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The Journal of Neuroscience, February 15, 2001, 21(4):1211-1217
Laminar Organization of the NMDA Receptor Complex within the
Postsynaptic Density
Juli G.
Valtschanoff and
Richard J.
Weinberg
Department of Cell Biology and Anatomy, University of North
Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
The NR2 subunit is an essential component of the NMDA
receptor. Recent biochemical research has identified a number of
molecules that can bind directly or indirectly to its cytoplasmic tail. These postsynaptic density (PSD) proteins play a role in intracellular signal transduction, and are implicated in synaptic plasticity and
memory mechanisms. We performed systematic electron microscopic immunogold analysis in rat neocortex to determine the spatial organization of NR2, in relation to six other proteins thought to be
involved in the NMDA receptor complex. Peak concentrations of each
protein were within the PSD but in different "layers" of the
density. In the axodendritic axis, gold particles coding for PSD-95 lay
an average of 12 nm cytoplasmic to the extracellular face of the plasma
membrane, very close to the C terminal of NR2. Nitric oxide synthase
lay 18 nm inside the membrane; the scaffolding proteins guanylate
kinase-associated protein and Shank lay 24-26 nm inside the membrane;
and CRIPT and dynein light chain, proteins that may link the
complex to cytoskeletal elements, lay on the cytoplasmic side of the
PSD, 29-32 nm inside the plasma membrane and extending into the spine
cytoplasm. The supramolecular organization of these molecules may
modulate intracellular transduction of NMDA-mediated signals.
Key words:
scaffolding protein; PDZ domain; excitatory synapse; postembedding immunocytochemistry; cerebral cortex; PSD-95; nitric
oxide synthase
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INTRODUCTION |
The NMDA receptor is an oligomer
comprising NR1 and NR2 subunits (Hollmann and Heinemann, 1994 ). Calcium
ions flowing through the receptor pore can trigger intracellular
biochemical cascades that underlie its key role in synaptic plasticity
(Collingridge and Watkins, 1994; Dingledine et al., 1999). Immunogold
electron microscopy reveals that NMDA receptor subunits concentrate at the postsynaptic membrane of most asymmetric synapses (Bernard and
Bolam, 1998; Kharazia and Weinberg, 1999; Petralia et al., 1999; Takumi
et al., 1999; Valtschanoff et al., 1999; Racca et al., 2000).
This postsynaptic clustering might be explained by anchoring molecules
found in the postsynaptic density (PSD) like PSD-95 (Cho et al., 1992;
Kistner et al., 1993).
PSD-95 contains three PSD-95, DLG, Zo-1 (PDZ) domains (sites for
protein-protein interaction) on its N-terminal side and a guanylate
kinase domain near its C terminal (Kennedy, 1997). Biochemical data
suggest that PSD-95 can bind, via its PDZ1 and 2, to the C terminal of
NR2 subunits (Kornau et al., 1995; Niethammer et al., 1996; Bassand et
al., 1999) and, via PDZ3, to the protein CRIPT, which may link the NMDA
receptor complex to the cytoskeleton (Niethammer et al., 1998;
Passafaro et al., 1999). Yeast two-hybrid screens using the guanylate
kinase domain of PSD-95 as bait pulled out guanylate kinase-associated
protein (GKAP) (Kim et al., 1997), a protein that modulates NMDA
channel conductance (Yamada et al., 1999). GKAP, in turn, can bind to
dynein light chain (DLC), a subunit of motor proteins implicated in
synaptic remodeling (Naisbitt et al., 2000), and to Shank, a protein
that links the NMDA receptor complex to metabotropic receptors, thus
orchestrating functional interactions between metabotropic and
ionotropic systems (Naisbitt et al., 1999; Tu et al., 1999).
PSD-95 knock-out mice retain functional postsynaptic NMDA receptors but
exhibit aberrant long-term potentiation and learning deficits (Migaud
et al., 1998). Thus, rather than merely anchoring the receptor, PSD-95
acts as a molecular scaffold to organize signaling cascades within the
NMDA receptor complex (Ziff, 1997; Craven and Bredt 1998; O'Brien et
al., 1998; Sprengel et al., 1998; Garner et al., 2000; Sheng and Pak,
2000) For example, the calcium-dependent enzyme neuronal nitric oxide
synthase (nNOS), implicated in synaptic plasticity, can bind to PSD-95
(Brenman et al., 1996; Christopherson et al., 1999). Might nNOS be
strategically positioned to detect Ca2+
entering through the NMDA pore?
