(Received for publication, March 10, 1997, and in revised form, May 29, 1997)
From the Departments of Oncology and
§ Molecular Neurobiology, Institute of Medical Science,
University of Tokyo, 4-6-1 Shirokanedai, Minato-ku,
Tokyo 108, Japan
The N-methyl-D-aspartate
(NMDA) receptor plays important roles in synaptic plasticity and brain
development. The NMDA receptor subunits have large intracellular
domains in the COOH-terminal region that may interact with
signal-transducing proteins. By using the yeast two-hybrid system, we
found that calmodulin interacts with the COOH terminus of the NR1
subunit and inactivates the channels in a
Ca2+-dependent manner. Here we show that
protein kinase C (PKC)-mediated phosphorylation on serine residues of
NR1 decreases its affinity for calmodulin. This suggests that
PKC-mediated phosphorylation of NR1 prevents calmodulin from binding to
the NR1 subunit and thereby inhibits the inactivation of NMDA receptors
by calmodulin. In addition, we show that stimulation of metabotropic
glutamate receptor 1, which potentiates NMDA channels through PKC,
decreases the ability of NR1 to bind to calmodulin. Thus, our data
provide clues to understanding the basis of cross-talk between two
types of receptors, metabotropic glutamate receptors and the NR1
subunit, in NMDA channel potentiation.
Glutamate is a major excitatory neurotransmitter in the central nervous system. Glutamate receptors are divided into two types of receptors, ionotropic receptors and metabotropic receptors, based on their structural and functional properties. N-Methyl-D-aspartate (NMDA)1 receptors, which belong to the family of ionotropic glutamate receptors, are implicated in synaptic plasticity, synaptogenesis, and excitotoxicity (1, 2). NMDA receptors consist of two distinct types of subunits, the principal subunit NR1 and modulatory subunits NR2A-2D (3, 4). Both types of subunits have a large COOH-terminal intracellular domain (5-7), suggesting that the large cytoplasmic domain may directly interact with proteins that regulate NMDA receptor function. For example, recent studies have suggested that postsynaptic density protein PSD95 directly interacts with NMDA receptors, although the biological significance of the interaction has not been established. The intracellular domain of NR1 contains serine residues that can be phosphorylated by protein kinase C (PKC) (8). It has also been reported that the NMDA receptor-mediated current is regulated by several protein kinases and phosphatases (9-11).
Calmodulin (CaM) is a ubiquitous molecule that regulates the function of a large number of proteins including Ca2+/CaM-dependent kinase II, adenylate cyclase, and phosphodiesterases. CaM also modulates the sensitivity of ion channels such as the olfactory cyclic nucleotide-gated channel and the retinal cyclic GMP-gated channel (12, 13). In addition, loading of CaM inhibitors at the postsynaptic neuron blocks the induction of long-term potentiation (LTP), a long-lasting enhancement of synaptic plasticity, and long-term depression, a long-lasting depression of synaptic plasticity, in the hippocampal CA1 region (14), suggesting that CaM is an important molecule for the induction of LTP and long-term depression.
We and Ehlers et al. (17) independently show that CaM binds to NR1 in a Ca2+-dependent manner and that the binding inactivates NMDA channels. NMDA receptors show high permeability to Ca2+. However, the rise of the intracellular Ca2+ level causes a reversible inactivation of the NMDA channels (15, 16). This feedback system regulates the amount of Ca2+ influx through the NMDA receptors. These data together suggest that Ca2+-dependent inactivation of NMDA receptors is mediated by CaM binding to NR1, namely, Ca2+ entering through the NMDA receptors binds to and activates CaM, and activated CaM, in turn, binds to NR1, resulting in inactivation of the NMDA channels.
The NMDA receptor-mediated current is potentiated during LTP (18, 19). In addition, DL-2-amino-4-phosphonovaleric acid, an antagonist of NMDA receptors, blocks the induction of LTP. Thus, induction of LTP should be accompanied by fine-tuning of the level of Ca2+ influx through the NMDA channels. Since Ca2+ influx results in inactivation of NMDA channels, there should be a mechanism by which the inactivation of the NMDA channels by Ca2+ influx is prevented so that potentiation of NMDA receptors results in the induction of LTP. Several studies suggest that induction of LTP requires concomitant activation of metabotropic glutamate receptors (mGluRs) and NMDA receptors (20) and that trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD), an agonist of mGluRs, potentiates NMDA currents in the hippocampus (21). Stimulation of mGluR1 and mGluR5 leads to the activation of PKC. There are reports that phorbol ester, an activator of PKC, enhances the amplitude of NMDA currents in Xenopus oocytes (22), hippocampus (21), and spinal dorsal horn neurons (23) and that intracellular injection of the selective PKC inhibitory peptide prevents the induction of LTP (24).
