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INTRODUCTION |
The NMDA1 receptor is a
subclass of ionotropic glutamate receptors in the mammalian brain. The
NMDA receptors exhibit several channel properties distinct from other
non-NMDA glutamate receptors, including a high
Ca2+/Na+ permeability ratio, a
voltage-dependent Mg2+ block, and a requirement
for glycine as co-agonist (1-4). The receptors are modulated by
phosphorylation catalyzed by protein kinase C (5, 6) and by tyrosine
kinases (7, 8). In brain, functional NMDA receptors are thought to
exist as heteromultimers of the
1 subunit (NR1) with
subunits
(NR2 subunits). Different combinations of these subunits exhibit
distinct channel properties and characteristic regional and
developmental expression in vivo (9-11). Disruption of the
1 gene is lethal in mice (12), whereas that of the
1 and
2
genes results in reduced channel activity and reduced LTP in the
hippocampus (13, 14). A number of evidences indicate that the
activation of NMDA receptors is essential for induction of LTP, which
underlies the formation and storage of some forms of memories (15).
NR2 subunits (
subunits) have been shown to interact specifically
with PSD-95 (postsynaptic
density-95) (16-19). The latter protein is a
member of the channel-associated proteins (PSD-95 family), including
SAP97 (synaptic associated protein
97)/hdlg (20, 21), chapsin-110/PSD-93 (22, 23), and SAP102
(24). This family of proteins is characterized by the presence of three domains with a length of ~90 amino acids (PDZ domain) in the
NH2-terminal region, followed by SH3 and guanylate
kinase-like domains. PSD-95 was shown to interact with the
COOH-terminal E(T/S)XV sequence motif of NMDA receptor
subunits (18, 19) and of K+ channels through the
NH2-terminal PDZ domains (25, 26). The interaction of these
proteins induces the clustering of the channel proteins (23, 25, 27).
Thus, PSD-95 plays an important role in the molecular organization of
NMDA receptors at postsynaptic sites. Although PSD-95 was shown to
enhance the channel activity of the inward rectifier K+
channel Kir4.1 (27), it is not known whether PSD-95 directly affects
the channel activities of the NMDA receptors.
In this study, we examined the effects of PSD-95 on
2/
1
heteromeric NMDA receptor channels expressed in Xenopus
oocytes. We found that PSD-95 modulates the channel activity of the
NMDA receptor. PSD-95 decreases the sensitivity of the channels to L-glutamate and inhibits the protein kinase C-mediated
potentiation of the channels.
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EXPERIMENTAL PROCEDURES |
Preparation of cRNA and Oocytes--
Plasmids pBKSA
1,
pBKSA
1, and pBKSA
2 containing cDNAs encoding mouse brain
1,
1, and
2 subunits of the NMDA receptor, respectively, were
kindly provided by Dr. Masayoshi Mishina (6, 28). Complementary RNAs of
NMDA receptors were synthesized in vitro with T3 RNA
polymerase using NotI-cleaved plasmids as templates. The
coding region of PSD-95 was amplified by polymerase chain reaction from
a rat brain cDNA library. The resulting product was subcloned into
pSP64 poly(A) vector (Promega, Madison, WI). The construction was
confirmed by dideoxynucleotide sequencing. The PSD-95 cRNA was
synthesized in vitro with SP6 RNA polymerase using
EcoRI-cleaved plasmid. Deletion and site-specific mutants of
the NMDA receptor
2 subunit and PSD-95 were constructed by polymerase chain reaction strategy using Pfu polymerase
(Stratagene, La Jolla, CA) (see Fig. 7A). The wild-type
2
subunit and PSD-95 were used as polymerase chain reaction templates for
mutation, except the C(3,5)S
PDZ(1+2) construct, for which the
PDZ(1+2) construct was used as a template. Mutations were verified
by dideoxynucleotide sequencing.
