Modulation of glycine potency in rat recombinant NMDA receptors containing
chimeric NR2A/2D subunits expressed in Xenopus laevis oocytes
Philip E. Chen, Matthew T. Geballe, Elyse Katz, Kevin Erreger, Matthew R. Livesey,
Kate K. O'Toole, Phuong Le, C. Justin Lee, James P. Snyder, Stephen F. Traynelis and
David J. A. Wyllie
J. Physiol. 2008;586;227-245; originally published online Oct 25, 2007;
DOI: 10.1113/jphysiol.2007.143172
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227
J Physiol 586.1 (2008) pp 227–245
Modulation of glycine potency in rat recombinant NMDA
receptors containing chimeric NR2A/2D subunits expressed
in Xenopus laevis oocytes
Philip E. Chen1 , Matthew T. Geballe2 , Elyse Katz3 , Kevin Erreger3 , Matthew R. Livesey1 , Kate K. O’Toole3 ,
Phuong Le3 , C. Justin Lee3 , James P. Snyder2 , Stephen F. Traynelis3 and David J. A. Wyllie1
1
Centre for Neuroscience Research, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK
Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA
3
Department of Pharmacology, Emory University School of Medicine, Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30322, USA
2
Heteromeric NMDARs are composed of coagonist glycine-binding NR1 subunits and
glutamate-binding NR2 subunits. The majority of functional NMDARs in the mammalian
central nervous system (CNS) contain two NR1 subunits and two NR2 subunits of which there
are four types (A–D). We show that the potency of a variety of endogenous and synthetic
glycine-site coagonists varies between recombinant NMDARs such that the highest potency is
seen at NR2D-containing and the lowest at NR2A-containing NMDARs. This heterogeneity is
specified by the particular NR2 subunit within the NMDAR complex since the glycine-binding
NR1 subunit is common to all NMDARs investigated. To identify the molecular determinants
responsible for this heterogeneity, we generated chimeric NR2A/2D subunits where we exchanged
the S1 and S2 regions that form the ligand-binding domains and coexpressed these with
NR1 subunits in Xenopus laevis oocytes. Glycine concentration–response curves for NMDARs
containing NR2A subunits including the NR2D S1 region gave mean glycine EC50 values similar
to NR2A(WT)-containing NMDARs. However, receptors containing NR2A subunits including
the NR2D S2 region or both NR2D S1 and S2 regions gave glycine potencies similar to those seen
in NR2D(WT)-containing NMDARs. In particular, two residues in the S2 region of the NR2A
subunit (Lys719 and Tyr735) when mutated to the corresponding residues found in the NR2D
subunit influence glycine potency. We conclude that the variation in glycine potency is caused
by interactions between the NR1 and NR2 ligand-binding domains that occur following agonist
binding and which may be involved in the initial conformation changes that determine channel
gating.
(Received 20 August 2007; accepted after revision 23 October 2007; first published online 25 October 2007)
Corresponding author D. J. A. Wyllie: Centre for Neuroscience Research, Hugh Robson Building, University of
Edinburgh, George Square, Edinburgh EH8 9XD, UK. Email: dwyllie1@staffmail.ed.ac.uk
In the mammalian central nervous system (CNS),
NMDARs are heteromeric glutamate receptor–channels
predominantly composed of two NR1 and two NR2
subunits, of which there are four subtypes (NR2A–D).
These receptors mediate the ‘slow’ component of
the excitatory postsynaptic current in neurones and
have been implicated in various physiological and
pathophysiological processes in the CNS. In contrast to
the ubiquitously expressed NR1 subunit, the expression
of the NR2 subunit is regulated temporally and spatially
within the mammalian brain. These NR2 subunits impart
a variety of distinct biophysical and pharmacological
This paper has online supplemental material.
properties on the NMDAR complex that characterize
different NMDAR subtypes (for reviews see Dingledine
et al. 1999; Cull-Candy et al. 2001; Erreger et al. 2004).
In addition, there are NR3A and B subunits that are
thought to assume a modulatory role within the NMDAR
complex. Each subunit is composed of a number of specific
functional regions: an amino-terminal domain, which is
the site of action for a number of modulatory agents; the
ligand-binding domain (LBD); the membrane-associated
domains, which form the ion channel pore; and an
intracellularly located carboxyl-terminal domain, which
allows the receptor to interact with various signalling
and scaffolding molecules. A cartoon depiction of the
structure of an NMDAR subunit is shown in Fig. 1A.
Uniquely among ionotropic receptors, NMDARs require
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P. E. Chen and others
both glutamate and the coagonist, glycine, to bind to
the receptor for channel activation to occur (Johnson
& Ascher, 1987; Kleckner & Dingledine, 1988). The
glycine binding site is located on the NR1 subunit and
the glutamate binding site on the NR2 subunit (for
reviews see Chen & Wyllie, 2006; Mayer, 2006) and
J Physiol 586.1
they are formed by residues encoded by regions within
the subunit cDNA, a region (S1) preceding the first
membrane-spanning domain and a region (S2) between
the second and third membrane-spanning domains. A
comparison of the amino acid sequences of these two
regions in NR2A and NR2D NMDAR subunits is shown in
Figure 1. Outline structure of an NMDAR subunit, sequences of the S1 and S2 regions in NR2A and NR2D
NMDAR subunits and pictorial representation of the various chimeras investigated
A, cartoon sketch of an ionotropic glutamate receptor subunit showing the proposed membrane topology of three
membrane spanning domains (M1, M3 and M4) and a re-entrant loop (M2) and the location of the amino terminal
domain (ATD) and carboxy terminal domain (CTD). The ligand binding domains (denoted D1 and D2) are formed
by the S1 and S2 regions of the protein which come together to form a hinged clamshell-like structure. B, amino
acid sequence of the S1 and S2 regions of NR2A and NR2D NMDAR subunits. The S1 region contains amino acids
that contribute mainly, but not exclusively, to Domain1 of the ligand-binding site, while those in S2 contribute
mainly, but not exclusively, to Domain2 of the ligand-binding site. Amino acids which are different in NR2A and
NR2D NMDAR subunits are highlighted in bold. C, linear representation of wild-type NR2A and NR2D NMDAR
subunits. The main structural-forming domains are shown in grey for NR2A and white for NR2D NMDAR subunits.
The various chimeric subunits that have been investigated in this study are shown together with the source of each
of the domains in the chimeric subunits and the nomenclature used to describe them. D, cartoon representation
of the constructs (i–vii, shown in C) illustrating how the various functional domains from the NR2D subunit are
incorporated into the five chimeric subunits investigated in this study.
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J Physiol 586.1
Control of glycine potency in NMDARs by NR2 binding domains
Fig. 1B while a comparison of the sequences of the S1 and
S2 regions for all four NR2 NMDARs is shown in the online
supplemental material, Supplemental Fig. 1. Recently,
X-ray crystallography has resolved the structures of the
glutamate and glycine binding pockets from the NR2A and
NR1 subunits and has found that in the NR1–NR2A S1S2
heterodimer both glutamate and glycine binding pockets
assemble in a ‘back-to-back’ configuration (Furukawa &
Gouaux, 2003; Furukawa et al. 2005). Since the initial
cloning of the NMDAR subunits, it has been observed
that glutamate potency differs among the NMDAR
subtypes (Kutsuwada et al. 1992; Ikeda et al. 1992; Monyer
et al. 1992; Laurie & Seeburg, 1994; Erreger et al. 2007).
The largest difference in glutamate potency is seen between
NR2A- and NR2D-containing NMDARs where glutamate
EC50 values vary by up to one order of magnitude. Indeed
this difference in potency is observed for a large number
of ligands acting at the NR2 binding site (Erreger et al.
2007). Similarly, the four types of heterodimeric NMDARs
also show variation in the potency with which glycine
acts at the NR1 coagonist binding site. Intriguingly, a
10-fold variation in glycine potency is also seen with
NR2A- or NR2D-containing NMDARs (Kuryatov et al.
1994; Wafford et al. 1995; Furukawa & Gouaux, 2003).
Furthermore, it is known that binding of glutamate to
its NR2 binding site influences the binding of glycine
to its NR1 binding site and vice versa (for example see
Vyklicky et al. 1990; Benveniste et al. 1990; Benveniste
& Mayer, 1991; Kemp & Priestley, 1991; Lester et al.
1993; Priestley & Kemp, 1994; Regalado et al. 2001).
