonic Acid Binding Site
Arachidonic acid, similar to nitric oxide (NO), is a small signaling moleculethat diffuses readily through both fluid and lipid phases and is generated inneurons in response to activation of NMDA receptors. In turn, arachidonicacid potentiates native NMDA receptor channels, by increasing the prob-ability of channel opening, with no change in open channel current (158).As a consequence, a positive feedback mechanism of NMDA receptorfunction is based on the activation of phospholipase A2 by Ca2þ (whichenters the cell through the NMDA receptor) and the consequent productionof arachidonic acid, which in turn potentiates NMDA receptor currents(159). The stimulatory effect of arachidonic acid is observed even with asaturating effect of agonists at the glutamate and glycine binding sites ofthe NMDA receptor and is not due to activation of protein kinase C or con-version of arachidonic acid to lipoxygenase or cyclooxygenase derivatives;in addition, it is independent from Mg2þ, Zn2þ, and polyamine bindingsites. Finally, it has been shown that arachidonic acid binds to a 131 aminoacid residue domain on the amino terminal of NR1 subunits, which has sig-nificant homology with fatty acid–binding proteins (160). These results sug-gest that arachidonic acid binds to a distinct allosteric site on the NMDAreceptor. The NMDA receptors containing the NR2A seem to be more sen-sitive to arachidonic acid than the NR2B subunit–containing channels (161).
The Felbamate Binding Site
Felbamate (FBM; 2-phenyl-1,3-propanediol dicarbamate) is a potent anti-convulsant used in the treatment of seizures associated with Lennox–Gastautsyndrome in children and complex partial seizures in adults. Initially foundto decrease NMDA-induced neuronal injury (but not kainate-induced neu-ronal injury) in cultured cortical neurons (162) and to reduce NMDAreceptor–mediated postsynaptic potentials in hippocampal slices (163),FBM was finally characterized as a noncompetitive NMDA receptorantagonist selective for receptors composed of NR1 and NR2B subunits(164,165). Similar to ifenprodil, FBM enhanced the affinity of agonists atthe glutamate binding site. However, FBM increases the binding of [3H]gly-cine to the NMDA receptor (166) and, unlike ifenprodil, pH did not affectits affinity for the NR1/NR2B receptor (164). A point mutation on the
NR2B subunit, which affects receptor sensitivity to ifenprodil, does not dra-matically change the affinity of FBM, suggesting that ifenprodil and FBMbind to different sites. It has been shown recently that FBM selectively bindsto the activated and desensitized states of NMDA receptors, with the resultthat the inhibitory effect of FBM is stronger with both higher NMDA con-centration and longer NMDA exposure (167). As a consequence of this use-dependent inhibitory mechanism, FBM is thought to antagonize the exces-sive activation that occurs during seizure discharges and preserve normalneuronal firing. In addition, FBM was shown to stabilize the desensitizedform of the NMDA receptor also in the absence of glutamate, possiblyhelping to prevent the formation of excessive NMDA currents that mayoccur in the presence of an intense rise in glutamate concentrations.
The Proton Modulatory Sites
The NMDA receptor appears to be highly sensitive to changes in the micro-environment: NMDA receptor responses are selectively inhibited by protons(Hþ), with IC50 values close to physiological pH. This suggests the existence,on the NMDA receptor, of one or more negative modulatory sites for Hþ
and implies that NMDA receptor channels are under tonic inhibition ofaround 50% at physiological pH (168,169). Many authors have demon-strated that the proton sensitivity of NMDA receptors is a point of conver-gence for the execution of many allosteric effects, including the high-affinity,voltage-independent inhibition by Zn2þ (104,170), the negative modulationby phenylethanolamines (128,129), and the glycine-independent enhance-
late neurotransmitter-induced gating through a mechanism of negativeallosteric coupling, with spermine increasing receptor activity by decreasingproton inhibition. On the contrary, ifenprodil (phenylethanolamines) andZn2þ inhibit NMDA receptors by enhancing proton inhibition (104).