Accumulating data on mechanisms underlying synaptic plasticity suggest
that the NMDA receptor complex acts as a "molecular machine"
(Malenka and Nicoll, 1999; Weng et al., 1999; Kennedy, 2000).
Investigation of the Drosophila photoreceptor points to the
functional significance of the supramolecular organization of analogous
protein assemblies (Scott and Zuker, 1998; Tsunoda and
Zuker, 1999). Molecular and biochemical studies can define the
topology of protein interactions emanating from the cytoplasmic tails
of NMDA receptor subunits, but they do not reveal its spatial geometry.
Light microscopy lacks adequate resolution, and x-ray crystallography
is poorly suited for the study of membrane-bound supramolecular
assemblies. By systematic use of quantitative immunogold electron
microscopy, we provide new insight into the architecture of the NMDA
receptor complex and demonstrate its laminar organization within the PSD.
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MATERIALS AND METHODS |
Tissue preparation. All procedures related to the
care and treatment of animals were according to institutional and
National Institutes of Health guidelines. Material from seven male
Sprague Dawley rats (Charles River, Wilmington, MA; 220-400 gm) was
used for this study. After deep anesthesia was induced with sodium pentobarbital (60 mg/kg, i.p.), rats were intracardially perfused with
heparinized saline followed by 500 ml of fixative. Fixative was a
mixture of freshly depolymerized 2-4% paraformaldehyde and 2-2.5%
glutaraldehyde in phosphate buffer (PB; 0.1 M), pH 7.4; in
some cases, 0.1% picric acid was added in an effort to improve membrane preservation. However, none of the modifications tested provided any clear advantage over 2% paraformaldehyde and 2%
glutaraldehyde. Brains were removed and stored in cold PB; brains were
post-fixed in the same fixative for 2-16 hr. Coronal 50 µm sections
were cut on a Vibratome and collected in cold PB.
EM immunolabeling. Sections were processed for osmium-free
embedment according to the method of Phend et al. (1995). For epoxy embedment, dehydrated sections were immersed in propylene oxide and
infiltrated with a mixture of Epon and Spurr resins [Electron Microscopy Sciences (EMS), Fort Washington, PA] and then sandwiched between strips of Aclar plastic film (EMS) and polymerized at 60°C
for 36 hr. For acrylic embedment, dehydrated sections were infiltrated
with Lowicryl HM-20 (EMS), sandwiched in Aclar, and polymerized at
 10°C for 48 hr under UV illumination. After polymerization, chips
including layers 2-3 of the somatic sensory cortex were cut from the
wafers and glued onto blank resin blocks with cyanoacrylate. Chips from
different animals were of consistent size and orientation.
Thin (~100 nm) sections were cut and collected on nickel mesh grids
and processed for immunogold labeling as described previously (Phend et
al., 1992, 1995). Briefly, after treatment with 4%
para-phenylenediamine in Tris-buffered saline [0.1
M Tris, pH 7.6, with 0.005% Tergitol NP-10 (TBST)], grids were incubated overnight at 37°C in
primary antibody (Table 1). Grids were
then transferred to TBST, pH 8.2, incubated for 1 hr in the F(ab)2
fragment of IgG conjugated to 10 nm gold particles (Jackson
ImmunoResearch, West Grove, PA; 1:25 in TBST, pH 8.2), and
counterstained with uranyl acetate and Sato's lead. When primary
antiserum was omitted as a control, virtually no gold particles could
be detected on the section; when normal serum was substituted for
immune serum, sparse gold particles scattered across the section showed
no discernible relationship to synapses.
Immunogold analysis. Grids were examined on a JEOL 200CX
electron microscope at 80 kV of accelerating voltage. At least three grids from three different rats were examined for each postsynaptic antigen considered (Table 2). Asymmetric
synapses that had clearly outlined synaptic membranes and were labeled
with at least one gold particle within 100 nm of the postsynaptic
membrane were randomly selected and photographed (Table 2). Micrographs
at 34,000× original magnification were digitized on a flatbed scanner. Locations of gold particles coding for each antigen were measured with
NIH Image software running on a Macintosh platform. To define "axodendritic" position, the distance between the center of each gold particle and the outer leaflet of the postsynaptic membrane was
measured (Fig. 1). Lateral synaptic
position of a gold particle was defined as the distance from each end
of the active zone to a line drawn perpendicular to the synaptic
membrane and running through the center of the particle (for details,
see Kharazia and Weinberg, 1997).
