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The Journal of Neuroscience, August 15, 1999, 19(16):6844-6854
Subtype-Dependence of NMDA Receptor Channel Open
Probability
Nansheng
Chen1,
Tao
Luo1, and
Lynn A.
Raymond1, 2, 3
1 Kinsmen Laboratory of Neurological Research,
Department of Psychiatry, 2 Department of Physiology, and
3 Division of Neurology, Department of Medicine, University
of British Columbia, Vancouver, British Columbia V6T 1Z3 Canada
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ABSTRACT |
NMDA receptor-mediated calcium transients play a critical
role in synaptogenesis, synaptic plasticity, and excitotoxicity. NMDA
receptors are heteromeric complexes of NR1A combined with NR2A, NR2B,
NR2C, and/or NR2D subunits. The NR2 subunits determine a variety of
electrophysiological and pharmacological properties of the NMDA
receptor complex. In this report, we provide evidence for the first
time that there is also a significant difference in peak channel open
probability (Po) between NMDA
receptors composed of NR1A/NR2A and those of NR1A/NR2B subunits. First,
whole-cell patch-clamp recordings from human embryonic kidney (HEK) 293 cells expressing NMDA receptors revealed that NR1A/NR2A-mediated peak current densities are approximately four times larger than those of
NR1A/NR2B. We show that this fourfold difference is unlikely caused by
differences in receptor surface expression, since these levels were
similar for the two subtypes by Western blot analysis. To determine
whether Po contributed to the difference in
peak current densities, we used two different open channel antagonists, MK-801 and 9-aminoacridine, in a variety of experimental paradigms. Our
results indicate that peak Po is
significantly higher (twofold to fivefold) for NR1A/NR2A than
NR1A/NR2B, with estimated values of ~0.35 and 0.07, respectively.
These results suggest that a change in the relative expression levels
of NR2A and NR2B can regulate peak amplitude of NMDA receptor-mediated
excitatory postsynaptic potentials and therefore may play a role in
mechanisms underlying synaptic plasticity.
Key words:
patch-clamp recording; transient transfection; recombinant receptors; use-dependent block; tail current; charge
transfer
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INTRODUCTION |
NMDA receptors have been the focus
of extensive investigation because of their central role in a number of
physiological processes, such as synaptogenesis and synaptic
plasticity, as well as pathological conditions, including Huntington's
disease and ischemic stroke (for review, see Chen et al., 1999 ;
Dingledine et al., 1999). Recent evidence indicates NMDA receptors are
heteromeric complexes of NR1A and the NR2A-NR2D subunits, resulting in
a tetrameric or pentameric complex (Dingledine et al., 1999).
Heterologous expression of NR1A with any one of the NR2 subunits yields
a recombinant receptor with remarkably unique pharmacological and
biophysical properties (Sucher et al., 1996). Since NR2 subunit
expression varies across different brain regions and neuronal types, as
well as with developmental stage (Monyer et al., 1994; Sheng et al., 1994), function and modulation of NMDA receptors are highly regulated both spatially and temporally.
A key determinant of the amplitude and spatial distribution of NMDA
receptor-mediated calcium transients is the channel open probability
(Po). Interestingly, this channel
property has been shown to be modified by a variety of physiological
processes, including phosphorylation/dephosphorylation (Kohr and
Seeburg, 1996; Wang et al., 1996; Lu et al., 1998), the polymerization state of the actin cytoskeleton (Rosenmund and Wesbrook, 1993), and
receptor interactions with calcium/calmodulin (Ehlers et al., 1996;
Zhang et al., 1998; Krupp et al., 1999). In addition,
Po can be altered by extracellular
agents that reduce or oxidize the disulfide bonds of the
receptor (Brimecombe et al., 1997). However, in each of these
reports, Po measurements were
relative, and no absolute numbers were given because of the difficulty
in determining the number of channels in the membrane during
single-channel recording.
