Dynamic and specific interaction between synaptic NR2NMDA receptor and PDZ proteins
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Citation
Bard, L., M. Sainlos, D. Bouchet, S. Cousins, L. Mikasova, C.
Breillat, F. A. Stephenson, B. Imperiali, D. Choquet, and L. Groc.
“Dynamic and specific interaction between synaptic NR2-NMDA
receptor and PDZ proteins.” Proceedings of the National
Academy of Sciences 107, no. 45 (November 9, 2010): 1956119566.
As Published
http://dx.doi.org/10.1073/pnas.1002690107
Publisher
National Academy of Sciences (U.S.)
Version
Final published version
Accessed
Wed May 25 22:08:53 EDT 2016
Citable Link
http://hdl.handle.net/1721.1/84594
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Detailed Terms
Dynamic and specific interaction between synaptic
NR2-NMDA receptor and PDZ proteins
Lucie Barda,b, Matthieu Sainlosa,c, Delphine Boucheta,b, Sarah Cousinsd, Lenka Mikasovaa,b, Christelle Breillata,b,
F. Anne Stephensond, Barbara Imperialic, Daniel Choqueta,b, and Laurent Groca,b,1
a
Laboratory for Cellular Physiology of the Synapse, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5091, 33077 Bordeaux, France;
Université de Bordeaux, 33077 Bordeaux, France; cDepartments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and
School of Pharmacy, University of London, London WC1N 1AX, United Kingdom
b
d
Edited by Richard L. Huganir, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 4, 2010 (received for review March
3, 2010)
|
lateral diffusion glutamate receptor
ligand development
|
| trafficking | biomimetic multivalent
T
he identification of the cellular mechanisms involved in the
regulation of glutamate receptor trafficking is crucial to our
understanding of synaptic maturation and plasticity. One common paradigm of these processes is the activation of the calciumpermeable postsynaptic NMDA receptors (NMDARs). In the
neocortex, the most abundant types of NMDARs are composed
of NR1 subunits associated with NR2A (enriched in synapses)
and/or NR2B subunits (1). Rapid changes in the synaptic 2A/2B
NMDAR ratio have been reported during connection refinements and synaptic plasticity (2), and several key molecular
interactions have been shown to control the trafficking of intracellular and membrane NMDARs (3–6).
The intracellular proteins that interact with the C terminus of
the subunits, through direct binding or modification of the
phosphorylation state, are likely candidates for regulating the
synaptic retention of NMDARs. Indeed, intracellular domains of
NR2 subunits provide a binding motif for proteins of the postsynaptic density such as PSD-95 and SAP102 (7–10). The binding
of the NR2B subunit C terminus to PDZ domain-containing
scaffold proteins regulates, in part, the synaptic retention of this
receptor (8, 9, 11–14). For the 2A-NMDARs, which make up the
majority of synaptic NMDARs, the role of such interactions in
synaptic retention remains controversial. Indeed, long-term expression of NR2A subunits with a truncated or mutated C terminus does not affect synaptic NMDAR currents in cerebellar or
hippocampal neurons (9, 15), whereas deletion of the NR2A
subunit C terminus sequence significantly reduces NMDAR
synaptic signaling (11, 14, 16, 17). Currently, there is no simple
explanation for this discrepancy, and the use of long-term expression of exogenous NR subunits and lack of good pharmacological tools to discriminate between 2A- or 2B-NMDAR
www.pnas.org/cgi/doi/10.1073/pnas.1002690107
signaling (18) render interpretation more difficult. Here, we apply
biomimetic divalent peptide-based competing ligands to acutely
interfere with the PDZ domain-containing scaffold proteins-2ANMDAR interaction and use single quantum dot (QD) tracking
to image, with subwavelength precision, the dynamics of surface
synaptic NMDARs.
Results
Design of a Biomimetic Multivalent Ligand to Disrupt the Interaction
Between NR2A Subunit and PDZ Domain-Containing Scaffold Proteins
(PDZ Proteins). The molecular mechanisms involved in the dy-
namic retention of 2A-NMDARs within postsynaptic membranes
are not defined. To investigate these mechanisms, we developed
a peptide-based ligand that strongly and acutely perturbs the
interaction between NR2A subunit and PDZ proteins (Fig. 1A).
Similar strategies have previously been used to dissociate the
PDZ scaffold–NMDAR interaction (19–21). In these studies,
disruption of the PDZ protein–NMDAR interaction was achieved by using monovalent peptide sequences that corresponded
to the last nine to 10 residues of a single subunit (NR2A or
NR2B). We reasoned that the efficiency of such an approach
could be improved by using synthetic ligands that would better
mimic the native interactions. Indeed, because (i) NMDARs are
heterodimeric complexes composed of NR2 subunit dimers and
(ii) the scaffold proteins (e.g., PSD-95, PSD-93 and SAP-102),
which interact with NMDARs, each contain clusters of PDZ
domains that recognize similar targets (22), we hypothesized that
a ligand composed of two NR2 C-terminal binding motifs would
more efficiently dissociate the native scaffold PDZ domain–
NMDAR interactions. In the current design, we conjugated two
of the 15 residue C-terminal sequences of the PSD-95 NR2A
binding motifs via their N-termini (Fig. S1 A and B). Homologous
monovalent sequences and a previously described nonsense sequence were used as controls (Fig. S1 A–D) (23). A series of
ligands incorporating a solvatochromic fluorophore was first used
to evaluate the binding constants with recombinant PSD-95 PDZ
domains 1 and 2 (23). The divalent ligand displayed a sevenfold
increase of affinity for the tandem domain in comparison with the
monovalent homolog (Fig. S1C), confirming the advantage of
divalency. The ligands were then appropriately modified for
specific experiments, e.g., for cell studies, by addition of a TAT
cell-transduction sequence to generate TAT-NR2A15 or TAT[NR2A15]2 and/or a labeling dye (BODIPY-fluorescein).
Author contributions: F.A.S., B.I., D.C., and L.G. designed research; L.B., M.S., D.B., S.C.,
and L.M. performed research; M.S., C.B., and B.I. contributed new reagents/analytic tools;
L.B. analyzed data; and L.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: laurent.groc@u-bordeaux2.fr.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1002690107/-/DCSupplemental.
PNAS Early Edition | 1 of 6
NEUROSCIENCE
The relative content of NR2 subunits in the NMDA receptor confers
specific signaling properties and plasticity to synapses. However,
the mechanisms that dynamically govern the retention of synaptic
NMDARs, in particular 2A-NMDARs, remain poorly understood.
Here, we investigate the dynamic interaction between NR2 C
termini and proteins containing PSD-95/Discs-large/ZO-1 homology
(PDZ) scaffold proteins at the single molecule level by using highresolution imaging. We report that a biomimetic divalent competing
ligand, mimicking the last 15 amino acids of NR2A C terminus,
specifically and efficiently disrupts the interaction between 2ANMDARs, but not 2B-NMDARs, and PDZ proteins on the time scale
of minutes. Furthermore, displacing 2A-NMDARs out of synapses
lead to a compensatory increase in synaptic NR2B-NMDARs, providing functional evidence that the anchoring mechanism of 2A- or
2B-NMDARs is different. These data reveal an unexpected role of
the NR2 subunit divalent arrangement in providing specific anchoring within synapses, highlighting the need to study such dynamic
interactions in native conditions.
A
NR2A
B
TAT-[NS15]2
QD
C
synapse
Potential PDZ Binding sites
Cumulative frequ
uency
NR2A
PDZ domain
binding motif
75
50
0
t1
t5
t10
0.01
0.1
1
Diffusion
Diff
i coefficient
ffi i t ((µm2/s)
/ )
E
% of synaptic
TAT-[NR2A15]2
TAT-[NS15]2
TAT-[NR2A15]2
t0
TAT-[NS15]2
TAT-[NR2A15]2
25
Biomimetic ligand
D
TAT-NS15
TAT-NR2A15
100
NR1
100
75
50
25
0
Synapse
QD
*
TAT-[NS15]2
TAT-[NR2A15]2
Before t1
t5
Time (min)
t10
Fig. 1. Acute disruption of the interaction between 2A-NMDARs and PDZ proteins using a NR2A-derived multivalent ligand. (A) Schematic representation of
a membrane NMDAR, a scaffold MAGUK protein, and the newly developed divalent ligand mimicking the C terminus (15 last amino acids) of two NR2A subunit
subunits (TAT-[NR2A15]2). (B) Representative trajectories of surface 2A-NMDARs, based on QD-coupled antibodies against an extracellular epitope of the NR2A
(Upper Left), after 10 to 20 min of incubation with TAT-[NS15]2 (Upper) or TAT-[NR2A15]2 (Lower). The green areas correspond to synaptic sites labeled with
Mitotracker. (Scale bar: 1 μm.) (C) Cumulative distribution of the instantaneous diffusion coefficient of 2A-NMDARs. The first point corresponds to the percentage
of immobile receptors (bin size, 0.0075 μm2/s). Note the higher increase in the mobility of 2A-NMDARs induced by the divalent TAT-[NR2A15]2 (n = 303 trajectories;
solid gray line) compared with monovalent TAT-NR2A15 (n = 170; dashed gray line) or TAT-NS15 (dashed and solid black lines; TAT-[NS15]2, n = 530 trajectories; TATNS15, n = 153 trajectories). (D) Displacement of individual NR2A-coupled QDs after incubation with TAT-[NR2A15]2. The neurons were incubated with Mitotracker
(green) and NR2A-coupled QDs (red spots). The localization of NR2A-coupled QDs was followed for 10 min after acute addition (arrow) of 5 μM TAT-[NS15]2
(Upper) or 5 μM TAT-[NR2A15]2 (Lower). (Scale bar: 1 μm.) (E) The synaptic localization of NR2A-coupled QDs decreased over the 10 min recording after incubation
with both TAT-[NS15]2 (n = 11) and TAT-[NR2A15]2 (n = 6). The reduction was significantly higher for TAT-[NR2A15]2 (*P < 0.05).
