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(Received for publication, November 29, 1995; and in revised form, January 24, 1996) From the
Ifenprodil is an atypical noncompetitive modulator of the N-methyl-D-aspartate (NMDA) receptor (NR) which
demonstrates a 140-fold preference for NR2B over NR2A subunits,
although the molecular basis for this subunit specificity is unknown.
We have made chimeric receptors by fusing the murine forms of NR2A
(
The N-methyl D-aspartate (NMDA) ( The cloning of
several subunits of the NMDA receptor has allowed investigation of drug
interactions at the molecular level. Rat NMDAR1 (NR1), NMDAR2A (NR2A),
NMDAR2B (NR2B), NMDAR2C (NR2C), and NMDAR2D (NR2D) have been
discovered(11, 14, 15) . In addition, eight
isoforms of the NR1 subunit can be generated by alternative RNA
splicing, (NR1A-H)(16, 17) . The murine forms of
NR1 ( Ifenprodil is a structurally unique modulator of the NMDA receptor
which exhibits subunit-specific affinity for NMDA
receptors(24, 26) . This phenylethanolamine derivative
noncompetitively antagonizes the NMDA receptor either by stabilizing
the closed-channel conformation of the ion channel or by causing a
modal shift in the gating of the ion pore(27, 28) . By
a different mechanism, ifenprodil can block NMDA receptors in a
voltage-independent manner(24) . Ligand binding experiments
have also suggested two sites for ifenprodil interaction. Inhibition of
both [ There is much controversy over whether ifenprodil and
polyamines interact at the same site on the NMDA
receptor(13, 27, 30, 31, 32) .
Polyamines such as spermidine or spermine interact with NMDA receptors
by at least three mechanisms. Polyamines stimulate NMDA receptors by
enhancing the receptor affinity for glycine (glycine-dependent
stimulation) and, in saturating glycine, increase the probability of
channel opening (glycine-independent stimulation)(25) . At
higher concentrations, polyamines can also block NMDA receptors at the
channel pore in a voltage-dependent manner(25) . Like
ifenprodil, glycine-independent stimulation by polyamines is dependent
on NR2 subunit expression. Electrophysiologic and binding paradigms
both demonstrate that in saturating concentrations of glycine,
polyamines stimulate NR1A/NR2B receptors whereas NR1A/NR2A combinations
are
polyamine-insensitive(25, 33, 34, 35, 36) .
Radioligand binding studies suggested additional linkage between
ifenprodil and polyamine sites. Spermidine displaces both We have investigated the molecular interactions of
ifenprodil at NMDA receptors to define the determinants of NR2B which
confer subunit-specific modulation, in an attempt to provide
biochemical evidence that ifenprodil and polyamines bind to discrete
sites on the NMDA receptor. We designed chimeric
Figure 1:
Design of the
Cleavage of the 1.7-kilobase fragment from
PCR3 with XhoI and AflII and subcloning into CH5 cut
with the same enzymes yielded CH84. The DNA sequences for all chimeras
were verified by double-stranded dideoxy sequencing.
The products from PCR1 and PCR2 were then digested with BclI
and ligated together. A third PCR product was generated using the
product of the ligation as the template and the primers: 5`, SP6; and
3`, AFLIIE23. This product was digested with SalI and AflII and cloned back into
Since the NMDA receptor subunits are large polypeptides
(about 1,500 amino acids) the characterization of the molecular
determinants of ifenprodil binding to NR2B was best accomplished using
chimeric NR2A-NR2B subunits. Because the protein sequences of NR2A and
NR2B are 50% identical(18, 19, 20) , their
sequences can be effectively fused and could be expected to retain the
functional properties of wild type receptors. The murine forms of
NR2A (
Figure 2:
Panel A, Western blot of NR1 splice
variants. The splice variants of NR1 are recognized by the monoclonal
antibody against NMDAR1. Membranes from 293 cells transfected with the
NR1 splice variants (A-G) were run on a 5% SDS-polyacrylamide gel
and transferred to nitrocellulose for Western blot analysis. Lane
1, untransfected 293 membranes; lanes 2-8,
NR1A-NR1G, respectively. The molecular mass markers are in kDa.
All lanes show the expected 118-kDa protein. The NR1 monoclonal also
detected fainter breakdown products of the NR1 splice variant subunits,
which vary in size based on isoform expression. Panel B,
Western blot of the NR2 subunits. The polyclonal antibodies raised
against NR2A were able to recognize NR2A, but not NR2B or NR2C. The
Western blot using the NR2A polyclonal shows cell membranes from
transfections of the following: vector DNA control lane 1), 1A
alone (lane 2), 1A/2A (lane 3), 1A/2B (lane
4), 1A/2C (lane 5), 1A/CH8 (lane 6), and 1A/CH25 (lane 7). Amounts loaded were: lanes 1-5, 150
µg; lanes 6 and 7, 400 µg. A single band
running at 165 kDa is found in the lanes with NR2A, 1A/CH8, and
1A/CH25, demonstrating that our polyclonal is specific for the rat form
of NR2A but recognizes both murine forms of NR2A and NR2B. Panel
C, expression levels of the NR2 chimeras. The mean total binding
of
Figure 3:
The
NH
Figure 4:
The high affinity ifenprodil binding
determinants are between amino acids 198 and 356 of
Figure 5:
Arg-337 of NR2B mediates high affinity
ifenprodil inhibition. All of the site-directed mutants of Arg-337
cause at least a 300-fold shift in the IC
Figure 6:
High
affinity ifenprodil inhibition is not dependent on NR1 splice variant
expression. The inhibition of
Further evidence for
ifenprodil insensitivity for the splice variants of NR1 was shown by
cotransfection of all eight splice variants with either CH8 or CH25. In
all cases, the splice variants that were transfected with CH8 had
IC
Although the determinants for glycine-independent
polyamine stimulation map to the same general region of NR2B, results
with the Arg-337 mutants gave the most conclusive evidence that
ifenprodil and polyamine sites were indeed distinct. All four mutants
at Arg-337 were found to be stimulated to the same extent as
Chimeric The expression of the murine forms of
NR2 was comparable to that of NR2A and NR2B. Western blot analysis
using a polyclonal antibody against NR2A demonstrated a high level of
protein expression of both native The
divergent effects of the splice variants of NR1 on both ifenprodil
inhibition and polyamine stimulation confirmed the distinct structural
determinants between these modulators. Polyamine stimulation in
electrophysiologic experiments depends on which splice variant of NR1
is
expressed(16, 17, 24, 46, 47) .
