(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
() 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.
The N-methyl D-aspartate (NMDA) ()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) .
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 () 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.
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 [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.
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 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.
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 (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.
Figure 1:
Design of the
/
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).
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 . 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.
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 () 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.
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 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.
Figure 3:
The
NH 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.
Figure 4:
The high affinity ifenprodil binding
determinants are between amino acids 198 and 356 of .
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.
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 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.
Figure 6:
High
affinity ifenprodil inhibition is not dependent on NR1 splice variant
expression. The inhibition of 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.
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 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.
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
( Fig. 5and Table 2), thus Arg-337 is
not a determinant of glycine-independent stimulation by polyamines.
Chimeric /
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.
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 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.
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-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.
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 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) .
Mutants at Arg-337 could potentially alter the affinity of
1A/ 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) .
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
(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.
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 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.
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 [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.
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.