Nevertheless, the overall potency (EC50 ) with which
an agonist acts is determined not only by equilibrium
constants governing binding reactions but also by the rate
constants controlling subsequent downstream channel
gating (including desensitization) (see Colquhoun, 1998).
Thus, differences in glycine potency at each of the four
NMDAR subtypes will be influenced not only by the
equilibrium constant for binding to the NR1 subunit but
also by the extent to which the NR1 NMDAR subunit
interacts with NR2 NMDAR subunits as part of the channel
gating process.
In order to understand the molecular determinants
underlying differences in glycine-site potency at NMDAR
subtypes, we have characterized the potencies of endogenous and synthetic ligands that act at the NR1 coagonist
binding site and have examined the pharmacology of
recombinant NMDARs expressing chimeric receptors
composed of regions from either NR2A or NR2D
subunits. Specifically, we have observed that the S2 region
that forms the majority of Domain2 of the LBD from the
NR2D subunit is a major structural determinant of glycine
potency. We have identified two residues in NR2A NMDAR
subunits that when mutated to those found in NR2D
NMDAR subunits result in a NR1/NR2D NMDAR-like
glycine potency. We contend that interactions between
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NR1 and NR2 LBDs that occur following agonist binding
and influence the channel-gating process are responsible
for the differences in glycine potencies observed at the
various NMDAR subtypes. Thus, distinct pharmacological
properties of different NMDAR subtypes are not only
dependent on the distinct intrasubunit binding pockets
(e.g. Chen et al. 2005; Erreger et al. 2007), but may also be
specified by interdomain interactions between the LBDs.
In an accompanying paper (Wrighton et al. 2008) we show
that the LBD, in addition to its effect on influencing glycine
potency, contributes to the potency of voltage-dependent
Mg2+ block at NMDARs.
Methods
Plasmid constructs, cRNA synthesis and receptor
expression in oocytes
The amino acid numbering system we use here is
consistent with our previous publications investigating
structure–function relationships in recombinant
NMDARs and refers to the position of residues in the
mature protein (i.e. residues up to and including Ala18 in
NR1, Arg19 in NR2A and Ala23 in NR2D are excluded in
our numbering system). The wild-type pSP64T-derived
expression plasmids for rodent NR1 and NR2 NMDA
receptor subunits were as previously described (Chen
et al. 2005; Wyllie et al. 2006). In this study we coexpressed
various NR2 receptor subunits with the NR1–1a (exon
5 lacking, exon 21, 22 containing) subunit (Hollmann
et al. 1993), which we will refer to as ‘NR1’. Chimeras
between NR2A and NR2D subunits (shown as constructs
(i) and (ii) in Fig. 1C and D, respectively) were introduced
using a PCR-based strategy. Chimeric NR2A/D subunits
were generated by replacing Val370–Val518 in the NR2A
subunit with Leu389–Val539 from the NR2D subunit
(referred to as the NR2A(2D-S1) chimera; shown as
construct (iii) in Fig. 1C and D) and by replacing
Glu638–Ile795 in the NR2A subunit with Glu659–Ile816
from the NR2D subunit (referred to as the NR2A(2D-S2)
chimera; shown as construct (iv) in Fig. 1C and D).
A chimera that replaced the NR2A S1 and S2 regions
with the equivalent sequences from the NR2D subunit is
referred to as NR2A(2D-S1S2) and shown as construct
(v) in Fig. 1C and D. In addition to these ‘binding site’
chimeras, we also generated a chimera in which the NR2A
M1, M2 and M3 membrane associated regions (residues
Ser519–Glu638) were replaced by those found in the NR2D
subunit (Arg541–Glu658). We refer to this chimera as
NR2A(2D-M1M2M3) and it is shown as construct
(vi) in Fig. 1C and D. Finally the NR2A(2DS1M1M2M3S2) chimera replaced both the NR2A
ligand-binding domain and first three membraneassociated regions with those found in the NR2D subunit
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P. E. Chen and others
(shown as construct (vii) in Fig. 1C and D). All inserted
PCR-generated DNA segments and subcloning sites
were confirmed by DNA sequencing. Site-directed
mutagenesis was performed using PCR-based strategies
and verified by sequencing. cRNA was synthesized as
runoff transcripts from restriction endonuclease (MluI
or NotI) linearized plasmid DNA using the Promega
RiboMax RNA synthesis kit (Promega, Madison, WI)
or mMessage Machine (Ambion, Warrington, UK).
Reactions were supplemented with 0.75 mm capping
nucleotide m7 G(5′ )ppp(5′ )G (Promega) in the presence of
1.6 mm GTP. cRNA amounts and integrity were estimated
by intensity of fluorescence in ethidium bromide-stained
agarose gels. NR1 and NR2 cRNAs were mixed at a
nominal ratio ranging from between 1 : 1 and 1 : 9, with
NR1 content being 5 ng.
Stage V–VI oocytes were obtained from Xenopus
laevis that had been anaesthetized by immersion in
a solution of 3-amino-benzoic acid ethylester (0.5%)
and then killed by injection of an overdose solution
of pentobarbital (0.4 ml of 20% solution) followed by
decapitation and exsanguation after the confirmation of
loss of cardiac output, for experiments carried out at
the University of Edinburgh. For experiments carried
out at Emory University, oocytes were obtained from
a ovarian lobe that had been surgically removed from
Xenopus laevis anaesthetized with 3-amino-benzoic acid
ethylester (Traynelis et al. 1998; Chen et al. 2005). All
procedures were carried out in accordance with current
UK Home Office requirements and the Emory University
IACUC. Prior to injection with cRNA mixtures of interest,
the follicular membranes of the oocytes were removed.
After injection oocytes were placed in separate wells of
24-well plates containing a modified Barth’s solution
with composition (mm): NaCl 88, KCl 1, NaHCO3 2.4,
MgCl2 0.82, CaCl2 0.77, Tris-Cl 15, adjusted to pH 7.35
with NaOH (Sigma-Aldrich, UK). This solution was
supplemented with 50 IU ml−1 penicillin and 50 μg ml−1
streptomycin (Invitrogen, UK). Oocytes were placed in
an incubator (19◦ C) for 24–48 h to allow for receptor
expression and then stored at 4◦ C until required for
electrophysiological measurements.
Electrophysiological recordings and solutions
Two electrode voltage clamp (TEVC) recordings were
made using a GeneClamp 500 or OC-725 amplifier
(Molecular Devices, Union City, CA, USA; Warner
Instruments, Hamden, CT, USA), from oocytes that were
placed in a solution that contained (mm) either: NaCl
115, KCl 2.5, Hepes 10, BaCl2 1.8, EDTA 0.01, pH 7.3
with NaOH (20◦ C) for experiments carried out at the
University of Edinburgh, or NaCl 90, KCl 3, Hepes 5,
BaCl2 0.5, EDTA 0.01, pH 7.3 with NaOH (20◦ C) for
experiments carried out at Emory University. The use of
J Physiol 586.1
either set of solutions resulted in comparable estimates for
agonist potencies and the data obtained from experiments
undertaken at either institution were pooled. Chemicals
were purchased from Sigma-Aldrich (Poole, UK or St
Louis, MO, USA). EDTA (10 μm) was added to chelate
contaminant extracellular divalent ions, including trace
amounts of Zn2+ . Current and voltage electrodes were
made from thin-walled borosilicate glass (GC150TF-7.5,
Harvard Apparatus, Kent, UK) using a PP-830 electrode
puller (Narashige Instruments, Japan) and when filled with
3 m KCl they possessed resistances of between 0.5 and
1.5 M. Oocytes were voltage-clamped at −40 mV. For
l-glutamate concentration–response measurements, the
recording solution was further supplemented with 50 μm
glycine and for glycine dose–response measurements this
solution was supplemented with 50 μm glutamate or
30 μm homoquinolinic acid. Application of solutions
was controlled either manually or through user-written
software controlling a rotary valve. Data were filtered
at 10 Hz and digitized at 100 Hz. Test solutions were
applied for 20–60 s or until a plateau to the agonist-evoked
response had been achieved. When investigating the
actions of various agonists acting at the coagonist binding
site on the NR1 subunit we use the term ‘relative efficacy’
to indicate the size of the maximum response evoked by a
ligand relative to the response to a saturating concentration
of glycine (nominally set to equal 100%; see Erreger
et al. 2007). Such relative efficacies can only be compared
within, and not between, different NMDAR subtypes
or chimeras since the maximum open probability of
an NMDAR is subunit dependent, as has been shown
from single-channel studies (Wyllie et al. 1998; Banke &
Traynelis, 2003; Popescu & Auerbach, 2003; Erreger et al.