Neither NR1B homomeric receptors nor NR1-NR2 combinations with the
NH The use of
chimeric receptors facilitated the discrete mapping of the site for
high affinity ifenprodil inhibition. A major determinant of high
affinity ifenprodil inhibition localizes to Arg-337 on the Mutants at Arg-337 could potentially alter the affinity of
1A/ Finally,
Arg-337 may interact directly with the NR1 subunit where the true
binding site for ifenprodil resides. NR1A mRNA injected into Xenopus oocytes yields functional homomeric channels with a
high affinity for ifenprodil (0.28 µM); thus the high
affinity binding site for ifenprodil was thought to reside on the NR1
subunit or the association of multiple NR1A subunits(26) .
Homomeric channels, although functional, lack many of the
characteristics of native NMDA receptors and have not been conclusively
shown to exist in
vivo(21, 26, 33, 34) . Multiple
ifenprodil binding sites, present on both NR1 and NR2 subunits, could
also exist. There is a considerable sequence homology between NR1 and
The 300-fold difference in ifenprodil affinity
between NR2A- and NR2B-containing receptors can best be explained by an
electrostatic interaction occurring at the high affinity ifenprodil
binding site. The chemical structure of ifenprodil contains no obvious
ionizable groups such as amines but does possess a phenyl ring with a
hydroxyl group attached. Tyrosyl-like groups may become phenolate ions
following the loss of a proton from the phenyl hydroxyl
group(52, 53, 54, 55, 56) .
The O Although some components of the
NR2B-specific effects of ifenprodil and polyamines overlap, results of
the NR1 splice variant experiments and the Arg-337 mutation experiments
provide biochemical evidence for distinct polyamine and ifenprodil
binding sites. The dissimilar structures of spermidine and ifenprodil
make competitive binding arguments unlikely. Even though the long
aliphatic chain and amine group of spermidine differ from the
phenylethanolamine structure of ifenprodil, some of the pharmacologic
properties of polyamines and ifenprodil overlap. Ifenprodil blocks the
stimulatory effects of polyamines on both TCP and MK-801 binding and
inhibits the increase in [ There is much current interest in the ability
of ifenprodil to act as a neuroprotective agent during focal cerebral
ischemia and as an anticonvulsive
agent(13, 25, 58) . The interaction between
ifenprodil and the NMDA receptor may underlie this neuroprotective
ability. Unlike many other neuroprotective agents, ifenprodil and the
derivative SL 82.0715, which has a better oral bioavailability, do not
cause any behavioral effects and have already been used clinically for
the treatment of hypertension and cerebral
ischemia(13, 58) . The location and mechanism of
ifenprodil action on the NMDA receptor are still not completely
understood. By identifying more residues like Arg-337, which directly
participate in modulating the function of NMDA receptors, and by
characterizing these modulatory sites at the molecular level, it will
be possible to design additional novel therapeutic agents to combat the
neurodegeneration that follows events such as stroke.
Acknowledgments-We give special thanks to Dr. Michael Robinson,
Dr. Brian Basckai, and Elfrida Grant for helpful comments on this
manuscript.
Dedicated to the memory of Dr. Dolan B. Pritchett for outstanding
mentorship, scientific intellect, creativity, and friendship.
Volume 271,
Number 16,
Issue of April 19, 1996 pp. 9603-9611
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
D
-aspartate Receptor (*)
) and NR2B (
) to localize the high
affinity determinants of ifenprodil inhibition on the 2B subunit.
Binding experiments with 
I-MK-801 implicated the region
between amino acids 198 and 356 of NR2B for high affinity ifenprodil
interaction. Site-directed mutants at Arg-337 showed that this residue
is absolutely required for high affinity ifenprodil inhibition.
Polyamines also modulate the NMDA receptor with a preference for NR2B
subunits, and the pharmacology of these agents overlaps with
ifenprodil. Although the determinants of the polyamine enhancement of
iodo-MK-801 binding also localize to the NH terminus of
NR2B, the point mutants at Arg-337 form receptors that are
polyamine-stimulated at wild type levels. In addition, polyamine
stimulation depends on the expression of NR1 splice variants, whereas
high affinity ifenprodil inhibition is independent of NR1 isoform
expression. These studies provide evidence that ifenprodil and
polyamines interact at discrete sites on the NR2B subunit.