Interaction Between 2A-NMDARs and PDZ Proteins Regulates NMDAR
Synaptic Retention. To investigate the ligand efficacy in neuronal
preparations, hippocampal cultured neurons were incubated with
a saturating (5–10 μM) nontoxic (i.e., no neuronal damage observed) concentration of TAT-[NS15]2 or TAT-[NR2A15]2. After
a 10-min incubation period, neurons were efficiently labeled by the
different BODIPY-containing ligands (SI Materials and Methods).
To investigate the specific impact of the ligands on the surface
2A-NMDAR anchoring, we used single QD tracking as a highresolution approach to estimate 2A-NMDAR surface diffusion
in live neurons (24). Native 2A-NMDARs were detected by using a
QD-antibody complex directed against the extracellular N terminus
of the NR2A subunit (Fig. 1B and SI Materials and Methods) and
their surface localization, i.e., onto a postsynaptic marker or outside synapse, determined during the recording session. The overall
surface diffusion of 2A-NMDARs was increased after monovalent
TAT-NR2A15 and divalent TAT-[NR2A15]2 ligand incubation, although to very different extents: (i) the cumulative distribution of
coefficient diffusion was highly shifted by TAT-[NR2A15]2 incubation; (ii) the diffusion coefficient medians were threefold and
27-fold increased by TAT-NR2A15 and TAT-[NR2A15]2, respectively [i.e., TAT-[NS15]2 median of 4.10−3 μm2/s, interquartile
range (IQR) of 0–2.10−2 μm2/s, n = 530 trajectories; TAT[NR2A15]2 median of 11.10−2 μm2/s, IQR of 6.10−4-5.10−1 μm2/s,
n = 303 trajectories; P > 0.05]; and (iii) the fraction of mobile 2ANMDARs (membrane diffusion >0.0075 μm2/s) increased by 2%
and 42% after TAT-NR2A15 and TAT-[NR2A15]2, respectively
(Fig. 1C). Similar results were obtained when examining solely
synaptic 2A-NMDARs (Fig. S2), indicating that disruption of the
2A-NMDAR anchoring increases the fraction of mobile receptors.
To investigate the impact of TAT-[NR2A15]2 on identified
single synaptic 2A-NMDARs, QD-2A-NMDAR complexes were
2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1002690107
tracked within synapse before and in the presence of TAT[NR2A15]2 or TAT-[NS15]2 (Fig. 1D). After TAT-[NS15]2 incubation (10 min) the fraction of 2A-NMDARs that remained
within synapses was unchanged, although a slight but not significant (P > 0.05) decrease is noted, consistent with the basal exchange rate of surface NMDARs between synaptic and extrasynaptic membranes (25–27). However, within the same time
frame in the presence of TAT-[NR2A15]2, approximately half of
the synaptic 2A-NMDARs escaped the synaptic area (Fig. 1 D
and E), indicating that anchoring of synaptic 2A-NMDARs by
PDZ scaffolds is a dynamic process. To further confirm the impact of the ligand on the surface NMDAR synaptic population,
and not only single receptor, we expressed the NR1 subunit
(obligatory subunit of surface NMDARs) fused to Super Ecliptic
pHluorin at its extracellular N terminus (SEP-NR1) to isolate
the surface fraction and quantify the average surface diffusion of
SEP-NR1-containing NMDARs using fluorescence recovery after photobleaching (FRAP; Fig. S3 A–C). The recovery of SEPNR1 fluorescence in dendrites was approximately 50%, whereas
it was only 20% in synapses (25). Consistently, a decrease of the
percentage of immobile synaptic receptors was observed, i.e.,
from 85% before incubation with TAT-[NR2A15]2 to 65% following 20 min incubation (Fig. 1E). The proportion of immobile
receptors outside synapses was not affected, suggesting that
TAT-[NR2A15]2 acts on synaptically enriched 2A-NMDARs (11,
28). Finally, these results were further confirmed with immunocytochemical staining of synaptic NR2A subunits (colabeled with
PSD-95), as, over a large fraction of synapses (TAT-[NR2A15]2,
n = 1,684 synapses; TAT-[NS15]2, n = 1,981), TAT-[NR2A15]2
consistently reduced the synaptic content of 2A-NMDARs (Fig.
S3 D and E).
Bard et al.
A
Cumul. freq.
Frequency (relative)
0.7
0.4
0.1
1
0.5
0
0.01 0.1
1
Diff. Coef. (µm2/s)
0.05
TAT-[NS15]2
TAT-[NR2A15]2
0
0
0.1
0.2 0.4 0.6
Diffusion coefficient (µm2/s)
WT
2B-WT
B
Mutant
2B-S1480A
t40s
t40s
synapse
D
0.6
0.4
0.2
**
0.0
2
1
0
E
t0
2B-WT
2B-S1480A
3
MSD (µm2)
0.8
0
0.05
0.1
Time (s)
Syn. dwell time (s)
F
2.0
1.0
0
**
Frequency (relative)
C
Syn. dwell time (s)
t0
1
- TAT-[NR2A15]2
+ TAT-[NR2A15]2
P>0.05 KS test
0.5
0
0
0.05
0.1
2A-S1462A diff. coef. (µm2/s)
Fig. 2. TAT-[NR2A15]2 incubation does not affect native 2B-NMDARs or
NR2A subunit mutant that does not bind to PDZ proteins. (A) Native 2BNMDARs were tracked using QDs coupled to antibodies directed against an
extracellular epitope of endogenous NR2B subunit in presence of TAT-[NS15]2
(n = 675 trajectories) or TAT-[NR2A15]2 (n = 442 trajectories). The frequency
distribution (cumulative; Upper Right) of diffusion coefficients revealed that
TAT-[NR2A15]2 did not significantly affect the diffusion of native 2B-NMDARs
(P > 0.05, Mann–Whitney test). (B) The surface diffusion of recombinant 2BNMDARs was assessed using recombinant flag-tagged NR2B subunits: WT
(2B-WT) or mutant form (2B-S1480A) that does not bind PDZ proteins. These
subunits were tracked using anti-Flag coupled QDs. Representative 40-s
trajectories of anti-Flag QDs tracking 2B-WT (Left) or 2B-S1480A (Right).
(Scale bar: 500 nm.) The starting and ending point are referred as t0 and t40s,
respectively. The green areas correspond to synapses. (C) Synaptic dwell time
was measured for 2B-NMDARs containing 2B-WT (n = 235 trajectories) or 2B-
Bard et al.