Recent scanned mutagenesis experiments of the NR1 subunit indicatethat residues that control proton inhibition are localized in discrete regions,namely, in the extracellular end of M3 and the adjacent linker leading to theS2 portion of the glycine binding domain (M3–S2 linker) and in the linkerbetween the S2 region and M4 (M4–S2 linker; 107). Interestingly, theM3–S2 linker partially overlaps the so-called ‘‘lurcher motif’’ (SYTAN-LAAF), a sequence conserved in all glutamate receptors that control recep-tor gating and channel opening, whereas the M4–S2 linker is downstreamfrom this motif. Also the M3–S2 and M4–S2 linkers of the NR2 subunitspresent the molecular determinants for proton sensitivity; nevertheless, theNR2A, NR2B, and NR2D subunits present, in the M4–S2 linker, a histidine(His) residue that is absent in the NR2C subunit and is likely to be an
Allosteric Interactions at the NMDA Receptor Channel Complex 111
ment of NMDA receptor function by polyamines (see section ‘‘The ZincBinding Sites,’’ ‘‘The Phenylethanolamines Binding Site,’’ and ‘‘The Polya-mine Binding Sites.’’). Protons and spermine (polyamines) seem to comodu-
essential determinant of the reduced pH sensitivity of NR1/NR2C receptors(108). Finally, it is worth pointing out that the M4–S2 linker partially over-laps the SMD1, recently discovered on the NR2B subunit (see section ‘‘TheSteroid Modulatory Sites’’). Interestingly, SMD1 is a critical determinant ofproton sensitivity in NR10XX/NR2B receptors (150). The SMD1 sequence isconserved between NR2A and NR2B, but significant differences existbetween NR2B (and NR2A) and NR2C and NR2D. Substituting theSMD1 sequence of the NR2B subunit with that of the NR2D subunitdecreases the proton sensitivity of NR1/NR2B receptors, suggesting thatNR2D (and NR2C) SMD1 domains lack part of the residues that conferproton sensitivity to the NR2B (and NR2A) subunit.
Other mutations that affect (although to a lesser extent) the sensitivityto protons are found in the ATD domain and S1 region of the N1 subunit(107), confirming previous findings that some molecular determinants ofproton inhibition may be located near the N1 insert site or in the ATD(108,124). Interestingly, NR1 subunits carrying the N1 insert (NR11XX)are less sensitive to proton inhibition and need a higher Hþ concentration(lower pH) to produce the same inhibition of NMDA receptor function(pH ¼ 6.6–6.8 and pH ¼ 7.2–7.4 for NR11XX and NR10XX, respectively).In addition, specific for the NR2A subunit, the protonation of some histi-dine residues in the ATD seems to inhibit NMDA receptor function (andpotentiate the inhibitory effect of Zn2þ), by increasing the rate of ATDopening and desensitized state formation (35,104).
Overall, these data suggest that the so-called ‘‘proton sensor,’’ pre-viously identified in the proximity of the N2 insert of the NR1 subunit, actu-ally resides in the M3–S2 and M4–S2 linkers of all NR1 and NR2 subunits.However, other proton modulatory sites, capable of modifying NMDAreceptor function, reside in the ATD of NR1 and NR2 subunits.
The Steroid Modulatory Sites
The neurosteroid pregnenolone sulfate (PS), one of the most abundant neu-rosteroids synthesized de novo in the nervous system, specifically potentiatesthe response of the NMDA receptor, while inhibiting GABA, glycine, andnon-NMDA iGluRs (171–173). In addition, it was shown that a varietyof sulfated steroids modulate the NMDA response in either a positive ora negative direction with a high degree of structural specificity. Surprisingly,the interaction between positive modulators, such as PS, and negative mod-ulators, such as pregnanolone sulfate (3a5bS) and epipregnanolone sulfate(3b5bS) is noncompetitive, suggesting the existence of distinct steroid posi-tive and negative allosteric sites on the NMDA receptor (174–176).