)subtype of glutamate receptor is a ligand-gated ion
channel that mediates the entry of Ca
into neurons of
the central nervous system and has been linked to neurologic disorders,
synaptic plasticity, and excitotoxic cell death (1, 2, 3, 4, 5) . In
addition to the natural agonist (glutamate) and coagonist (glycine),
many other compounds affect NMDA receptor function, including the
channel blocking agents phencyclidine (PCP)(6) , N-1-(thienyl)cyclohexyl)piperidine
(TCP)(7, 8) , and dizolcilpine (MK-801) (6, 9) and the competitive antagonists D-3-(2-carboxy-piperazine-4-yl)-propyl-1-phosphonic acid (CPP) (10) and cis(±)-4-phosphonomethyl-2-piperidine
carboxylic acid (CGS 19755)(11) . Modulatory sites, distinct
from both the glutamate and glycine sites, for polyamines (12) (such as spermidine) and for the atypical, noncompetitive
antagonist ifenprodil (13) have also been characterized. Novel
therapeutic agents that interact favorably at noncompetitive sites
could control the excitotoxic Ca influx mediated by
NMDA receptor overstimulation without the psychotic side effects
exhibited by channel-blocking agents(5) .
) and NR2 (/
) have more
than 99% amino acid homology with rat NR2
subunits(18, 19, 20) , allowing functional
coexpression of mouse and rat subunits(21) . Receptors formed
by coexpression of different heteromeric combinations of NR1 and NR2
subunits exhibit distinct pharmacologies and can mimic regional and
developmental expression in
vivo(14, 15, 18, 19, 22, 23, 24, 25) .
Since recombinant receptors can be selectively mutated, they provide
excellent tools for molecular mapping of drug binding sites. 
H]MK-801 and I-MK-801 and
[H]TCP binding to rat brain by ifenprodil is
biphasic(24, 27, 29) , suggesting the
presence of both high and low affinity ifenprodil sites. The high
affinity site for ifenprodil, measured electrophysiologically, has a K
in the submicromolar range (0.2-1
µM), whereas the K for the
low affinity site is 140-300-fold higher (60-100
µM)(24) . Binding and electrophysiologic studies
show that ifenprodil exhibits a 140-fold preference for NR1A/NR2B
() receptors over NR1A/NR2A ()
combinations(24) . Therefore, the expression of NR2 subunits
may underlie the biphasic nature of ifenprodil interactions with NMDA
receptors.I- and [H]ifenprodil from rat
brains, although direct competition is
unclear(37, 38, 39, 40) . The
polyamine stimulation of I-MK-801 and
[H]CPP binding can also be reduced by increasing
ifenprodil concentrations (13, 39) , further
suggesting overlapping sites. Conversely, electrophysiologic studies of
ifenprodil and polyamine effects on NMDA receptors expressed in Xenopus oocytes have ruled out simple competitive interactions
of ifenprodil and
polyamines(24, 26, 27, 28) . The
molecular determination of the ifenprodil and polyamine sites on the
NMDA receptor could aid in understanding the interactions between these
modulators. (NR2A)/ (NR2B) subunits, coexpressed them with
NR1 subunits, and measured the dose-dependent inhibition of I-MK-801 binding by ifenprodil for these receptors in
order to localize the binding determinants of ifenprodil interaction on
the NR2B subunit. The NH-terminal 464 amino acids of NR2B
contained determinants for both ifenprodil and polyamine interactions,
and additional chimeras permitted localization of high affinity
ifenprodil inhibition to amino acids 198-356. Site-directed
mutants of were then characterized to define the
ifenprodil modulatory site at the molecular level. All substitutions at
Arg-337 render the receptor low affinity for ifenprodil, whereas these
mutant receptors maintain wild type polyamine stimulation, providing
biochemical evidence for discrete modulatory sites. The distinction of
polyamine and ifenprodil binding sites was corroborated by additional
experiments with NR1 splice variants. Unlike
polyamines(25, 36) , ifenprodil inhibition was shown
to be unaffected by NR1 isoform expression, further suggesting distinct
binding mechanisms. The molecular characterization of modulatory sites
on the NMDA receptor could provide vital information for the design of
novel therapeutic agents for the prevention of the neurodegeneration
following cerebral ischemia.
Materials
(+)-3-I-MK-801 was purchased from DuPont
NEN. Spermidine, (±)-ketamine, 
-mercaptoethanol, and
polyethylenimine were obtained from Sigma. (+)-MK-801 (hydrogen
maleate) and ifenprodil (tartrate) were from Research Biochemicals
International. Restriction enzymes and Taq DNA polymerase were
purchased from either Life Technologies, Inc. or New England Biolabs.
Sequencing was performed using the Sequenase II kit from U. S.
Biochemical Corp. Monoclonal antibodies recognizing the NH terminus of NMDAR1 were purchased from Pharmingen (San Diego).
Goat anti-rabbit and anti-mouse peroxidase-conjugated secondary
antibodies were purchased from Boehringer Mannheim. ECL
chemiluminescence reagents were obtained from Amersham Corp. HEK293
cells were purchased from American Type Culture Collection (ATCC). All
other reagents used were of the highest purity available and were
obtained from standard commercial sources.Chimeric NMDAR2 Subunit Construction
DNA encoding and (the
murine forms of NMDAR2A and NMDAR2B, respectively) were subcloned into
the SalI/EcoRI sites of the vector pRK7 downstream of
the cytomegalovirus promoter, which yields high level expression of
each of these subunits in mammalian cell culture
systems(21, 33, 34, 41) . The first
chimera (CH8) (see Fig. 1) was constructed by replacing the
first 1393 base pairs of with the corresponding
region of by digesting with SalI and AflII and ligating the 1.6-kilobase fragment
into the same sites of . The complementary chimera
(CH25) was constructed by ligating the SalI/AflII
fragment of into . A chimera (CH5)
containing the first 198 amino acids of and the rest
being was constructed using PCR. The two primers, SP6
(5` primer) and REMXHO13, which introduces an XhoI site into
, (5`-GTGATCACGTTCTCGAGATCCCAGCCC-3`), were used to
amplify (95 °C, 1 min; 55 °C, 1 min; 72 °C, 2 min; 30
cycles) the 5` region of . The 795-base pair PCR
product was digested by SalI and XhoI and ligated
into the corresponding sites of (cloned into the
vector pRK7). CH6 was constructed by digesting CH5 with SalI/AflII and ligating the resulting 1.6-kilobase
fragment into SalI/AflII-cut . An
/ chimera containing the
NH-terminal 356 amino acids of , (CH48),
was created using a three-step PCR method using overlapping primers.