Huettner and Bean (1988) were the first to report a quantitative value
for neuronal NMDA receptor Po. They
used MK-801, a slowly reversible open channel antagonist, to determine
a Po of 0.002 from whole-cell
recordings of NMDA-evoked currents in cultured rat neocortical neurons.
However, subsequent studies using MK-801 have reported widely differing
results for Po, up to as high as 0.3 (Jahr, 1992;
Hessler et al., 1993; Rosenmund et al., 1993, 1995). Dzubay and Jahr
(1996) have argued that some of this variability may be attributable to
differences in experimental preparation and protocol, which may affect
NMDA receptor inactivation. As well, two studies have shown that
measurements of neuronal NMDA receptor Po made
from recordings in the outside-out patch mode are significantly larger
than those made in the whole-cell mode (Benveniste and Mayer,
1995; Rosenmund et al., 1995).
We propose that NMDA receptor subunit composition may play an important
role in determining Po. To test this
idea, we assessed the relative Po for NR1A/NR2A
and NR1A/NR2B expressed in human embryonic kidney (HEK) 293 cells. Because we observed a fourfold difference in peak macroscopic
whole-cell current between these receptor subtypes, our experimental
protocols and data analysis were designed to assess
Po at the peak of the current response (i.e., maximal Po) in the whole-cell
recording mode. From our results we conclude that peak
Po for NR1A/NR2A is twofold to
fivefold higher than that for NR1A/NR2B.
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MATERIALS AND METHODS |
Cell culture and transfection. Culture and
transfection of HEK 293 cells (CRL 1573; ATCC, Rockville, MD) were as
described previously (Chen et al., 1997). Briefly, HEK 293 cells were
routinely maintained in incubators set at 37°C and 5%
CO2, with culture media prepared from minimum
essential medium (Life Technologies, Burlington, Ontario,
Canada), containing Earle's salts (Life Technologies), supplemented
with 2 mM L-glutamine, 1 mM sodium
pyruvate (Life Technologies), 100 U/ml penicillin/streptomycin (Life
Technologies), and 10% fetal bovine serum (Hyclone, Logan, UT). Cells
were passaged once every 2-4 d. For calcium phosphate transfection
(Chen and Okayama, 1987), cells were plated at a density of
1 × 106 cells/ml in 10 cm culture
dishes (Falcon; Becton Dickinson, Franklin Lakes, NJ). For the purpose
of this project, cells were transfected with cDNAs encoding NR1A [a
gift from Dr. S. Nakanishi, Kyoto University, Kyoto, Japan;
nomenclature of Sugihara et al. (1992); also known as NR1A-1a (Hollmann
et al., 1993)], NR2A (from mouse brain, also called  1, a gift from
Dr. M. Mishina, University of Tokyo, Tokyo, Japan) or NR2B (from Dr. S. Nakanishi), and  -galactosidase (-gal) at a ratio of 1:1:1. A
total of 12 µg of plasmid cDNA was used for transfection of a 10 cm
culture plate. The transfection efficiency was assessed using the
-gal staining procedure (Raymond et al., 1996). HEK 293 cells were
transfected for ~8 hr in a 3% CO2 incubator.
After transfection, 1 mM (±)-2-amino-5-phosphonopentanoic acid (APV; Research Biochemicals, Natick, MA) and/or 100 µM memantine (Research Biochemicals) were added
to the culture media, and the cells were transferred onto glass
coverslips in 35 mm culture plates (Falcon). For Western blot analysis,
cells were replated in 10 cm culture dishes precoated with 10 µg/ml
poly-D-lysine.
Electrophysiology. The whole-cell patch clamp recording
technique and recording solutions were essentially the same as
previously described (Chen et al., 1997). At 24-36 hr after the start
of transfection, the cells were transferred from 35 mm culture plates (Falcon) to the recording chamber on the stage of an inverted microscope (Aviovert 100; Carl Zeiss, Thornburg, NY). Agonist-evoked currents were recorded in the whole-cell mode under voltage clamp (VH =  60 mV). Recording pipettes
were pulled from borosilicate glass (Warner Instruments, Hamden, CT)
with the Narishige (Tokyo, Japan) PP-83 electrode puller. Electrodes
with open tip resistances of 1-5 M were used. After establishing
the whole-cell mode, cells were lifted from the coverslip. Ultra-fast
application of agonists was achieved by a piezo-driven  -tube
(Hilgenburg, Malsfeld, Germany) (see also, Chen et al., 1997). Control
and agonist solutions were continuously gravity-fed through the two
sides of the -tube. The 10-90% rise time for solution exchange was
0.5-0.8 msec (Fig. 1A1)
at the open tip of a recording electrode and 3.0-3.9 msec (Fig.