2005a,b; Schorge et al. 2005). Glutamate- and glycine-site
agonists and antagonists were purchased from Tocris
Bioscience (Bristol, UK or Ellisville, MO, USA).
Data analysis for dose–response curves
Concentration–response curves were fitted individually
for each oocyte with the Hill equation:
I = Imax / 1 + (EC50 /[A])n H
where nH is the Hill coefficient, I max is the maximum
current, [A] is the concentration of agonist, and EC50 is the
concentration of agonist that produces a half-maximum
response. Each data point was then normalized to the fitted
maximum of the dose–response curve. The normalized
values were then pooled and averaged for each construct
and fitted again with the Hill equation, with the maximum
and minimum for each curve being constrained to
asymptote to 1 and 0, respectively. A similar protocol was
used to determine the concentration of the NMDA-glycine
site antagonist 5,7-dichlorokynurenic acid (5,7-DCKA)
required to inhibit a glycine-evoked response by 50%
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J Physiol 586.1
Control of glycine potency in NMDARs by NR2 binding domains
(IC50 ). In these experiments the glycine concentration was
set to the concentration required to evoke a half-maximal
response in each of the constructs being investigated.
Throughout this study we use the term ‘potency’ to
describe the relative differences in EC50 or IC50 values
for ligands acting at the various NMDAR constructs we
have investigated. Thus increases in potency correspond
to decreases in EC50 (or IC50 ) values and vice versa. The
level of ‘contaminating’ glycine in our recording solutions
was determined using the method described by Johnson
& Ascher (1992) and Traynelis et al. (1995). We estimated
these levels to be < 10 nm.
Measurement of competitive antagonist equilibrium
binding constants using Schild analysis
5,7-DCKA antagonism on wild-type NR2A-, NR2D- and
NR2A(2D-S1S2)-containing NMDARs was also examined
using the Schild method (Arunlakshana & Schild, 1959;
Wyllie & Chen, 2007). Partial, concentration–response
curves were obtained from two concentrations (<< EC50 )
of glycine in the absence of antagonist. Then, partial
concentration–response curves were determined in the
presence of increasing concentrations of 5,7-DCKA, by
applying higher concentrations of glycine in order to
produce approximately equivalent responses. 5,7-DCKA
was preapplied for 120 s before glycine application in order
to ensure that the antagonist was in equilibrium with the
receptors. As predicted by the low concentration limit of
the Hill equation, these partial concentration–response
curves produced a series of straight lines when plotted
on a log–log scale and the slope of the initial
(antagonist-free) curve was fitted on the remaining
two-point concentration–response curves, generating a
series of parallel lines. From these lines the dose ratio (r,
ratio of glycine concentrations needed to produce the same
response in the presence and absence of 5,7-DCKA) could
be calculated for each 5,7-DCKA concentration used. The
mean dose ratios were used to generate a Schild plot,
of log (r – 1) versus log [B], where [B] is the antagonist
concentration. In the first instance, the data was fitted as
a ‘free’ fit with a linear regression. The Schild equation,
(r – 1) = [B]/K B , predicts that the slope of the Schild plot
should be 1 for a competitive antagonist at equilibrium.
Therefore if the slope of the ‘free’ fit was sufficiently close to
unity, the data were taken to be consistent with the Schild
equation, and the data were refitted with the slope fixed at
1. The intercept on the x-axis of the line with a fixed slope
of 1 gives the log of the equilibrium constant for antagonist
binding, K B (see Wyllie & Chen, 2007).
Molecular modelling
Starting from the crystal structure of the NR1/NR2A
agonist binding domain dimer (PDB code 2A5T), a
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model of the NR2D glutamate binding domain has
been constructed as previously described (Erreger et al.
2007). Agonist binding domains of NR2A and NR2D
show 72% sequence identity and 82% homology in
their alignment. The structure of NR2A was modified
from that of 2A5T to represent the wild-type peptide
sequence, and the models were energy optimized with
the OPLS-2005 force field in Maestro (Schrödinger,
Portland, OR, USA; Kaminski et al. 2001) to remove strain.
Subsequently, the glycine and glutamate ligands were left
in their crystallographic positions prior to unrestrained
molecular dynamics simulation in which both protein and
ligand can move. These complexes were then prepared
for molecular dynamics (MD) simulation as previously
described (Erreger et al. 2007). After a 50 ps simulation
with ligand and protein restrained to allow for water
equilibration, a 10 ps simulation was performed at 50 K to
remove any gross steric clashes within the structures. The
simulation was then restarted at 50 K and the temperature
was increased linearly to 300 K over 250 ps (1 K ps−1 ) and
continued at 300 K for 10 ns. All MD simulations were
performed with NPT conditions (Erreger et al. 2007), while
the corresponding figures were produced using Visual
Molecular Dynamics (VMD; Humphrey et al. 1996).
Results
Glycine-site agonist potencies at heterodimeric
NR1/NR2 NMDARs
We initially investigated the potencies of a series
of glycine-site agonists at NR1/NR2A, NR1/NR2B,
NR1/NR2C and NR1/NR2D NMDARs. These included
endogenous ligands such as glycine, d- and l-isomers of
serine and alanine and synthetic halogenated and cyclic
derivatives that also act as coagonists at the NR1 NMDAR
subunit. Figure 2 illustrates TEVC currents recorded
from oocytes expressing either NR1/NR2A (Fig. 2A) or
NR1/NR2D (Fig. 2B) NMDARs. The currents were evoked
by applying (cumulatively) increasing concentrations of
glycine in the presence of a saturating concentration
of glutamate (50 μm). Figure 2C shows the mean data
points obtained for each of the four receptor subtypes
which have been fitted with the Hill equation. As is
shown in Fig. 2C and in the summary data from all
our experiments (Fig. 3) all agonists studied displayed
the same rank order of potency, regardless of their
relative efficacy or structure. Thus, the order of
potency for all agonists is (highest potency, lowest
EC50 ) NR2D > NR2C > NR2B > NR2A (lowest potency,
highest EC50 ). Differences in potency ratios between
NR2A- and NR2D-containing NMDARs ranged between
8-fold (d-serine) and 28-fold (β-fluoro-dl-alanine).
When comparing relative efficacies, where glycine was
denoted a value of 100% (see Methods), we can see
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P. E. Chen and others
that most of the ligands studied were in the range
of ∼80–110% of this value. The determination of a
particular ligand’s ‘relative efficacy’ or ‘potency’ does
not provide us with any direct measure of equilibrium
constants underlying either binding or gating reactions
(Colquhoun, 1998). Indeed measurements of macroscopic
responses are in general difficult to interpret in terms
of the underlying rate constants that are altered as a
J Physiol 586.1
result of different ligands being used to activate a receptor
without additional information that can be obtained from
single-channel studies (for example see Erreger et al.
2005b). Nevertheless mutations in the ligand binding
site of NR2A and NR2D NMDAR subunits that lead to
1000-fold shifts in glutamate’s potency can be accounted
for by changes in the dissociation rate constant (Wyllie
et al. 2006) suggesting distinct functional domains within
the NMDAR may influence binding and gating reactions
separately (see Gibb, 2006). Therefore since the greatest
difference in potency of ligands acting at the glycine site
is seen with NR2A- and NR2D-containing NMDARs, we
have investigated which structural elements contained
within these two NR2 subunits might be responsible
for the observed difference in agonist potencies at the
ligand-binding site in the NR1 subunit.
Glycine potency at recombinant NMDARs containing
chemeric NR2A/D subunits
Figure 2. Example TEVC current traces showing responses
evoked by increasing concentrations of glycine for NR1/NR2A
and NR1/NR2D NMDARs
A, example TEVC current recording obtained from an oocyte
expressing NR1/NR2A(WT) NMDARs. In the presence of glutamate
(50 μM), increasing concentrations of glycine (30 nM to 30 μM) were
applied cumulatively. B, as A but recorded from an oocyte expressing
NR1/NR2D(WT) NMDARs and with the range of glycine concentrations
applied being 10 nM to 10 μM. Note the increased glycine potency at
NR2D-containing compared to NR2A-containing NMDARs. C, mean
glycine concentration–response curves for each of the four NMDAR
subtypes. The rank order of glycine potency (highest potency, lowest
EC50 ) is NR2D > NR2C > NR2B > NR2A with the mean EC50 values
and Hill slopes for each of the NMDAR combinations given in Fig. 3.