NR2 subunits are thought to associate with PSD-95 via a Cterminal 4-aa sequence, which is identical in NR2A and NR2B
subunits. Other upstream amino acid sequences that differ between NR2A and NR2B subunits have also been implicated in
PSD-95 binding (29), and there is some evidence that, at least for
potassium channels, binding to PSD-95 tandem PDZ domains
involves up to 12 C-terminal residues (30). Interestingly, the
amino acid sequence homology decreases to only 60% after
alignment of NR2A and NR2B subunit 15 C-terminal residues
(Fig. S1). Although most studies on isolated PDZ domains and
minimal peptides derived from the C-termini of binding partners
tend to limit the ligand interacting residues to the C-terminal 4
aa, we anticipated that the native interactions might achieve
higher specificity by engaging additional residues, constituting
the rationale for using the last 15 aa of the NR2A subunit. We
first analyzed the effect of TAT-[NR2A15]2 on native 2BNMDAR surface diffusion [measured in young hippocampal
cultured neurons that do not express 2A-NMDAR (25)]. Strikingly, TAT-[NR2A15]2 had no effect on the surface diffusion of
native 2B-NMDARs (Fig. 2A). The diffusion coefficient distributions were superimposed, indicating that TAT-[NR2A15]2
did not affect the anchoring of 2B-NMDARs. Because the synaptic anchoring of 2B-NMDARs may not depend on the interaction with PDZ proteins, we compared the surface trafficking
of NR2B WT (2B-WT) and a NR2B mutant (i.e., 2B-S1480A),
which does not coimmunoprecipitate with PSD-95 (9). The diffusion coefficient was significantly higher for 2B-S1480A (median of 0.57 μm2/s, IQR of 0.24–1.08 μm2/s, n = 694 trajectories)
than for 2B-WT (median of 0.32 μm2/s, IQR of 0.13–0.69 μm2/s,
n = 344 trajectories) and the synaptic dwell time, defined as the
mean time spent by a mobile receptor in the synaptic area, was
significantly higher for 2B-WT compared with 2B-S1480A (Fig.
2C). Both 2B-WT and 2B-S1480A were confined within the
synapse but to a significantly lower degree for 2B-S1480A (Fig.
2D). These results demonstrate that the synaptic retention of
surface 2B-NMDARs is dynamically regulated by the interaction
with PDZ proteins (9) and insensitive to TAT-[NR2A15]2 ligand.
In addition, incubating the neurons with TAT-NR2B15 (5 μM, 10
min) that mimics the last 15 aa of the NR2B subunit C terminus
increased the surface diffusion of synaptic 2B-NMDARs without
affecting the one of 2A-NMDARs (Fig. S4), consistent with
previous biochemical reports using similar ligands (23, 24). Finally, a monovalent ligand containing only the last 6 aa of the
NR2 C-terminus sequence (TAT-NR2X15), which is identical for
NR2A and NR2B subunits, increased the surface diffusion of
both 2A- and 2B-NMDARs (Fig. S5), indicating that the PDZ
binding sequence (last few amino acids of the C terminus) is
indeed necessary to anchor the receptor in the synapse, and
upstream amino acid sequence(s) provide a specificity motif for
NR2 subunit.
To further test the specificity of the ligand, we then reasoned
that if TAT-[NR2A15]2 competes specifically against the NR2A
S1480A (n = 532 trajectories). Note the reduction in the time spent by 2BS1480A within the synapse (**P < 0.01, t test). (D) Plot of the mean square
displacement (MSD) versus time for synaptic receptors containing the subunit
2B-WT or 2B-S1480A. The curves exhibit a negative curvature characteristic of
a confined behavior. Note the higher degree of confinement for 2B-WT
subunits. (E) Synaptic dwell time was measured for 2A-NMDARs containing
either 2A-WT (n = 487 trajectories) or mutant 2A-S1462A (n = 474 trajectories). Note the reduction in the time spent by the mutant within the synapse
(**P < 0.01, t test). (F) The frequency distribution of diffusion coefficients of
mutant 2A-S1462A in absence (n = 58 trajectories; black squares) or presence
of TAT-[NR2A15]2 (n = 94 trajectories; gray squares). No significant difference
was observed (P > 0.05, Mann–Whitney test).
PNAS Early Edition | 3 of 6
NEUROSCIENCE
NR2A-Derived Ligand Does Not Interfere with 2B-NMDAR, Kv
Potassium Channel, or GABAA Receptor Surface Trafficking. The
2B-NMDAR
Specific immunoreactivity
Buffer
+TAT-[NS15]2
+TAT-[NR2A15]2
1.5
1.0
0.5
0
Fig. 3. TAT-[NR2A15]2 specifically blocks the interaction between PSD-95
and NR2A subunit. HEK 293 cells were cotransfected in triplicate with NR11a/NR2A or NR1-1a/NR2B with or without PSD-95 and cell surface expressed
NMDARs measured by ELISA using either anti-NR2A 44–58 Cys or anti-NR2B
46–60 Cys affinity-purified antibodies. The results are expressed as the ratio
of absorbance and expressed as the means ± SEM (n = 2 independent
transfections for each combination). As previously shown, PSD-95 enhanced
cell surface delivery of 2A- and 2B-NMDARs. These effects were then examined after incubation with TAT-[NS15]2 (10 μM; open bar) or TAT[NR2A15]2 (10 μM; gray bar). Note that PSD-95 failed to increase surface 2ANMDAR expression in the presence of TAT-[NR2A15]2.
C-terminal domain for the binding to its PDZ proteins, it would
have no additional effect on the diffusion of 2A-NMDARs
containing a 2A-S1462A mutation in the C terminus that prevents NR2A/PSD-95 coimmunoprecipitation (co-IP) (9). The
2A-S1462A displayed a threefold higher surface diffusion (P <
0.001) and 1.4-fold shorter dwell time (Fig. 2E) than WT 2ANMDARs. TAT-[NR2A15]2 did not increase the surface diffusion of 2A-S1462A (Fig. 2F), indicating that the TAT[NR2A15]2-induced increase in 2A-NMDAR surface diffusion
was occluded by the 2A-S1462A-induced increase in 2ANMDAR surface diffusion. We then tested the specificity of
TAT-[NR2A15]2 on other membrane proteins by imaging the
surface trafficking of the native potassium channel Kv1.3, endogenously expressed in hippocampal neurons (31), because its
C terminus contains a PDZ binding site similar to those of
NR2A and NR2B subunits (Fig. S6A) (32). Remarkably, TAT[NR2A15]2 produced no change in the diffusion pattern, the
mobile fraction, the global diffusion coefficient, or the surface
distribution of Kv1.3 channels (Fig. S6 B–E). Thus, although
NR2A and Kv1.3 channel bind PDZ site with similar affinities
(32) and exhibit a high similarity in their C-terminus amino acid
sequence, TAT-[NR2A15]2 specifically acts on surface 2ANMDARs and not on surface Kv1.3 channel. In addition, we
report that TAT-[NR2A15]2 does not impact on the surface
GABAA receptor (Fig. S6F), which is not anchored in synapse by
a PDZ domain-binding motif (33).
Interaction Between NR2A Subunits and PSD-95 Is Specifically
Disrupted by the NR2A Ligand. Because the PDZ-containing scaf-
folding proteins change during development, i.e., PSD-95 is the
dominant scaffolding protein in mature neurons and SAP102 is the
dominant scaffolding protein in immature neurons, the possibility
that the TAT-[NR2A15]2 ligand better interacts with certain PDZ
proteins remains to be tested. For this, we first measured, from
forebrain homogenates, the impact of the NR2A ligand on the
interaction between PSD-95 and NR2A or NR2B subunits using
co-IP. The PSD-95/2A subunit interaction was specifically affected
by the ligand whereas the PSD-95/2B subunit interaction remains
unaffected (Fig. S7). Furthermore, we used a heterologous cell
system to further determine the impact of the ligand on the interaction between NR2 subunit and the most abundant PDZ
proteins, PSD-95. In heterologous cells, 2A- and 2B-NMDARs
coimmunoprecipitate with the four PSD-95 MAGUK family of
4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1002690107
scaffolding proteins (34). In addition, PSD-95 enhances 2A- and
2B-NMDAR cell surface expression through a process that
requires the NR2 C terminus sequence -ESDV (34). To test the
specificity of TAT-[NR2A15]2 on 2A- and 2B-NMDAR trafficking, we then measured the effect of PSD-95 on cell surface 2A- or
2B-NMDAR expression, as previously described (34). We first
observed that either TAT-[NS15]2 or TAT-[NR2A15]2 had no effect per se on the basal expression of the subunit (Fig. 3). PSD-95
enhanced the cell surface expression of both 2A- and 2BNMDARs (Fig. 3). The incubation with TAT-[NR2A15]2 completely blocked the PSD-95-induced 2A-NMDAR surface expression, whereas TAT-[NR2A15]2 had no effect on PSD-95induced 2B-NMDAR surface expression (Fig. 3). In all conditions,
TAT-[NS15]2 incubation had no significant effect on the PSD-95induced NR2-NMDAR surface expression. All together, these
data demonstrate, in neuronal and heterologous systems, that the
TAT-[NR2A15]2 divalent ligand specifically blocks the interaction
between 2A-NMDARs (no effect on 2B-NMDARs) and the most
abundant protein of the postsynaptic density, PSD-95.
Rapid Redistribution of 2A- and 2B-NMDARs in Excitatory Synapses.