1A2) for the whole-cell response,
as measured by jumps between NaCl- and N-methylglucamine
(NMG)-containing extracellular recording solutions. Extracellular
recording solution contained (in mM) 145 NaCl,
5.4 KCl, 0.2 CaCl2, 11 glucose, and 10 HEPES,
titrated to pH 7.35 with 10 M NaOH. In all
experiments, 50 µM glycine was added to both
control and glutamate-containing extracellular solutions. Glutamate,
glycine, MK-801 (Research Biochemicals), and 9-aminoacridine (Aldrich,
Milwaukee, WI) stock solutions were stored at 20°C; aliquots were
thawed on ice for each day's experiment and diluted into the
extracellular recording solution just before use. The intracellular
recording solution contained (in mM) 145 KCl, 5.5 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic
acid, 4 MgATP, and 10 HEPES, titrated to pH 7.25 with KOH.
Currents were sampled at 2 kHz and acquired and analyzed using pClamp
software and the Axopatch 200A amplifier (Axon Instruments, Foster
City, CA). Current amplitude measurement and kinetics fitting were
conducted with Clampfit software.
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Figure 1.
Peak current density of agonist-evoked responses
is larger for recordings from cells transfected with NR1A/NR2A than
NR1A/NR2B. A1,
A2, Representative traces of open tip
recording (A1) or whole-cell current
(A2) made by switching between
NMG-containing and NaCl-containing extracellular solutions.
B, Representative current traces in response to 20 msec
application of 1 mM glutamate (in continuous presence
of 50 µM glycine) recorded from a cell transfected
with either NR1A/NR2A (thick line) or NR1A/NR2B
(thin line). C, NR1A/NR2B current trace
(thin line) normalized to the peak of an NR1A/NR2A
current trace (thick line) to illustrate difference in
activation time courses. D, Bars show mean ± SEM for
10-90% rise-time (n = 10 for both subtypes;
**p < 0.01 by unpaired t test).
E, Mean ± SEM for 10-90% decay time
(n = 10 for both subtypes; **p < 0.01 by unpaired t test). F, Bars
indicate mean ± SEM for current responses to 100 µM
glutamate (in the continuous presence of 50 µM
glycine) recorded from n = 60 (NR1A/NR2A) or
n = 106 (NR1A/NR2B) different cells.
**p < 0.001 by unpaired t
test.
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Western blot analysis. The analyses of overall NMDA receptor
expression level and surface expression level were carried out essentially as described in Chen et al. (1999). HEK 293 cells transfected with NR1A/NR2A or NR1A/NR2B were washed twice with warm PBS
and incubated with the membrane-impermeable reagent
N-hydroxysuccinimide-SS-biotin (NHS-SS-biotin; Pierce,
Rockford, IL) at a concentration of 1.5 mg/ml in PBS containing
Mg2+/Ca2+ for
30 min at 4°C with mild shaking. The cells were washed twice with
Mg2+/Ca2+-containing
PBS supplemented with 100 mM glycine, and
incubated with this solution for 30 min in order to remove any
NHS-SS-biotin not bound to protein. After washing the cells with
Mg2+/Ca2+-containing
PBS three times, cells were scraped with 1 ml of ice-cold harvest
buffer (1 mM EGTA, 1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, and 100 U/ml aprotonin in PBS) into eppendorf tubes prechilled on ice,
sonicated (10 sec), and centrifuged (14,000 rpm for 30 min at 4°C).