Stage V and VI oocytes were obtained from anesthetized Xenopus
laevis and incubated for 2 h at room temperature with 2 mg/ml collagenase in Barth's medium (88 mM NaCl, 1 mM KCl, 0.33 mM
Ca(NO3)2, 0.41 mM
CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3, and 7.7 mM Tris-HCl, pH
7.2) containing 18 units/ml penicillin and 18 µg/ml streptomycin without Ca2+. The follicular cell layer was then removed
with forceps. The oocytes were injected with
2 and
1 cRNAs at a
molar ratio of 1:2. The total amounts of cRNA injected were 5 ng/oocyte. For co-expression of PSD-95, the synthesized PSD-95 cRNA
(1-25 ng) was injected 24 h after injection of the NMDA receptor
cRNAs. Before recording, oocytes were incubated at 19 °C for 18-26
h in Barth's medium.
Electrophysiological Recordings--
Currents were recorded with
two-electrode voltage-clamp techniques using a CA-1a high performance
oocyte clamp (Dagan Corp., Minneapolis, MN). Electrodes were filled
with 3 M KCl and had resistances of 1-5 megaohms. Oocytes
were perfused by a constant stream of Ba2+ Ringer solution
(115 mM NaCl, 2.5 mM KCl, 1.8 mM
BaCl2, and 10 mM HEPES, pH 7.2) at
23-25 °C. The oocyte membrane was voltage-clamped at
70 mV. In
the standard assay, currents were evoked by bath perfusion of
Ba2+ Ringer solution containing 100 µM
L-glutamate and 10 µM glycine for 20 s,
followed by a washout with standard Ba2+ Ringer solution.
Current signals were digitized for analyses; statistical significance
was determined using Student's t test.
Immunoblotting--
Polyclonal antibodies against the NMDA
receptor
2 and
1 subunits were obtained from Calbiochem and Santa
Cruz Biotechnology (Santa Cruz, CA), respectively. Monoclonal antibody
against the central region (amino acids 353-504) of rat PSD-95 was
obtained from Transduction Laboratories (Lexington, KY). Polyclonal
antibodies against PSD-95 were raised in rabbits using a glutathione
S-transferase fusion protein with the
NH2-terminal region (amino acids 4-404) of rat PSD-95 as
immunogen and were affinity-purified. For immunoblotting of PSD-95,
oocytes were solubilized with Laemmli gel sample buffer containing 2%
SDS (50 µl/oocyte) (29). For immunoblotting of the NMDA receptor, the
total membrane fraction was prepared from oocytes. After measurement of
current responses, oocytes were homogenized with 100 mM
NaCl, 5 mM EDTA, and 20 mM Tris-HCl, pH 7.5, and then centrifuged at 1000 × g for 10 min. The
supernatant was centrifuged at 200,000 × g for 30 min.
The obtained pellet (total membrane fraction) was solubilized with
Laemmli gel sample buffer. The samples were separated by
SDS-polyacrylamide gel electrophoresis on 5 or 7.5% gels and
transferred to nitrocellulose sheets. After blocking with 5% skim milk
and 0.05% Tween 20 in Tris-HCl-buffered saline, the sheets were
incubated with primary antibodies in the blocking solution. Labeled
bands were visualized using enhanced chemiluminescence (Pierce). The
bands were analyzed by densitometric scanning using Densitograph
AE-6900M (Atto Corp., Tokyo, Japan). The amounts of the proteins were
quantified from the intensity of the bands, which has a linearity to
the amounts of the samples applied to the gel.
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RESULTS |
Characterization of
2/
1 Heteromeric NMDA Receptor Channels in
Oocytes Co-expressing PSD-95--
To investigate the effects of PSD-95
on the channel activities of the
2 (NR2B)/
1 (NR1a) heteromeric
NMDA receptor, we expressed the NMDA receptor and PSD-95 in
Xenopus oocytes by injection of in vitro
synthesized cRNAs. When the expression of the proteins was monitored by
immunoblotting, the level of the NMDA receptor reached a plateau ~40
h after injection of the cRNAs and was then constant for several days
(data not shown). On the other hand, the level of PSD-95 rapidly
increased to the maximum within 24 h after injection and then
decreased (data not shown). Therefore, in this study, PSD-95 cRNA was
injected into oocytes that were already expressing the NMDA receptor,
and the effects of PSD-95 on the NMDA receptor channels were examined
18-26 h after injection of PSD-95 cRNA, measuring current responses to
100 µM L-glutamate and 10 µM
glycine in Ba2+ Ringer solution under voltage clamp at
70
mV. However, the results obtained with these oocytes were qualitatively
the same as those obtained with oocytes in which cRNAs of the NMDA
receptor and PSD-95 were co-injected.