Here we show that PKC-mediated phosphorylation on serine residues of
NR1 decreases its CaM binding affinity. Moreover, we provide evidence
that activation of mGluR1 decreases the affinity of NR1 for CaM.
These results suggest that PKC-mediated phosphorylation of NR1 through
mGluR1
decreases NR1-CaM interaction and prevents inactivation of
NMDA channels by Ca2+.
The NR1 cDNA
encoding residues between the fourth transmembrane domain and the COOH
terminus (Glu834-Ser938) was amplified by
polymerase chain reaction with two primers: 5-CCTCGAATTCATTGAGATCGCC-3
, incorporating the
EcoRI site (underlined), and
5
-GGGCGGGTCGACTCAGCTCTCCC-3
, incorporating the
SalI site (underlined). The amplified fragment was inserted
into the EcoRI-SalI site of pBTM116. The
resulting plasmid encodes a fusion protein of the LexA DNA-binding
domain and the COOH-terminal domain of NR1. The yeast reporter strain
L40 containing this plasmid was transformed with a mouse cDNA
library in pVP16 by the lithium acetate method. The transformants were
plated on medium lacking Trp, His, Ura, Leu, and Lys and containing 5 mM 3-aminotriazole. Nitrocellulose filters were placed on
the plates and assayed for
-galactosidase expression as described
(25).
DNA sequencing was performed by the Sanger chain termination method, employing a BcaBEST sequencing kit (Takara).
Cell CultureHEK 293T cells (simian virus 40 large T
antigen-expressing human embryonic kidney cells) were grown in
Dulbecco's modified Eagle's medium containing 2 mM
glutamine. Cell culture reagents were from Life Technologies, Inc.
mGluR1-expressing Chinese hamster ovary cells were grown in
Dulbecco's modified Eagle's medium containing 2 mM
glutamine (26).
The NR1 expression
plasmid (pNR1) contains NR1 cDNA from pN60 (3) at the
EcoRI-NotI site of pME18S (27). The two mutagenic primers used were as follows. Primer 1 (5-CTGGCCTCAAGCTTCCAGCAACAACAGTCCTCCAAA-3
, nucleotides
2659-2694) has a HindIII site (underlined) without changing
the original amino acid sequence of NR1. Primer 2 (5
-ACTAGTCGCGAGAAAAAACCTC-3
) carries the same sequence as
that of the multicloning site of pME18S, but contains an
NruI site (underlined) instead of the original
XbaI site. Site-directed mutagenesis was carried out according to the method of Deng and Nickoloff (28) using the above two
primers and pNR1 as a template. Introduction of the mutations in NR1
was confirmed by DNA sequencing.
HEK 293T cells in a 10-cm culture dish were transfected with 10 µg of NR1 expression plasmid by the standard calcium phosphate method. After 48 h, the cells were lysed with 500 µl of buffer A (10 mM Tris-HCl (pH 7.5), 1.0% Nonidet P-40, 0.15 M NaCl, and 10 µg/ml aprotinin) containing 1.0 mM CaCl2 for 1 h at 4 °C. The lysates were centrifuged at 15,000 rpm for 30 min, and 30 µl of CaM-agarose (Sigma) was added to the supernatants. After incubation for 1 h at 4 °C, the CaM-agarose was washed with the same buffer. For immunoblotting, proteins in the CaM-agarose complex were resolved by 6.0% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane filter was treated with blocking reagents containing 5.0% bovine serum albumin and probed with an antibody to the COOH terminus of NR1 (Chemicon International, Inc.).
ImmunoprecipitationHEK 293T cells were transfected with 20 µg of NR1 expression plasmid. After 48 h, the cells were treated with or without 100 nM TPA for 30 min. The cells were lysed with buffer B (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM CaCl2, and 1% Nonidet P-40), and the lysate was immunoprecipitated with anti-CaM antibody (Upstate Biotechnology, Inc.).