Functional studies on recombinant NMDA receptors demonstratedthat residues on the NR2 subunit are key determinants of modulation ofPS and 3a5bS (177). Indeed, very recently, Jang et al. (150) have identifieda 78-aa segment on the subunit NR2B that mediates the selective
potentiation of PS on NMDA receptors, the so-called SMD1. Interestingly,SMD1 is conserved within the iGluR family and corresponds to the aminoacid sequence involved in regulating the gating kinetics and binding of apositive modulator, cyclothiazide, to the AMPA receptor (178). Thedomain contains two a-helices, J and K, located in the S2 region of thebilobate structure of the glutamate binding site and the contiguous fourthtransmembrane domain (M4). More precisely, J and K helices reside at thedimerization interface between two AMPA subunits, and binding ofcyclothiazide promotes, by rearrangement of structures at this interface,dimer formation and alleviates desensitization. Molecular modeling, basedon AMPA receptor structure, suggests that SDM1 of the NR2B subunitmay contribute residues to a hydrophobic pocket capable of accommodat-ing PS (150). Within the NR2 subunit family, SMD1 is highly conserved,but significant differences exist. This reflects the fact that PS potentiatesthe response of recombinant receptors containing the NR2A and NR2Bsubunits, while it inhibits the response of receptors containing theNR2C and NR2D subunits (177). Modifying the amino acid sequence nearthe inner interface on NR2B to that of NR2D eliminates positive allostericmodulation by PS.
SMD1 is not involved in the negative modulation by 3a5bS, but par-
(replaced with the corresponding region of the NR2D subunit), are less sen-sitive to tonic proton inhibition and need more protons (lower pH) to pro-duce the same inhibition of NMDA receptor function (pH¼ 6.6 and 7.5 formutated and wild-type NR1/NR2B receptors, respectively). In addition,they lose spermine potentiation, which depends on the relief of tonic protoninhibition. Therefore, SMD1 is important for PS potentiation, proton inhibi-tion, and spermine potentiation. It should be underlined, however, that PSstimulates NMDA receptor channels via a route independent of the protonsensor; rather, it involves the SMD1, although the proton sensor andSMD1 may share some common elements (the M4–S2 linker). This isproven by the fact that the potentiating effect of PS is abolished by thelack of SMD1.
The ATP Modulatory Site
Adenosine 5’-triphosphate (ATP) has both inhibitory and facilitating effectson NMDA receptor activity, depending on the ATP concentration. Inhi-bition of NMDA receptors by guanine nucleotides has been reportedpreviously using radioligand binding assays (179,180). Indeed, recentelectrophysiological experiments on native and recombinant heteromericNR1/NR2A and NR1/NR2B (but not NR1/NR2C) receptors expressedin Xenopus laevis oocytes have revealed that, at a low concentration of glu-tamate, ATP and other nucleotides behave as competitive antagonists of
Allosteric Interactions at the NMDA Receptor Channel Complex 113
ticipates in controlling proton sensitivity [see Section ‘‘The Proton Modula-tory Sites’’ (150)]. Mutated NR1/NR2B receptors, lacking the SMD1 insert
the glutamate binding site (181,182). However, at concentrations of gluta-mate high enough to displace ATP from the NMDA binding site, NMDAcurrent is potentiated in the presence of ATP, indicating that ATP bindsas a positive allosteric modulator of NMDA receptor function. Interestingly,the affinity of ATP for its modulatory site on the NMDA receptor is 15-foldlower than that for the glutamate binding site, suggesting that a high concen-tration of ATP is needed to produce its potentiating effect. As a conse-quence, potentiation of NMDA receptor function by ATP is revealed inthe presence of high concentrations of both glutamate and ATP, whereasthe inhibitory effect of ATP can be revealed only at low concentrations ofboth glutamate and ATP. If we consider that ATP might be co-released withglutamate into the synaptic cleft, the final action of ATP may consist infocusing and enhancing the effects of glutamate at regions near the transmit-ter release sites (182). Finally, at some synapses in which zinc ions are alsoreleased into the synaptic cleft, potentiation of NMDA receptor functionby ATP may be further increased by chelation of Zn2þ ions and relief oftonic Zn2þ inhibition of NMDA receptor function (see earlier paragraphs).Overall, the evidence that ATP can enhance NMDA receptor function isin line with the finding that ATP promotes the induction of LTP via adirect action on NMDA receptors, with no involvement of P2X or P2Yreceptors (183).
