Allosteric Interactions at The NMDA

2006 by Taylor & Francis Group, LLC
5
Allosteric Interactions at the NMDA
Receptor 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 mammalian
central nervous system (CNS) and mediates neurotransmission across most
excitatory synapses (1). Soon after the earliest description of the marked
excitatory action of L-glutamic acid on the general electrical activity of the
mammalian cerebral cortex (2), it became evident that a variety of amino
acids had a depolarizing effect on single neurons of the CNS (3), the most
potent of which was the synthetic derivative of the D-form of the naturally
occurring L-aspartic acid, N-methyl-D-aspartic acid (NMDA) (4). In the
following decades, a huge number of electrophysiological and pharmacologi-
cal studies, together with the development of selective antagonists, led to the
unequivocal distinction of NMDA receptors, within the wide variety of
glutamate receptors, as those selectively activated by NMDA (5).
Indeed, the NMDA receptor was the rst class of glutamate-gated
ion channels to be clearly identied, 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
93
postsynaptic level in the vast majority of fast excitatory synapses of CNS.
NMDA receptors, in particular, are ubiquitously distributed in the brain
and play a fundamental role in normal CNS function. Their peculiar charac-
teristics, like calcium (Ca
2
) permeability (6) and voltage-dependent blockade
by magnesium (Mg
2
) ions (7), make NMDA receptors critical for important
physiological mechanisms, like long-term potentiation (LTP) and synaptic
plasticity (the mechanisms underlying learning and memory), as well as
pathological events like excess elevation of intracellular Ca
2
, which is
thought to be the cause of cell death in many neurodegenerative diseases
(8,9). Competitive agonists and antagonists, i.e., compounds that activate
or block NMDA receptors through direct interaction with the neurotransmit-
ter binding site, have proved to be neurotoxic or to have strong side
effects, respectively. For this reason, positive and negative allosteric modula-
tors are preferred to restore NMDA receptor activity to physiological
levels, without causing excitotoxicity or complete blockade of normal
receptor functions.
In the 1970s and 1980s, the NMDA receptor channel complex was
extensively 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 composition
of 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 interactions
for these proteins), the present chapter will deal with a description of the
pharmacology and the structural determinants of the most important allo-
steric modulatory sites of the NMDA receptor channel complex, in the
context of its molecular diversity. Finally, the therapeutic potential of com-
pounds designed for selectively targeting the different modulatory sites of
the NMDA receptor will be discussed.
Molecular Biology of NMDA Receptors
So far, three families of NMDA receptor (NR) subunits have been identied
by molecular cloning: the NR1, which is composed of eight isoforms gener-
ated by alternative splicing of a single gene; the NR2, which contains four
subunits (NR2A, NR2B, NR2C, and NR2D) encoded by four different
genes; and the NR3 family, which is formed by two subunits (NR3A and
NR3B) encoded by two genes (1220). NR1 variants differ according to
the presence or absence of three different amino acid cassettes: N1 (exon
5), a segment of 21 amino acids in the N-terminus domain, and C1 (exon
21) and C2 (exon 22), segments of 37 and 38 amino acids, respectively, in
the C-terminus domain. In the present review, NR1 splice variants will be
identied according to the nomenclature proposed by Durand et al. (21), in
which three subscripts (one for every cassette) indicate the presence (1) or the
94 Mugnaini
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). NR1
011
, for
example, indicates the splice variant lacking the N1 cassette but containing
both C1 and C2 cassettes, whereas NR1
1XX
is used to indicate all splice var-
iants containing the N1 cassette, independently of the presence or absence of
C1 and C2 cassettes.
Table 1 Allosteric Sites on NMDA Receptor
Localization
Allosteric site Effect
a
Subunit Domain
Glycine " NR1 LBD
Polyamines (glycine
independent)
" NR1
0XX
/NR2B
interface
ATD (R2)
Polyamines (glycine
dependent)
" NR1 Not determined
Histamine " NR1
0XX
, NR2B Not determined
Arachidonic acid " NR1, NR2A Not determined
Steroid positive
modulatory site
" NR2A, NR2B SMD1
Adenosine triphosphate " NR2A, NR2B Not determined
Zn
2
(very high afnity,
voltage independent)
" NR1
0XX
Not determined
Polyamines (low
glutamate
concentration)
# NR2B Not determined
Steroid negative
modulatory site
# NR2 Not determined
Zn
2
(high afnity,
voltage independent)
# NR2A ATD
Phenylethanolamines # NR2B ATD
Felbamate # NR2B Not determined
Proton modulatory
sites, primary
# NR1, NR2 M3S2 linker,
M4S2 linker
Proton modulatory
sites, secondary
# NR1
0XX
, NR2A ATD
Redox modulatory site,
primary
" # NR1 LBD
Redox modulatory site,
secondary
" # NR1
0XX
, 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
increase or decrease NMDA receptor function.
Abbreviations: NMDA, N-methyl-D-aspartic acid; NR, NMDA receptor; ATD, amino terminal
domain; LBD, ligand binding domain; SMD1, rst steroid modulatory domain; M3, third
hydrophobic domain; M4, fourth hydrophobic domain; R2, second regulatory region; S2,
second polypeptide segment.
Allosteric Interactions at the NMDA Receptor Channel Complex 95
NMDA receptor subunits share the same structural features of other
iGluR subunits: a large extracellular N-terminus domain; four hydrophobic
domains (M1, M2, M3, and M4); and an intracellular C-terminus domain
transmembrane spanning segments, whereas one, M2, makes a re-entrant
loop within the membrane, which, together with the M2 regions of the other
subunits forming the ion channel complex, lines the pore (22). Finally,
NMDA subunits possess an additional extracellular amino acid segment
located between M3 and M4 transmembrane spanning regions, and two
intracellular 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 around
150 amino acids, termed S1) and a portion of the segment between M3 and
M4 (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 a
closed conformation (24). The neurotransmitter glutamate binds to the LBD
of the NR2 subunits (2527), whereas glycine binds to the LBD of NR1 sub-
units (2831). At present, it is not clear if the LBD of NR3 subunits, which
has structural homology to the LBD of both NR1 and NR2 subunits, binds
glutamate, glycine, or a yet unidentied ligand.