As TAT-[NR2A15]2 specifically destabilizes synaptic 2ANMDARs, we investigated the functional consequences of such
an effect by first measuring NMDAR-mediated synaptic currents. We report that, in the presence of [NR2A15]2 (within recording pipette), the kinetics of NMDAR miniature excitatory
postsynaptic currents (mEPSCs) were significantly increased
whereas AMPAR mEPSC remained unchanged (Fig. S8). We
then recorded evoked NMDA excitatory postsynaptic currents
(eEPSC) from CA1 pyramidal neurons (P16–20; Fig. 4A) and
found that a 15- to 20-min dialysis of [NR2A15]2 significantly
increased the Ro 25-6981 (NR2B subunit antagonist, 1 μM)-induced inhibition of NMDAR current (Fig. 4A), consistent with
an increased contribution of 2B-NMDARs to synaptic currents.
Interestingly, the amplitude of both mEPSCs and eEPSCs
remained unchanged in the presence of [NR2A15]2, indicating
that the [NR2A15]2-induced removal of 2A-NMDARs from
synapse was compensated by the insertion of other NMDARs
with slower kinetics (Fig. 4B). To gain insight in the NR2NMDAR trafficking at identified synapses, the fluorescence intensity of surface NR2A (SEP-NR2A) and NR2B (SEP-NR2B)
subunits was measured over time before and after incubation
with TAT-[NS15]2 or TAT-[NR2A15]2 (Fig. 4C). Synaptic and
extrasynaptic NMDARs were distinguished by coexpressing the
synaptic marker Homer 1C-DsRed. First, the intensity of extrasynaptic surface NMDARs (SEP-NR1, SEP-NR2A, SEP-NR2B)
was not significantly altered by the presence of TAT-[NS15]2 or
TAT-[NR2A15]2. Within synapses, the intensity of 2A-NMDAR
clusters was decreased as demonstrated by the significant left
shift of the cluster distributions (P < 0.001; Fig. 4D) or by the
significant decrease of average values (Fig. 4E). Surprisingly,
under the same conditions the intensity of 2B-NMDAR synaptic
clusters was increased significantly (e.g., right shift of the distribution; P < 0.001; Fig. 4 D and E). The effect was observed 15 to
20 min after the ligand incubation and was stable over time. In
such mature synapses, TAT-[NR2A15]2 reduced the surface diffusion of synaptic 2B-NMDARs and increased their synaptic
dwell time, consistent with a higher retention of these receptors.
These data demonstrate thus that 2A- and 2B-NMDARs rapidly
redistribute within synaptic areas. In addition, displacing 2ANMDARs out of synapses by preventing the interaction of 2ANMDARs and PDZ proteins is compensated by the increase
contribution of other NMDAR subtypes, indicating that an unexpected level of specificity between NR2 subunits and PDZ
proteins is present in postsynaptic densities.
Bard et al.
[NS15]2
0
t20
100
50
0
TAT-[NS15]2
D 0.50
TAT-[NR2A15]2/SEP-NR2A
TAT-[NR2A15]2/SEP-NR2B
0.25
0
0
1
2
Fluo. intensity (norm.)
1.50
1.25
1.00
***
***
0.75
3
Fig. 4. Dynamic regulation of surface 2A- and 2B-NMDAR content in postsynaptic areas. (A) Evoked NMDAR EPSCs (recorded at +30 mV) averaged at 0 to 2
min (black trace) or 18 to 20 min (red trace) after dialysis with Ro 25-6981 (1 μM, 2B-NMDAR antagonist; Left). (Horizontal scale bar: 100 ms.) The Ro 25-6981
incubation significantly reduced the NMDAR eEPSC amplitude (Right). In te presence of [NS15]2 (n = 9 neurons) or [NR2A15]2 (n = 8 neurons), Ro 25-6981
reduced the NMDAR eEPSC amplitude by 35% and 47%, respectively. (B) The amplitude (normalized) of NMDAR eEPSCs remained stable in presence of
[NR2A15]2 ligand ([NS15]2, n = 6 neurons; [NR2A15]2, n = 7 neurons). (C) The fluorescence intensity of synaptic SEP-NR2A (Upper) and SEP-NR2B (Lower) clusters
colocalized with Homer 1C was followed over a period of 20 min after acute addition of 5 μM of TAT-[NR2A15]2 (Scale bar: 1 μm.) (D) Left: Frequency distribution of the fluorescence intensity of SEP-NR2A and SEP-NR2B clusters after 20 min in the presence of TAT-[NS15]2 or TAT-[NR2A15]2. The Gaussian fit is
centered on 1 after incubation with TAT-[NS15]2 (n = 563 clusters; dashed black line) indicating that the receptor content within the cluster did not change
over time. Note the shift of the curve toward the left for SEP-NR2A (n = 451 clusters; solid gray line) and toward the right for SEP-NR2B (n = 309 clusters; full
black line) after the 20 min incubation with TAT-[NR2A15]2 showing, respectively, a decrease and an increase in the receptor content. Right: Normalized mean
fluorescence intensity of the clusters before and after a 20-min incubation with TAT-[NS15]2 or TAT-[NR2A15]2 (P < 0.001, paired t test).
Discussion
Although synaptic NR2-NMDARs play a key role in synaptic refinement (2), the molecular mechanisms as well as the dynamics
that govern their surface distribution and rapid trafficking are
largely unknown. To shed new light on this issue, we developed
a biomimetic divalent ligand that acutely and efficiently blocks the
interaction between PDZ proteins and native 2A-NMDARs
(enriched at synapses). We unravel an unexpected role of the
divalent arrangement of the NR2 subunits in providing efficient
anchoring within synapses and strengthen the need to dynamically
study such interactions in native conditions. Indeed, by using
mono- or divalent ligands, we now identified that the binding efficacy is highly dependent on the divalent structure of the 2ANMDAR complex, and the specific binding of 2A-NMDAR (versus 2B-NMDAR for instance) relies on amino acid sequence(s)
upstream to the C terminus, whereas the last C terminus amino
acids are implicated in the direct binding to PDZ scaffold proteins
(Fig. S9). Thus, NR2 subunits associate with PDZ proteins via
a C-terminal 4-aa sequence (7–10), which is identical in NR2A and
NR2B subunits, and other upstream amino acids that are within 15
aa of the C terminus, and as previously proposed, in more upstream
sequences (29). Although the binding mechanism of these domains remains poorly understood (35), it suggests that 2A- or 2BNMDARs are engaged in different sets of interactions within the
scaffold environment. Consistently, the NR2A-ligand-induced
rapid exit of 2A-NMDARs from postsynaptic densities was paralleled by a compensatory increase in 2B-NMDAR content, indicating that the 2A/2B-NMDAR synaptic ratio is dynamically
regulated. Functionally, long-term potentiation of hippocampal
ACKNOWLEDGMENTS. We thank Laurent Ladépêche, Beatrice Tessier,
Arnaud Frouin, and Christophe Blanchet for technical assistance; Robert
Wenthold (National Institutes of Health, Bethesda, MD) for providing NR2
cDNA plasmids and constructive discussions; and Antoine Triller [Ecole Normale Superieure (ENS), Paris] for providing antibody. This work was supported by Centre National de la Recherche Scientifique/Agence Nationale
de la Recherche Grant JC08_329238 (to L.G.), Chem-Traffic (M.S. and D.C.),
Human Frontier Science Program Grant MRGP0007/2006-C (to B.I., M.S., and
D.C.), Fondation pour la Recherche Médicale (L.B., D.C.,and L.G.), Conseil
Régional d’Aquitaine, Marie Curie postdoctoral fellowship (PICK-CPP to M.S.),
Ministère de l’Enseignement Supérieur et de la Recherche, European Research Council Advanced Research Grant Nano-Dyn-Syn (to D.C.), and the
UK Biotechnology and Biological Sciences Research Council (F.A.S.).
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Materials and Methods
Complete discussions of ligand synthesis, cell culture, immunocytochemistry,
synaptic live cell staining, protein expression, single particle (QD) tracking,
fluorescence recovery after photobleaching (FRAP), electrophysiology, immunoprecipitation, and in vitro cell surface assays are in SI Materials
and Methods. The transduction and cell distribution of the ligand are detailed in Fig. S10. The impact of the ligand and its vehicle on receptor
trafficking is detailed in Fig. S11.
PNAS Early Edition | 5 of 6
NEUROSCIENCE
20
t0
Fluo. intensity (norm.)
40
B
Ampl. (norm.)
% Ro 25-6981 inhib.
+ Ro 25-6981
*
Homer 1C
SEPN R2B
50 pA
60
C
SEPNR2A
[NR2A15]2
Frequency
A
9.
10.
11.
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Bard et al.