To characterize the channels in oocytes co-expressing PSD-95, we first
examined the effects of ifenprodil on the channel activity. Ifenprodil
is known to selectively block
2/
1 heteromeric NMDA receptor
channels (30). Ifenprodil completely inhibited the channels in oocytes
co-expressing the receptor and PSD-95, as in oocytes expressing the
receptor alone, whereas it had no effect on other subtypes of NMDA
receptor channels such as the
1/
1 heteromeric receptor (Fig.
1). These results indicate that the current responses in oocytes co-expressing the receptor with PSD-95 are
indeed through
2/
1 NMDA receptor channels. The average of the
current responses from 15 oocytes was 1.34 ± 0.56 µA (mean ± S.D., n = 15) in oocytes expressing the NMDA
receptor and 2.56 ± 1.00 µA (mean ± S.D.,
n = 15) in oocytes co-expressing PSD-95. When the
expression of the receptor was compared in these oocytes by
immunoblotting, the expression level of the receptor was proportional to the current response, and the ratio between
2 and
1 subunits was not changed by PSD-95 (Fig. 2). These
results indicate that PSD-95 does not inhibit the expression or plasma
membrane insertion of the receptor. A higher expression level of the
receptor in oocytes co-expressing PSD-95 might be due to the
stabilization of the receptor by interacting with PSD-95. However, the
amplitudes of evoked currents varied from oocyte to oocyte and could
not simply be compared between oocytes expressing the receptor and co-expressing PSD-95. In this study, we focused on the qualitative differences in the channel properties between them. When the channel activities were measured at various concentrations of glutamate, the
dose-response curve shifted to the right in oocytes co-expressing the
receptor and PSD-95 (Fig. 3). The
EC50 was increased from 1.39 to 5.76 µM by
injection of PSD-95 cRNA. Thus, PSD-95 significantly decreased the
sensitivity of the channels to glutamate. On the other hand, PSD-95 did
not change other basic properties of the NMDA receptor such as a
requirement for glycine as co-agonist, the current-voltage
relationship, and a voltage-dependent Mg2+
block of the channels (data not shown).

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Fig. 1.
Effects of ifenprodil on
L-glutamate-induced current responses in Xenopus
oocytes expressing NMDA receptors and PSD-95. Current
responses were measured in oocytes injected with in vitro
synthesized cRNAs of the 2/ 1 NMDA receptor (A), the
2/ 1 NMDA receptor and PSD-95 (B), or the 1/ 1
NMDA receptor (C). In B, 25 ng of PSD-95 cRNA was
injected into an oocyte 24 h after injection of NMDA receptor
cRNAs. The responses were measured in Mg2+-free
Ba2+ Ringer solution at 70-mV membrane potential.
Representative tracings are presented. Bars show the
duration of application of 100 µM L-glutamate
and 10 µM glycine. In the traces on the right, 100 µM L-glutamate and 10 µM
glycine were applied in the presence of 10 µM ifenprodil.
Inward current is downward.
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Fig. 2.