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Figure 1.
A, An axospinous cortical synapse
of the simple (unperforated) asymmetric type, which exhibits a
prominent PSD. Scale bar, 100 nm. B,
Higher-magnification view of another asymmetric synapse. The good
ultrastructural preservation achieved with the osmium-free fixation
protocol used here permits study of the spatial relationship of the
elements of the synapse at high resolution. Measurements of immunogold
labeling were performed relative to the extracellular face of the
postsynaptic membrane (0 nm), within the region 25 nm "outside" the
postsynaptic membrane (25 nm) and 75 nm "inside" the membrane
(dashed lines). dp, Presynaptic dense
projections; prm, presynaptic membrane;
psd, postsynaptic density; psm,
postsynaptic membrane; sc, synaptic cleft;
ssw, subsynaptic web; sv, synaptic
vesicle.
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Despite its excellent spatial localization, immunogold has an inherent
error because of the size of the particle and the antibody bridge,
along with errors associated with the ~100 nm section thickness; we
estimate the maximal likely extent of these errors to be ~25 nm
(Kellenberger and Hayat, 1991). Because the PSD has a typical thickness
of ~50 nm (Martone et al., 1999), we restricted measurements of
lateral position to gold particles lying within a 100-nm-wide band,
running between a line 25 nm presynaptic to the postsynaptic plasma
membrane and a parallel line 75 nm cytoplasmic to the postsynaptic
membrane (see Fig. 1B, dashed lines).
Excel, Kaleidagraph, and CricketGraph software packages were used to generate graphs; Data Desk was used to compute statistics.
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RESULTS |
Material processed according to the osmium-free protocol we use
for immunocytochemistry exhibited good ultrastructural preservation. Plasma membranes were sharply defined, and synaptic specializations were prominent and well delineated. In the forebrain, most of these
specializations exhibited the prominent postsynaptic densities typical
of excitatory glutamatergic synapses (Ottersen and Landsend, 1997). In
supragranular layers of cerebral cortex, the large majority of synapses
were of the axospinous type (Fig. 1A). At high
magnification, features commonly described in osmium-treated material
could be readily identified, including synaptic vesicles, presynaptic
dense projections, and the subsynaptic web extending from the PSD into the cytoplasm (Fig. 1B). The postsynaptic density is
especially prominent in this material, typically extending 25-50 nm
into the cytoplasm beyond the postsynaptic membrane.
Gold particles coding for seven different proteins contributing to the
biochemically defined NMDA receptor complex accumulated mainly over the
PSD (Fig. 2). In contrast, particles
coding for synaptophysin, a protein associated with synaptic vesicles,
accumulated over the presynaptic terminal (Fig. 2), documenting the
specificity of this postsynaptic labeling. Each of the postsynaptic
proteins was also associated with somatic endoplasmic reticulum and
dendritic microtubules (data not shown). Several of the proteins,
especially DLC, were also found over the spine apparatus (Fig. 2)
(Naisbitt et al., 2000).
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Figure 2.
Postembedding immunogold labeling for the proteins
studied here. Each of the NMDA receptor-related proteins shows labeling
associated with the PSD, in contrast to synaptophysin
(SYN; upper left). Labeling for DLC
(lower right) is also over the spine apparatus. Scale
bars, 100 nm.
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Close examination of Figure 2 hints at subtle differences in the
synaptic organization of these seven proteins. To provide objective
evidence of possible differential organization, we examined the
distribution of immunogold particles compiled from a randomly selected
sample of asymmetric synapses. We first wanted to determine whether
labeling for each protein was coextensive with the synapse. We reasoned
that if antigen were associated with the plasma membrane per se,
there should be just as high a density of particles associated with the
plasma membrane away from the synaptic specialization as at the
synapse. Aside from modest variations in background noise and labeling
efficiency between Epon-embedded and Lowicryl-embedded material, there
were no obvious differences among animals in the pattern of labeling.
We therefore pooled immunogold data from all seven animals. For each of
the seven putative postsynaptic antigens tested, gold particles close
to the synaptic membrane concentrated at the synaptic specialization,
declining rapidly past the lateral edge of the PSD (Fig.
3A).
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Figure 3.