The Redox Modulatory Site
Reducing agents like dithiothreitol (DTT) potentiate NMDA receptor chan-nels, while oxidizing agents are inhibitory (184), suggesting the existence of aredox modulatory site in equilibrium between a fully reduced state (thiolateanion, RS�) and an oxidized state (disulfide, RS–SR). The effect of DTT hastwo components, a reversible potentiation, which disappears spontaneouslyby washout of DTT, and a persistent (irreversible) potentiation, which isabolished only by an oxidizing agent (185). The reversible potentiationis present only in the NR1/NR2A combination, suggesting that it may bemediated by DTT chelation of Zn2þ, which selectively inhibits the functionof NMDA receptors containing the NR2A subunit. The persistent potentia-tion relies on reduction of two cysteine residues located in the S2 extracellu-lar loop region (between segments M3 and M4) of the NR1 subunit, whichconstitute the proper redox modulatory site of the NMDA receptor (186).These residues are located in the hinge of the cleft of NR1 and thereforeaffect the movement of the clam shell–like LBD. When the disulfide bondis reduced, the structure is more flexible and the closure of S1 and S2domains around glycine is facilitated (31).
There is increasing evidence, however, that cysteine residues of theATD of NR10XX and NR2A subunits also play a role in sensitivity to redoxagents (187). These residues might modulate the flexibility of the bilobate
structure of the ATD, as occurs in the LBD. Alternatively, some authorshave suggested the existence of cysteine bridge links between the R1domains of different subunits (35).
NO–producing agents also inhibit NMDA receptor channels, prob-ably via donation of NOþ ions to the cysteine thiolate anions (RS�) ofthe redox modulatory site of the NMDA receptor. This reaction, termedS-nitrosylation, leads to the formation of unstable S-nitrosothiols (RSNO),which more easily form disulfide bonds, resulting in a persistent block of theNMDA receptor channel (188–190). Interestingly, a negative feedbackmechanism of NMDA receptor function has been proposed, which consistsof stimulation of nitric oxide synthase (NOS) by calcium (which enters thecell following the opening of the NMDA receptor channel), with productionof NO, which in turn inhibits NMDA receptor function via interaction withthe redox modulatory site (159,188).
OTHER SUBSTANCES MODULATING NMDARECEPTOR FUNCTION
In addition to the substances regulating NMDA receptors through the inter-action with the allosteric modulatory sites described above, a variety ofother compounds have been reported to modify NMDA receptor functionthrough different (indirect) or still unknown mechanisms. This section willdescribe the effect of substances modulating NMDA receptor functionthrough nonallosteric mechanisms, or for which a clear interaction withan allosteric site on the NMDA receptor has not yet been produced.
Ethanol
Ethanol inhibits NMDA receptor channels in a concentration-dependentmanner (191–195). The sensitivity to ethanol seems to be independent ofthe NR1 splice variant, whereas NMDA receptors containing the NR2Aand NR2B subunits are more sensitive to ethanol than those containingthe NR2C subunit. Interestingly, ethanol sensitivity is enhanced by acalcium-dependent process that involves the interaction of the intracellularC0 domain of the NR1 subunit (a region of the cytoplasmic tail common toall NR1 splice variants) with proteins of the actin cytoskeleton (196). Inaddition, many reports suggest that ethanol inhibition of NMDA receptorfunction may occur through ethanol regulation of the activity of differentprotein kinases and phosphatases (which in turn change NMDA receptorfunction by phosphorylation or dephosphorylation) rather than through adirect interaction with an allosteric site on the NMDA receptor activation(197–200). A few studies, however, also suggest that ethanol may interactwith specific amino acids in the M3 and M4 domains, which are involvedin transducing agonist binding to channel opening and desensitization
Allosteric Interactions at the NMDA Receptor Channel Complex 115
(201,202). In support of this theory, the kinetics of blockade ofNMDA-gated currents by ethanol seem to be too rapid to be explained onlyin terms of phosphokinase activation (203).