Expression of the
2/ 1 NMDA receptor in
Xenopus oocytes. Oocytes were injected with cRNAs
of the 2/ 1 NMDA receptor (lanes 1) and 2/ 1 NMDA
receptor and PSD-95 (lanes 2). PSD-95 cRNA (25 ng) was
injected into oocytes expressing the 2/ 1 NMDA receptor. After
measurement of current responses, 15 oocytes were combined, and the
total membrane fraction was prepared as described under "Experimental
Procedures." The samples were separated by SDS-polyacrylamide gel
electrophoresis on a 5% gel. Immunoblotting was performed with
antibodies against the 2 (A) and 1 (B)
subunits of the NMDA receptor. The average of the currents evoked by
100 µM L-glutamate and 10 µM
glycine was 1.34 ± 0.56 µA (mean ± S.D.,
n = 15) in oocytes expressing the NMDA receptor and
2.56 ± 1.00 µA (mean ± S.D., n = 15) in
oocytes co-expressing PSD-95. The amounts of expressed NMDA receptor
were estimated by densitometric scanning of the immunoblot. The ratios
of the amounts of expressed 2 and 1 subunits were 1:1.97 and
1:2.05, respectively, between oocytes expressing the NMDA receptor and
co-expressing PSD-95.
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Fig. 3.
Dose-response curves for
L-glutamate in oocytes expressing the
2/ 1 NMDA receptor and
PSD-95. Various concentrations of L-glutamate with 10 µM glycine were applied, and the steady-state currents
were measured in oocytes injected with 2/ 1 NMDA receptor cRNAs
(closed circles) and with 2/ 1 NMDA receptor and PSD-95
cRNAs (open circles). PSD-95 cRNA (25 ng) was injected
24 h after injection of NMDA receptor cRNAs. Each point represents
the mean ± S.E. of the current amplitudes obtained from five
oocytes. Theoretical curves were drawn according to the equation
I = Imax/(1 + (EC50/A)n), where I
represents the current response, Imax is the
maximum response, A is the concentration of
L-glutamate, and n is the Hill coefficient. The
EC50 values were 1.39 and 5.76 µM for oocytes
expressing the 2/ 1 NMDA receptor and co-expressing PSD-95,
respectively, and the Hill coefficient values were 1.29 and 1.07, respectively.
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Effects of PSD-95 on the Protein Kinase C-mediated Potentiation of
the Channels--
It was reported that the channel activity of the
2/
1 NMDA receptor in oocytes is markedly potentiated by treatment
with TPA, due to activation of protein kinase C (28, 31, 32). Under our
assay conditions, the evoked currents were ~4-fold increased by 1 µM TPA in oocytes expressing the NMDA receptor (Figs.
4 and 5).
In oocytes co-expressing PSD-95, however, little potentiation of the
channel activity (~1.3-fold) was observed with TPA treatment (Figs. 4
and 5). This inhibitory effect of PSD-95 on the potentiation of the
channels by TPA was dependent on the amounts of PSD-95 cRNA injected
into an oocyte (Fig. 5). To rule out the possibility that PSD-95
directly modulates protein kinase C in oocytes, we examined the effects
of PSD-95 on the metabotropic glutamate receptor. The activity of
metabotropic glutamate receptors in oocytes can be estimated by the
channel activity of Ca2+-activated chloride channels. When
expressed in oocytes, metabotropic glutamate receptor-5 showed
pronounced desensitization in response to an activation by 1 mM glutamate (data not shown), as reported previously (33).
This desensitization has been shown to require protein kinase
C-catalyzed phosphorylation of the receptor (33). Indeed, a brief
treatment with TPA after activation by glutamate prolonged the
desensitization. Co-expression of PSD-95 had no effect on the
desensitization upon activation by 1 mM glutamate or on the
prolonged desensitization by TPA (data not shown), indicating that
expression of PSD-95 does not change the activity of protein kinase C
in oocytes.

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Fig. 4.
Effects of TPA on current responses in
oocytes expressing the
2/ 1 NMDA receptor and
PSD-95. Current responses were measured in oocytes expressing the
2/ 1 NMDA receptor (A) and co-expressing PSD-95
(B) before and after bath application of 1 µM
TPA for 10 min. Bars show the duration of application of 100 µM L-glutamate and 10 µM
glycine.
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Fig. 5.
Dose-dependent effects of PSD-95
on the protein kinase C-mediated potentiation of NMDA receptor
channels. Various amounts (1-25 ng/oocyte) of PSD-95 cRNA were
injected into oocytes expressing the 2/ 1 NMDA receptor. The
potentiation of the current response after TPA treatment (at 1 µM for 10 min) was plotted against the amounts of PSD-95
cRNA injected into an oocyte. The data shown are the mean ± S.E.
of four oocytes.