The rst part (around 400 amino acids) of the large extracellular
N-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 bilobate
structure, with two regulatory domains (R1 and R2) that facilitate the
accommodation of the ligand in a clam shelllike feature. Many allosteric
modulators of the NMDA receptor bind to the ATD, which is thought to
translate binding of these agents into alterations of the LBD and/or stabi-
lize specic receptor conformations, therefore changing NMDA receptor
function (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 an
ATD 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 cell
lines. Homomeric expression of NR1 subunits in Xenopus oocytes gives
96 Mugnaini
(Fig. 1). Three of the four hydrophobic domains, M1, M3, and M4, are
Figure 1 Schematic representation of a tetrameric NMDA receptor composed of
two 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 the
C-terminal cassettes C1 and C2 and is therefore termed NR1
011
, according to
the nomenclature proposed by Durand et al. (21). Every subunit contains a large
extracellular N-terminus, four hydrophobic domains (M1, M2, M3, and M4), and
an 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 other
subunits, lines the pore. The extracellular portion of every subunit contains two
bilobate structures: an LBD, with the two polypeptide segments S1 and S2, and
an ATD, with two regulatory regions (R1 and R2). The neurotransmitter glutamate
binds 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 allosteric
modulators Zn
2
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 NR1
and NR2B subunits. Abbreviations: NMDA, N-methyl-D-aspartic acid; NR, NMDA
receptor; LBD, ligand binding domain; ATD, amino terminal domain; PS, pregne-
nolone sulfate; SMD, steroid modulatory domain.
functional receptors, but it is not clear if this is due to the association of
mammalian NR1 subunits with Xenopus glutamate receptor subunit XenU1
(36). However, the contemporary coexpression of at least a member of the
NR1 family and a member of the NR2 family gives functional receptors
with much bigger currents in both Xenopus oocytes and mammalian cell
lines (3739). When the NR3 subunit is present in the complex, the result
is 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 never
been reported. Similar to other iGluRs (41), NMDA receptors are thought
to assemble with a preliminary step of family-specic subunit dimerization
[to form, for example, (NR1)
2
or (NR2A)
2
], followed by dimerization of
these 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 this
hypothesis (4244), conrming previous reports suggesting a tetrameric
structure 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 rst time by Monod et al. (46)
to dene, in enzymes, accessory sites topographically distinct from the
substrate-binding site or isosteric site (the terms were derived from
the ancient Greek words allos and sos, which mean other and
equal, respectively). Binding of a ligand to the allosteric site was able to
induce a conformational change in the protein and modulate the binding
of the substrate to the isosteric site, with the result of a modication in enzy-
matic activity. The term was later introduced to dene any binding site, on a
receptor 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 ortho s, straight), with the result
of a change in receptor activity (47,48). The ability of the allosteric site to
modulate the binding properties of the orthosteric site was termed allo-
steric interaction or allosteric modulation.
According to this denition, the term allosteric can be used also to
describe interactions between multiple, identical sites present on the same
receptor protein, a phenomenon better known as cooperative interaction.
It is the case of many ligand-gated ion channels, for which more molecules
of the same ligand bind to the same receptor and binding of one molecule to
one site can affect the binding properties to the other site [as occurs for the
two c-aminobutyric acid (GABA) molecules, which bind to the same
GABA
A
receptor (49), or two acetylcholine (Ach) molecules, which bind
to the same Ach receptor (50)]. In the present review, to avoid confusion,
98 Mugnaini
receptor is shown in Figure 1 (only one NR1 subunit is shown).
allosteric sites are dened as sites topographically and morphologically
distinct from the primary ligand binding site(s), or orthosteric site(s). In
parallel, the term allosteric interaction will be used to identify the inter-
actions between allosteric and orthosteric sites and cooperative interac-
tion or cooperativity to dene the interactions between binding sites
topographically distinct (they are either orthosteric or allosteric sites) but
identical in terms of ligand specicity.
The term allosteric has also been widely used in the literature to
describe the properties of proteins that exist in an equilibrium mixture of
different conformational states, corresponding to different pharmacological
activities (51). An example of allosteric proteins are nicotinic Ach receptors,
oligomers composed of ve subunits that may shift, through so-called allo-
steric transitions, between resting, active, quickly desensitized, and slowly
desensitized conformations (52). Every binding site of the receptor protein,
irrespective of whether it is an orthosteric or an allosteric site, has different
binding properties depending on the conformational state. A specic ligand
might have a greater afnity 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 the
term receptor isomerization, rather than allosteric transition, to de-
scribe the conversion of receptors between multiple conformations. The way
in which a ligand, by binding to a preexisting major conformational state,
can shift the equilibrium toward that conformation is called conformational
selection, whereas the mechanism by which a ligand modies the conforma-
tion through the binding process, as occurs during the allosteric interactions
and the cooperative interactions, is termed conformational induction.
It is reasonable to think that in NMDA receptors, being ligand-gated
ion 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 identied
mate binding sites are present in a single NMDA receptor (53) and although
initial studies seemed to suggest that the occupancy of one site does not
affect the afnity at the other site (54,55), the absence of cooperative inter-
actions cannot be completely excluded. Finally, the presence of multiple
closed and open states has been revealed by many authors (5659). Further
complication derives from the fact that glycine, similar to glutamate, is an
essential requirement for NMDA receptor activation (60) and binds to the
LBD rather than other structures of the NR1 subunit, so that many authors
consider 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
on the NMDA receptor channel complex (Table 1). Moreover, two gluta-
neurotransmitter (being released from the presynaptic terminals in an
activity-dependent manner, while glycine is present in the extracellular uid
at more constant levels), the glutamate (or NMDA) binding site will be con-
sidered as the orthosteric site and the glycine binding site as the allosteric
site. In addition, the effect of all compounds modulating NMDA receptor
function in a noncompetitive manner will be described. This can be broadly
classied as (i) compounds binding to an allosteric site and changing
NMDA receptor activity through a mechanism of conformational induction
(i.e., proper allosteric interaction); (ii) compounds binding to an allosteric
site and changing NMDA receptor activity with a mechanism of conforma-
tional selection; (iii) compounds changing NMDA receptor activity in an
indirect 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 unidentied
routes. Substances like ()-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)
cyclohepten-5,10-imine (MK801), which, rather than modulating NMDA
receptor function, completely block the receptor activity through binding
to 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 Mg
2
, block by high concentra-
tions of Ca
2
, glycine-independent desensitization, and Ca
2
-dependent
inactivation. Also, modulation of NMDA receptor function by mechanisms
of phosphorylation, dephosphorylation, and interaction with intracellular
regulatory 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 rst
made by Johnson and Ascher in 1987 (61). In the following years, several
functional studies [reviewed by Thomson (62)] demonstrated that glycine
interacted with a distinct recognition site, later found to reside on NR1 sub-
units. The positive allosteric nature of this interaction was further supported
by 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 afnity of glutamate for its binding site on
NMDA receptors (63,64) and that this effect was reciprocal, with glutamate
increasing the afnity of glycine (65,66).
It became even more interesting when glycineglutamate interactions
were investigated by means of fast perfusion systems, which made feasi-
ble the direct measurement of activation and desensitization kinetics of
the NMDA receptor channel complex. These techniques made possible the
100 Mugnaini
discovery of the so-called glycine-dependent desensitization, a time-
dependent loss of NMDA receptor function (in the continuous presence
of glutamate or NMDA) that could be almost completely overcome by
increasing glycine concentration (67). Glycine potentiation of NMDA-
evoked current was detected both during the initial (peak) response and at
equilibrium conditions (i.e., during the steady-state response recorded after
the onset of desensitization), therefore conrming both previous functional
studies reporting a glycine-induced increase in NMDA response at equilib-
rium conditions (60,61) and binding studies reporting an increase of NMDA
or glutamate afnity at equilibrium conditions (63,64). Glycine potentiation
of the steady state was much larger than that of the peak response, with the
result that, by increasing glycine concentration, the difference between the
steady state and the peak response (in other words, desensitization) progres-
sively diminished. This effect was found to depend on the ability of glycine
to dramatically speed up the rate of the recovery from desensitization (67).