Single Channel Recording of the NMDA CurrentsHEK 293T cells were cotransfected with a DNA mixture containing NR1, NR2A, and a mutant (S65T) of green fluorescent protein (GFP) expression plasmids (0.1, 0.3, and 0.3 µg/cm2, respectively) (29). After 12 h, 50 µM DL-amino-5-phosphonopentanoic acid was added to the medium to prevent cell death caused by NMDA receptor activation. After 12-48 h of transfection, the expression of GFP was confirmed with an epifluorescence microscope, and cells expressing GFP were used for electrophysiological recordings. Fire-polished borosilicate glass pipettes (4-5 megohms) filled with an extracellular solution (110 mM Na2SO4, 5 mM Cs2SO4, 10 mM HEPES, 10 mM D-glucose, 1 µM pyruvic acid, and 1 mM tetrodotoxin (pH 7.25)) containing 1 mM CaCl2, 10 µM NMDA, and 3 µM glycine were used for single channel recording. Inside-out patches were excised from HEK 293T cells in the extracellular solution and moved into the flow of an intracellular solution (140 mM cesium gluconate, 10 mM HEPES, and 1 µM CaCl2 (pH 7.25)). For analysis of the effect of CaM, the flow was changed to the intracellular solution containing 250 nM CaM (Sigma). Currents through the NMDA channels were measured by using the voltage-clamp technique at room temperature (22-25 °C). The single channel currents were amplified by a patch-clamp amplifier (EPC-9, HEKA Electronics) and stored on a videocassette tape recorder through a PCM converter system (VR10B, Instrutech Corp.) digitized at 37 kHz. Data were reproduced and low-pass-filtered at 2 kHz by a filter with Bessel characteristics (48 db/octave) (Model 3625, NF Instruments), sampled at 10 kHz, and analyzed on a Macintosh computer by using Patch Analyst Pro software (Version 1.16, MT Corp.). Single channel amplitudes were determined by fitting gaussian curves to the amplitude histograms. The threshold used to judge the open state was set at half of the single channel current. The open probability was noted as NPo, where N is the number of channels and Po is the open probability of a single channel. Continuous recordings (50-110 s) of each experiment (150-800 opening events in the absence of CaM and 200-950 opening events in the presence of CaM) were analyzed to obtain the open probability. Under the above condition, the absolute NPo values in the absence of CaM were in the range 0.006-0.030.
Nondenaturing Polyacrylamide Gel Shift AssaysCaM-peptide
complexes were formed in buffer C (10 mM Tris-HCl (pH 7.5),
1.0% Nonidet P-40, and 0.15 M NaCl) containing either 2 mM CaCl2 or 5 mM EDTA for 30 min at
4 °C. The complexes were then resolved on nondenaturing 15%
polyacrylamide gels containing 2 mM CaCl2 and
visualized by Coomassie Blue staining. To phosphorylate the peptide,
3000 pmol of peptides was incubated with 0.3 milliunits of PKC (Pierce)
in buffer D (20 mM Tris-HCl (pH 7.5), 0.5 mM
CaCl2, 5 mM Mg(OAc)2, 50 µg/ml
phosphatidylserine, and 2 mM [-32P]ATP)
for 3 h at room temperature. The peptide that was incubated with
PKC in the above solution without [
-32P]ATP was used
as a nonphosphorylated peptide.
To
identify proteins that interact with the COOH-terminal intracellular
domain of NR1, we employed the yeast two-hybrid screening system (30,
31). To prepare a bait, we constructed a plasmid that expresses a
fusion protein consisting of LexA and the COOH terminus
(Glu834Ser938) of NR1. The yeast reporter
strain L40 containing the bait plasmid was transformed with a mouse
cDNA library constructed in pVP16. By screening ~106
yeast transformants, we obtained 177 positive clones that activate expression of both the selection marker His and LacZ. Of these, 50 clones were subjected to nucleotide sequence analysis. The inserts of
16 clones encode the COOH-terminal amino acid sequence of CaM that
corresponds to the amino acids between Lys77 and
Lys148.
Most
CaM-binding proteins contain conserved amino acid sequences that can
form the amphiphilic -helix (32, 33). Inspection of the NR1 sequence
revealed the presence of the amphiphilic structure within a cytoplasmic
domain of NR1 between Lys875 and Thr900 (Fig.
1A). To examine whether this
region is responsible for CaM binding, we constructed a mutant of NR1
(MNR1) with Gln892, Gln893, Gln894,
and Gln895 instead of Lys892,
Arg893, Arg894, and Arg895,
respectively, by site-directed mutagenesis (Fig. 1A). These mutations would disrupt the amphiphilic structure. The MNR1-CaM binding
assay revealed that the amount of CaM binding to the mutant receptor
was significantly decreased compared with the amount of CaM binding to
the wild-type receptor (Fig. 1B). Thus, we concluded that
this region (Lys875-Thr900) is indeed a major
CaM-binding site on NR1. The small amounts of CaM binding to MNR1
observed may be due to the existence of other CaM-binding sites on the
NR1 molecule.