PCR , (PCR1), used the primers SP6 (5`) and the
overlapping primer OAPAL3E1 (5`- GCACGATCACC
ACAAGCCTGGGGTGCACCTGGTAGCCATCTTCACTGAAGG-3`), with as
the template. The second PCR reaction (PCR2) used the overlapping
primer OAPAL5E2
(5`-CCTTCAGTGAAGATGGCTACCAGGTGCACCCCAGGCTTGTGGTGATCGTGC-3`), and a 3`
primer downstream of the AflII site, E1AFLII3
(5`-GTTCTGGACAGTT TCTTAAGGATGTC-3`), with as the
template. The products of these PCR reactions anneal across a 44 base
pair region containing the chimeric fusion point at the center. 1
µL of each of the PCR reactions (PCR1 and PCR2) are diluted 1/10 in
PCR reaction buffer and are combined, allowing homologous regions to
anneal. After 4 cycles of PCR, a double stranded template is formed
from the annealed products. The outside primers SP6 and E1AFLII3 are
then added, and a 1.7-kilobase fragment is amplified. The final
fragment is ligated into the SalI/AflII sites of
. A chimera that substitutes the amino acid region
199-356 of into (CH58) was
derived from CH48 by replacing the SalI/XhoI fragment
of CH48 with the SalI/XhoI fragment of CH5. A chimera
encoding only at amino acids 356-464 was
created using the same overlapping primer method with the following
primers and templates.
/ chimeras. The schematic primary
sequences depict how portions of (white regions) and (black regions) are fused
to form chimeric NR2 subunits. The IC
values for
ifenprodil inhibition were calculated using the best of either one- or
two-sited fits using PROPHET. In all cases of two-sited fits the
percent high affinity represented 65% of the total inhibition; low
affinity was 35%. Data are representative of 4-14 experiments.
Polyamine stimulation is shown by either + + +
(150-170% stimulation over baseline), + (50% stimulation),
- (<10% spermidine enhancement), or - - -
(0% polyamine stimulation).
PCR1
The primers were as follows. 5`: E1XHOI53
(5`-GTGGGCTGGGATATGCAGAACGTGATC-3`); 3`: OAPAL3E2 (5`-GAAGGATTAT
CACCAGCTTCGGGTGCACCTGATAGCCTTCCTCAGTGAAG-3`). The template was
.PCR2
The primers were as follows. 5`: OAPAL5E1
(5`-CTTCACTGAGGAAGGCTAT CAGGTGC ACC CGAAGCTGGTGATAATCC-3`); 3`:
AFLIIE23 (5`-CTTAGAAATTTTCTTAAGG ATATCAATAC-3`). The template was
.PCR3
The primers were as follows. 5`: E1XHOI53;
3`: E1AFLII3. The templates were products of PCR1+PCR2 elongated
by four cycles of PCR.Site-directed Mutagenesis
The point mutations at Arg-337 were constructed using a
similar three-step PCR technique. Mutagenic primers were constructed
with a degeneracy at the codon for Arg-337 to allow simultaneous
generation of a panel of mutations.PCR 1
The primers were as follows. 5`: SP6; 3`: E2ARGX35
(5`-GTGACGTTGATCAGATAT(TG)(CTG)A TTCAGCATGTTAGACTG-(3`). The template
was in pRK7.PCR2
The primers were as follows. 5`: E2ARGX53,
(5`-CAGTCTAACATG CTGAAT(GAC)(AC)ATA TCTGATCAACGTCAC-3`); 3`: AFLIIE23
(see above). The template was in pRK7.. Sequencing the
final product confirmed the mutants R337A, R337P, R337K. The mutant
R337Q was made using the same multistep PCR technique with the more
restrictive primer, E2R337EQ (5`-GTGACGTTGATCAGATA(GC)TCATTCAGCAT GTTA
GACTG-3`, substituted for the primer E2ARGX35 above.Cell Culture and Transfection of HEK 293 Cells
Human embryonic kidney cells (HEK293) were propagated in
minimal Eagle's medium containing 5% horse serum and 5% fetal
bovine serum, 2 mM glutamine, and 1 
penicillin/streptomycin in a humid 95% air, 5% CO atmosphere. Cells were transfected by the calcium phosphate
precipitation method of Chen and Okayama with the modification that 10
µM MK-801 is present during transfection to protect cells
from NMDA-induced excitotoxicity(41, 42) .Polyclonal Antibody Production
Polyclonal antibodies were raised against the COOH terminus
of the rat NMDAR2A protein. The sequence encoding amino acids
1115-1465 was subcloned into the pRSET vector (Invitrogen) at the
unique BglII site. The expression construct was transformed
into the Escherichia coli strain BL21(DE3), and high levels of
fusion protein were induced with
isopropyl-1--D-thiogalctopyranoside, (Boehringer
Mannheim). Fusion proteins were purified by nickel affinity
chromatography, with elution in 6 M guanidine. Following
further purification by SDS-polyacrylamide gel electrophoresis,
isolated bands were crushed and eluted in 100 mM NaCl, 50
mM Tris buffer, pH 7.5, and subsequently sent to Rockland Inc.
for polyclonal antibody production. Initial boosts of between 250 and
500 µg of fusion protein in complete Freund's adjuvant were
followed at 2-week intervals by 200-µg injections in incomplete
Freund's adjuvant. Antibodies in the sera were capable of
detecting 20-50 ng of fusion protein by enzyme-linked
immunosorbent assay screening. Further purification of the polyclonal
antibodies was accomplished by affinity chromatography. Affinity
columns were made by coupling 200 µg of fusion protein to 6
Reacti-Gel (Pierce) in 50 mM sodium borate buffer, pH 8.5.