Considering that the afnity of glycine during the peak response was higher
than during the steady state (68,69), these data suggest that the afnity of
glycine for the resting and open states of the NMDA receptor was higher
than its afnity for the desensitized state of the NMDA receptor and that
glycine favored the nondesensitized states of the NMDA receptor for a
mechanism of conformational selection.
To explain glycine reduction of desensitization, some authors have
hypothesized the existence of further NMDA receptor states: a glutamate-
(or NMDA-) unbound, closed conformation, with high afnity for glycine,
and a glutamate- (or NMDA)-bound, closed conformation with low afnity
for glycine, but in rapid equilibrium with an open (active) conformation
(57). This theory was later supported by results from electrophysiological
experiments on recombinant NR1/NR2A receptors, showing that glycine
afnity was higher in the absence of glutamate (70). The model described
the loss of glycine afnity upon glutamate binding in terms of negative
allosteric interaction (or negative cooperativity, following the authors ter-
minology) between the NMDA and the glycine binding sites, which might
seem somewhat misleading in the light of the more common reports of
positive allosteric interaction or coagonism between these two sites. A model
considering the coexistence of major discrete quaternary transitions (i.e.,
receptor isomerization) between clear conformational states (e.g., resting
state, open state, desensitized state) and, within each state, local reorganiza-
tion at the subunit level more directly linked to fractional glycine and
NMDA (or glutamate) binding (in other words, conformational induction)
might help in interpreting these high-resolution electrophysiological data
and reconciling them to more classical studies performed at equilibrium
conditions. Unfortunately, how fractional binding of glycine and glutamate
can affect quaternary organization at each conformational state is still a
matter of debate.
The fact that NMDA receptors are hetero-oligomers of four subunits
and 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 afnities, and/or glycineglutamate
allosteric interactions. Monaghan et al. (71) were the rst to hypothesize the
presence of NMDA receptor subtypes of this kind in rat brain. These
authors noticed the different regional distribution, in rat brain sections, of
radiolabeled NMDA site agonists and antagonists and initially explained
this difference with the possible existence of agonist- and antagonist-
preferring NMDA receptor subtypes, i.e., NMDA receptors with a rela-
tively higher afnity for NMDA site agonists and antagonists, respectively.
In the same study, the authors also noticed that glycine potentiation of
[
3
H]glutamate binding was regionally different in rat brain sections (with
striatum, for example, less sensitive than the cerebral cortex) and that
glycine, while increasing the binding of NMDA site agonists, had the
opposite effect on the binding of NMDA site antagonists. Following these
observations, these authors (71) theorized the possibility that agonist- and
antagonist-preferring NMDA receptor subtypes might have, in addition
to (or as an alternative to) intrinsic differences in agonist and antagonist
afnities, also different glycineglutamate allosteric interactions and there-
fore be differently regulated (in terms of binding at the NMDA site) by
the endogenous glycine, which is difcult to be properly washed away in
brain sections. This fact could explain why, in regions like the septum and
striatum, for the predominance of NMDA receptors with a more efcient
glycine allosteric modulation, there was relatively more NMDA-sensitive
[
3
H]glutamate binding (because of a greater glycine-induced potentiation
of binding) and relatively less binding of [
3
H]-3-(()-2-carboxypiperazin-
4-yl) propyl-1-phosphonic acid (CPP), an NMDA site antagonist (because
of a greater glycine-induced inhibition of [
3
H]-CPP binding). On the con-
trary, in other regions like the thalamus and the cerebral cortex, possibly
for a less efcient glycineglutamate allosteric interaction, there was a
relatively lower [
3
H]glutamate binding (corresponding to a smaller glycine-
induced potentiation) and relatively higher [
3
H]-CPP binding (correspond-
ing to a smaller glycine-induced inhibition). These differences could not
be explained in terms of regionally different concentrations of endogenous
glycine, as revealed by microdialysis studies (72,73).
The theory that the differentially distributed receptor subtypes
revealed by Monaghan et al. (71) differed in the efciency of the glycine
glutamate interaction was not demonstrated until the discovery of [
3
H]-D,
L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP39653) (74).
The high afnity and selectivity of this NMDA site antagonist, as well as
its interaction with a single binding site, made [
3
H]CGP39653 the radioli-
gand of choice for detecting NMDA receptors in studies of receptor binding
and autoradiography (64,75,76). It was therefore possible to determine that
102 Mugnaini
(i) glycine, while increasing the afnity of glutamate, was able to decrease
the afnity of [
3
H]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 [
3
H]CGP39653 binding (similar to glycine
enhancement of [
3
H]glutamate binding) was regionally different in rat brain
sections (with striatum, for example, less sensitive than the cerebral cortex),
suggesting the presence of regionally different NMDA receptor subtypes
with different glycineglutamate allosteric interactions; (iv) also, reversal
of glycine inhibition by 7-CKA or GV150526A was regionally different in
rat 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 site
agonists and antagonists; (v) in brain membranes (in which the levels of
endogenous glycine are much lower in comparison to brain sections) striatal
and cerebral cortical NMDA receptors did not present signicant differ-
ences in terms of glycine or glutamate afnity, or the afnity of any other
ligand used to demonstrate the existence of different allosteric interactions
(Table 2); and (vi) the potency of glycine allosteric inhibition of
[
3
H]CGP39653 binding was greater in the striatum than in the cerebral cor-
tex (apparent pK
i
7.48 and 6.98, respectively).
In other words, the existence of the regionally distinct NMDA
receptor subtypes reported previously was conrmed (71) and it was proven
Table 2 Afnity of Glycine and NMDA Site Ligands for Their Respective Binding
Sites and Potency of Glycine in GlycineGlutamate Allosteric Interaction, in the
Striatal and Cerebral Cortical Membranes
Striatum Cortex
Glycine binding site
[
3
H]glycine pK
D
7.08 0.08 (3) 7.01 0.04 (3)
7-CKA (vs. [
3
H]glycine) pK
i
6.89 0.09 (3) 6.71 0.07 (5)
GV150526A (vs. [
3
H]glycine) pK
i
8.47 0.10 (3) 8.49 0.02 (3)
Glutamate binding site
[
3
H]CGP39653 pK
D
7.80 0.05 (4) 7.91 0.08 (8)
Glutamate (vs. [
3
H]CGP39653) pK
i
6.66 0.06 (3) 6.51 0.05 (5)
Glycineglutamate interaction
Glycine (vs. [
3
H]CGP39653) App pK
i
(high) 7.48
a
0.05 (3) 6.98 0.02 (7)
App pK
i
(low) 3.89 0.06 (3) 3.49 0.11 (7)
a
Signicantly different from the cerebral cortex.