Ca2+-dependent Interaction of NR1 with CaM
To investigate the Ca2+ dependence of the NR1-CaM interaction, we performed the binding assay between CaM and a synthetic peptide containing the CaM-binding site. We synthesized a peptide (DPKKKATFRAITSTLASSFKRRRSSKDT) corresponding to the sequence from Asp873 to Thr902 of NR1 that includes the CaM-binding site. CaM (300 pmol) was incubated with various amounts of the peptide in a solution with or without Ca2+. As shown in Fig. 1C, the CaM-peptide complex was found in the presence of Ca2+. However, the CaM-peptide complex was not detected in the absence of Ca2+ (Fig. 1C, EDTA). Therefore, we concluded that the NR1-CaM interaction is Ca2+-dependent.
Decrease in NR1-CaM Interaction by TPA StimulationThe four
serine residues in the CaM-binding site on NR1 can be phosphorylated
with PKC by TPA stimulation (Fig.
2A) (8). PKC-induced
phosphorylation of these serine residues alters their hydrophilicity
and therefore might disrupt the amphiphilic structure of the
CaM-binding site. Consequently, CaM would be unable to bind to
phosphorylated NR1. To investigate this possibility, HEK 293T cells
expressing NR1 were treated with 100 nM TPA. Using anti-phosphoserine antibodies, we detected the increment in serine phosphorylation of NR1 upon TPA treatment (data not shown) (8). The
cells were then lysed, and the NR1-CaM binding assay was performed using CaM-agarose. As expected, TPA treatment decreased the ability of
NR1 to bind CaM (Fig. 2B). In addition, when CaM was
immunoprecipitated with anti-CaM antibody from the lysate of the
TPA-treated cells, the amount of coimmunoprecipitated NR1 was decreased
as compared with that from untreated cells (Fig. 2C). The
residual NR1 coimmunoprecipitated may be due to CaM binding to another
CaM-binding site on NR1 that does not contain serine residues for PKC
phosphorylation. The results suggest that the affinity of NR1 for CaM
binding is decreased by PKC-mediated phosphorylation of NR1.
Quantitative Analysis of CaM Binding to NR1 Phosphorylated by PKC
To quantitate CaM binding to NR1 phosphorylated by PKC, we
performed the binding assay between CaM and the NR1 peptide
phosphorylated at the CaM-binding site. The peptide corresponding to
the sequence from Asp873 to Thr902 of NR1 that
includes the CaM-binding site was incubated with PKC to achieve its
phosphorylation. Phosphorylation of the peptide was confirmed by
radiolabeling (data not shown). CaM (300 pmol) was incubated with
various amounts of peptide either nonphosphorylated or phosphorylated
by PKC. As shown in Fig. 3B,
the CaM-nonphosphorylated peptide complex was seen at a peptide/CaM
ratio of 1:10. However, the CaM-phosphorylated peptide complex could be
seen at a peptide/CaM ratio of 1:1. The data suggest that the
phosphorylated peptide had a significantly lower affinity for CaM than
the nonphosphorylated peptide. Thus, PKC-mediated phosphorylation of
NR1 decreased the affinity of NR1 for CaM.
Inactivation of the NMDA Receptor by CaM Binding
To
investigate the functional regulation of NMDA channels by CaM, we
measured the single channel activity from HEK 293T cells cotransfected
with NR1, NR2A, and GFP expression plasmids. Fig. 4A shows a typical single
channel current detected in inside-out patches excised from the HEK
293T cells expressing GFP. The slope conductance of the channel was
48 ± 3 picosiemens (n = 4), which corresponded
closely to the single NMDA channel conductance (~50 picosiemens)
measured in adult rat dentate gyrus granule cells (11). The current was
detected in patches from GFP-expressing cells, whereas it was not
detected in untransfected cells (n = 4). Under the
condition in which a saturating concentration (3 µM) of
glycine was applied in the pipette solution, the current did not show a
substantial rundown for at least 5 min.