The precipitates, which correspond to the membrane fraction, were
redissolved by sonication (10 sec) in solubilization buffer (harvest
buffer with 1% Triton X-100) followed by end-over-end mixing for 30 min in 4°C cold room. After centrifugation (14,000 rpm, 30 min at
4°C), supernatants were saved and stored at 80°C for further
analysis. Protein concentrations were determined by bicinchoninic acid
protein assay (BCA kit; Pierce).
For analysis of NMDA receptor overall expression, identical amounts (1, 2, and 4 µg) of protein from the membrane fraction of each cell
lysate were loaded to 8% SDS-PAGE. For the assessment of surface
receptor expression level, the remainder of each cell lysate was
incubated with 100 µl (spun down from 200 µl suspension) neutravidin-linked beads (Pierce) by end-over-end rotation for 2 hr at
4°C. Beads were extensively centrifuged and washed to isolate
bead-bound proteins. These proteins were eluted by incubating the beads
with dithiothreitol-containing SDS-PAGE loading buffer and loaded to
8% SDS-PAGE.
After overnight transfer of gels to PVDF membranes, the membranes were
blotted with anti-NR1A polyclonal antibodies (Upstate Biotechnology,
Lake Placid, NY) at 1 µg/ml concentration. The membranes were then
incubated with horseradish peroxidase-conjugated donkey anti-rabbit
secondary antibody (Amersham, Arlington Heights, IL) diluted 1:5000.
Bands were visualized by enhanced chemiluminesence (ECL; Amersham).
Band intensities were determined by densitometry, and protein
quantity-band density relation standard curves were generated from
measurements of bands representing the 1, 2, and 4 µg aliquots of the
total membrane lysates. Surface protein expression level was calculated
as a percentage of total membrane protein.
Materials. All chemicals, unless otherwise stated, were
purchased from Sigma (St. Louis, MO)
Data analysis and presentation. Results were presented as
mean ± SEM. Different sets of results were compared using the
Student's t test. Significant differences were determined
at 95% confidence intervals. Figures were created with Origin or
Photoshop software.
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RESULTS |
To determine the relative channel open probability of NMDA
receptor subtypes, we have compared macroscopic currents recorded from
HEK 293 cells transfected with either NR1A/NR2A or NR1A/NR2B. We chose
to study peak Po using the whole-cell
recording mode because this configuration preserves the integrity of
cytoskeleton and membrane-associated proteins better than the
outside-out patch mode, and therefore may better reflect the
physiological state. Morevoer, ultrafast superfusion of agonists and
antagonists can be achieved for whole-cell recordings from HEK 293 cells by lifting cells from the chamber floor to within 100 µm of the
openings of the piezo-controlled application -tube (see Materials
and Methods; exchange time at the open tip of a recording electrode is
illustrated in Fig. 1A). The relation between the macroscopic NMDA
receptor-mediated peak current amplitude (I
peak) and peak Po is given by:
therefore,
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(1)
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(2)
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where i is the unitary current and N is the
number of functional surface receptors. Single-channel conductances
have been shown to be identical (with a main conductance of 50 pS and a subconductance of 38 pS) for these two subtypes of NMDA receptors expressed in HEK 293 cells (Stern et al., 1992, 1995). Therefore, if
relative I peak and N are
measured, then the relative Po can be determined.
Macroscopic currents mediated by NR1A/NR2A are significantly larger
than those of NR1A/NR2B
After transient transfection with either NR1A/NR2A or NR1A/NR2B,
whole-cell currents were recorded under voltage-clamp (60 mV) from
HEK 293 cells in response to brief (20 msec) applications of a
saturating concentration of agonist (Fig. 1B). As
described previously (Monyer et al., 1992, 1994; McBain and Mayer,
1994; Vicini et al., 1998; Chen et al., 1999), NR1A/NR2A and NR1A/NR2B mediate currents with dramatic kinetic differences. The 10-90% rise
time was significantly slower for NR1A/NR2B- than NR1A/NR2A-mediated currents, although this difference was small (6.4 ± 0.2 msec, n = 10 for NR1A/NR2A; 10.0 ± 0.4 msec, n = 10 for NR1A/NR2B; p < 0.01 by unpaired t
test; Fig. 1C,D). As well, the time courses of
desensitization, inactivation, and deactivation of NR1A/NR2A currents
were significantly faster than those of NR1A/NR2B (Krupp et al., 1996;
Chen et al., 1997, 1999; see Fig.