Abbreviations: NMDA, N-methyl-D-aspartic acid; 7-CKA, 7-chlorokinurenic acid; App, apparent.
Source: From Refs. 64, 75, 76, and 78.
that the difference between these receptor subtypes (at least for the striatum
and the cerebral cortex) resided in the potency of glycineglutamate allo-
steric interaction rather than in signicant differences in glycine or gluta-
mate afnity for their respective binding sites.
It is difcult to explain the features of these allosterically different
NMDA receptor subtypes in terms of possible differences in subunit compo-
sition. In functional studies, the sensitivity of recombinant NR1/NR2A
receptors to glycine is around 10-fold lower than that of NR1/NR2B,
NR1/NR2C, and NR2D receptors (7982). To some extent, binding studies
also show similar results, with the afnity 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 signicant differences in terms of glycine afnity,
despite their relatively greater abundance in NR2A and NR2B subunits,
Following the nding that NMDA site antagonists and agonists have
a slightly higher afnity for NR1/NR2A and NR1/NR2B recombinant
receptors, respectively (79,80), some authors have suggested that these sub-
unit combinations might represent the antagonist- and agonist-preferring
NMDA receptor subtypes previously found with receptor binding studies
in native tissues (84,85). This result, however, was not conrmed by other
authors, who found a higher afnity of both agonists and antagonists for
the NR1/NR2A rather than the NR1/NR2B combination (25). In addition,
receptor binding studies suggest that regions containing antagonist- and
agonist-preferring NMDA receptor subtypes (namely, the cerebral cortex
and the striatum) do not present signicant differences in terms of
agonist and antagonist afnities at the NMDA binding site (Table 2). More-
over, the relatively higher abundance of the NR2B mRNA in the striatum
than the cerebral cortex (with respect to the NR2A) might suggest that this
subunit confers agonist-preferring characteristics on NMDA receptors, but
this is not true for the thalamus, which also contains relatively more NR2B
than NR2A mRNA (14,86). Finally, the afnity of [
3
H]glutamate was only
slightly increased by glycine in the NR1/NR2B combination, when com-
pared with the NR1/NR2A combination (84), which agrees with the smaller
glycine-induced increase of [
3
H]glutamate binding and smaller glycine-
induced decrease of [
3
H]CGP39653 binding in regions containing agonist-
preferring receptors, like the striatum. This nding, however, does not
explain why the same region presented the greatest enhancement of
[
3
H]CGP39653 binding by 7-CKA or GV150526A.
Interestingly, NR1/NR2A recombinant receptors appeared to form
more efciently than other combinations and looked very similar to native
receptors in terms of binding properties (i.e., NMDA site agonist and
antagonist afnity) and allosteric interactions, with glycine increasing the
afnity of [
3
H]glutamate and inhibiting [
3
H]CGP39653 binding (25,84,87).
104 Mugnaini
respectively (Table 2).
Given the widespread distribution of the NR2A subunit mRNA in rat brain
(which overlaps that of NR2B, NR2C, and NR2D subunits mRNA), it is
reasonable to imagine that NR1/NR2A receptors may provide a core to
which the other subunits can attach and inuence the properties of the
whole receptor. Indeed, the coexistence of more than one member of
the NR2 family in the same receptor has been revealed by several authors
(8890). However, there are no reports of studies describing glycine
glutamate allosteric interactions in such complex combinations of NMDA
receptor subunits (such as NR1/NR2A/NR2B, NR1/NR2A/NR2C, or
NR1/NR2A/NR2D), so that a comparison with the NR1/NR2A combi-
nation and native receptors is not possible. In addition, different splice
variants of the NR1 subunit family might account for the different glycine
glutamate allosteric interactions observed in native receptors.
Glycine afnity in native cerebellar NMDA receptors is lower than
that of the cerebral cortex, especially at cerebellar granule cells (91). In addi-
tion, glycine did not potentiate NMDA-induced currents in cerebellar
mRNA-injected oocytes, while, as expected, it potentiated cerebral mRNA-
injected oocytes (92). These data might suggest that native NMDA receptors
containing the NR2C subunit (which is highly expressed in the granular
layer of cerebellum) have a lower afnity for glycine and/or lower potency
in the glycineglutamate interactions. Functional and binding studies on
heteromeric NR1/NR2 receptors, however, do not support this hypothesis,
with glycine afnity for NR1/NR2C receptors being similar or higher than
that for NR1/NR2A and NR1/NR2B receptors (14,70,80,84). Glycine
afnity in recombinant NMDA receptors in a tri-heteromeric combination
containing the NR2C subunit (e.g., NR1/NR2A/NR2C), however, has
never been determined.
The Zinc (Zn
21
) Binding Site
Many experimental results have demonstrated that zinc ions (Zn
2
) inhibit
NMDA receptor function by interaction with a specic site, distinct from
the Mg
2
binding site and located on the extracellular surface of the recep-
tor (9396). In cultured neurons, Zn
2
inhibits NMDA receptor function in
a voltage-independent manner, at concentrations as low as 110 mM, and
in a voltage-dependent manner, at concentrations of 10100 mM (97,98).
These two effects of Zn
2
on NMDA receptor function have been termed
as high-afnity, voltage-independent inhibition and low-afnity, voltage-
dependent inhibition, respectively.
Among heteromeric NR1/NR2 receptors, the highest sensitivity for
the high-afnity, voltage-independent Zn
2
inhibition is given by those con-
taining the NR2A subunit, suggesting the presence of a specic Zn
2
nega-
tive modulatory site responsible for this activity on this subunit (99102).
Indeed, more recent studies have revealed that Zn
2
resides in the cleft of
the bilobate structure of the NR2A subunit (103105) and that inhibition of
Zn
2
is involved in the fast desensitization of NR1/NR2A receptors (106).
Recent molecular modeling studies suggest that Zn
2
stabilizes the open
state of the ATD, which corresponds to a desensitized form of the NMDA
receptor (35). The sensitivity for the high-afnity, voltage-independent Zn
2
inhibition of NR1/NR2A receptors is higher in the presence of protons (low
pH), 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
Zn
2
inhibition is determined also by the presence of the N1 insert in the NR1
subunits, which, in fact, decreases both voltage-independent Zn
2
inhibition
and proton (H
) inhibition of the NR1/NR2A and NR1/NR2B combina-

tions, suggesting that Zn
2
and H
may share similar structural determinants

(101,102). In line with this nding, glycine-independent spermine potentiation
of NMDA receptor function relieves both voltage-independent Zn
2
and H
inhibition (102,108).
The low-afnity, voltage-dependent inhibition by Zn
2
is likely to occur
within the ion channel pore and seems to involve the same residue on the M2
segment of the NR1 subunit implicated in Mg
2
block and Ca
2
permeability
of the receptor channel (109111).