We then tested the effect of CaM on the currents detected in the
inside-out patches from GFP-expressing HEK 293T cells. As previously
reported (17), application of 250 nM CaM in the
intracellular solution containing 1 mM Ca2+
reduced the probability of channel opening (NPo)
to 49.4 ± 25.3% (mean ± S.D.) of the control values
(n = 9) (Fig. 4, A and B). CaM
did not significantly affect the single channel conductance in all
patches measured: 3.23 ± 0.47 pA (n = 9) at 60
mV in the absence of CaM and 3.49 ± 0.42 pA (n = 8) at
60 mV following the addition of CaM. Therefore, CaM decreases
the activity of NMDA channels by reducing the frequency of channel
openings. The NMDA receptors have high permeability to
Ca2+, and Ca2+ influx through the channels, in
turn, inactivates the NMDA receptors (15, 16). Since CaM binds to NR1
in a Ca2+-dependent manner (17), inactivation
of the NMDA receptors by Ca2+ influx may be through the
NR1-CaM interaction.
NMDA receptor-mediated currents are potentiated during LTP
(18, 19). The potentiation of NMDA currents during LTP is opposed to
inactivation of NMDA channels by Ca2+-dependent
NR1-CaM interaction. Therefore, there should be a mechanism by which
inactivation of the NMDA channels by Ca2+ influx is
prevented during LTP. LTP is regulated by both mGluRs and NMDA
receptors. In addition, stimulation of mGluRs by t-ACPD, an
agonist of mGluRs, evokes a long-lasting enhancement of NMDA currents
(34). Since stimulation of mGluR1 leads to activation of PKC, the
activated PKC may be utilized to phosphorylate NR1, which prevents the
interaction between NR1 and CaM. To investigate this, we examined the
affinity of NR1 for CaM after stimulation of mGluR1
by
t-ACPD. Chinese hamster ovary cells stably expressing mGluR1
were transfected with NR1 expression plasmids. The cells were
then treated with or without 10 mM t-ACPD, and
the NR1-CaM binding assay was performed using CaM-agarose. As expected,
stimulation of mGluR1
decreased the affinity of NR1 for CaM (Fig.
5). These results suggest that the
decrease in the affinity of NR1 for CaM underlies the potentiation of
NMDA channels. Phosphorylation of NR1 by PKC may be one of the
mechanisms for NMDA receptor potentiation.
We have shown that CaM inactivates the NMDA channels by binding to NR1 and that PKC-mediated phosphorylation of NR1 decreases the affinity of NR1 for CaM. Therefore, when the NMDA receptor is activated and permits Ca2+ influx, CaM binds to NR1 in a Ca2+-dependent manner and inactivates NMDA channels. However, when serine residues of NR1 are concomitantly phosphorylated by PKC, CaM cannot bind to NR1 and thus is unable to inactivate the NMDA channels, and this may result in the potentiation of LTP. Previous reports also show that CaM binding to target proteins is regulated by phosphorylation. Neuromodulin is a molecule that is present in neurons in the spinal cord, retina, and brain (35, 36). Neuromodulin releases CaM in response to its phosphorylation by PKC: neuromodulin binds to and localizes CaM at specific sites within the cell in the resting state and releases CaM locally in response to its phosphorylation by PKC (37, 38). Thus, in addition to Ca2+, phosphorylation also regulates the interaction of CaM and CaM-binding proteins.
To date, at least eight splice variants of NR1 have been identified (39-41). These variants are generated by three exons: an exon near the N terminus and two exons near the C terminus. These variants differ in their sensitivity to factors such as Mg2+, glycine, polyamines, and protein kinases/phosphatases. Our study shows that CaM plays a role in regulating NMDA channel activity through the interaction of CaM with the sequence (Lys875-Thr900) in the C1 cassette (Asp864-Thr900) in the COOH-terminal region of NR1. Among the NR1 splice variants, there are variants that do not contain this cassette, suggesting that these variants have a different sensitivity to CaM. Indeed, Ehlers et al. (17) reported that splice variants that lack the C1 exon cassette, e.g. NR1-c and NR1-e, are much less sensitive to CaM than the variants that have the C1 cassette. They also reported that CaM binds to two sites of NR1: the lower affinity region (Lys838-Gln863) as well as the higher affinity region (Lys875-Lys898). Consistently, our data demonstrate that the NR1 mutant (MNR1) incorporating Gln instead of Lys or Arg at positions 892, 893, 894, and 895 significantly reduces the affinity for CaM, but still can bind to CaM very weakly (Fig. 1B). This residual ability of MNR1 to bind CaM may be due to the other CaM-binding site (Lys838-Gln863) of NR1. Although the sensitivities to CaM are different among these variants at a low concentration of CaM, the effect of CaM on inactivation of the channels is not different at a high concentration of CaM (17). A high concentration of Ca2+/CaM that inactivates all the splice variants might be the limit of the intracellular Ca2+ concentration that induces excitotoxicity. Thus, whereas the low affinity CaM-binding site (Lys838-Gln863) might contribute to the prevention of excitotoxicity, phosphorylation and dephosphorylation of the high affinity CaM-binding site (Lys875-Lys898) in the C1 exon cassette might be important primarily in determining the sensitivity of channels to CaM.