Several studies have shown that when glutamate binds to
NMDARs this causes the rate of dissociation of glycine
from its binding site to increase and hence a subsequent
reduction in its affinity (for example see, Vyklicky et al.
1990; Benveniste et al. 1990; Benveniste & Mayer, 1991;
Kemp & Priestley, 1991; Lester et al. 1993; Priestley &
Kemp, 1994; Regalado et al. 2001). Thus, we hypothesized
that the variation in the potency of coagonists acting at the
glycine (NR1) site we see with NR1/NR2A and NR1/NR2D
NMDARs may be specified by amino acid residues within
the S1 and S2 domains of the NR2 (glutamate binding)
since earlier studies have demonstrated glutamate–glycine
binding interactions. Although the amino acid residues
that hydrogen-bond with glutamate when it occupies
the NR2 ligand-binding site are conserved among NR2A
and NR2D subunits (Furukawa et al. 2005), sequence
alignments of the entire S1 and S2 regions show a number
of important differences (Fig. 1B; Supplemental Fig. 1). In
addition, molecular dynamics simulations of NR1/NR2A
and NR1/NR2D suggest additional differences between
the NR2 subunit (Erreger et al. 2007). We generated three
chimeric subunits by inserting the NR2DS1, NR2DS2 and
both NR2DS1 and S2 domains into the NR2A subunit
using PCR-based mutagenesis, and the schematic location
of the NR2A/NR2D domain exchanges are shown in
Fig. 1C and D. In vitro transcribed cRNA from all chimeras
produced robust glutamate-evoked inward currents when
expressed with NR1 cRNA.
We have previously reported the effects on glutamate
potency of exchanging S1 and S2 regions in the NR2A
subunit with the equivalent region from the NR2D
subunit (Erreger et al. 2007). Briefly, replacement of either
the S1 or S2 region in NR2A with the corresponding NR2D
region produces an NMDAR with a glutamate EC50 that
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Control of glycine potency in NMDARs by NR2 binding domains
is intermediate between that seen with NR2A(WT)- or
NR2D(WT)-containing NMDARs. Replacement of both
S1 and S2 regions in NR2A subunits with the respective
regions from NR2D subunits results in a chimeric NMDAR
where glutamate potency is equivalent to that seen in
NR2D(WT)-containing NMDARs (Erreger et al. 2007).
Recent structural studies have shown that the glycine
and glutamate binding pockets on the NR1 and NR2
subunits interact via their hinge regions in a ‘back-to-back’
manner (Furukawa et al. 2005). We examined the effect
233
these NR2A/D chimeras had on glycine potency by
measuring glycine concentration–response curves in the
presence of a saturating concentration of glutamate
(50 μm). Figure 4 shows voltage clamp currents recorded
in response to the application of 30 nm, 300 nm, 3 μm
and 30 μm glycine from oocytes expressing NR1/NR2A
(Fig. 4A), NR1/NR2D (Fig. 4B) or NR1/NR2A(2D-S1S2)
(Fig. 4C) NMDARs. Mean concentration–response curves
for these receptor combinations and the NR2A(2D-S1)
and NR2A(2D-S2) chimeras are shown in Fig. 4D.
Figure 3. Summary of the potencies and ‘relative efficacies’ of ligands acting at the NR1 coagonist
binding site for each of the four heterodimeric NMDARs
Mean EC50 and Hill slope values obtained from fitting concentration–response relationships for a series of NR1
agonists. Efficacy denotes the maximal current response to the test agonist relative to the maximal response to
glycine. The ratio of the EC50 at NR1/NR2A compared to NR1/NR2D NMDARs is given to indicate the agonist
selectivity between these two NMDAR subtypes. The structures of the various agonists are also illustrated together
with the subunit dependence of the EC50 for each agonist. Notice that for each agonist studied, greatest potency
is seen at NR2D-containing NMDARs and the least at NR2A-containing NMDARs.
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P. E. Chen and others
As reported previously, glycine is approximately
10-fold more potent at NR1/NR2D-containing
receptors (EC50 = 133 ± 7 nm) than those containing
NR2A (EC50 = 1.31 ± 0.08 μm). Glycine potency of
NMDARs containing the NR2A(2D-S1) chimera
Figure 4. Glycine concentration–response data for wild-type
and chimeric NMDARs
A, representative TEVC current recordings obtained from an oocyte
expressing NR1/NR2A(WT) NMDARs, elicited by increasing
concentrations of glycine (30 nM to 30 μM; in the presence of
glutamate (50 μM)). B, representative TEVC current recordings
obtained from an oocyte expressing NR1/NR2D(WT) NMDARs, elicited
by the same concentrations of glycine (and glutamate) as illustrated in
A. Notice that in contrast to the recordings shown in A, responses to
3 μM and 30 μM glycine evoke similarly sized currents indicating that
the potency of glycine is greater at NR2D-containing NMDARs.
C, representative TEVC current recordings obtained from an oocyte
expressing NR1/NR2A(2D-S1S2) NMDARs, elicited by the same agonist
concentrations as shown in A and B. D, mean concentration–response
curves for glycine acting at NR1/NR2A(WT) and NR1/NR2D(WT)
NMDARs as well as each of the three ‘binding’ domain chimeric
NMDARs. Mean EC50 values and Hill slopes for each of the NMDAR
combinations are given in Fig. 6. The mean Hill slopes and mean
maximal currents recorded were for NR2A(WT): 1.66 ± 0.08,
1.7 ± 0.4 μA; NR2D(WT): 1.32 ± 0.07, 0.4 ± 0.06 μA; NR2A(2D-S1):
1.16 ± 0.04, 1.6 ± 0.2 μA; NR2A(2D-S2): 0.99 ± 0.04, 2.1 ± 0.6 μA;
and NR2A(2D-S1S2): 1.48 ± 0.05, 1.9 ± 0.2 μA.
J Physiol 586.1
(EC50 = 1.02 ± 0.06 μm) is close to the NR1/NR2A
NMDAR value. However, the NR2A(2D-S2) and
NR2A(2D-S1S2) chimeras each show glycine potencies
close to that of NR2D(WT) receptors (EC50 values
219 ± 13 nm and 233 ± 10 nm, respectively). The
corresponding Hill slope values for each of these receptor
constructs together with the equivalent data for other
glycine-site agonists are shown in Fig. 6.
We next examined whether the shift in potency in
the NR2A(2D-S1S2) construct was also observed with
Figure 5. Concentration–response curves for D-serine,
β-fluoro-DL-alanine and ACPC acting at wild-type and chimeric
NMDARs
A, mean concentration–response curves for D-serine acting at
NR1/NR2A(WT) and NR1/NR2D(WT) NMDARs as well as each of the
three chimeric NMDARs. B, mean concentration–response curves for
β-fluoro-DL-alanine. C, mean concentration–response curves for
ACPC. For each agonist, replacing the S1 and S2 region of the NR2A
subunit with the corresponding regions from the NR2D subunit
resulted in an increase in agonist potency. Mean EC50 , Hill slope and
‘relative efficacy’ values for each of the agonists acting at the various
NMDAR combinations are given in Fig. 6.
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Control of glycine potency in NMDARs by NR2 binding domains
other ligands that bind to the NR1 coagonist site.
Figure 5 shows the mean concentration–response curves
for d-serine (Fig. 5A), β-fluoro-dl-alanine (Fig. 5B) and
1-amino-cyclopropyl carboxylic acid (ACPC; Fig. 5C)
acting at wild-type and chimeric NMDARs. As we have
observed in the case of glycine, replacement of the S1
region in the NR2A subunit with the corresponding region
from NR2D has little effect on the potency of each of these
NR1 agonists. In contrast to what we have observed with
glycine, exchanging the S2 region of NR2A with that of
NR2D also has little effect on the potency of d-serine and
ACPC. Replacing the NR2A S2 region with the NR2D
S2 region increases β-F-dl-alanine potency by ∼9-fold,
similar to glycine. Swapping both S1 and S2 domains leads
to further increases in NR1 agonist potency for d-serine
and ACPC; however, for β-F-dl-alanine its potency at
the NR2A(2D-S1S2) chimera was similar to that seen at
the NR2A(2D-S2) chimera. The extent of potency shifts
are shown in Fig. 6.