A, Graph shows "lateral"
position of gold particles, tangentially along the postsynaptic
membrane, for each protein studied. Only gold particles lying in a band
running between 25 and +75 nm from the plasma membrane of the
postsynaptic profile were considered. Lateral positions were
normalized, so that 0 corresponds to the center of the synapse and 1.0 corresponds to its edge. Data shown were put into six bins from 0 to
2.0; thus, for example, the third data point includes all particles
between 0.67 and 1.0. Labeling for all seven antigens was mainly within
the lateral borders of the synapse. B, Axodendritic
distributions of each of the seven proteins studied are shown (for
clarity, two graphs are used). Only gold particles within the width of
the PSD and lying in a band running between 25 and +75 nm from the
postsynaptic plasma membrane were considered. Data were put into 5 nm
bins; for graphing, data were smoothed with a digital filter
(3-point-weighted running average). All seven antigens concentrate in
the PSD; some (especially DLC) exhibit broader distribution than the
others, extending into the spine cytoplasm.
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We wanted to test whether each antigen concentrated just inside the
postsynaptic plasma membrane (rather than, e.g., uniformly within the
dendritic cytoplasm or preferentially within the synaptic cleft).
Therefore, we examined the axodendritic distribution of gold particles
lying within the lateral borders of the synaptic specialization, for
each of the seven antigens tested. Figure 3B documents that
each of the antigens was at the highest level in the region just
cytoplasmic to the postsynaptic membrane. Although the peak for each
graph is broadened by measurement error, it is apparent that some
proteins (especially DLC) exhibited broader distributions than did the
others. Likewise, although all of the peaks were inside the
postsynaptic membrane, careful inspection reveals that some lay closer
to the plasma membrane than did others.
Notwithstanding the intrinsic measurement noise, we reasoned that as
long as this error was random and unbiased, averaging would reduce its
magnitude, thus substantially increasing the accuracy of estimates of
axodendritic position. Results of this procedure are given in Table
3. These data reveal a laminar
organization of proteins within the PSD; some (PSD-95 and NR2) lay
close to the plasma membrane, others (CRIPT, GKAP, Shank, and DLC) lay markedly farther away, and still others (nNOS) lay in a position intermediate between the two. The exact level of significance for these
data depends on the assumptions of the statistical model, but a robust
nonparametric analysis suggests that many of the differences observed
were unlikely to have arisen by chance (Table 4), supporting a laminar organization of
the proteins within the PSD.
The SEs of mean positions of the antigens listed in Table 3 range from
1.2 to 3.2 nm, substantially smaller than the dimensions of many
proteins. This measurement precision suggests that the axodendritic
data might enable us to infer details of the organization of the NMDA
receptor complex and in favorable cases might even give clues as to the
orientation of individual protein molecules within the complex. We
therefore used these data, along with published data on molecular
weights, binding partners, and binding sites, to construct a physically
realistic model of the architecture of the protein assembly (Fig.
4). Although some aspects of this diagram
are arbitrary, the diagram is constrained by available data. For
example, because no data are available that directly address the
orientation of PSD-95 within the complex, it may seem that its
depiction pointing away from the membrane is purely arbitrary. However,
the orientation is constrained by (1) its N-terminal palmitoylation (Craven et al., 1999), (2) the immunogold-defined location of its antigenic region, and (3) the measured location of
CRIPT, one of its binding partners. Although schematic and incomplete
(at least 10 more proteins biochemically associated with the NMDA
receptor complex have been described previously), this diagram may
provide a more accurate view of the supramolecular architecture of the
NMDA complex than has been available previously.
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Figure 4.
Left, On this side of the scale is
the mean (± SE) axodendritic position of gold particles coding for the
seven proteins studied. Note that the majority of gold particles lie
outside the SE bars (see Fig. 3B). Only gold particles
within the region from 25 and +75 nm of the postsynaptic membrane and
laterally within the width of the PSD were included in computations
(see Fig. 2); data are from Table 3. PSD-95 and the C terminal of NR2
lie just inside the postsynaptic membrane; nNOS lies in the central
part of the PSD; and Shank, GKAP, CRIPT, and DLC lie on the cytoplasmic
side of the PSD. Right, On this side of the scale is a
model of the architectural layout of the seven proteins studied based
on our pooled data. The proteins are drawn as ellipses of uniform
shape, depicting prolate spheroids whose volumes are proportional to
their molecular weights; thus, the length of each ellipse is
proportional to (molecular weight)1/3. The N terminal of
each protein is light; the C terminal is dark. Proteins reported to
bind directly to each other in vitro are shown touching.