Antiserum was applied to 3-ml columns in 20 mM Hepes, pH 7.5,
and the purified antibodies were eluted with 6 M guanidine, pH
3.0, directly into 1 M Tris to elevate the pH rapidly. The
yield of purified antibody from 100 ml of antiserum was approximately
100 µg.Western Blotting
Membranes from 293 cells transfected with combinations of NR1
and NR2 subunits were prepared by 2-3-s sonication in isotonic
medium (0.32 M sucrose, 4 mM Hepes, pH 7.5, 1
µM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 1 µM leupeptin), followed by
centrifugation at 14,000 rpm for 20 min to remove the nuclear fraction.
Samples were boiled in SDS sample buffer containing
-mercaptoethanol and loaded on 6% polyacrylamide gels. Protein was
transferred to nitrocellulose using a submarine transfer unit (Hoeffer
Scientific), and blots were incubated in 2.5% dry milk in 100 mM NaCl, 50 mM Tris (1 TBS), pH 7.5, for 30 min
before incubation with primary antibody. Blots were incubated in either
polyclonal NMDAR2A or monoclonal NMDAR1 antibodies (3 µg/ml in TBS,
2.5% dry milk) for 4-6 h with constant agitation. After excess
primary antibody was removed (once, 5 min, 0.01% Triton X-100/TBS;
twice, 5 min, TBS), peroxidase-conjugated goat anti-rabbit (or mouse)
secondary antibody was added at a 1/30,000 dilution in 1% dry milk, 1
TBS. Multiple washes in 0.01% Triton, 0.1% Tween 20, and TBS
were followed by detection with the ECL chemiluminescence detection
system.Radioligand Binding
Cells were prepared, as described
previously(33, 34) , for I-MK-801
binding by washing in 100 µM glycine, 100 µM glutamate, 300 µM Mg, 20 mM Hepes, pH 7.5 (three times, 30 min, 32 °C) to remove the
unlabeled MK-801 used to reduce cell death during transfection. Binding
to membranes was performed in the presence of I-MK-801
(150 pM), glycine (100 µM), Mg (300 µM), glutamate (100 µM), and
spermidine (100 µM) to facilitate channel opening and
ligand binding. Increasing concentrations of either ifenprodil or
spermidine were added as appropriate. Incubations were for 3 h at 37
°C. Each concentration was measured in duplicate with a
corresponding blank containing 10 µM unlabeled MK-801.
Specific binding was measured on a Beckman gamma counter (model 5500B)
following rapid filtration (Brandel).
) an NR2B () were chosen for
chimera construction due to the presence of unique restriction sites
and because bacterial synthesis of the NR2B gene consistently yields
low quantities of DNA. ()Even though there is a 99% identity
at the amino acid level between the mouse and rat
subunits(18, 19, 20) , we needed to eliminate
the possibility of dissimilar pharmacologies. Cotransfection of rat
NR1A subunits with either or creates receptors with similar pharmacology as pure rat channels,
consistent with previous work with NR1A/ receptors(21) . The K
values of the
receptors NR1A/ and NR1A/ for I-MK-801 were within statistical error with pure rat
receptors(34, 35) , both exhibiting values of 150
pM (data not shown). In addition, these same receptors retain
the subunit specific effects of ifenprodil inhibition and polyamine
stimulation. NR1A/ receptors have an IC for ifenprodil inhibition of 59 µM; NR1A/ receptors have an IC of 0.16 µM,
comparable to previous results for pure rat combinations (34) (Fig. 1). 1A/ receptors were also
not polyamine-stimulated, unlike 1A/ combinations.
Since the murine NR2 subunits retain the properties of their rat
homologs, this validates the strategy of designing chimeric
/ subunits to map the
subunit-specific determinants of ifenprodil and polyamine interaction.
In the present study we have designed seven chimeric
/ receptors (Fig. 1), which
were used to map the determinants of ifenprodil inhibition on the NR2B
subunit.Expression of NMDA Receptor Subunits by Western
Blot
Western blot analysis demonstrated that NR1, NR2,
, , and the
/ chimeras all exhibit high levels of
expression in our transfection system (Fig. 2, A-C). The splice variants of NR1A-G are all recognized
by the monoclonal antibody against NR1A, and membranes analyzed by
Western blot demonstrate comparable levels of expression of all forms (Fig. 2A). The monoclonal against NR1 also detects the
fainter breakdown products of the NMDAR1 protein, exhibiting different
patterns depending on the splice variant transfected. A polyclonal
antibody raised against the COOH terminus of the NR2A was used to
verify NR2 subunit expression. This antibody recognizes NR2A but not
NR2B or NR2C or any of the splice variants of NR1 (Fig. 2B). Fig. 2B shows a Western blot
of two of the chimeras: CH8, which contains the COOH terminus of
, and CH25, which contains the COOH terminus of
. Bands running at the predicted molecular mass (165
kDa) demonstrated high level expression of both chimeras. All of the
other five chimeric NR2 subunits showed similar levels of expression by
Western blot (data not shown). The total I-MK-801 binding
for all chimeras, which ranged from 32 to 55 fmol/mg protein (with 150
pMI-MK-801), was comparable to wild type
receptors (1A/, 58 fmol/mg protein;
1A/, 87 fmol/mg protein), demonstrating high level
expression for chimeric NR2 subunits (Fig. 2C). Based
on total radioligand binding, all eight splice variants have expression
levels comparable to combinations containing NR1A (Table 1).