In addition to its well-known inhibitory effect at high concentrations,
Zn
2
potentiates agonist-induced currents at submicromolar concentra-
tions (EC
50
0.5 mM) in a voltage-independent manner (112). This effect,
however, is seen only in homomeric receptors made of subunits lacking
the N1 insert (NR1
0XX
) and is absent in all kinds of NR1/NR2 hetero-
meric receptors, independently of the N1 insert in the NR1 subunit. The
presence of native functional homomeric NMDA receptors, however, has
never been demonstrated and the physiological relevance of the very high
afnity, voltage-independent stimulation of NMDA receptors is still to
be resolved.
The Phenylethanolamines Binding Site
Ifenprodil, a phenylethanolamine derivative, is a noncompetitive NMDA
site antagonist, initially thought to have antagonistic effect at the polyamine
positive modulatory sites (113115). In line with the glycine-dependent stim-
ulatory effect of polyamines, which is present only with NR1/NR2B
recombinant receptors, ifenprodil selectively inhibitedNMDAreceptors com-
posed of the NR2B subunit, with a 400-fold higher afnity 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 polyamine
binding site (118120). In fact, residues on the NR2B subunit important
106 Mugnaini
LBD and the transmembrane region (see section The Proton Modulatory
Site), but also for the protonation of histidine residues in the ATD, which
for polyamine stimulation are not required for ifenprodil inhibition and
vice versa. In addition, ifenprodils effect is independent of the presence of
the N1 cassette in NR1 splice variants, unlike polyamine stimulation.
Rather, polyamine and ifenprodil binding sites on the NR2B subunits seem
to 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 NR2B
subunit (123125). Functional studies suggest that ifenprodil (and
structurally related compounds, like Ro 84304) antagonizes NMDA recep-
tor function with an activity-dependent mechanism (126,127) and by
increasing fast desensitization (106). Similar to the action of Zn
2
at the
NR2A subunit, ifenprodil is thought to bind to the open state of the
ATD domain of the NR2B subunit and stabilize the desensitized form of
the NMDA receptor (35). As with Zn
2
inhibition, ifenprodil inhibition
of NMDA receptor function is also potentiated by protons (128,129). How-
ever, mutation of several residues in the ATD of the NR1 subunit also
affects sensitivity to ifenprodil, suggesting that some molecular determinants
of the ifenprodil binding site may also be located on the NR1 subunits, or
that these residues are important for the stabilization of the binding site
or subunitsubunit interactions (124).
Interestingly, neonatal rat brain NMDA receptors have a uniformly
high afnity for ifenprodil, which decreases during development because
of the appearance of a lower afnity component (117). This nding corre-
lates well with the fact that NR2B subunits are expressed much earlier in
development than NR2A subunits (130).
The Polyamine Binding Sites
Modulation of NMDA receptor activation by polyamines was rst reported
by Ransom and Stec (131), who showed that spermine and spermidine
increased the binding of [
3
H]()-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)
cyclohepten-5,10-imine (MK801) to rat brain membranes, an index of
NMDA receptor channel activation (132134). 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
(135137). Nevertheless, the most relevant effect of spermine as an endoge-
nous modulator remains its facilitatory inuence on NMDA receptor
mediated neurotransmission (138), especially under pathological conditions
such as brain ischemia, where its production is dramatically enhanced (139).
The effect of polyamine on the afnity of the primary neurotransmitter glu-
tamate on native receptors is still uncertain, with studies reporting a small
decrease of glutamate afnity by 100 mM spermine (140) and higher
concentrations of spermine (up to 1 mM) increasing the binding of
interaction site has been hypothesized for this class of compounds (Table 1)
[
3
H]glutamate (135). In contrast, receptor binding studies on native recep-
tors clearly support the evidence that polyamines increase glycine afnity
to its binding site (65,141).
Electrophysiological studies have proved the existence of two different
mechanisms 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 linked
to the spermine-induced increase of the afnity for glycine at its modulatory
site (see earlier paragraphs). Glycine-independent stimulation involves an
increase of channel opening frequency and a decrease of the onset rate of
glycine-independent desensitization (136,142147). This effect, which can be
revealed only at saturating concentrations of glycine, probably corresponds
to the increase of [
3
H]MK801 binding observed in initial studies and is
likely to be the most relevant effect of spermine on NMDA receptor
function 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 of
tonic H
and Zn
2
inhibition of NMDA receptor function (108) and is
absent in NR1 splice variants carrying the N1 insert. This suggests that this
segment, for its structural similarities to polyamines, might relieve NMDA
receptors from tonic H
and Zn
2
inhibition by binding to the same site of
polyamines. More recently, it has been shown that two residues critical for
spermine potentiation are in the ATD of the NR1 subunit, very close to the
site of the N1 insert, suggesting that this spermine binding site resides in
the ATD of the NR1 subunit and that N1 insert constitutively occupies this
binding site in NR
1XX
subunits. However, only receptors containing the
NR2B subunit present glycine-independent stimulation by spermine, sug-
gesting that this subunit also carries part of the binding site specic for the
allosteric effect of polyamines (119,148,149). Recently, it has been proposed
that spermine binds to residues between the R2 lobes of NR1 and NR2B sub-
units, stabilizing the dimer interface and the closed conformation of the ATD
of these subunits, therefore favoring the open and resting states of the NMDA
receptor and diminishing the number of receptors in the desensitized state (35).
Another hypothetical site of spermine binding is the rst steroid modulatory
domain (SMD1), another segment that may be involved in dimer formations
that glycine-independent spermine stimulation of NMDA receptor function is
reduced by 80% if the SMD1 of the NR2B subunit is substituted with the
corresponding sequence of the NR2D subunit, suggesting that SMD1 also
plays a key role in this specic allosteric effect of polyamines. Finally, it is
worthwhile to mention that Mg
2
ions, which block NMDA receptor channels
in a voltage-dependent manner, at very high, millimolar concentrations, can
potentiate 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 with
NR1 splice variants lacking the N1 cassette and only with the NR1/NR2B
combination), suggesting that Mg
2
may be the physiological agonist acting
at the NR2B subunitspecic spermine site (151).
Glycine-dependent stimulation of NMDA receptor function is seen
only for homomeric NR1 channels and NMDA receptors carrying the
NR2A and/or the NR2B subunits (21,33). Overall, these data are in line
with the nding that the binding of [
3
H]MK801 to NMDA receptors in
the cerebellum (which should contain a predominance of NMDA receptors
carrying the NR2C subunits) appears to be less sensitive to the action of
polyamines than is the case with forebrain receptors in which the NR2A
and NR2B subunits are highly expressed (152,153).
Electrophysiological studies on recombinant receptors have also con-
rmed the existence of mechanisms of negative allosteric modulation by
spermine, 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 concentration
of glutamate (or NMDA) in the case of NR1/NR2B (but not NR1/NR2A)
receptors: at low NMDA or glutamate concentration, spermine induced a
small decrease in NMDA, and glutamate afnity, which counteracted the
stimulatory effect of spermine, resulting in little net effect of spermine
(148). These results suggest that endogenous polyamines might act as a
bidirectional gain control at some native NMDA receptors, by dampening
the response at low concentrations of glutamate and enhancing the response
at high concentrations of glutamate. Less relevant under physiological con-
ditions is a mechanism of voltage-dependent inhibition of NMDA receptor
function, which occurs only at hyperpolarized potentials and in the absence
of extracellular Mg
2
, possibly representing a direct block of the ion channel
by spermine (142,144,146,154). This effect was present at NR1/NR2A and
NR1/NR2B receptors but was absent on NR1/NR2C (81).