Supporting Information
Bard et al. 10.1073/pnas.1002690107
SI Materials and Methods
Ligand Synthesis and in Vitro Characterization. The design and
synthesis (M.S., B.I.) of peptide-based ligands is schematically
described in this paragraph. Briefly, the last 15 residues of the
ligand binding motifs were first assembled by standard Fmoc-based
solid phase peptide synthesis and capped with a mixture of azideand alkyne-derived acids. The ligation was conducted by click
chemistry (1) on resin. For the 4-DMAP-containing ligand, the
fluorophore was inserted post ligation on resin. The TAT sequence was coupled as a C-terminal thioester to the purified ligand N-terminal Cys residue by native chemical ligation.
BODIPY-FL was coupled using a maleimide derivative to the
same Cys residue. Recombinant PSD-95 tandem PDZ domains
expression and fluorescence titrations were performed as previously described (2).
Single Particle (QD) Tracking and Surface Diffusion Calculation. QD
655 Goat F(ab′)2 anti-Rabbit or anti-mouse IgG (Invitrogen)
were first incubated for 30 min with the polyclonal antibodies
against NR2A (1 μg) and NR2B subunits (1 μg), the monoclonal
anti-Flag (Stratagene), the anti GABAA α2 (gift from A. Triller,
Ecole Normale Superieure, Paris), and the Kv1.3 antibodies
(Alomone Labs; epitope location corresponds to the first extracellular loop between domains S1 and S2 amino acid, i.e., residues
263–276 of Kv1.3). For this Kv1.3 antibody, specificity control has
been obtained only with Western blot (Alomone Labs). Nonspecific binding was blocked by additional casein (Vector Laboratories) to the QD 15 min before use. For experiments using
TAT peptides, neurons were first incubated for 10 min at 37 °C in
culture medium with precoated QDs (final dilution 1:2,000 for
anti-NR2A and anti-NR2B coupled QDs, 1:10,000 for anti-GABAA coupled QDs, 1:5,000 for anti-kv1.3 coupled QDs), then
for 1 min with 20 nM Green Mitotracker (Molecular Probes), for
5 min with 50 μM pyrene butyrate (3) and finally for 10 min with
5 μM TAT-[NS15]2 or TAT-[NR2A15]2 (or TAT-NR2B15 when
specified).
We first examined the penetration of the TAT-[NR2A15]2 in
neurons. As shown in Fig. S10, the ligand efficiently penetrates cultured neurons and was observed in all dendritic compartments, including postsynaptic densities. We next tested the nonspecific effects
of pyrene butyrate or TAT-[NS15]2 on the mobility of 2A-NMDARs
(Fig. S11). Whereas pyrene butyrate had no effect per se, TAT[NS15]2 increased the proportion of immobile receptors. Importantly, such effect of TAT-[NS15]2 was observed on 2B-NMDARs,
GluR2-AMPARs, GABAARs, and reproduced using the monomeric TAT-[NS15]. This indicates that TAT-[NS15]2 slightly reduced
the surface trafficking of neurotransmitter receptors, irrespective of
the nature of the receptors or the structure of the TAT ligand.
For single particle tracking of Flag-tagged NR2B subunits, neurons were incubated for 10 min with precoated anti-Flag QDs (final
dilution, 1:10,000). QDs were detected by using a mercury lamp and
appropriateexcitation/emissionfilters.Imageswereobtainedwithan
integration time of 5 to 30 ms with up to 2,000 consecutive frames.
Signals were detected using a CCD camera (Quantem; Roper Scientific). QDs were followed on randomly selected dendritic regions
for up to 20 min. QD recording sessions were processed with Metamorph software (Universal Imaging). The instantaneous diffusion
coefficient, D, was calculated for each trajectory, from linear fits of
the first four points of the MSD versus time function:
Bard et al. www.pnas.org/cgi/content/short/1002690107
MSDðtÞ ¼ < r2 > ðtÞ ¼ 4Dt
[S1]
Synaptic dwell time was calculated for exchanging receptors and
defined as the mean time spent within the synaptic area. The 2D
trajectories of single molecules in the plane of focus were constructed by correlation analysis between consecutive images using
a Vogel algorithm.
Cell Culture, Immunocytochemistry, Synaptic Live Staining, and
Protein Expression. Cultures of hippocampal neurons were pre-
pared from E18 Sprague–Dawley rats following a previously described method (4–6). Briefly, cells were plated at a density of 100
to 200 × 103 cells per milliliter on poly-lysine precoated coverslips.
Cultures were maintained in serum-free neurobasal medium
(Invitrogen) and kept at 37 °C in 5% CO2 for 20 d in vitro at
maximum. For immunostaining, surface 2A-NMDARs were
specifically stained using a polyclonal anti-NR2A subunit antibody (1:100; F. A. Stephenson, London, United Kingdom) for 15
min on live neurons at 37 °C in culture medium. The specificity of
the antibody was previously described (5). Briefly, neurons were
then fixed with 4% paraformaldehyde/4% sucrose for 15 min,
washed, and incubated with secondary antibodies anti-rabbit
Alexa 488 antibodies (1:1,000, 45 min; Molecular Probes). To
label postsynaptic areas, neurons were permeabilized using 0.1%
Triton X-100, incubated with a primary rabbit polyclonal antishank antibody (1:750, 45 min; Abcam), and finally incubated with secondary antibody anti-rabbit Alexa 568 antibodies
(1:1,500, 30 min; Molecular Probes). Neurons were washed and
mounted and preparations were kept at 4 °C until quantification.
For the quantification of surface NR2A staining within individual
shank cluster, the shank-synaptic staining served as a mask filter
to isolate surface NR2A staining in individual shank clusters. The
integrated fluorescence level over the shank-cluster area was then
measured for each cluster. The fluorescence analysis was realized
using imaging tools from Metamorph software (Universal Imaging). To label synapses live cultured neurons were incubated for 1
to 2 min at room temperature with 10 nM Mitotracker (Deep
Red-Fluorescent Mitotracker; Molecular Probes) prior to imaging experiments. Neurons were transfected at 7 to 10 d in vitro
with Homer1c-DsRed or Homer1c-GFP alone or with SEP-NR1,
SEP-NR2A, or SEP-NR2B using the Effectene transfection kit
(Qiagen). We mixed 2 μg of DNA with 25 μL of Effectene and 8
μL of enhancer in 150 μL of reaction buffer, and then added the
mixture to cultured neurons, which were transferred to serumfree neurobasal medium 10 min beforehand. After an incubation
period of 45 min, neurons were placed in the old medium again.
Postsynaptic Localization of NMDARs. Surface diffusion of postsynaptic NMDARs has been described using electrophysiological
and high-resolution imaging approaches (5, 7–9). It is noteworthy
that the diffusion of presynaptic NMDARs has also been described in synapses from the entorhinal cortex at early stages of
development (10). Because single particle tracking on endogenous
NMDARs does not distinguish between pre- and postsynaptic
receptors, we investigated whether presynaptic tagged NMDARs
were present in our hippocampal system. However, we found no
evidence for surface NMDARs in presynaptic (VAMP-2-positive
cluster) terminals, indicating that in our neuronal preparation
surface NMDARs are mostly located in the postsynaptic area.
Electrophysiology. Neurons were continuously perfused with extra-
cellular solution containing (in mM): 145 NaCl, 2.5 KCl, 10 Hepes,
10 glucose, 2 CaCl2, and 0.1 mM Mg2+, pH 7.3, osmolarity 300
1 of 12
mOsm/L. Patch pipettes (3–5 MΩ) were filled with (in mM): 140
cesium methanesulfonate, 2 MgCl2, 4 NaCl, 5 phosphocreatine, 10
Hepes, 2 Na2-ATP, 0.33 Na-GTP, 5 QX-314, and 0.2 EGTA, pH
7.3, osmolarity 290 mOsm/L. Recordings in voltage clamp mode
were performed with an EPC 10 double patch-clamp amplifier
(HEKA). Recordings included for analysis were collected during
periods of stable series resistance. Data were acquired and stored
using Pulse-Pulse fit software, version 8.62. Miniature NMDAR
EPSCs were recorded at +40 mV and isolated in the presence of
TTX (1 μM), bicuculline (20 μM; Ascent Scientific), and NBQX
(10 μM, Ascent Scientific). Miniature AMPAR EPSCs were recorded at −50 mV and isolated in the presence of TTX (1 μM),
bicuculline (20 μM; Ascent Scientific), and AP5 (25 μM; Ascent
Scientific) (11). Miniature EPSCs were analyzed by using the MiniAnalysis program (version 6.0.7; Synaptosoft) and IGOR (WaveMetrics). The limit of detection was greater than 5 pA. The decay
times of NMDA-mEPSCs were fitted using two exponentials.
Hippocampal slices were prepared from P16-20 C57Bl6 mice.