The C1 exon cassette is also important in determining the subcellular distribution of NR1 (42). NR1 splice variants containing the C1 exon are aggregated and located in discrete, highly concentrated regions associated with the plasma membrane. NR1 splice variants lacking this exon cassette are distributed throughout the cell. Furthermore, PKC phosphorylation of serine residues within the exon disrupts the formation of the NR1 aggregation at the plasma membrane. The CaM-binding site (Lys875-Thr900) of NR1 has high homology to the CaM-binding site (effector domain) of the cytoskeletal organizing protein MARCKS (myristoylated alanine-rich C kinase substrate). The effector domain of MARCKS binds both CaM and actin and contains the major site of PKC phosphorylation. PKC phosphorylation inhibits CaM and actin binding, and CaM binding prevents PKC phosphorylation and actin binding. Our results show that CaM binds to the sequence in the region encoded by the C1 exon and that PKC-mediated phosphorylation of the serine residues regulates the NR1-CaM interaction. Therefore, as with the MARCKS protein, PKC phosphorylation and CaM binding may also contribute to the subcellular distribution of NR1 by interacting with cytoskeletal proteins.
We have shown that stimulation of mGluR1, which can induce NMDA
receptor potentiation, decreased the affinity of NR1 for CaM. Since
stimulation of mGluR1 and mGluR5 activated PKC and since PKC-mediated
phosphorylation of NR1 decreased the affinity of NR1 for CaM (Figs. 2
and 3), it is likely that phosphorylation of NR1 by activated PKC
through mGluR1
results in inhibition of the NR1-CaM interaction.
There are reports that stimulation of µ-opioid receptors or
muscarinic receptors increases the NMDA currents through activation of
PKC (21, 43). Therefore, activation of PKC through these G
protein-coupled receptors may also contribute to phosphorylation of NR1
at sites within the CaM-binding region.
Although activation of NMDA channels is necessary every time LTP is generated, activation of mGluRs is not: activation of mGluRs is necessary the first time LTP is generated, and thereafter, mGluRs are no longer necessary to generate further LTP (44). Thus, mGluRs may activate some sort of "molecular switch." Once activated, the molecular switch stays on during induction of LTP. Our results suggest that PKC-mediated phosphorylation of NR1 may be a molecular switch activated by mGluRs. Once serine residues of NR1 are phosphorylated by PKC, CaM cannot inactivate the NMDA channels by binding to NR1 until those serine residues are dephosphorylated. In the NR1 phosphorylated state, NMDA receptors remain potentiated without additional activation of mGluRs. If this hypothesis is correct, phosphorylation of NR1 should increase during LTP. Recent reports indicated that phosphorylation of NR2B increases during LTP (45, 46). It would be worthwhile to examine whether the level of phosphorylation of NR1 increases during LTP.
Ca2+ influx through NMDA receptors affects synaptic plasticity and excitotoxicity. The extent of Ca2+ influx through NMDA channels is proposed to be responsible for the establishment of LTP and long-term depression: a large Ca2+ influx through NMDA channels results in LTP, and a small Ca2+ influx results in long-term depression. Furthermore, excessive Ca2+ influx through NMDA channels causes neuronal death. Ca2+-dependent inactivation of NMDA channels and release from inactivation by NR1 phosphorylation would play important roles in these phenomena.
We thank S. Nakanishi for providing the NMDA
receptor 1 and 2A cDNAs and mGluR1-expressing Chinese hamster
ovary cells; R. Y. Tsien for GFP expression plasmids; P. Bartel,
S. Fields, and S. Hollenberg for pBTM116 and the mouse cDNA library
for the yeast two-hybrid system; S.-T. Li for technical help in
patch-clamp recording; K. Kato and A. Miyawaki for fruitful discussion;
and L. G. Sayers for critical reading of the manuscript.