The Histamine Binding Site
The nding that histamine can modulate currents gated by the NMDA
receptor dates back to 1984 (155). Later, it was found that the potent
enhancement 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, to
the glycine-independent stimulation by spermine: the ability of histamine
to positively modulate NMDA receptor function is present only with NR1
subunits 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,
the stimulatory effect of histamine, unlike that of spermine, is characterized
by a rapidly developing increase in magnitude followed by marked and slow
desensitization to a steady-state level, indicating that histamine acts at a
novel recognition site distinct from that of spermine (157).
At highconcentrations (1 mM), histamine alsocauses avoltage-dependent
inhibition of NMDA currents, also at receptors that are not sensitive to
stimulation 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 molecule
that diffuses readily through both uid and lipid phases and is generated in
neurons in response to activation of NMDA receptors. In turn, arachidonic
acid 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 receptor
function is based on the activation of phospholipase A
2
by Ca
2
(which
enters the cell through the NMDA receptor) and the consequent production
of arachidonic acid, which in turn potentiates NMDA receptor currents
(159). The stimulatory effect of arachidonic acid is observed even with a
saturating effect of agonists at the glutamate and glycine binding sites of
the 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 Mg
2
, Zn
2
, and polyamine binding
sites. Finally, it has been shown that arachidonic acid binds to a 131 amino
acid residue domain on the amino terminal of NR1 subunits, which has sig-
nicant homology with fatty acidbinding proteins (160). These results sug-
gest that arachidonic acid binds to a distinct allosteric site on the NMDA
receptor. The NMDA receptors containing the NR2A seem to be more sen-
sitive to arachidonic acid than the NR2B subunitcontaining 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 LennoxGastaut
syndrome in children and complex partial seizures in adults. Initially found
to decrease NMDA-induced neuronal injury (but not kainate-induced neu-
ronal injury) in cultured cortical neurons (162) and to reduce NMDA
receptormediated postsynaptic potentials in hippocampal slices (163),
FBM was nally characterized as a noncompetitive NMDA receptor
antagonist selective for receptors composed of NR1 and NR2B subunits
(164,165). Similar to ifenprodil, FBM enhanced the afnity of agonists at
the glutamate binding site. However, FBM increases the binding of [
3
H]gly-
cine to the NMDA receptor (166) and, unlike ifenprodil, pH did not affect
its afnity for the NR1/NR2B receptor (164). A point mutation on the
110 Mugnaini
NR2B subunit, which affects receptor sensitivity to ifenprodil, does not dra-
matically change the afnity of FBM, suggesting that ifenprodil and FBM
bind to different sites. It has been shown recently that FBM selectively binds
to the activated and desensitized states of NMDA receptors, with the result
that 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 normal
neuronal ring. In addition, FBM was shown to stabilize the desensitized
form of the NMDA receptor also in the absence of glutamate, possibly
helping to prevent the formation of excessive NMDA currents that may
occur 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
(H
), with IC
50
values close to physiological pH. This suggests the existence,
on the NMDA receptor, of one or more negative modulatory sites for H
and implies that NMDA receptor channels are under tonic inhibition of
around 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-afnity,
voltage-independent inhibition by Zn
2
(104,170), the negative modulation
by phenylethanolamines (128,129), and the glycine-independent enhance-
late neurotransmitter-induced gating through a mechanism of negative
allosteric coupling, with spermine increasing receptor activity by decreasing
proton inhibition. On the contrary, ifenprodil (phenylethanolamines) and
Zn
2
inhibit NMDA receptors by enhancing proton inhibition (104).
Recent scanned mutagenesis experiments of the NR1 subunit indicate
that residues that control proton inhibition are localized in discrete regions,
namely, in the extracellular end of M3 and the adjacent linker leading to the
S2 portion of the glycine binding domain (M3S2 linker) and in the linker
between the S2 region and M4 (M4S2 linker; 107). Interestingly, the
M3S2 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 M4S2 linker is downstream
from this motif. Also the M3S2 and M4S2 linkers of the NR2 subunits
present the molecular determinants for proton sensitivity; nevertheless, the
NR2A, NR2B, and NR2D subunits present, in the M4S2 linker, a histidine
(His) residue that is absent in the NR2C subunit and is likely to be an
ment of NMDA receptor function by polyamines (see section The Zinc
Binding 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 M4S2 linker partially over-
laps the SMD1, recently discovered on the NR2B subunit (see section The
Steroid Modulatory Sites). Interestingly, SMD1 is a critical determinant of
proton sensitivity in NR1
0XX
/NR2B receptors (150). The SMD1 sequence is
conserved between NR2A and NR2B, but signicant differences exist
between NR2B (and NR2A) and NR2C and NR2D. Substituting the
SMD1 sequence of the NR2B subunit with that of the NR2D subunit
decreases the proton sensitivity of NR1/NR2B receptors, suggesting that
NR2D (and NR2C) SMD1 domains lack part of the residues that confer
proton sensitivity to the NR2B (and NR2A) subunit.
Other mutations that affect (although to a lesser extent) the sensitivity
to protons are found in the ATD domain and S1 region of the N1 subunit
(107), conrming previous ndings that some molecular determinants of
proton inhibition may be located near the N1 insert site or in the ATD
(108,124). Interestingly, NR1 subunits carrying the N1 insert (NR1
1XX
)
are less sensitive to proton inhibition and need a higher H
concentration
(lower pH) to produce the same inhibition of NMDA receptor function
(pH 6.66.8 and pH 7.27.4 for NR1
1XX
and NR1
0XX
, respectively).
In addition, specic for the NR2A subunit, the protonation of some histi-
dine residues in the ATD seems to inhibit NMDA receptor function (and
potentiate the inhibitory effect of Zn
2
), by increasing the rate of ATD
opening and desensitized state formation (35,104).
Overall, these data suggest that the so-called proton sensor, pre-
viously identied in the proximity of the N2 insert of the NR1 subunit, actu-
ally resides in the M3S2 and M4S2 linkers of all NR1 and NR2 subunits.
However, other proton modulatory sites, capable of modifying NMDA
receptor 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, specically potentiates
the response of the NMDA receptor, while inhibiting GABA, glycine, and
non-NMDA iGluRs (171173). In addition, it was shown that a variety
of sulfated steroids modulate the NMDA response in either a positive or
a negative direction with a high degree of structural specicity. 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 (174176).