Mice were decapitated and the brain was removed and placed in
ice-cold solutioncomposedof(in mM): 87NaCl,2.5 KCl,10 glucose,
75 sucrose, 25 NaHCO3, 1.25 NaH2PO4, 0.5 CaCl2, and 7 mM
Mg2+. Transverse hippocampal slices (350 μm) were cut using
a vibrating tissue slicer (Leica) and transferred to a holding
chamber and stored at 33 °C. For recording, slices were individually
transferred to a recording chamber where they were perfused at
30 °C to 35 °C. The extracellular solution contained (in mM): 125
NaCl, 2.5 KCl, 25 glucose, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2,
and 1 mM Mg2. GABAAR postsynaptic currents were blocked by
20 μM bicuculline. AMPAR EPSCs were blocked by 10 μM NBQX.
CA1 pyramidal cells were visually identified using IR-DIC videomicroscopy and whole-cell patch-clamp recordings were performed
with an EPC-10 patch-clamp amplifier (HEKA). The pipette solution contained (in mM): 140 cesium methanesulfonate, 2 MgCl2, 4
NaCl, 5 phosphocreatine, 10 Hepes, 2 Na2-ATP, 0.33 Na-GTP, 5
QX-314, and 0.2 EGTA, pH 7.3, osmolarity 290 mOsm/L. Patch
pipettes were pulled using a horizontal puller (Sutter Instruments)
and their resistance was 3 to 5 MΩ. The series resistance, which was
continuously monitored during the experiments using a 5-mV hyperpolarizing pulse. Recordings included for analysis were collected
during periods of stable series resistance. Recordings with series
resistance greater than 20 MΩ were discarded. Responses were
sampled at 10 kHz. A glass pipette filled with extracellular solution
(NaCl 0.9%) was used to evoke unitary NMDA EPSCs. The pipette
was placed in the stratum radiatum of the CA1 area. Stimulations
were applied at 0.05 Hz and the cells were held at +30 mV.
with Homer1c-DsRed and either SEP-NR2A or SEP-NR2B.
Clusters were imaged over a period of 20 min after acute addition of 5 μM TAT-[NS15]2 or TAT-[NR2A15]2. Fluorescence
intensity was measured using Metamorph software (Universal
Imaging) and corrected for photobleaching and background
noise.
Immunoprecipitation and Western Blot Analysis. A frozen adult rat
brain (approximately 1.6 g) was thawed in 16 mL ice cold SHC
buffer (320 mM sucrose, 1 mM Hepes, 1 mM MgCl2, 1 mM
NaHCO3) containing a protease inhibitor mixture (1:1,000;
Calbiochem) for 5 min and cut into small pieces. The tissue was
homogenized using a Teflon-glass homogenizer and the homogenate was spun at 1,000 × g for 10 min at 4 °C. The resulting
supernatant was spun at 10,000 × g for 15 min to obtain a P2
crude membrane fraction. P2 pellets were divided into aliquots
and stored at −80 °C until solubilization and immunoprecipitation reactions were performed.
P2 fractions were solubilized with RIA buffer containing 1% SDS
(200 mM NaCl, 10 mM EDTA, 10 mM Na2HPO4, 0.5% Nonidet P40, 1% SDS) for 5 min at 4 °C, followed by dilution to 0.1% SDS.
The resulting lysate (200 μg of protein) was diluted in RIA buffer
containing 0.1% SDS, followed by incubation under constant agitation at 37 °C for 15 min with ligand TAT-[NR2A15]2 (1.5 μM),
TAT-[NS15]2 (1.5 μM), or a similar volume of dH2O. NR2A (0.6 μg,
AGC-002; Alomone Labs) or NR2B (0.8 μg, polyclonal antibody
described in ref. 7) antibodies were incubated under constant agitation at 37 °C for 15 min with 10 μL of prewashed Protein A beads
(Dynabeads Protein A; Invitrogen). The lysates were added to the
antibody-bead mixtures and incubated under constant agitation
overnight at 4 °C. Immunoprecipitates were separated by SDS/
PAGE and analyzed by Western blotting using a rabbit monoclonal
antibody to NR2A (04-901, clone A12W; Millipore), a rabbit
polyclonal antibody to NR2B (A-6474; Molecular Probes), or
a mouse monoclonal antibody to PSD-95 (MA1-046; Thermo Scientific). Detection was performed using the SuperSignal West
Femto Maximum Sensitivity Substrate detection kit (Pierce), revealed with a Chemigenius system (Syngene). Quantification of
bands intensity was performed using Genetools software (Syngene).
In Vitro Cell Surface Assay. HEK 293 cells were cotransfected in
triplicate in 24-well tissue culture plates with either pCIS vector,
NR1-1a/NR2A or NR1-1a/NR2B NMDA receptors in the presence and absence of PSD-95 (2 μg of total DNA per well). Posttransfection (24 h), cells were incubated with either vehicle control, TAT-[NR2A15]2 or control TAT-[NS15]2 (10 μM) for 1 h at 37
°C. Cell surface NMDA receptor expression was determined by
ELISA with affinity-purified antibodies directed against extracellular epitopes of NR2A and NR2B, i.e., anti-NR2A 44–58 Cys
(0.25 μg/mL) or anti-NR2B 46–60 Cys (0.5 μg/mL).
FRAP and Follow-Up of Cluster Fluorescence Intensity. SEP-NR1 and
Homer1c-DsRed cotransfected neurons were placed on the
heated stage (37 °C) of an inverted confocal spinning-disk microscope (Leica). To test the population of surface SEP-NR1, we
used low-pH solution adjusted to pH 5.4, which quenched all of
the fluorescence indicating that SEP allows the specific visualization of surface receptors. Fluorescence was excited using a
monochromator controlled by Metamorph software (Universal
Imaging). To photobleach locally, we used a sapphire laser 488–
20 to 50% power to avoid photo damage. The laser was coupled
to the microscope via a galvometric mirror, which allowed us to
photobleach several regions within a short time window. Recovery from photobleaching was monitored by two consecutive
acquisition periods at 2 and 0.5 Hz acquisition rates, respectively.
Recovery curves were corrected for continuous photobleaching
and background noise. For the follow-up of cluster fluorescence
intensity to assess receptor content, neurons were cotransfected
Data and Statistical Analysis. The instantaneous diffusion coefficient
is reported as the median ± 25% to 75% (i.e., IQR). All of the other
group values are expressed as mean ± SEM. Comparisons between
groups for instantaneous diffusion coefficients were performed using
Mann–Whitney test (pair comparison) or Kruskal-Wallis followed by
Dunn multiple-comparison test (group comparison). All of the other
comparisons between groups were performed using parametric
statistical tests, Student t test (pair comparison), ANOVA followed
by Newman-–Keuls multiple comparison test (group comparison),
or Kolmogorov–Smirnov test (distribution comparison). Significance levels were defined as P < 0.05, P < 0.01, and P < 0.001.
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6. Tardin C, Cognet L, Bats C, Lounis B, Choquet D (2003) Direct imaging of lateral
movements of AMPA receptors inside synapses. EMBO J 22:4656–4665.
7. Groc L, et al. (2006) NMDA receptor surface mobility depends on NR2A-2B subunits.
Proc Natl Acad Sci USA 103:18769–18774.
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8. Tovar KR, Westbrook GL (2002) Mobile NMDA receptors at hippocampal synapses.
Neuron 34:255–264.
9. Zhao J, et al. (2008) Synaptic metaplasticity through NMDA receptor lateral diffusion.
J Neurosci 28:3060–3070.
10. Yang J, Chamberlain SE, Woodhall GL, Jones RS (2008) Mobility of NMDA
autoreceptors but not postsynaptic receptors at glutamate synapses in the rat
entorhinal cortex. J Physiol 586:4905–4924.
11. Groc L, Gustafsson B, Hanse E (2002) Spontaneous unitary synaptic activity in CA1
pyramidal neurons during early postnatal development: Constant contribution of
AMPA and NMDA receptors. J Neurosci 22:5552–5562.
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Fig. S1. Structure and amino acid sequence of the competing ligands. (A) Structure of the monovalent and divalent ligands (X: O for NR2A15 or NH for NS15).
(B) Sequences used for the peptide-based ligands of panel A and C-terminus residues of NMDAR subunits. φ, Dab(4-DMAP); λ, norleucine; x. . .x, PEG spacer (20
atoms; reference 01–63-0141; Novabiochem). Critical residues at positions 0 and −2 are highlighted (green for the common residues found in PSD-95 ligands,
red otherwise). (C) Fluorescence titrations of the 4-DMAP-containing ligands with recombinant GST-PSD95-1+2. (D) Peptide-based ligands characterization.