I-MK-801, expressed as fmol/mg of protein (n = 4-8), is shown. All seven chimeric subunits, when
coexpressed with NR1A, show comparable levels of expression with wild
type receptors. Error bars are
shown.
The NH
The region of NR2B
which most likely mediates subunit specificity is in the more divergent
NH Terminus of Is the
Site of High Affinity Ifenprodil Interaction-terminal region, which is proposed to create the
extracellular face of the receptor(28, 36) . The first
chimeras (CH8 and CH25) were constructed by exchanging the
NH-terminal thirds of and (Fig. 1). I-MK-801 binding to receptors
formed by cotransfection of NR1A/CH8, which contains the
NH-terminal third of , is stimulated by
spermidine, whereas 1A/CH25 (the complementary chimera) expression
produced polyamine-insensitive receptors. Similarly, coassembly of CH8
with NR1A led to high affinity ifenprodil displacement (1.6
µM) I-MK-801 binding, whereas NR1A/CH25
receptors exhibited low affinity inhibition (65 µM) (Fig. 3). These results implicated the NH-terminal
third of in both glycine-independent polyamine
stimulation and high affinity ifenprodil inhibition.
terminus of NR2B contains determinants for high affinity
ifenprodil inhibition and polyamine stimulation. Ifenprodil inhibition
and polyamine stimulation of I-MK-801 binding are
governed by the NH-terminal third of the subunit. The top panel compares the ifenprodil
inhibition curves for the receptors 1A/CH8 (
), 1A/CH25 (
),
1A/ (
), and 1A/ (
), which
were determined by nonlinear least squares analysis by the PROPHET
computer program. The bottom panel shows the modulation of I-MK-801 binding for the same receptors by increasing
spermidine concentrations.
Localization of the Determinants of High Affinity
Ifenprodil Binding
The determinants of high affinity ifenprodil
inhibition were localized using five chimeric
/ subunits (Fig. 1). NR1A/CH5
receptors had IC values for ifenprodil of 2.6
µM; NR1A/CH6 had an IC of 3.3
µM. The retention of high affinity inhibition by CH6
localized the -specific determinants of ifenprodil
inhibition to amino acids 198-464. Three additional chimeras,
CH48, CH58, and CH84, further defined the region of NR2B mediating high
affinity ifenprodil inhibition. The ifenprodil inhibition curves for
these chimeras are shown in Fig. 4. The IC values
for CH48, CH58, and CH84 were 4.2, 18, and 72 µM,
respectively ( Fig. 1and Fig. 4). Only a 5-fold loss in
affinity of NR1A/CH48 over NR1A/ was observed in
inhibition assays, whereas NR1A/CH58 had a 5-fold greater apparent
affinity for ifenprodil than 1A/, implicating amino
acids 198-356 as an important region for high affinity ifenprodil
inhibition. NR1A/CH84 receptors have an even lower IC for
ifenprodil (72 µM), than 1A/ receptors
(59 µM), further implicating amino acids 198-356 for
high affinity ifenprodil modulation.
.
Five additional chimeras were used to localize high affinity ifenprodil
binding determinants to amino acids 198-356. The top panel shows the inhibition of I-MK-801 binding by
ifenprodil for the receptors 1A/CH48 (
), 1A/CH58 (),
1A/CH84 (), 1A/ (), and 1A/ (). Theoretical curves are shown for 1A/ (CH48), 1A/CH58, and 1A/ (1A/CH84) and have been
calculated as is described in the legend to Fig. 3. The bottom panel shows the polyamine modulation by the same
receptor combinations. The decrease in spermidine enhancement of I-MK-801 binding for the receptor series
1A/, 1A/CH48, 1A/CH58, 1A/CH84, and 1A/ is shown.
High Affinity Ifenprodil Inhibition Is Mediated by
Arg-337
An important determinant for high affinity ifenprodil
inhibition was discovered by site directed mutagenesis of
. Ifenprodil may act as an acid by losing a phenolic
hydrogen in physiologic conditions; thus, efficacious ifenprodil
binding could be mediated by a positively charged residue on the NR2B
subunit such as lysine or arginine. The region of between amino acids 198 and 357 contains a single positively
charged amino acid that was conserved in both and 2B,
but not in or 2A, namely Arg-337. Site-directed
mutants were then created at Arg-337 by the use of PCR mutagenesis, and
all of these mutants showed expression levels comparable to wild type
receptors (Table 2). Mutation of Arg-337 to lysine (NR1A/R337K)
exhibited an IC for inhibition of I-MK-801
binding by ifenprodil of 120 µM, even lower affinity than
( Table 2and Fig. 5). Arg-337 was also
changed to alanine (R337A), proline (R337P), and glutamine (R337Q),
which is the conserved amino acid in and NR2A. The
IC values for R337A, R337P, and R337Q were 83, 100, 65
µM, respectively. As any residue other than arginine at
position 337 attenuates high affinity ifenprodil inhibition, Arg-337 is
a functional determinant of high affinity ifenprodil binding. Although
the differences in IC between and both
and the Arg-337 mutants for ifenprodil are too great
to be explained only by differences in their affinities for I-MK-801, saturation analysis of 1A/,
1A/, and R337Q was performed. The K values for I-MK-801 of 1A/, 1A
, and R337Q were 124, 133, and 145 pM,
respectively, demonstrating that the observed changes in ifenprodil
potency do not result from changes in the affinity for I-MK-801 and that receptor integrity is preserved in
these mutants.
of Ifenprodil
inhibition from wild type receptors. The ifenprodil inhibition curves
for 1A/ (), 1A/ (),
1A/R337A (
), 1A/R337K (
), and 1A/R337Q () are shown.