Functional studies on recombinant NMDA receptors demonstrated
that residues on the NR2 subunit are key determinants of modulation of
PS and 3a5bS (177). Indeed, very recently, Jang et al. (150) have identied
a 78-aa segment on the subunit NR2B that mediates the selective
112 Mugnaini
potentiation of PS on NMDA receptors, the so-called SMD1. Interestingly,
SMD1 is conserved within the iGluR family and corresponds to the amino
acid sequence involved in regulating the gating kinetics and binding of a
positive modulator, cyclothiazide, to the AMPA receptor (178). The
domain contains two a-helices, J and K, located in the S2 region of the
bilobate structure of the glutamate binding site and the contiguous fourth
transmembrane domain (M4). More precisely, J and K helices reside at the
dimerization interface between two AMPA subunits, and binding of
cyclothiazide promotes, by rearrangement of structures at this interface,
dimer formation and alleviates desensitization. Molecular modeling, based
on AMPA receptor structure, suggests that SDM1 of the NR2B subunit
may contribute residues to a hydrophobic pocket capable of accommodat-
ing PS (150). Within the NR2 subunit family, SMD1 is highly conserved,
but signicant differences exist. This reects the fact that PS potentiates
the response of recombinant receptors containing the NR2A and NR2B
subunits, while it inhibits the response of receptors containing the
NR2C and NR2D subunits (177). Modifying the amino acid sequence near
the inner interface on NR2B to that of NR2D eliminates positive allosteric
modulation 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 (pH6.6 and 7.5 for
mutated and wild-type NR1/NR2B receptors, respectively). In addition,
they lose spermine potentiation, which depends on the relief of tonic proton
inhibition. Therefore, SMD1 is important for PS potentiation, proton inhibi-
tion, and spermine potentiation. It should be underlined, however, that PS
stimulates NMDA receptor channels via a route independent of the proton
sensor; rather, it involves the SMD1, although the proton sensor and
SMD1 may share some common elements (the M4S2 linker). This is
proven by the fact that the potentiating effect of PS is abolished by the
lack of SMD1.
The ATP Modulatory Site
Adenosine 5-triphosphate (ATP) has both inhibitory and facilitating effects
on NMDA receptor activity, depending on the ATP concentration. Inhi-
bition of NMDA receptors by guanine nucleotides has been reported
previously using radioligand binding assays (179,180). Indeed, recent
electrophysiological experiments on native and recombinant heteromeric
NR1/NR2A and NR1/NR2B (but not NR1/NR2C) receptors expressed
in Xenopus laevis oocytes have revealed that, at a low concentration of glu-
tamate, ATP and other nucleotides behave as competitive antagonists of
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, NMDA
current is potentiated in the presence of ATP, indicating that ATP binds
as a positive allosteric modulator of NMDA receptor function. Interestingly,
the afnity of ATP for its modulatory site on the NMDA receptor is 15-fold
lower 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 in
the presence of high concentrations of both glutamate and ATP, whereas
the inhibitory effect of ATP can be revealed only at low concentrations of
both glutamate and ATP. If we consider that ATP might be co-released with
glutamate into the synaptic cleft, the nal action of ATP may consist in
focusing and enhancing the effects of glutamate at regions near the transmit-
ter release sites (182). Finally, at some synapses in which zinc ions are also
released into the synaptic cleft, potentiation of NMDA receptor function
by ATP may be further increased by chelation of Zn
2
ions and relief of
tonic Zn
2
inhibition of NMDA receptor function (see earlier paragraphs).
Overall, the evidence that ATP can enhance NMDA receptor function is
in line with the nding that ATP promotes the induction of LTP via a
direct action on NMDA receptors, with no involvement of P2X or P2Y
receptors (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 a
redox modulatory site in equilibrium between a fully reduced state (thiolate
anion, RS
) and an oxidized state (disulde, RSSR). The effect of DTT has

two components, a reversible potentiation, which disappears spontaneously
by washout of DTT, and a persistent (irreversible) potentiation, which is
abolished only by an oxidizing agent (185). The reversible potentiation
is present only in the NR1/NR2A combination, suggesting that it may be
mediated by DTT chelation of Zn
2
, which selectively inhibits the function
of 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, which
constitute the proper redox modulatory site of the NMDA receptor (186).
These residues are located in the hinge of the cleft of NR1 and therefore
affect the movement of the clam shelllike LBD. When the disulde bond
is reduced, the structure is more exible and the closure of S1 and S2
domains around glycine is facilitated (31).
There is increasing evidence, however, that cysteine residues of the
ATD of NR1
0XX
and NR2A subunits also play a role in sensitivity to redox
agents (187). These residues might modulate the exibility of the bilobate
114 Mugnaini
structure of the ATD, as occurs in the LBD. Alternatively, some authors
have suggested the existence of cysteine bridge links between the R1
domains of different subunits (35).
NOproducing agents also inhibit NMDA receptor channels, prob-
ably via donation of NO
ions to the cysteine thiolate anions (RS
) of
the redox modulatory site of the NMDA receptor. This reaction, termed
S-nitrosylation, leads to the formation of unstable S-nitrosothiols (RSNO),
which more easily form disulde bonds, resulting in a persistent block of the
NMDA receptor channel (188190). Interestingly, a negative feedback
mechanism of NMDA receptor function has been proposed, which consists
of stimulation of nitric oxide synthase (NOS) by calcium (which enters the
cell following the opening of the NMDA receptor channel), with production
of NO, which in turn inhibits NMDA receptor function via interaction with
the redox modulatory site (159,188).
OTHER SUBSTANCES MODULATING NMDA
RECEPTOR FUNCTION
In addition to the substances regulating NMDA receptors through the inter-
action with the allosteric modulatory sites described above, a variety of
other compounds have been reported to modify NMDA receptor function
through different (indirect) or still unknown mechanisms. This section will
describe the effect of substances modulating NMDA receptor function
through nonallosteric mechanisms, or for which a clear interaction with
an allosteric site on the NMDA receptor has not yet been produced.
Ethanol
Ethanol inhibits NMDA receptor channels in a concentration-dependent
manner (191195). The sensitivity to ethanol seems to be independent of
the NR1 splice variant, whereas NMDA receptors containing the NR2A
and NR2B subunits are more sensitive to ethanol than those containing
the NR2C subunit. Interestingly, ethanol sensitivity is enhanced by a
calcium-dependent process that involves the interaction of the intracellular
C0 domain of the NR1 subunit (a region of the cytoplasmic tail common to
all NR1 splice variants) with proteins of the actin cytoskeleton (196). In
addition, many reports suggest that ethanol inhibition of NMDA receptor
function may occur through ethanol regulation of the activity of different
protein kinases and phosphatases (which in turn change NMDA receptor
function by phosphorylation or dephosphorylation) rather than through a
direct interaction with an allosteric site on the NMDA receptor activation
(197200). A few studies, however, also suggest that ethanol may interact
with specic amino acids in the M3 and M4 domains, which are involved
in transducing agonist binding to channel opening and desensitization
(201,202). In support of this theory, the kinetics of blockade of
NMDA-gated currents by ethanol seem to be too rapid to be explained only
in terms of phosphokinase activation (203).