General Anesthetics
Recent work has implicated NMDA receptor inhibition as a major source ofCNS depression with general anesthetics, from gaseous anesthetics [e.g.,nitrous oxide (N2O) and xenon (Xe)] to volatile anestethics (VAs; e.g., isoflur-ane) and intravenous anesthetics (e.g., ketamine; 204–208). Recently, it hasbeen shown that the NMDA receptor is an essential requirement for thebehavioral action of N2O (but not of VAs) in Caenorhabtidis elegans (209).
Apart from dissociative anesthetics, like ketamine, which produce anopen channel block of NMDA receptor function (like MK801), the molecu-lar determinants of many anesthetics remain to be identified. Recent evi-dences, however, suggest that many different VAs produce a similarinhibition of NMDA-gated currents and that the kinetics for these agentsare inconsistent with an open channel block or an effect mediated by phos-phokinases; rather there is an interaction with an allosteric site (203).
Interleukin-2
Interleukin-2 (IL-2) is a brain-derived glycoprotein that influences mesocor-ticolimbic dopamine release. Recently, it has been shown that, in voltage-clamped neurons freshly isolated from the ventral tegmental area, IL-2, atphysiologically relevant concentrations (0.01–10 ng/mL), inhibits NMDA-induced currents in a voltage-independent manner, while at higher doses(>50 ng/mL) it significantly increases NMDA-induced currents in avoltage-dependent fashion (210). The inhibitory effect was competitive forthe glutamate binding site of the NMDA receptor, whereas the obligatoryrequirement of intracellular ATP for the stimulatory effect suggests that thisphenomenon may be determined by the ability of the neuron to maintainintracellular phosphorylation and therefore not be mediated by direct inter-action of IL with an allosteric site on the NMDA receptor.
THERAPEUTIC POTENTIAL OF ALLOSTERIC MODULATORS OFNMDA RECEPTORS
Given their abundant and widespread distribution in mammalian neuronaltissue and their importance in excitatory transmission and normal CNSfunctioning, it is reasonable to imagine that NMDA receptors are involvedin a variety of neuropsychiatric diseases and that drugs targeting this class ofglutamate-gated ion channels may have great therapeutic potential. How-ever, overstimulation of NMDA receptors, as occurs in the presence of highconcentrations of glutamate or other competitive agonists, leads to excess
intracellular calcium elevation and excitotoxicity (9). On the other hand,complete block of NMDA receptors, obtained in the presence of competi-tive NMDA site antagonists or channel blockers, may disrupt normal brainfunctioning and produce a series of adverse effects such as learning andmemory impairment, neurotoxicity, disturbances of motor coordination,hallucinations, centrally mediated increase of blood pressure, catatonia,and anesthesia (211–218). Drugs acting at the different allosteric sites ofthe NMDA receptor may have the advantage of modulating NMDA recep-tor function without dramatically changing its basal activity; in addition,given the heterogeneity of allosteric modulations among recombinant recep-tor subtypes, they may selectively exploit their action through specificneuronal pathways.
Positive Allosteric Modulators
The hypothesis that hypofunction of the glutamatergic system might occurin schizophrenia was first made by Carlsson and Carlsson (219), whonoticed how NMDA channel blockers, like phencyclidine and ketamine,caused a schizophrenic-like syndrome in humans, recapitulating both thepositive and the negative symptoms of this disease. In the following years,clinical and preclinical evidence strongly suggested that potentiation ofNMDA receptor function may improve memory and cognition, and there-fore be beneficial in cognitive disorders and schizophrenia (220). Morerecently, it has been shown that enhancement of NMDA receptor functionmay facilitate extinction of conditioned fear, and therefore be beneficial alsoin anxiety disorders (221).
Glycine site agonists, or compounds acting through the steroid, poly-amines, and ATP positive modulatory sites of the NMDA receptors mayhave beneficial effects in treating these disorders. Indeed, glycine and glycinesite agonists, like D-serine and D-cycloserine, have proven efficacy whengiven in addition to standard antipsychotic therapy for the treatment ofschizophrenia (222,223). Interestingly, overexpression of the NR2B subunitimproves learning and memory in mice (224), suggesting that NR2Bsubunit–containing NMDA receptors may play a special role in the patho-genesis of this disease. In light of this hypothesis, targeting the site respon-sible for the glycine-independent stimulation of the NMDA receptor byspermine, located between the NR1 and the NR2B subunits, might be ofspecial interest for treating schizophrenia.