Purity was assessed by analytical reverse-phase HPLC (YMC C18, ODS-A 5/120, 250 × 4.6 mm) using a standard gradient (5% acetonitrile containing 0.1% TFA
for 5 min followed by 5–95% acetonitrile containing 0.1% TFA over 50 min in water containing 0.1% TFA at a flow rate of 1 mL min−1). All peptide-based
ligands were more than 95% pure as judged by analytical HPLC. [atR indicates retention time; bpeptide-based ligands identity was confirmed by MALDI-TOF MS
(Voyager; PerSeptive Biosystems) using DHB as a matrix in linear or reflective modes.]
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Neurons > 15 d.i.v.
A
TAT-[NS15]2
B
TAT-[NR2A15]2
synapse
75
50
25
0.002
C
D
*
0.4
0.2
MSD (μm²)
0.6
mobile
0.0
E
0.02
0.2
2
4
3
2
1
0
TAT-[NS15]2
TAT-[NR2A15]2
0
0.1
0.2
Time lag (sec)
F
75
50
TAT-[NS]2 (n = 43)
TAT-[NR2A]2 (n = 84)
P < 0.001
25
Dwe ll ti me ( s )
C u mu la ti v e f r e q .
100
NR2B-QD
TAT-[NS15]2 (n = 50)
TAT-[NR2A15]2 (n = 40)
Diffusion coefficient (μm2/s)
Diffusion coefficient
(μm2/s)
NR2A-QD
Cumulative freq.
100
2.0
*
1.0
0.0
0
0.0
0.5
1.0
1.5
NR2B diffusion coefficient (μm2/s)
Fig. S2. TAT-[NR2A15]2 increases the mobility of synaptic 2A-NMDARs and decreases the mobility of 2B-NMDARs in mature (>15 d in vitro) synapses. (A)
Representative trajectories of synaptic QDs coupled to NR2A after 10 to 20 min of incubation with TAT-[NS15]2 (Right) or TAT-[NR2A15]2 (Left). The gray regions
correspond to synaptic sites labeled with Mitotracker. (Scale bar: 500 nm.) (B) Distribution of the diffusion coefficients of synaptic trajectories (bin size, 0.0075
μm2/s). The first point of the curve corresponding to the proportion of immobile receptors shows the increase in mobility of 2A-NMDARs following incubation
with TAT-[NR2A15]2. (C) The diffusion coefficient of mobile synaptic receptors is significantly increased in the presence of TAT-[NR2A15]2 (n = 13 trajectories/
group; *P < 0.05, Mann–Whitney test). (D) Plot of the MSD (in μm2) versus time lag (in s) for synaptic 2A-NMDARs in presence of TAT-[NS15]2 or TAT-[NR2A15]2.
(E) Distribution of the diffusion coefficients of NR2B synaptic trajectories. Note the significant shift toward lower values. (F) The synaptic 2B-NMDAR dwell time
was significantly increased in presence of TAT-[NR2A15]2.
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C
B
dendrite
0
10
20
30
25
0
40
TAT-[NR2A15]2
0
50
TAT-[NS15]2
0.5
TAT-[NR2A15]2
75
% immobile
Fluorescence(relat.)
SEP-NR1
***
100
synaptic
1.0
TAT-]NS15]2
A
Time (s)
D
TAT [NS15]2
TAT-[NS
TAT [NR2A15]2
TAT-[NR2A
E
PSD-95
Cumulative frequency
100
50
TAT-[NS15]2
TAT-[NR2A15]2
0
Surface NR2A
0
5
Fluo. intensity (a.u.)
10
Fig. S3. Acute disruption of the interaction between 2A-NMDARs and PDZ proteins increases the surface diffusion of surface NR1-NMDARs and decreases the
synaptic content of NR2A subunits. (A and B) Clusters of SEP-NR1 were photobleached and the recovery of the fluorescence was followed over a period of 50 s
and expressed as recovery versus time on synaptic and dendrite areas. Note the higher proportion of immobile receptors within the synaptic compartment
compared with the dendrite. (C) The average immobile fraction of synaptic and dendritic NMDARs are quantified after incubation with TAT-[NS15]2 (n = 52
synaptic clusters; n = 21 dendritic clusters) or TAT-[NR2A15]2 (n = 61 synaptic clusters; n = 20 dendritic clusters). Note the specific decrease of the proportion of
immobile receptors within the synaptic area compared with the dendrite (***P < 0.001, t test). (D) Immunostaining of PSD-95 (synapse) and surface NR2A
subunits in the presence of TAT-[NR2A15]2 or TAT-[NS15]2. Arrowheads represent synapses, i.e., PSD-95 cluster. (Scale bar: 5 μm.) (E) Fluorescence intensity of
surface NR2A subunits in synapses (TAT-[NR2A15]2, n = 1,684 synapses; TAT-[NS15]2, n = 1,981).
Fig. S4. TAT-NR2B15 ligand specifically alters the surface diffusion of 2B-NMDARs without affecting the one of 2A-NMDARs. (A) Distribution of the diffusion
coefficients of synaptic surface NR2B-NMDARs in presence of the nonsense or TAT-NR2B15 ligand (5 μM, 10–15 min). Note the significant shift of the distributions in presence of TAT-NR2B15, consistent with an increase diffusion (nonsense, n = 57; TAT-NR2B15, n = 99 trajectories; P < 0.001). (B) Distribution of the
diffusion coefficients of synaptic surface NR2A-NMDARs in presence of the nonsense or TAT-NR2B15 ligand (5 μM, 10–15 min). No significant effect was observed (nonsense, n = 71; TAT-NR2B15, n = 68 trajectories; P > 0.05).
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A
TAT-NR2A15
TAT-NR2X15
B
TAT-[NS15]2
TAT-NR2X15
(5 μM)
C
synapse
Cumu lative f req u e n c y
NR2A-QD
100
80
60
40
TAT-[NS15]2
TAT-NR2X15
20
0
0.01
0.1
1
2
Diffusion coefficient (μm /s)
E
D
Cumulative frequency
NR2B-QD
100
80
60
40
TAT [NS15]2
TAT-
20
TAT-NR2X15
0
0.01
0.1
1
Diffusion coefficient (μm2/s)
Fig. S5. TAT-NR2X15 increases in a nonspecific manner the mobility of synaptic 2A- and 2B-NMDARs. (A) Comparison of TAT-NR2X15 and TAT-NR2A15 ligands.
The TAT-NR2X15 ligand comprises the last six residues of the C terminus of both NR2A and NR2B subunits (−SIESDV), a linker composed of PEG and glycine
residues that provides a neutral backbone preventing any specific amino acid side chain interactions while maintaining the TAT sequence at a similar distance
to that used for the other NR2A- and NR2B-derived ligands. (B) Representative trajectories of synaptic QDs coupled to NR2A after 10 to 20 min of incubation
with TAT-[NS15]2 (Left) or TAT-NR2X15 (Right). The green regions correspond to synaptic sites labeled with Mitotracker. (Scale bar: 500 nm.) (C) Cumulative
distribution of the diffusion coefficients of synaptic trajectories (bin size, 0.0075 μm2/s). The first point of the curve corresponds to the proportion of immobile
receptors. Note the significant increase mobility of 2A-NMDARs following incubation with TAT-NR2X15 ligand (TAT-[NS15]2, n = 54; TAT-NR2X15, n = 105
trajectories; P < 0.001). (D) Representative trajectories of synaptic QDs coupled to NR2B after 10 to 20 min of incubation with TAT-[NS15]2 (Left) or TAT-NR2X15
Legend continued on following page
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(Right). The green regions correspond to synaptic sites labeled with Mitotracker. (Scale bar: 500 nm.) (E) Cumulative distribution of the diffusion coefficients of
synaptic trajectories (bin size, 0.0075 μm2/s). The first point of the curve corresponds to the proportion of immobile receptors. Note the significant increase
mobility of 2B-NMDARs following incubation with TAT-NR2X15 ligand (TAT-[NS15]2, n = 108; TAT-NR2X15, n = 47 trajectories; P < 0.01).
Sequence C-term
Name
NR2B [rat]
-LNSCS NRRVY KKMPS IESDV-COOH
-FNGSS NGHVY EKLSS IESDV-COOH
Kv1.3 [rat]
- CTTNN NPNSC VNIKK IFTDV-COOH
NR2A [rat]
QD
Kv1.3
B
Kv1.3 MSD (μm²)
A
1.0
0.8
0.6
0.4
TAT-[NS15]2
TAT
TAT-[NR2A15]2
0.2
0
0
0.50
1
1.5
Time lag (sec)
D
Kv1.3 % mobile
100
80
60
40
20
0
F
E
0.5
0.4
0.3
0.2
0.1
0
TAT-[NS15]2
Kv1.3 surf. distribution
(% of particule)
C
Kv1.3 diff. coef. (μm²/s)
C-term
100 syn./peri extrasyn.