General Anesthetics
Recent work has implicated NMDA receptor inhibition as a major source of
CNS depression with general anesthetics, from gaseous anesthetics [e.g.,
nitrous oxide (N
2
O) and xenon (Xe)] to volatile anestethics (VAs; e.g., isour-
ane) and intravenous anesthetics (e.g., ketamine; 204208). Recently, it has
been shown that the NMDA receptor is an essential requirement for the
behavioral action of N
2
O (but not of VAs) in Caenorhabtidis elegans (209).
Apart from dissociative anesthetics, like ketamine, which produce an
open channel block of NMDA receptor function (like MK801), the molecu-
lar determinants of many anesthetics remain to be identied. Recent evi-
dences, however, suggest that many different VAs produce a similar
inhibition of NMDA-gated currents and that the kinetics for these agents
are 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 inuences mesocor-
ticolimbic dopamine release. Recently, it has been shown that, in voltage-
clamped neurons freshly isolated from the ventral tegmental area, IL-2, at
physiologically relevant concentrations (0.0110 ng/mL), inhibits NMDA-
induced currents in a voltage-independent manner, while at higher doses
(>50 ng/mL) it signicantly increases NMDA-induced currents in a
voltage-dependent fashion (210). The inhibitory effect was competitive for
the glutamate binding site of the NMDA receptor, whereas the obligatory
requirement of intracellular ATP for the stimulatory effect suggests that this
phenomenon may be determined by the ability of the neuron to maintain
intracellular 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 OF
NMDA RECEPTORS
Given their abundant and widespread distribution in mammalian neuronal
tissue and their importance in excitatory transmission and normal CNS
functioning, it is reasonable to imagine that NMDA receptors are involved
in a variety of neuropsychiatric diseases and that drugs targeting this class of
glutamate-gated ion channels may have great therapeutic potential. How-
ever, overstimulation of NMDA receptors, as occurs in the presence of high
concentrations of glutamate or other competitive agonists, leads to excess
116 Mugnaini
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 brain
functioning and produce a series of adverse effects such as learning and
memory impairment, neurotoxicity, disturbances of motor coordination,
hallucinations, centrally mediated increase of blood pressure, catatonia,
and anesthesia (211218). Drugs acting at the different allosteric sites of
the 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 specic
neuronal pathways.
Positive Allosteric Modulators
The hypothesis that hypofunction of the glutamatergic system might occur
in schizophrenia was rst made by Carlsson and Carlsson (219), who
noticed how NMDA channel blockers, like phencyclidine and ketamine,
caused a schizophrenic-like syndrome in humans, recapitulating both the
positive and the negative symptoms of this disease. In the following years,
clinical and preclinical evidence strongly suggested that potentiation of
NMDA receptor function may improve memory and cognition, and there-
fore be benecial in cognitive disorders and schizophrenia (220). More
recently, it has been shown that enhancement of NMDA receptor function
may facilitate extinction of conditioned fear, and therefore be benecial also
in anxiety disorders (221).
Glycine site agonists, or compounds acting through the steroid, poly-
amines, and ATP positive modulatory sites of the NMDA receptors may
have benecial effects in treating these disorders. Indeed, glycine and glycine
site agonists, like D-serine and D-cycloserine, have proven efcacy when
given in addition to standard antipsychotic therapy for the treatment of
schizophrenia (222,223). Interestingly, overexpression of the NR2B subunit
improves learning and memory in mice (224), suggesting that NR2B
subunitcontaining 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 by
spermine, located between the NR1 and the NR2B subunits, might be of
special interest for treating schizophrenia.
Negative Allosteric Modulators
It is now generally accepted that cell death caused by sustained or prolonged
NMDA receptor overactivation is the primary mechanism of neuronal
death following cerebral ischemia (139) and may be an important cofactor
of neuronal damage in many neurodegenerative diseases such as
Parkinsons, Huntingtons, and Alzheimers diseases (219,225228). In
addition, it is now well established that overactivity of excitatory pathways
may be the source of epilepsy and neuropathic pain (229232). There is also
growing evidence that inhibition of NMDA receptor function may be
benecial in counteracting different aspects of substance use disorders, from
the expression and maintenance of morphine dependence (233,234), the
acquisition 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 efcacious in several animal
effects reported for competitive NMDA site antagonists and NMDA chan-
nel blockers (244). Unfortunately, up to now, clinical trials with glycine
antagonists have failed to meet preclinical expectations, showing little or
no therapeutic benet (245247). The difculties associated with the inter-
pretation of clinical data for complex pathologies like stroke and traumatic
brain injury, together with the initial concern regarding brain penetration
and brain availability of glycine antagonists, might have been the reason
for these negative results. Hopefully, further trials with compounds with
better pharmacokinetic proles, supported by positron emission tomogra-
phy studies monitoring the levels of NMDA receptor occupancy in healthy
volunteers and patients, will reveal the potential therapeutic value of
these compounds.
NR2B subunitselective negative allosteric modulators of NMDA
receptor function can be obtained with compounds binding at the phenyl-
ethanolamine binding site (like ifenprodil). These compounds may have
special interest, given the abundance of the NR2B subunit in the dorsal horn
of the spinal cord and in the caudate putamen, for neuropathic pain and
Parkinsons disease, respectively. Indeed, there is signicant preclinical
evidence of the antinociceptive and antiparkinsonian efcacy of NR2B-
selective compounds (248250).
Interestingly, mice decient in the NR2A subunit or the NR2C sub-
unit show attenuation of focal ischemic brain injury (251,252), suggesting
that NR2A-selective negative modulators, such as compounds acting
through the high-afnity Zn
2
binding site, or NR2C-selective inhibi-
tors may be particularly efcient neuroprotectants in cerebral stroke and
similar 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. Their
heteromeric structure, in terms of subunit composition and the large number
of subunits cloned so far, suggests that many NMDA receptor subtypes may
118 Mugnaini
models of these diseases [see reviews (241243)] without displaying the side
exist that play different roles in distinct neuronal pathways. The great
number and heterogeneity of allosteric sites on NMDA receptors offer
the potential to design drugs that selectively modulate the function of distinct
NMDA receptor subtypes, or act on many NMDA receptors at the same time.
Establishing the subunit composition of native NMDA receptors and
their signicance in normal and disease states remains the major challenge at
this point. Immunoprecipitation and purication of native NMDA recep-
tors, followed by their pharmacological characterization, may help in clar-
ifying their subunit composition, whereas a more extended phenotypic
characterization of knockout mice would be useful to understand the role
played by different subunits in different pathologies. Especially the genera-
tion of conditional and tissue-restricted knockdown mice, with the use of
RNA interference technology, would be of great help to study the function
of specic NMDA receptor subunits and their involvement in the etiology of
specic CNS diseases (253).
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2006 by Taylor & Francis Group, LLC

) inhibition of the NR1/NR2A and NR1/NR2B combina-

may share similar structural determinants

) and an oxidized state (disulde, RSSR). The effect of DTT has

ions to the cysteine thiolate anions (RS

binding to NMDA receptors. Neuron 2000; 25:683694.

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