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Analyses of the Channel Modulation by PSD-95 Using the Deletion
Mutant of the NMDA Receptor--
PSD-95 has been shown to interact
with the COOH terminus of the NMDA receptor
2 subunit through the
first two PDZ domains (18, 19, 25). To clarify a role of this
interaction in the modulation of glutamate sensitivity, the effects of
PSD-95 were examined on the mutant NMDA receptor that lacks the
COOH-terminal four amino acid residues of the
2 subunit. The change
in the sensitivity to glutamate was monitored by the ratio of current response at 100 µM glutamate to that at 10 µM. In accord with the dose-response curves (Fig. 3),
expression of PSD-95 increased the ratio from 1 to ~1.5 in oocytes
expressing the wild-type NMDA receptor (Fig.
6). In oocytes expressing the mutant
receptors, however, the ratio remained ~1 regardless of PSD-95
expression (Fig. 6). These data indicate that the interaction of the
COOH-terminal region of the
2 subunit with the PDZ domain of PSD-95
is essential for a decrease in sensitivity to glutamate.

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Fig. 6.
Effects of PSD-95 on the glutamate
sensitivity of wild-type and mutant NMDA receptor channels. PSD-95
cRNA (25 ng) was injected into oocytes expressing the wild-type
2/ 1 NMDA receptor and into the mutant 2/ 1 NMDA receptor
lacking the COOH-terminal four amino acid residues of the 2 subunit.
A ratio of current responses between 100 and 10 µM
L-glutamate in the presence of 10 µM glycine
is presented. The data shown are the mean ± S.E. of the number of
oocytes indicated in parentheses. *, p < 0.01 compared with oocytes expressing the 2/ 1 NMDA receptor
without PSD-95.
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Analyses of the Channel Modulation by PSD-95 Using Mutant
PSD-95--
The modulation of glutamate sensitivity by PSD-95 was
further analyzed using a series of PSD-95 mutants (Fig.
7A). The expression of these
constructs was compared with that of wild-type PSD-95 by immunoblotting
with antibodies against the NH2-terminal region (amino
acids 4-404) and against the central region (amino acids 353-504) of
rat PSD-95. When 25 ng of cRNA was injected into an oocyte for each
mutant, there was no significant difference in the expression of these
constructs at the protein level (Fig. 7, B and
C). The deletion mutant lacking the SH3 domain (
SH3), the
COOH-terminal half-region including the SH3 and guanylate kinase-like
domains (
C-half), or the PDZ1 domain (
PDZ1) had the same effect
on the glutamate sensitivity of the channels as full-length PSD-95
(Fig. 8). Deletion of the PDZ2 domain
(
PDZ2) significantly decreased the ratio of current responses
between 100 and 10 µM L-glutamate from that
of full-length PSD-95. A double PSD domain deletion mutant lacking both
PDZ1 and PDZ2 domains (
PDZ(1+2)) and a triple PDZ domain deletion
mutant (
PDZ(1+2+3)) further decreased the ratio to that observed in
oocytes expressing the channels without PSD-95 (Fig. 8). Since two
cysteine residues near the NH2 terminus at positions 3 and
5 in PSD-95 have been shown to be critical for the in vivo
interaction with the K+ channel (34, 35), we examined the
role of these two cysteine residues in the channel modulation by PSD-95
using the mutant in which both of these cysteine residues were
substituted to serine (C(3,5)S). This construct partially decreased the
ratio from that of wild-type PSD-95 (Fig. 8). Deletion of both PDZ1 and
PDZ2 domains from this mutant (C(3,5)S
PDZ(1+2)) further decreased
the ratio to the level without PSD-95 (Fig. 8). Thus, the PDZ2 domain
is critical for the action of PSD-95 on the glutamate sensitivity of
the channels, although the PDZ1 domain and the NH2-terminal two cysteine residues play an additional role in it.

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Fig. 7.
Expression of mutant PSD-95 in oocytes.