235
Glycine potency is not influenced by the nature
of the agonist acting at the NR2-ligand-binding site
In AMPARs, the efficacy of channel activation is correlated
to the degree of domain closure of the LBD (Jin et al.
2003). Although it is unclear whether this is the case
for agonist activation at the NR2-glutamate binding
pocket, we examined whether using a partial agonist to
activate the glutamate binding pocket rather than a full
agonist such as glutamate would alter glycine potency.
Supplemental Fig. 2 shows the effect of using the NR2A
partial agonist, homoquinolinate (Erreger et al. 2005b;
30 μm) rather than glutamate to activate the NMDAR
constructs. Glycine potencies with homoquinolinate were
similar to those obtained when glutamate was used as
the agonist for NR1/NR2A and NR1/NR2D receptors.
Glycine concentration–response curves had EC50 values
of 1.91 ± 0.14 μm (n = 9) for NR1/NR2A, 194 ± 14 nm
(n = 6) for NR1/NR2D(WT), and 136 ± 7 nm (n = 14)
for NR1/NR2A(2D-S1S2). Thus using a partial agonist
Figure 6. Summary of the pharmacological properties and ‘relative efficacies’ of ligands acting at the
NR1 coagonist binding site for wild-type and chimeric NMDARs
Mean EC50 and Hill slope values obtained from fitting concentration–response relationships for a series of NR1
agonists acting at wild-type and chimeric NMDARs. Efficacy denotes the maximal current response to the test
agonist relative to the maximal response to glycine. The ratio of the EC50 at NR1/NR2A(2D-S1S2) compared to
NR1/NR2D NMDARs is given to indicate the extent to which incorporation of the S1 and S2 regions from the NR2D
NMDAR subunit results in a more ‘NR2D-like’ agonist potency. For comparison the ratio of EC50 at NR1/NR2A
to NR1/NR2D NMDARs is indicated in parentheses. The dependence of the EC50 for each agonist at the various
constructs is illustrated as bar graphs to the right of these ratios.
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P. E. Chen and others
to activate NMDARs does not alter the glycine potency
obtained when the corresponding receptor is activated by
a full NR2 agonist.
Chimeric NR2A subunits containing NR2D M1, M2
and M3 membrane domains display no change in
glycine potency
Macroscopic measurements such as the EC50 value
of an agonist are determined by microscopic rate
constants that describe both binding and gating reactions
(see Colquhoun, 1998). NR2D-containing NMDARs
have characteristically different biophysical properties
compared to those containing NR2A subunits (Monyer
et al. 1994; Wyllie et al. 1996, 1998; Vicini et al. 1998).
For example, NR2D-containing NMDARs exhibit
‘low-conductance’ single-channel openings compared
to the ‘high-conductance’ NR2A-containing receptors.
In addition, the overall probability that a channel
is open during an activation is considerably less for
NR2D-containing NMDARs (Wyllie et al. 1998). The
pore-forming regions of ligand-gated ion channels are
likely to control/influence rate constants that describe
‘gating’ reactions. We therefore examined whether
transferring the M1, M2 and M3 membrane associated
domains of the NR2D subunit into NR2A-containing
NMDARs similarly altered glycine potency. Supplemental
Fig. 3 shows mean concentration–response curves for
glutamate and glycine acting at NR2A(2D-M1M2M3)
and NR2A(2D-S1M1M2M3S2) chimeras. When
only the M1, M2 and M3 regions were transferred
from NR2D into NR2A neither glutamate potency
(3.28 ± 0.16 μm, n = 13; Suppl. Fig. 3A) nor glycine
potency (1.36 ± 0.10 μm, n = 9; Suppl. Fig. 3B)
were different from the corresponding values seen
with NR2A(WT)-containing NMDARs. By contrast,
increases in both glutamate and glycine potencies were
observed when NR2D membrane associated domains
as well as S1 and S2 regions were included in the
chimeric construct (EC50 (glut) = 429 ± 24 nm, n = 8;
EC50 (gly) = 311 ± 17 nm, n = (11)).
Potency of the glycine-site antagonist, 5,7-DCKA at
wild-type and chimeric NMDARs
In addition to the actions of agonists at the NR1 coagonist
binding site and the influence of NR2 S1 and S2 regions
on their potencies we also considered whether the action
of competitive antagonists could be affected by the nature
of the NR2 subunit present in the heteromeric NMDAR
complex. We examined the effect of 5,7-dichlorokynurenic
acid (5,7-DCKA, Baron et al. 1990; McNamara et al.
1990) by measuring the concentration of antagonist
required to inhibit responses by 50% for NR2A(WT)-,
J Physiol 586.1
NR2D(WT)-, NR2A(2D-S1)-, NR2A(2D-S2)- and
NR2A(2D-S1S2)-containing NMDARs. In addition, we
measured the equilibrium constant, K B , by the Schild
method for this antagonist at NR2A(WT)-, NR2D(WT)and NR2A(2D-S1S2)-containing NMDARs.
Figure 7A shows a TEVC current recording obtained
from an oocyte expressing NR1/NR2A NMDARs.
Currents were evoked by a saturating concentration of
glutamate (50 μm) and the EC50 concentration of glycine
(1.5 μm) for this receptor combination. Increasing the
concentration of 5,7-DCKA from 10 nm to 3 μm results
in a concentration-dependent decrease in the magnitude
of the current recorded. Figure 7B shows mean inhibition
curves for this receptor and the others studied. As would
be anticipated if glycine had different affinities for these
two receptor subtypes, 5,7-DCKA is most potent at
inhibiting responses mediated by NR1/NR2A NMDARs
(IC50 = 96 ± 7 nm, n = 5) and least potent when
acting at NR1/NR2D NMDARs (IC50 = 1.7 ± 0.15 μm,
n = 6; glycine concentration = 150 nm). Chimeric
NR2A receptor subunits showed intermediate IC50
values for 5,7-DCKA that were 226 ± 46 nm (n = 12;
glycine concentration = 1 μm) for NR1/NR2A(2D-S1),
269 ± 21 nm (n = 6; glycine concentration = 250 nm)
for NR1/NR2A(2D-S2), and 322 ± 32 nm (n = 8; glycine
concentration = 250 nm) for NR1/NR2A(2D-S1S2).
IC50 values for antagonist action at NMDARs are
dependent on the concentration and identity of the
agonist used (for example see Frizelle et al. 2006;
Wyllie & Chen, 2007). Thus, although we carried out
our measurements of IC50 values using equipotent
concentrations of glycine for each receptor combination
examined, differences in rate constants governing binding
and gating reactions may also influence the observed IC50 .
In this regard, we also determined the affinity of 5,7-DCKA
for its binding site in the NR1 subunit by performing
Schild analysis to obtain K B values for this antagonist
acting at NR2A-, NR2D- and NR2A(2D-S1S2)-containing
NMDARs. Supplemental Fig. 4 shows examples of
two-point concentration–response curves, for each of
these receptor combinations, generated in the absence
and in the presence of increasing concentrations of
5,7-DCKA. The Schild plots obtained by pooling data
from several such experiments are illustrated in Fig. 7C–E.
The fitted lines in each case show the fit of the data
points with the Schild equation and the intercept with
the abscissa gives the K B for 5,7-DCKA (see Methods).
No significant differences were observed for any of
the receptor combinations examined and the mean K B
values were 80 ± 2 nm for NR1/NR2A (n = 6; Fig. 7C),
70 ± 3 nm for NR1/NR2D (n = 6; Fig. 7D), and 79 ± 4 nm
for NR2A(2D-S1S2) (n = 5; Fig. 7E). Indeed, the estimates
of the K B for 5,7-DCKA obtained in the present study
are in good agreement with a previously published
value obtained from Schild analysis of its antagonism
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Control of glycine potency in NMDARs by NR2 binding domains
of NMDA receptor-mediated responses recorded from
oocytes expressing whole-brain mRNA (McNamara et al.
1990) but indicate a higher affinity of 5,7-DCKA binding
than has been reported for the NR1 S1S2 fusion protein
(540 nm; Furukawa & Gouaux, 2003). These data suggest
that 5,7-DCKA does not sense the same structural
differences in the NR2 subunits that glycine does, which
may reflect different degrees of domain closure induced by
ligand binding to NR1 (Furukawa & Gouaux, 2003). This
may also reflect predominant contact residues in the NR1
237
Domain1 (Furukawa & Gouaux, 2003), which is likely to
be minimally influenced by structural changes in Domain2
for NR2D versus NR2A.