The lower panel shows that the mutants 1A/R337A and R337K are
both polyamine-stimulated to the same extent as 1A/,
providing direct biochemical evidence that ifenprodil and polyamines
interact at discrete sites on the NR2B subunit.
Splice Variants of NMDAR1 Distinguish between Ifenprodil
and Polyamines
Although the -specific
properties of polyamine stimulation and high affinity ifenprodil
interaction map to similar regions, experiments with the splice
variants of NR1 suggest that these agents bind to distinct sites. Since
NR1 subunits containing the NH-terminal insert, such as
NR1B, are polyamine-insensitive(25) , we studied the effects of
NR1 isoform expression on ifenprodil inhibition to determine whether
both ifenprodil and polyamines share splice variant-specific
properties. We found that NR1B/ and NR1B/ receptors share the same subunit-specific effects as NR1A
combinations, with IC values for ifenprodil inhibition of
37 and 0.52 µM, respectively ( Table 1and Fig. 6). Experiments with NR1C and NR1G yielded similar results (Table 1), suggesting that ifenprodil interactions are not
dependent on NR1 splice variant expression.
I-MK-801 binding by
ifenprodil for 1B/ and 1B/ receptors
shows the same 100-fold difference in apparent ifenprodil affinity as
1A/ and 1A/. Binding data and
theoretical curves for 1A/ () and 1B/ () both demonstrated low affinity ifenprodil inhibition,
whereas 1A/ () and 1B/ () were high affinity. Conversely, neither of the
1B-containing receptors showed polyamine enhancement of iodo-MK-801
binding (lower panel), demonstrating that polyamine
stimulation is dependent on NR1 isoform expression, whereas high
affinity ifenprodil inhibition is not.
values close to 1 µM, whereas all splice
variant combinations with CH25 demonstrated half-maximal inhibition
between 46 and 89 µM (Table 1), providing evidence
that the high affinity ifenprodil site is not affected by the
expression of any NR1 splice variant even with chimeric receptors.
Unlike polyamines, the inhibition of MK-801 binding by ifenprodil is
not affected by NR1 isoform expression.NR2 Chimeras and R337 Mutants Demonstrate Discrete
Modulatory Sites
The chimeras CH5, CH6, CH48, CH58, and CH84
were used to determine if polyamine stimulation and ifenprodil
inhibition localize to the same region of , although
the results were not as straightforward as for ifenprodil inhibition. I-MK-801 binding to 1A/CH5 and 1A/CH48 receptors is
stimulated by spermidine to the same extent as 1A/CH8, whereas 1A/CH6
and 1A/CH58 are stimulated only 50% as much as wild type receptors (Fig. 4). This suggests that the determinants of polyamine
stimulation, as for ifenprodil, are found in the region between amino
acids 198 and 356, although a significant loss in polyamine stimulation
occurs when the chimera fusion point is at amino acid 198.
Surprisingly, there was a slight spermidine stimulation of 1A/CH84
receptors, which suggests that the binding determinants of polyamine
stimulation may involve more regions of NR2B than for ifenprodil
inhibition. ( Fig. 5and Table 2), thus Arg-337 is
not a determinant of glycine-independent stimulation by polyamines.
/ subunits,
coupled with site-specific mutagenesis, permitted the localization of
high affinity inhibition to the NH terminus of NR2B and
distinguished it from glycine-independent polyamine stimulation. To
utilize the murine forms of NR2 subunits, we had to demonstrate that
they exhibit the same pharmacology as their rat homologs. The murine
forms show the same magnitude and subunit specificity for the
ifenprodil inhibition and polyamine stimulation of I-MK-801 binding as pure rat
receptors(24, 25, 35) . The and forms of NR2 subunits thus proved to be the
ideal tool for chimera construction and characterization of the effects
of ifenprodil and polyamines. and receptors and of all the chimeric NR2 subunits characterized. Our
NR2A polyclonal was unable to discriminate between and , which may be due to 33% homology between
and NR2A in the polyclonal recognition
region(18, 19, 20) . The peptide sequences of
NR2A and NR2B in this region are less than 20% similar and share no
homology to NR2C or D, possibly explaining antibody specificity for the
rat form of NR2A. By preabsorbing the antibodies in our polyclonal
mixture on an affinity column it may be possible to
isolate a population that demonstrates total specificity for NR2A and
, obtaining a valuable tool for the biochemical
characterization of NMDA receptors. Definitive evidence for high level
expression was confirmed by the level of MK-801 binding, which was
between 30 and 110 fmol/mg of protein for all forms of murine receptors
when coexpressed with rat NR1A. Thus, expression of all of the chimeric
subunits and the site-directed mutants was comparable
to wild type receptors. Since we have shown previously that NR1A
homomeric receptors bind insignificant levels of I-MK-801(34) , which is generally consistent with
[H]MK-801 binding
experiments(43, 44, 45) , our wild type
binding levels confirm that our chimeric and mutant NR2 subunits
efficiently coassemble to create intact MK-801 binding sites.-terminal insert (such as NR1B) display
glycine-independent stimulation by polyamines. The effects of
ifenprodil on receptors expressed with different splice variants had
not been well characterized. We have shown that the high affinity
inhibition by ifenprodil is not dependent on the NR1 subunit but is
regulated by the NR2 subunit. All of the NR1 splice variants
(A-H), when coexpressed with NR2 subunits, formed receptors whose
modulation was governed only by NR2 expression. Conversely, polyamine