A, schematic diagram of the wild-type and mutant constructs
of PSD-95 used in this study. The domains of PSD-95 are indicated.
S·S, substitution of cysteines 3 and 5 with
serine. B and C, immunoblotting of wild-type and
mutant PSD-95 expressed in oocytes. Wild-type or mutant PSD-95 cRNA (25 ng) was injected into an oocyte expressing the 2/ 1 NMDA receptor.
Immunoblotting was performed as described under "Experimental
Procedures" using polyclonal antibodies against the
NH2-terminal region (amino acids 4-404) (B) and
a monoclonal antibody against the central region (amino acids 353-504)
(C) of rat PSD-95.
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Fig. 8.
Effects of wild-type and mutant
PSD-95 on the glutamate sensitivity of
2/ 1 NMDA receptor
channels. Wild-type or mutant PSD-95 cRNA (25 ng) was injected
into an oocyte expressing the 2/ 1 NMDA receptor. A ratio of
current responses is presented between 100 and 10 µM
L-glutamate in the presence of 10 µM glycine.
The data shown are the mean ± S.E. of the number of oocytes
indicated in parentheses. *, p < 0.05, and
**, p < 0.01 compared with oocytes co-expressing the
NMDA receptor with wild-type PSD-95.
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DISCUSSION |
PSD-95 has been shown to induce clustering of the
2 (NR2B) NMDA
receptor (23). In addition to this function of PSD-95, the present
study demonstrated that it functionally modulates the channel activity
of the
2/
1 heteromeric NMDA receptor. PSD-95 decreased the
sensitivity of the channels to L-glutamate (Fig. 3) and
inhibited the protein kinase C-mediated potentiation of the channels
(Figs. 4 and 5). Furthermore, we demonstrated that the interaction
between the COOH terminus of the
2 subunit and the PDZ2 domain of
PSD-95 plays a critical role in the decrease in glutamate sensitivity
(Fig. 8). On the other hand, other basic channel properties of the
2/
1 NMDA receptor such as a voltage-dependent Mg2+ block, a requirement for glycine as co-agonist, and
inhibition of the channels by ifenprodil are not significantly altered
by PSD-95. Our results indicate that PSD-95 has inhibitory effects on
2/
1 NMDA receptor channels, in contrast to the inward rectifier K+ channel Kir4.1. The channel activity of the latter
channel is enhanced by PSD-95 in co-expressed cells (27). For
2/
1
NMDA receptor channels, PSD-95 not only makes signal transmission more efficient by clustering the channels at postsynaptic sites, but also
protects neuronal cells from excitotoxicity by decreasing the glutamate
sensitivity and by inhibiting the protein kinase C-mediated potentiation.
Our mutational studies indicate that a direct interaction between the
COOH terminus of the NMDA receptor
2 subunit and the PDZ2 domain of
PSD-95 is responsible for the decrease in the glutamate sensitivity of
the channels because PSD-95 could not decrease the sensitivity of the
mutant NMDA receptor lacking the COOH-terminal four amino acid residues
(Fig. 6) and because the activity to decrease the sensitivity was
significantly reduced in any mutants of PSD-95 lacking the PDZ2 domain
(Fig. 8). It has been reported that the
2 subunit (NR2B) has a
strong preference for the PDZ2 domain over the PDZ1 domain (19). In
accord with this, deletion of the PDZ2 domain did not completely
abolish the effect of PSD-95, whereas a double PSD domain deletion of
both PDZ1 and PDZ2 domains did (Fig. 8). The interaction between the
COOH terminus of the channels and the PDZ2 domain also induces channel
clustering (23, 34). Does channel clustering decrease glutamate
sensitivity? The NH2-terminal two cysteine residues of
PSD-95 have been shown to be essential for clustering of Shaker-type
K+ channel Kv1.4, but not for the binding of the COOH
terminus of the channel to the PDZ1 or PDZ2 domain (34). If this is
true of the NMDA receptor, our results suggest that the binding (but not the clustering) decreases glutamate sensitivity because the activity to decrease the glutamate sensitivity was still retained to
some extent (Fig. 8) when cysteines 3 and 5 of PSD-95 were substituted
with serine, which completely abolishes the channel clustering activity
(34). This intermediate effect of the cysteine substitution might be
explained by the finding that cysteines 3 and 5 are palmitoylated,
which is essential for membrane targeting of PSD-95 (35). The PSD-95
mutant, which is not recruited to the plasma membrane, might not
efficiently exert its action on the channels even though it has a PDZ2 domain.