These data suggest that the identity of the NR2
subunit coexpressed with the NR1 subunit does not cause
global structural changes within this binding site that lead
to reduction in affinity of all ligands since antagonists are
insensitive to the identity of the NR2 subunit present in
the NMDAR complex. Rather, it seems likely that interactions between NR1 and NR2 subunits that occur after
Figure 7. Antagonism of wild-type and chimeric NMDAR mediated responses by 5,7-DCKA
A, representative TEVC current recording of the inhibition by 5,7-DCKA of a NR1/NR2A(WT) NMDAR-mediated
response. In this recording the glycine concentration was set to be equal to the EC50 concentration at this receptor
combination (1.5 μM) whereas the glutamate concentration was set at a saturating level (50 μM). B, mean inhibition
curves for 5,7-DCKA antagonism of responses mediated by NR1/NR2A(WT) and NR1/NR2D(WT) NMDARs and each
of the three ‘binding’ domain chimeric NMDARs. C–E, Schild analysis of 5,7-DCKA antagonism. The mean dose
ratios, r, measured from the parallel shifts of two-point concentration–response curves (Supplemental Fig. 4) are
plotted on a log–log scale as (r – 1) versus antagonist concentration, [B]. If the slope of the linear regression fit of
the data points was sufficiently close to 1 (as is to be expected of competitive antagonism), the data were refitted
with the Schild equation (r – 1) = [B]/K B , where the slope of the line is unity and the equilibrium constant for
antagonist binding, K B , is given by the intercept on the x-axis. Similar K B values for 5,7-DCKA were obtained for
NR2A(WT), NR2D(WT) and NR2A(2D-S1S2) NMDARs.
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P. E. Chen and others
ligand binding and lead to channel opening are responsible
for differences in the glycine-site agonist potency
observed.
Comparison of NR2A and NR2D ligand-binding
domains and identification of residues in NR2
subunits that influence glycine potency
In order to understand how the identity of the NR2
subunit influences glycine’s actions at NR1, we evaluated
J Physiol 586.1
hydrated models of NR2A and NR2D using molecular
dynamics (see Methods). Figure 8A shows the bi-lobar
structure of the NR1/NR2A agonist binding domain
dimer (grey). Superimposed on this is the equivalent
structure of the NR1/NR2D agonist binding domain
dimer (purple). Figure 8B shows a superimposition of
the NR1 ligand binding sites (with glycine bound) at
the end of a 10 ns simulation of the heterodimeric
NR1–NR2A and NR1–NR2D dimer–dimer. These
simulations demonstrate that glycine is predicted to
Figure 8. Comparison of NR1/NR2A and NR1/NR2D glycine binding pockets and identification of NR2
residues that influence glycine potency
A, superimposition of NR1/NR2A (grey) and NR1/NR2D (purple) agonist binding domain dimers. The locations of the
glutamate and glycine binding sites are highlighted by the yellow dashed-line boxes, with the glycine binding site
shown in greater detail in panel B. The blue and red dashed-line boxes highlight the regions in the NR2A and NR2D
D2 domains that are shown in greater detail in C and D, respectively. B, expanded view of the NR1 glycine binding
site when associated with NR2A (grey) or NR2D (purple) showing that despite different potencies, both protein
backbone and sidechains of the glycine binding site are predicted to be quite similar after 10 ns of simulation.
C, illustration of the locations of Lys719 (NR2A, orange) and Met740 (NR2D, light green) in Domain2 of each NR2
NMDAR subunit. These residues are distant from the NR1–NR2 interface (indicated by the blue dashed-line box in
A seen from a different orientation). Likewise, the carboxylate group of glutamate (when it occupies its binding
site) and Lys719 are separated by more than 10 ˚A. D, illustration of the locations of Tyr735 (NR2A, orange) and
Lys756 (NR2D, green) in Domain2 of each NR2 NMDAR subunit, positioned in the lower interface between NR1
and NR2 (indicated by the red dashed-line box in A seen from a different orientation).
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Control of glycine potency in NMDARs by NR2 binding domains
adopt a very similar orientation in the NR1/NR2A
and NR1/NR2D glycine binding pockets, even though
differences develop in the positions of amino acid residues
that interact directly with glutamate in each of these NR2
NMDAR subunits (see Erreger et al. 2007). However, these
differences in the positioning of amino acids in the NR2
binding pocket per se are unlikely to account for NR2–NR1
interactions that ultimately lead to differences in glycine
potencies seen at NR1/NR2A and NR1/NR2D NMDARs.
Rather, the simulations suggest that the differences in
Domain2 of the NR2 are well positioned to influence NR1
function.
Inspection of the sequence throughout the S2 region
(Fig. 1B; Suppl. Fig. 1) in NR2A and NR2D subunits that
encodes most of Domain2 reveals several differences in
amino acid sequence. Moreover, as glycine potency is also
greater at NR2B- and NR2C-containing NMDARs than
in NR2A-containing NMDARs, we examined whether
these amino acid differences were also observed between
NR2A and these two other NMDAR subunits. There are
two residues within the S2 region that are conserved in
NR2B, NR2C and NR2D subunits but are different in
the NR2A subunit and which result in charged/uncharged
residue exchanges in these subunits. These are Lys719 in
NR2A (equivalent to Met740 in NR2D) and Tyr735 in
NR2A (equivalent to Lys756 in NR2D). The positions of
each of these sites are highlighted in Fig. 1B and Suppl.
Fig. 1. Figure 8C and D shows the location of each of
these residues in our superimposed hydrated models
of NR1/NR2A and NR1/NR2D agonist binding domain
dimers. As discussed below, at least one of these NR2
residues appears to mediate with the NR1 subunit and may
contribute, in part, to the differences in glycine potency
seen at various NMDARs.
We created point mutations in the NR2A subunit to
generate sequences that contained the NR2D subunit
residue to determine whether either of these residues
was responsible for the difference in glycine potency
at NR2A- and NR2D-containing NMDARs. Figure 9A
shows the NR1/NR2A agonist binding domain dimer
with the location of Tyr735 in NR2A indicated along
with the locations of glycine and glutamate in their
respective binding sites. In Fig. 9B, a higher resolution
image illustrates how Tyr735 in the NR2A subunit is
predicted to form a hydrogen-bonding network with
Glu786 and Lys790 in the NR1 subunit as well as Tyr679
in NR2A. A representative TEVC current trace showing
responses to increasing concentrations of glycine and
obtained from an oocyte expressing NR1/NR2A(K719M
Y735K) NMDARs is illustrated in Fig. 9C. Figure 9D
shows mean glycine concentration–response curves
for the NR2A(K719M) and NR2A(Y735K) mutations
(mimicking the residues found in NR2D subunits). Each of
these mutations resulted in an increase in glycine potency
with EC50 values for glycine of 231 ± 10 nm (n = 33)
239
and 362 ± 18 nm (n = 12) for NR2A(K719M) and
NR2A(Y735K), respectively. However, the introduction
of the two point mutations into the NR2A subunit,
NR2A(K719M Y735K), did not result in a further increase
in glycine potency and gave a mean EC50 value of
277 ± 7 nm (n = 12). In addition, replacing the lysine
residue at position 719 in NR2A with a glutamate residue
(K719E) to alter the charge at this position also gave rise
to an NMDAR with increased glycine potency (Suppl.
Fig. 5A). Each of these point mutations also caused, albeit
smaller, shifts in glutamate potency (Suppl. Fig. 5B and
C). We therefore presume that while the NR2A Met719
and Lys735 mutant residues are important for modulating
glycine potency, the wild-type sequence of the subunit is
also critical for the overall effect.
We also examined the effects of introducing the residues
present in NR2A subunits into the NR2D subunit.
Figure 10A shows the NR1/NR2D agonist binding domain
dimer with the location of Lys756 in NR2D indicated.
The positions of glycine and glutamate in their respective
binding sites are also indicated. Figure 10B shows the
predicted interaction of Lys756, in the NR2D subunit
with Glu786 in NR1. Unlike Tyr735 in NR2A, the NR2D
residue Lys756 develops a different set of interactions
with Glu786 in NR1 and Val757 in NR2D while no
interaction with the side-chain of the Lys790 residue
in NR1 is predicted as seen in Fig. 9B. This change in
the hydrogen-bonding network at the interface between
domains may correlate with the effect on glycine potency.