It is not yet clear whether all these proteins are found at the same
synapse. The NMDA receptor is depicted as a tetramer containing NR1 and
NR2 subunits. Ellipsoids are oriented so that the protein regions
recognized by the antibodies (white bars) lie at
distances from the postsynaptic membrane experimentally measured by
immunogold (left side of figure). Positions of domains
within molecules correspond to their location in the primary sequence
and do not necessarily represent their true tertiary structure. The
model also includes available biochemical data about molecular
interactions. For example, the N terminal of PSD-95, which is
palmitoylated, is shown touching the postsynaptic membrane; GKAP, which
binds with its C terminal to the PDZ domain of Shank (amino acids
541-602 of 1740), is depicted touching Shank at a point approximately
one-third of the way from its N terminal.
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DISCUSSION |
Elucidation of intermolecular interactions at the synapse is a
central task of molecular neurobiology (Kennedy, 2000). X-ray crystallography can solve protein structures at the atomic scale but is
poorly suited to study membrane-bound protein complexes vital for
synaptic function. High-resolution cryoelectron microscopy has been
used to visualize the opening of the acetylcholine receptor (Unwin,
1995), but this technically challenging method requires purified
in vitro samples. A recent study harnessed multiple
approaches, including preembedding immunogold EM, to reveal the
supramolecular architecture of the nuclear pore complex in yeast (Rout
et al., 2000). We here use postembedding immunoelectron microscopy to elucidate aspects of the mesoscale organization of the NMDA receptor complex in the adult neocortex. Although this method has limited spatial resolution, it can in principle be applied to most protein assemblies, including those intimately bound to membranes.
Qualitatively similar immunogold results have been reported previously
for individual antigens by our laboratory and others. In the present
work, we studied the organization of multiple proteins, analyzing data
from each antigen concurrently. By thus eliminating several potential
sources of systematic error, it becomes possible to compare
axodendritic positions for each antigen directly. The embedding method
we have used may be less sensitive than techniques involving slam
freezing and low-temperature embedding in Lowicryl resins, although
both methods yield generally similar results (Valtschanoff et al.,
1998; Petralia et al., 1999). However, for our purpose, sensitivity is
less important than unbiased sampling of antigen. The strong aldehyde
fixation we used causes massive cross-linking, and therefore proteins
within the PSD are unlikely to migrate significantly during embedding
and immunoprocessing. Therefore, the methods used here are likely to
provide a reasonably accurate picture of antigen distribution in
vivo.
The expected error intrinsic to the immunogold method may be up to ±25
nm (Kellenberger and Hayat, 1991). Assuming unbiased sampling, the law
of large numbers suggests that the error in mean position should be
reduced by an order of magnitude by averaging data from 100 gold
particles; this estimate is consistent with the SEs reported in Table
3. Even greater accuracy might be possible with larger samples or
further refinements of technique (e.g., smaller gold particles, direct
conjugation of the primary antibody). However, biological variability
in the location of epitopes also contributes to the variance of the
measurements. This biological variability will be lost in the averaging
procedure, and thus the graph in Figure 3B reveals features
of the supramolecular organization not apparent in Table 3, like the
greater dispersion of DLC than the other proteins studied. We interpret
this to imply that significant fractions of DLC may not be bound to
GKAP. Indeed, electron micrographs show substantial labeling of the
spine apparatus, which lies at some distance from the PSD (Fig. 2) (cf.
Naisbitt et al., 2000).
The diagram shown in Figure 4 is only an initial effort to provide a
spatially accurate impression of the organization of the NMDA receptor
complex. Several sources of axodendritic error remain. For example, the
tortuous three-dimensional shape of a spine implies that the distance
measured is not truly orthogonal to the plane of synaptic apposition,
especially for antigens far from the postsynaptic membrane, consistent
with the generally larger SDs observed for antigens lying
farther from the PSD (Table 3). Likewise, DLC is found at appreciable
concentrations beyond the PSD, and inadvertent inclusion of some of
this labeling may exaggerate its mean axodendritic distance. The
epitope data provided in Table 1 are only approximate, leading to
further inaccuracies. Finally, the protein structures given are
primarily guesswork; we eagerly await x-ray crystallographic data that
can be used to refine the model.