The EC50 of NMDA receptor channels for
L-glutamate in neurons has been measured in cultured
neurons. The reported values vary from 2.3 to 16 µM (36,
37). Lower EC50 values (0.8-1.5 µM) were
reported, including this study, in oocytes expressing
2/
1 (or
NR1/NR2B) NMDA receptor channels (28, 38). These values cannot simply
be compared because a neuron may not express only one type of NMDA
receptors. Furthermore, the kinetics of the channel in neurons are not
simple since the desensitization occurs during measurement. Despite
these uncertainties, the EC50 obtained from oocytes
co-expressing the
2/
1 NMDA receptor with PSD-95 (Fig. 3) is in
good agreement with EC50 values obtained from neurons. We
do not know the physiological significance of this effect of PSD-95 on
the channels. PSD-95 may play a protective role in neuronal excitotoxicity, lowering the sensitivity of the NMDA receptor channels
to glutamate.
In this study, we demonstrated that PSD-95 suppresses the protein
kinase C-mediated potentiation of NMDA receptor channels (Figs. 4 and
5). It is unlikely that PSD-95 directly inhibits the kinase activity of
protein kinase C in oocytes because PSD-95 had no effect on the
desensitization of metabotropic glutamate receptor-5 in oocytes
co-expressing both proteins, which was shown to require phosphorylation
of metabotropic glutamate receptor-5 by protein kinase C (33).
Further study is necessary to elucidate how the NMDA receptor
channels are potentiated by protein kinase C and how this potentiation
is modulated by PSD-95.
Although
2/
1 NMDA channels in oocytes are potentiated by
the treatments that activate protein kinase C (Fig. 4) (31, 32), it is
still controversial whether phosphorylation by protein kinase C
potentiates the NMDA receptor channels in vivo. The protein kinase C-mediated potentiation of the channels is observed in dorsal
horn neurons in spinal cord and caudal brain stem (5, 39), whereas
activation of the kinase does not potentiate NMDA receptor-mediated
responses in CA1 hippocampal neurons (40) where the
2 subunit is
highly expressed (9, 10, 28). The latter observation can be explained
in part by our finding that PSD-95 suppresses the protein kinase
C-mediated potentiation of
2/
1 NMDA receptor channels.
The induction of LTP at synapses requires Ca2+
entry into the postsynaptic dendritic spine via NMDA receptors (15).
LTP is triggered by delivering synchronous high frequency stimulation, a tetanus, to the pathway. In the widely accepted model, tetanic stimulation sufficiently depolarizes the postsynaptic membranes, which
reduces the extent of the Mg2+-induced block of NMDA
receptors and allows Ca2+ influx (15). Consistent with this
model, the PSD-95-interacting NMDA receptors also showed a
voltage-dependent Mg2+ block of the channels
(data not shown). A number of evidences indicate that the activation of
protein kinase C is necessary for induction of LTP (15, 41). If the
activation of protein kinase C is induced by tetanic stimulation,
PSD-95 may play a protective role regarding the NMDA receptor channels,
preventing further potentiation of the channels and excess
Ca2+ influx.
In this study, we demonstrated that PSD-95 functionally modulates the
channel activity of the
2/
1 heteromeric NMDA receptor. Recently,
a number of proteins were reported to bind to PSD-95. GKAP
(guanylate kinase-associated
protein) or SAPAP
(SAP90/PSD-95-associated protein)
binds to the guanylate kinase-like domain (42-44). Neuroligin (45) and
CRIPT (cysteine-rich interactor of
PDZ three) (46) bind to the PDZ3 domain. It
remains to be seen whether these PSD-95-binding proteins affect the
channel modulation by PSD-95.