Figure 10C shows an example of a TEVC current trace
illustrating the increase in the response to cumulative
applications of increasing concentrations of glycine,
obtained from an oocyte expressing NR1/NR2D(K756Y).
The initial response shows the effect of perfusing the
oocyte with a solution containing only glutamate (no
added glycine) and allows us to estimate the levels of
‘contaminating’ glycine in our solutions (Johnson &
Ascher, 1992; Traynelis et al. 1995). We estimated the level
of this contamination to be < 10 nm (6.7 ± 1 nm; n = 9).
As can be seen from this example TEVC current trace and
the mean concentration–response curves (Fig. 10D), the
NR2D(K756Y) point mutation increases the sensitivity
of the receptor to glycine. The (uncorrected) mean
EC50 value for this construct is 26 ± 1 nm (n = 21)
suggesting a ‘true’ EC50 of around 20 nm. Mutation of
Met740 to a lysine residue, NR2D(M740K), however,
did not result in any change in glycine potency
when compared to NR2D(WT)-containing NMDARs
(EC50 = 149 ± 7 nm, n = 20; Fig. 10D). The glutamate
sensitivity of NR2D(M740K)-containing NMDARs was
not different from NR2D(WT) though a slight increase
in glutamate potency is observed with the NR2D(K756Y)
mutation (Suppl. Fig. 5D).
Thus, both our molecular modelling and mutagenesis
studies would seem to suggest that these different
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P. E. Chen and others
intersubunit contacts may provide qualitatively different
associative interactions between the D2 domains of NR1
and NR2A than between NR1 and NR2D, a potential basis
of why differences in glycine potencies exist in various
NR1/NR2 NMDARs.
Discussion
The most important result of this work is the identification
of Domain2 of the NR2 subunit as a molecular
determinant of glycine potency. These data complement
previous studies that have established that amino acid
residues located in Domain 1 and 2 on the NR2 subunits
contribute to the glutamate-binding pocket of NMDARs
J Physiol 586.1
(see Chen & Wyllie, 2006; Mayer, 2006). Furthermore,
these findings help provide new information on how
the variation in agonist potencies is observed among
different recombinant NMDAR subtypes. Specific residues
within Domain2 are highlighted as critical mediators of
long-range intraprotein interactions that control glycine
potency.
Structural interactions from the NR2D-S2 domain can
influence glycine potency in recombinant NMDARs
Several studies have examined the differences in amino
acid sequence between NR2 subunits in order to identify
NMDAR subtype specific ligands (Feng et al. 2004;
Figure 9. Analysis of the effects of point mutations in the S2 region of the NR2A subunit on glycine
potency
A, view of the NR1/NR2A agonist binding domain dimer. The positions of glycine and glutamate in their respective
binding sites are indicated, together with the Tyr735 residue located in Domain2 of NR2A. B, a frame from
the MD simulation of NR1/NR2A agonist binding domain dimer showing the hydrogen–bond interactions of
Tyr735 in the NR2A subunit with residues Glu786 and Lys790 in the NR1 subunit and Tyr679 of NR2A. The
location of glutamate in its binding site is also indicated. C, example TEVC current trace obtained in responses
to applications of increasing concentrations of glycine (0.01–10 μM) and recorded from an oocyte expressing
NR1/NR2A(K791M Y735K) NMDARs. D, mean glycine concentration–response curves for NR1/NR2A(K719M),
NR1/NR2A(Y735K) and NR1/NR2A(K719M Y735K) NMDARs. Each of the point mutations result in a shift to the
left in glycine potency to values that are similar to those seen for NR2D(WT)-containing NMDARs. Mean Hill slopes
and mean maximal currents recorded were for NR2A(K719M): 1.08 ± 0.02, 1.9 ± 0.1 μA; for NR2A(Y735K):
1.05 ± 0.04, 3.0 ± 0.2 μA; and for NR2A(K719M Y735K): 1.16 ± 0.02, 3.8 ± 0.3 μA. The dashed and dotted
lines show the corresponding glycine concentration–response curve for NR2A(WT)- and NR2D(WT)-containing
NMDARs, respectively.
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J Physiol 586.1
Control of glycine potency in NMDARs by NR2 binding domains
Kinarsky et al. 2005; Erreger et al. 2007). Other studies
have shown that specific residues that contact ligand in
crystal structures control glutamate and glycine potency
in functional receptors (Kuryatov et al. 1994; Laube et al.
1997; Anson et al. 1998, 2000; Chen et al. 2004, 2005;
Erreger et al. 2007). Interestingly, mutations of residues
that contact glutamate within the NR2 LBD and reduce
glutamate potency have little effect on glycine potency
(Anson et al. 1998; Chen et al. 2005; for review see Chen &
Wyllie, 2006), suggesting that the effects of the NR2 subunit on glycine potency are unlikely to involve differences
in glutamate binding. Similarly, point mutations within
the NR1 glycine binding pocket that alter glycine potency
have little effect on glutamate potency (Kuryatov et al.
241
1994; Wafford et al. 1995; Williams et al. 1996). Thus,
impaired binding within one subunit does not seem to
greatly influence potency of agonist binding to the other
subunit. This is supported by our observation that using
either a full or partial agonist to activate the NR2-glutamate
binding site has little influence on glycine potency. This
raises the idea that other features of the agonist binding
domains or their function must, respectively, influence
agonist potency.
Evidence has shown that in terms of agonist efficacy both
NR1 and NR2 agonist binding pockets may rely on distinct
mechanisms (Furukawa & Gouaux, 2003; Erreger et al.
2005b; reviewed in Kristensen et al. 2006). For example,
in contrast to the correlation between domain closure
Figure 10. Analysis of the effects of point mutations in the S2 region of the NR2D subunit on glycine
potency
A, view of the NR1/NR2D agonist binding domain dimer The positions of glycine and glutamate in their respective
binding sites are indicated, together with the Lys756 residue located in Domain2 of NR2D. B, a frame from the
MD simulation of NR1/NR2D agonist binding domain dimer showing the hydrogen bonding between Lys756 in
the NR2D subunit and residues Glu786 in the NR1 subunit and Val757 of NR2D. The location of glutamate in its
binding site is also indicated. C, example TEVC current trace obtained in responses to applications of increasing
concentrations of glycine (0.01–3 μM) and recorded from an oocyte expressing NR1/NR2D(K756Y) NMDARs.
Note that the initial response is obtained in the presence of no added glycine. Such recordings were used to
estimate the levels of ‘contaminating’ glycine in our recording solutions. D, mean glycine concentration–response
curve for NR1/NR2D(M740K) and NR1/NR2D(K756Y) NMDARs. The NR2D(M740K) mutation causes no change in
glycine potency compared to NR2D(WT). Mean Hill slopes and mean maximal currents recorded for NR2D(M740K):
1.23 ± 0.06, 0.35 ± 0.15 μA. In contrast the NR2D(K756Y) mutation resulted in an increase in glycine potency.
Mean Hill slopes and mean maximal currents recorded for NR2D(K756Y): 0.99 ± 0.04, 0.48 ± 0.09 μA. The dashed
line shows the corresponding glycine concentration–response curve for NR2D(WT)-containing NMDARs.
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242
P. E. Chen and others
and agonist efficacy for the GluR2 and GluR5/6 pockets
(Jin et al. 2003; Mayer, 2005; Nanao et al. 2005), partial and
full agonists for the NR1-glycine binding pocket induce
the same degree of domain closure (Inanobe et al. 2005).
It is still unclear whether there is a correlation between
agonist efficacy and the degree of domain closure for the
NR2-glutamate binding pocket. However, the insertion
of steric clashes within the NR2B-glutamate pocket has
suggested that the domain closure may be correlated with
agonist efficacy for certain agonists (Hansen et al. 2005).
Our results suggest that the influence of the NR2D-S2
region on glycine potency is mediated by interactions
between the glutamate and glycine LBDs from the NR1
and NR2 subunits. Because NMDARs containing chimeras
with either the NR2D S1 or S2 regions showed increased
glutamate potency but only the NR2A(2D-S2) chimera
influenced glycine potency, modification of glutamate
potency (in either direction) seems unlikely to be the
predominant cause of changes in glycine potency. These
data suggest that interactions either directly or indirectly
induced by the conformational arrangement of the
NR2D-S2 domain with the other subunits are able to sway
the receptor’s sensitivity to glycine. Consistent with this
idea, the S2 region of the NR2 subunit shows the largest
predicted divergence in structure in molecular dynamics
simulations of NR1/NR2A and NR1/NR2D (Erreger et al.