stimulation was not observed for either NR1B/ or
NR1B/, whereas the NR1B/ receptor
exhibited the same 140-fold greater affinity for ifenprodil as seen for
combinations with NR1A. Clearly, the mechanisms by which ifenprodil and
polyamines interact with the NMDA receptor differ. subunit. There are at least three possibilities for the mechanism
of action for Arg-337. First, ifenprodil may directly bind to Arg-337
of the subunit. The localization of high affinity
determinants to NR2B is consistent with the dramatic differences in
affinity between 1A/ and 1A/ receptors and by the lack of NR1 splice variant-specific
modulation. The fact that both in situ hybridization studies
of NR2B mRNA and radiolabeled ifenprodil experiments show a strong
correlation between high affinity ifenprodil binding and the
developmental and regional profiles of NR2B expression also strengthens
the argument that residues of NR2B interact directly with
ifenprodil(26, 39, 48) . Electrophysiologic
evidence using outside-out patches has demonstrated that the high
affinity ifenprodil site is located on the extracellular portion of the
NMDA receptor(28) . Theoretical models of the transmembrane
architecture of the subunit are consistent with
Arg-337 being present on this extracellular surface(36) . for either the agonist (glutamate) or coagonist
(glycine), affecting the association of MK-801 to open channels. This
is ruled out by the fact that our assay system utilizes high excess
concentrations of both glutamate and glycine and that the K for I-MK-801 of all our chimeras
and point mutants were identical. The binding of MK-801 acts as a good
internal control for receptor integrity. Functional high affinity
MK-801 binding requires the presence of both functional glycine and
glutamate sites and a structurally intact channel
pore(33, 34) ; thus the changes in ifenprodil
inhibition mediated by Arg-337 must be distinct from effects on either
agonist or coagonist sites. Significant reduction of either glycine or
glutamate affinity in mutant receptors results in receptors that do not
bind I-MK-801(34, 35, 49) . The
glycine coagonist site has recently been localized to the aromatic
residues 390, 392, 466 and the charged residues 481 and 483 of the NR1
subunit. These residues are not only distal to Arg-337, but also are
present exclusively on NR1(50, 51) . (NR2B) near
Arg-337(11, 14, 18, 19, 20) .
An arginine residue exists in NR1 (Arg-344) at the comparable position
of Arg-337 in and may be the site of high affinity
ifenprodil inhibition found in homomeric receptors. New chimeras and
site-directed mutants of both NR1 and NR2 subunits will help gain
future insight into the mechanism by which high affinity ifenprodil
binding occurs.
is stabilized through conjugation with the
double-bond structure of the phenyl ring. An electrostatic interaction
between ionized ifenprodil and one or more basic amino acid residues of
the NMDA receptor could be proposed. The energy loss from the
disruption of an electrostatic interaction is believed to be
approximately 3-5 kcal (57) . This change in apparent
binding energy would account for a change in K of
approximately 150-4,000-fold. The 300-fold difference in
IC is consistent therefore with the loss of a weak
electrostatic interaction in -bearing receptors.
Arg-337 is the only basic amino acid residue that is conserved in both
and 2B between amino acids 198 and 356, whereas
glutamine is found at this position in and 2A.
Surprisingly, even substitution of the basic residue lysine at position
337 renders the receptor low affinity, suggesting that not only is a
positively charged residue necessary at residue 337, but specifically
arginine. Since the orientations of the positively charged moieties of
lysyl and arginyl side chains differ, it is likely that the precise
positive charge alignment of Arg-337 is required for efficacious high
affinity ifenprodil binding. Glutamine substitution at this position
exhibits the least detrimental effect on the IC of
ifenprodil inhibition, presumably because the glutamine side chain has
a surface volume most similar to that of arginine. Additional point
mutations will be necessary to define further the involvement of
Arg-337 in NMDA receptor modulation.H]CPP binding
facilitated by spermidine(39) . Polyamines antagonize the
partial displacement of [H]glycine by
ifenprodil(32) . These overlapping effects may be explained by
the determinants of polyamine stimulation on the subunit being between amino acids 198 and 293, which is
potentially close to the site of high affinity ifenprodil binding.
Curiously, the homologous region of NR1 (amino acids 190-211) is
the location for the 63-residue insertion that renders splice variants
such as NR1B
polyamine-insensitive(16, 17, 25) .
Site-directed mutants of this region of NR1A eliminate polyamine
stimulation(46) . Although the determinants of polyamine
stimulation on have not yet been characterized at the
amino acid level, it seems likely that the region from amino acids 198
to 464 will include some component of the glycine-independent polyamine
stimulation region of the subunit. The binding sites
for ifenprodil and polyamines are biochemically distinct, although
their determinants on the NR2B subunit are at least allosterically
linked if not overlapping. Since the binding of polyamines and possibly
ifenprodil involves both the NR1 and NR2 subunits, biochemical
information about NR1-NR2 interactions could be studied by closer
examination of the allosteric linkage between ifenprodil inhibition and
polyamine stimulation.
))
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