80
60
40
20
0
TAT-[NR2A15]2
QD-GABAA
QD
α2-GABAA
Fig. S6. Impact of TAT-[NR2A15]2 on potassium Kv1.3 channel and GABAA receptor surface trafficking. (A) Amino acid sequences of NR2A, NR2B, and Kv1.3
C-termini (Rattus norvegicus). Note the high similarity among the three sequences in the last 5 aa, in the known PDZ binding site. The tracking of endogenous
Kv1.3 potassium channel was done by detecting an extracellular epitope of the channel using an antibody-QD complex (schematic representation, Lower). For
the experiments below, we analyzed 116 trajectories in presence TAT-[NS15]2 and 209 trajectories in presence TAT-[NR2A15]2. (B) Plot of the MSD (in μm2) versus
time lag (in s) of surface Kv1.3 channel in presence of TAT-[NS15]2 or TAT-[NR2A15]2. No significant effect (P > 0.05). (C) The percent of mobile surface Kv1.3
channel remain statistically similar in presence of TAT-[NS15]2 or TAT-[NR2A15]2 (P > 0.05). (D) The distributions (median, 25–75% range) and medians of Kv
diffusion coefficient were not significantly changed in presence of TAT-[NS15]2 or TAT-[NR2A15]2 (P > 0.05). (E) The surface distribution of Kv1.3 channels was
examined using single particle live distribution. The Kv channels are mostly extrasynaptic (approximately 80%). Incubations with TAT-[NS15]2 (n = 6 dendritic
fields) or TAT-[NR2A15]2 (n = 10 dendritic fields) did not affect the Kv1.3 channel surface distributions. (F) GABAA receptor surface diffusion was assessed using
QDs coupled to an antibody directed against the α2 subunit of the receptor. Representative trajectories of single QDs show no effect on the mobility of GABAA
receptors after 10 to 20 min of incubation with TAT-[NS15]2 or TAT-[NR2A15]2. (Scale bar: 1 μm.)
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Fig. S7. TAT-[NR2A15]2 specifically blocks the interaction between PSD-95 and NR2A subunit in rat brain homogenates. (A) Representative gels of co-IP of PSD95 with NR2 subunits in rat forebrain fractions treated with ligands. (B) TAT-[NR2A15]2 reduced the OD ratio of PSD-95:NR2A whereas induced no significant
effect on OD ratio of PSD-95:NR2B. Values are means ± SEM (n = 5 for TAT-[NS15]2 and n = 8 for control and TAT-[NR2A15]2). Of note, although the specific
effect of the divalent ligand could be monitored in brain lysates with solubilized protein complexes, we also observed for this approach a high sensitivity with
respect to the nature of the detergents used in particular with the existence of a fine balance between conditions allowing for co-IP of the protein complexes
of interest and conditions compatible with ligand-induced competition.
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A
B
NMDAR mEPSCs
20 pA
15-20 min [NR2A15]2
5 pA
0-5 min
+ AP-5
C
30
50 ms
50 ms
[NR2A15]2
[NS15]2
D
40
0
20
10
0
20 pA
15-20 min [NR2A15]2
100 ms
Amplitude (pA)
F
0-5 min [NR2A15]2
20
10
0
0 5 15-20
0-5
15 20
0 5 15
0-5
15-20
20
Time (min)
E
30
0 5 15-20
0-5
15 20
0 5 15
0-5
15-20
20
Time (min)
40
20
Freque
ency (Hz)
**
10
Ampl. (pA)
20
1
(ms)
30
30
20
10
0
0-5 15-20
Time (min)
15
10
5
0
0-5 15-20
Time (min)
Fig. S8. The [NR2A15]2 functionally impacts only on miniature synaptic NMDAR currents. (A) Representative recordings of NMDAR mEPSCs at +30mV in the
presence of Ca2+/Mg2+ (2/2 mM), NBQX (10 μM; AMPAR antagonist), and bicuculline (20 μM; GABAA antagonist) in the whole cell configuration. The addition of
AP-5 (25 μM) into the bath solution rapidly abolished NMDAR mEPSCs (Lower). The NMDAR mEPSC frequency range between 0.2 and 0.9 Hz, indicating that, on
average, one event was detected every 2 s and overlap between events (example, Upper) was rare. (B) Averaged traces of NMDAR mEPSCs for two different
time intervals: 0 to 5 min (black) and 15 to 20 min (gray) in the presence of [NR2A15]2 (5 μM) in the pipette solution. Note the slower decay after 15 to 20 min
infusion compared with 0 to 5 min. (C) Time constants, τ1, of the two time intervals (0–5 and 15–20 min). The decay was fitted using an exponential fit with
a fast τ1 and a slower τ2 components. In the presence of [NR2A15]2 (n = 6 neurons) τ1 was significantly increased (**P < 0.01, paired t test) whereas no effect was
observed in the presence of [NS15]2 (n = 5 neurons; P > 0.05, paired t test). The same results were obtained with the slower τ2 component. (D) Amplitude of
NMDAR mEPSCs for the two time intervals (0–5 and 15–20 min) in the presence of [NR2A15]2 or [NS15]2 (P > 0.05, paired t test). (E) Representative traces of
AMPAR mEPSCs recorded at −50 mV and isolated with 1 μM TTX, 20 μM bicuculline, and 25 μM AP5 during two time intervals: 0 to 5 min (Upper) and 15 to 20
min (Lower) after whole cell configuration. (F) Neither the amplitude nor the frequency of AMPAR mEPSCs was affected by [NR2A15]2 (n = 6 neurons).
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A
Subunit
NR2A15
NR2B15
NR2X15
Monovalent
Monovalent
Divalent
Monovalent
NR2A
+
+++
-
+
NR2B
-
-
+
+
NR2AS1462A
-
GABAA
-
Kv1.3
-
(-) No detectable change in diff.
(+) Higher diffusion
(+++) Very high diffusion
B
Fig. S9. Working model of TAT-[NR2A15]2. (A) Summary table of the impact of NR2-derived ligands on the synaptic diffusion of various surface subunits. (B) In
basal conditions, 2A-NMDARs interact with MAGUK proteins like PSD-95 through their C-terminal PDZ binding domain. This interaction strongly retains 2ANMDARs within the synapse. The divalent ligand (TAT-)[NR2A15]2 containing two binding motifs competes with 2A-NMDARs for the binding to specific interactors. This highly specific competition induces a destabilization of 2A-NMDARs.
Fig. S10. Transduction of the BODIPY-TAT-[NR2A15]2 ligand in hippocampal cultured neurons. (A) Neurons were incubated for 5 min with 50 μM pyrene
butyrate and for 10 min with 5 μM Bodipy-TAT-[NR2A15]2. Note the high transduction efficiency of the ligand within almost all processes. (Scale bar: 10 μm.) (B)
Colocalization of TAT-[NR2A15]2 ligand with the postsynaptic marker shank. Neurons were incubated with 50 μM pyrene butyrate followed by 10 min with 5
μM TAT-[NR2A15]2. After a 20-min wash, cells were fixed in PFA 4%/sucrose 4% in PBS solution for 15 min. They were then permeabilized using Triton 0.1% in
PBS solution. Neurons were incubated for 30 min with a rabbit anti-shank 3 antibody and for 30 min with a secondary anti-rabbit antibody coupled to an Alexa
568. (C) Consistent with the use of a saturating ligand concentration, no significant synaptic enrichment of both ligands was observed (TAT-[NS15]2, n = 939
synapses; TAT-[NR2A15]2, n = 426 synapses).
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Syn.
QD-NR2 Ab
< 0.2 μg/ml
10-15 min
Mitotracker
20 nM
Pyr. But.
TAT-peptide
Pyrene
Butyrate
50 μM
1-5 μM
8.10-3
Diffusion coefficient
(µm2/s)
QD-NR2A Ab
4.10-3
0
500 nm
QD-Ab
alone
QD-Ab
+ Py.Bu.
QD-Ab
+ Py.Bu.
+ TAT-[NS15]2
Fig. S11. Effect of pyrene butyrate and TAT peptide on basal NR-NMDAR surface diffusion. The nonspecific effects of pyrene butyrate or TAT-[NS15]2 was
tested on the mobility of 2A-NMDARs. Whereas pyrene butyrate had no effect per se, TAT-[NS15]2 increased the proportion of immobile receptors. Such effect
of TAT-[NS15]2 was observed on 2B-NMDARs, GluR2-AMPARs, GABAARs, and reproduce using the monomeric TAT-[NS15]. This indicates that TAT-NS slightly
reduced the surface trafficking of neurotransmitter receptors, irrespective of the nature of the receptors or the structure of the TAT ligand.
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