2007).
Interestingly, the insertion of the NR2D-S2 sequence
into NR2A does not change the affinity of a competitive
antagonist at the NR1-glycine binding pocket, suggesting
that the contact residues have not undergone any
significant structural changes. This idea is supported
by our molecular dynamics simulations, which predict
little difference between the NR1/NR2A and NR1/NR2D
glycine binding sites. In further support of this idea is
the fact that we observed shifts in potencies at chimeric
NMDARs with d-serine, β-F-dl-alanine and ACPC when
they were used as NR1 agonists. As each of these
agonists will adopt different conformations within the
NR1 LBD and interact with different contact residues it
would seem unlikely that structural changes in the NR2
subunit would give rise to changes at the NR1 binding
pocket that result in the changes in potency we have
observed. Thus, we conclude that cross-subunit effects
must be occurring downstream of the initial binding event
at the pocket, perhaps being mediated by conformational
changes in the protein as it translates the ligand binding
event into the opening of the ion pore. Our data
show that the NR2D ion pore itself has little influence
on the changes in glycine potency we have observed
(i.e. M1–M3; Suppl. Fig. 3), suggesting that the LBD
and the ion pore functional domains act independently
in this respect. The crystal structure of the NR1/NR2A
LBD heterodimer has suggested that like the non-NMDAR
GluR2 S1S2 homodimer, a number of interpocket
interactions exist (Furukawa et al. 2005) and that these
J Physiol 586.1
may have functional consequences on receptor function.
Furukawa et al. (2005) observed that the conformation of
S2 domains showed the greatest dissimilarities upon superimposition of the NMDAR and non-NMDAR binding
pockets. These data are consistent with our molecular
dynamics analysis. Thus the regions of the LBDs encoded
by the S2 region may adopt a structural conformation
unique to NMDARs that may influence the interaction
between the glycine and glutamate binding pockets. The
identification of two specific residues in different parts
of Domain2 of NR2 subunits that affect glycine potency
indicates a complex interaction between the Domain2 of
NR2 and NR1. One of these residues (Tyr735 (in NR2A),
Lys756 (in NR2D); Figs 9 and 10) is at the interface between
Domain2 of NR1 and Domain2 of NR2 and could play a
role in the equilibrium of that interface or in interacting
with linker regions during a gating event. Moreover, linker
regions connecting the LBD to the transmembrane regions
are not represented in crystal structures, but are encoded by
S1 and S2 regions of the cDNA, and seem likely candidates
for influencing the ability of agonist binding to open the
permeation pore. Our results, however, indicate that the
introduction of the corresponding NR2A residues into the
NR2D subunit did not decrease glycine potency. Indeed,
in the case of the NR2D(K756Y) mutation we generated
an NMDAR with a lower EC50 for glycine than that seen
with NR2D(WT)-containing NMDARs. The reasons for
this increase in glycine potency are unclear, but it suggests
that converting NR2D-containing NMDARs to receptors
with lower sensitivities to glycine is likely to involve other,
as yet unidentified regions of the NR2D subunit.
The contribution of interlobe atomic interactions to
glutamate potency has recently become relevant for
non-NMDARs (Robert et al. 2005; Weston et al. 2006)
and in NMDARs (Maier et al. 2007; Erreger et al. 2007).
Even though the NR1–NR2A cocrystal structure is now
available (Furukawa et al. 2005), little is known about
the importance of interpocket interactions for agonist
potency. Coupling occurs between the glutamate and
glycine binding pockets in NMDARs (Vyklicky et al. 1990;
Benveniste et al. 1990; Benveniste & Mayer, 1991; Kemp &
Priestley, 1991; Lester et al. 1993; Priestley & Kemp, 1994)
and part of the S1 region of the NR2A subunit has been
suggested to influence glycine-dependent desensitization
(Regalado et al. 2001). However, the relationship between
the two pockets in influencing agonist potency has not
been examined in great detail.
Heterogeneity of glycine potencies among different
NMDAR subtypes
It is unclear whether differences in agonist potency
influence
physiological
and
pathophysiological
mechanisms in neurons. Nevertheless, one would
predict receptors at which glutamate and glycine have
high affinity may be candidates for detecting trophic
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J Physiol 586.1
Control of glycine potency in NMDARs by NR2 binding domains
effects of glutamate. This could be consistent with the
early developmental expression of NR2D. Receptors
with higher affinities for glutamate might also be
more optimal for detecting spillover glutamate from
the cleft or glutamate release by glial cells, which can
reach concentrations of only a few micromolar (Lee
et al. 2007). Similarly, receptors at which glutamate
has lower affinity would mean that their activation
would be more dependent on the high concentrations
of glutamate released at synaptic sites. Indeed, in this
respect, receptors with the highest sensitivity to glutamate
(NR2D-containing NMDARs) have, as yet, not been
found at synaptic locations. Although concentrations
of glycine within the cerebrospinal fluid have been
estimated at micromolar levels that would saturate the
glycine site, the actual concentration of glycine in the
synaptic cleft remains unknown. Saturation of the glycine
site may be synapse dependent, influenced not only
by NR2 subunit expression but also by the activity of
local glycine transporters. The concentrations of other
endogenous amino acids that are high affinity agonists at
the glycine site, such as d-serine, will also have an impact
on this question (Thomson et al. 1989; Supplisson &
Bergman, 1997; Berger et al. 1998; Bergeron et al. 1998).
Thus, the NR2 receptor control of glycine potency needs
to be considered in circumstances where the glycine
concentration is low. Indeed receptors displaying lower
sensitivity to glycine are likely to be those targeted by
glycine transport inhibitors.
Conclusion
NMDARs are unusual in that they require two different
ligands to occupy unique binding sites within the
receptor–channel. The basis of why glycine potency
varies at different NMDAR subtypes is less clear when
each of these contains the same glycine-binding (NR1)
subunit. We have identified residues within Domain2
of the NR2 subunit that influence glycine potency
and, in part, account for the heterogeneity in glycine
potencies. Protein–protein interactions at the interfaces
of non-NMDAR subunits control desensitization and
deactivation and it is clear from our data that such
interactions between NR1 and NR2 subunits are key
to regulating receptor function. Together with our
accompanying paper (Wrighton et al. 2008) these studies
demonstrate that the LBD and its interaction with other
functional domains influences two defining characteristics
of this receptor family, namely glycine (coagonist) potency
and voltage-dependent Mg2+ block.
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Acknowledgements
This work was supported by a grant from the Biotechnology
and Biological Sciences Research Council (BB/D001978/1),
the Undergraduate Pharmacology Honours programme at the
University of Edinburgh and the National Institutes of Health
(NS36654; S.F.T.), NARSAD (S.F.T.), and the Michael J Fox
Foundation (S.F.T.).
Authors’ present addresses
K. E. Erreger: Department of Molecular Physiology and
Biophysics, Vanderbilt University, 465 21st Avenue, Nashville,
TN 37232, USA.
M. R. Livesey: Neurosciences Institute, Division of Pathology and
Neuroscience, Ninewells Hospital and Medical School, Dundee,
DD1 9SY, UK.
C. J. Lee: Center for Neural Science, Division of Life Sciences,
Korea Institute of Science and Technology, Seoul, Korea.
Supplemental material
Online supplemental material for this paper can be accessed at:
http://jp.physoc.org/cgi/content/full/jphysiol.2007.143172/DC1
and
http://www.blackwell-synergy.com/doi/suppl/10.1113/jphysiol.
2007.143172
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Modulation of glycine potency in rat recombinant NMDA receptors containing
chimeric NR2A/2D subunits expressed in Xenopus laevis oocytes
Philip E. Chen, Matthew T. Geballe, Elyse Katz, Kevin Erreger, Matthew R. Livesey,
Kate K. O'Toole, Phuong Le, C. Justin Lee, James P. Snyder, Stephen F. Traynelis and
David J. A. Wyllie
J. Physiol. 2008;586;227-245; originally published online Oct 25, 2007;
DOI: 10.1113/jphysiol.2007.143172
This information is current as of January 14, 2008
Updated Information
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including high-resolution figures, can be found at:
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Fezal Ozdemir
Ege University
Branka Vasiljevic
University of Belgrade
Monica Ballarino
Università degli Studi "La Sapienza" di Roma
Sabina Passamonti
Università degli Studi di Trieste