The diagram gives a sense of crowding, accentuated by the many
components of the NMDA receptor complex not shown in this diagram, including  -actinin, tubulin, spectrin, neuroligin, calmodulin, yotiao, CaM kinase II, Raf-1, ErbB-4, SynGAP, synaptic scaffolding molecule, nArgBP2, mammalian LIN-7, adenomatous polyposis coli tumor
suppressor, and Homer, among others (Adam and Matus, 1996; Irie et al.,
1997; Wyszynski et al., 1997, 1998; Chen et al., 1998; Gardoni
et al., 1998; Hirao et al., 1998; Kim et al., 1998; Lin et al., 1998;
Jo et al., 1999; Kawabe et al., 1999; Tu et al., 1999; Garcia et
al., 2000; Yanai et al., 2000; Yao et al., 2000). Although additional
elements of the NMDA receptor complex continue to be identified, it
will be important also to define its stoichiometry and its
heterogeneity to achieve a satisfactory understanding of its
organization. The receptor complex in vivo exists in three
dimensions, helping to relieve the crowding. Likewise, not every
NMDA-related protein is present at every PSD; for example, the PSD
protein Citron is selectively expressed by GABAergic neurons (Zhang et
al., 1999). Recent data on the distribution of AMPA receptor-binding proteins suggest possible heterogeneity of the NMDA
receptor complex even for adjacent synapses onto the same dendrite
(Burette et al., 2001).
On the other hand, each of the NMDA receptor subunits is likely to be
associated with a cytoplasmic assembly of similar complexity; moreover,
some of the cytoplasmic proteins studied here may be oligomers in
vivo [e.g., NOS (Hallmark et al., 1999)]. Thus, it seems likely
that a sizable fraction of the protein packed within the PSD is
associated with the NMDA receptor complex. We presume that this dense
packing accounts for the observed electron density of the PSD; the less
electron-dense postsynaptic specialization of GABAergic inhibitory
synapses suggests that GABA receptors contain a far less complex
intradendritic assembly. Exceptionally dense packing of proteins at the
excitatory PSD may explain the difficulties observed in preembedding
immunostaining of the synapse, except after weak fixation or
proteolytic treatment (Burette et al., 1999; Fukaya and Watanabe, 2000;
Valtschanoff et al., 2001).
The topological organization of the receptor complex as defined by
yeast two-hybrid screens and coimmunoprecipitation (Kim and Huganir,
1999; Sheng and Pak, 2000) is consistent with our data. However, these
biochemical methods cannot provide the spatial information provided in
the present study. Although the data assembled in Figure 4 are only
preliminary, this model may provide new insights into the trafficking
and function of the NMDA receptor complex. It is intriguing, for
example, that CRIPT [shown recently to be essential for the clustering
of PSD-95 and GKAP in cultured neurons (Passafaro et al., 1999)] is on
the cytoplasmic side of the PSD. It is not surprising the DLC lies at
the cytoplasmic edge of the complex, considering its likely role in
trafficking, but it may seem puzzling that DLC (identical to protein
inhibitor of nNOS) has been proposed as an endogenous inhibitor
of nNOS (Jaffrey and Snyder, 1996; but see Rodriguez-Crespo et al.,
1998). In fact, our data support an unusually wide distribution of DLC,
so that although its highest concentration is at the cytoplasmic side of the PSD (extending into the spine cytoplasm), a significant fraction
is also found closer to the membrane, permitting a possible interaction
with nNOS. Our data are also consistent with the possibility that nNOS
may be positioned close to the NMDA receptor pore, where it may be
directly exposed to calcium fluxes. This orientation, which would be
predicted by evidence from cultured neurons that nNOS is selectively
activated by NMDA receptor activation, over other stimuli that also
raise intracellular [Ca2+] (Kiedrowski
et al., 1992), points to the likely importance of supramolecular
organization in the regulation of calcium-signaling pathways.
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FOOTNOTES |
Received May 22, 2000; revised Nov. 20, 2000; accepted Nov. 21, 2000.
This work was supported by National Institutes of Health Grants NS29879
and 39444 to R.J.W. We thank K. Phend for expert histological support
and M. Sheng for his encouragement and careful critique of this manuscript.
Correspondence should be addressed to Dr. Juli Valtschanoff, Department
of Cell Biology and Anatomy, CB# 7090, University of North
Carolina, Chapel Hill, NC 27599. E-mail: jval{at}med.unc.edu.
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ABSTRACT
INTRODUCTION