Negative Allosteric Modulators
It is now generally accepted that cell death caused by sustained or prolongedNMDA receptor overactivation is the primary mechanism of neuronaldeath following cerebral ischemia (139) and may be an important cofactorof neuronal damage in many neurodegenerative diseases such as
Allosteric Interactions at the NMDA Receptor Channel Complex 117
Parkinson’s, Huntington’s, and Alzheimer’s diseases (219,225–228). Inaddition, it is now well established that overactivity of excitatory pathwaysmay be the source of epilepsy and neuropathic pain (229–232). There is alsogrowing evidence that inhibition of NMDA receptor function may bebeneficial in counteracting different aspects of substance use disorders, fromthe expression and maintenance of morphine dependence (233,234), theacquisition of and relapse to cocaine addiction (235,236), and reverse toler-ance to cocaine and amphetamine (237), to nicotine sensitization (238,239)and to ethanol withdrawal symptoms (240).
Glycine site antagonists have proven to be efficacious in several animal
effects reported for competitive NMDA site antagonists and NMDA chan-nel blockers (244). Unfortunately, up to now, clinical trials with glycineantagonists have failed to meet preclinical expectations, showing little orno therapeutic benefit (245–247). The difficulties associated with the inter-pretation of clinical data for complex pathologies like stroke and traumaticbrain injury, together with the initial concern regarding brain penetrationand brain availability of glycine antagonists, might have been the reasonfor these negative results. Hopefully, further trials with compounds withbetter pharmacokinetic profiles, supported by positron emission tomogra-phy studies monitoring the levels of NMDA receptor occupancy in healthyvolunteers and patients, will reveal the potential therapeutic value ofthese compounds.
NR2B subunit–selective negative allosteric modulators of NMDAreceptor function can be obtained with compounds binding at the phenyl-ethanolamine binding site (like ifenprodil). These compounds may havespecial interest, given the abundance of the NR2B subunit in the dorsal hornof the spinal cord and in the caudate putamen, for neuropathic pain andParkinson’s disease, respectively. Indeed, there is significant preclinicalevidence of the antinociceptive and antiparkinsonian efficacy of NR2B-selective compounds (248–250).
Interestingly, mice deficient in the NR2A subunit or the NR2C sub-unit show attenuation of focal ischemic brain injury (251,252), suggestingthat NR2A-selective negative modulators, such as compounds actingthrough the high-affinity Zn2þ binding site, or NR2C-selective inhibi-tors may be particularly efficient neuroprotectants in cerebral stroke andsimilar pathologies.
CONCLUDING REMARKS
NMDA receptors are present ubiquitously in mammal CNS and are prob-ably involved in a large variety of neurologic and psychiatric diseases. Theirheteromeric structure, in terms of subunit composition and the large numberof subunits cloned so far, suggests that many NMDA receptor subtypes may
118 Mugnaini
models of these diseases [see reviews (241–243)] without displaying the side
exist that play different roles in distinct neuronal pathways. The greatnumber and heterogeneity of allosteric sites on NMDA receptors offerthe potential to design drugs that selectively modulate the function of distinctNMDA receptor subtypes, or act on many NMDA receptors at the same time.
Establishing the subunit composition of native NMDA receptors andtheir significance in normal and disease states remains the major challenge atthis point. Immunoprecipitation and purification of native NMDA recep-tors, followed by their pharmacological characterization, may help in clar-ifying their subunit composition, whereas a more extended phenotypiccharacterization of knockout mice would be useful to understand the roleplayed by different subunits in different pathologies. Especially the genera-tion of conditional and tissue-restricted knockdown mice, with the use ofRNA interference technology, would be of great help to study the functionof specific NMDA receptor subunits and their involvement in the etiology ofspecific CNS diseases (253).
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