1B,E for deactivation). Interestingly, mean peak current amplitude was approximately fourfold larger for NR1A/NR2A-transfected cells (data not shown). Because cell
surface membrane area can affect numbers of surface receptors, we
normalized peak current amplitude to cell capacitance (current density,
picoamperes per picofarads). Mean peak current density was also
approximately fourfold larger for NR1A/NR2A-transfected cells (Fig.
1F; 543 ± 56 pA/pF, n = 60 for
NR1A/NR2A vs 130 ± 9 pA/pF, n = 106 for NR1A/NR2B;
p  0.01, unpaired t test). Therefore, the ratio
of mean Ipeak for NR1A/NR2A to that for
NR1A/NR2B was ~4. Based on this ratio, we reasoned that peak channel
open probability and/or receptor surface expression level must be
significantly higher for NR1A/NR2A than NR1A/NR2B.
NR1A/NR2A- and NR1A/NR2B-transfected cells show similar total and
surface expression of NR1A
To investigate the relative mean numbers of surface receptors for
NR1A/NR2A- and NR1A/NR2B-transfected cells, we compared total and
surface expression of these receptors in the membrane fraction of
transfected HEK 293 cell lysates by Western blot analysis. Assuming
that the two subtypes share identical stoichiometry (see Discussion),
we used anti-NR1A polyclonal antibodies to probe the expression of both
NR1A/NR2A and NR1A/NR2B. Figure 2 shows representative Western blots and the pooled data from four different experiments. Although total expression of NR1A was approximately twofold higher in NR1A/NR2B- than NR1A/NR2A-transfected cells (Fig.
2A,C), the fraction of NR1A
expressed at the cell surface was twofold higher for
NR1A/NR2A-transfected cells (Fig. 2A,B). Thus,
relative NR1A surface expression levels for these two subtypes were not
significantly different (Fig. 2D; p = 0.12, paired t test). Assuming that NR1A expression levels
accurately reflect numbers of NR1A/NR2A and NR1A/NR2B receptors (see
Discussion), then mean numbers of surface receptors for NR1A/NR2A- and
NR1A/NR2B-transfected cells were very similar. These data taken
together with the results from analysis of peak macroscopic current
densities suggested that Po
(NR1A/NR2A)/Po (NR1A/NR2B) should be
approximately 4. In order to test this conclusion more directly, we
examined the relative peak Po of
NR1A/NR2A and NR1A/NR2B using two use-dependent antagonists: MK-801 and
9-aminoacridine.
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Figure 2.
Cells transfected with NR1A/NR2A or NR1A/NR2B show
similar levels of NR1A surface expression. A,
Representative Western blot of membrane fraction of lysates from
NR1A/NR2A- and NR1A/NR2B-transfected cells probed with anti-NR1A
antibody. 1, 2, and 4
represent micrograms of protein loaded in each lane;
Surface represents neutravidin bead-precipitated
receptors. B-D, Band intensities from Western blots
were analyzed by densitometry, and bars represent mean ± SEM from n = 4 experiments. In B,
the fraction of total NR1A protein expressed at the cell surface in
each experiment was calculated by comparing "surface" band
intensity to band intensities in lanes loaded with total membrane
protein (1, 2, and
4). *p < 0.05 by unpaired
t test. In C, band intensities measured
for lanes 1, 2, and 4 of NR1A/NR2B cell
lysates in each experiment were normalized to the values measured for
lanes loaded with equal amounts of membrane protein from
NR1A/NR2A-transfected cells. *p < 0.05 by paired
t test. In D, the fraction of total
NR1A/NR2B expressed at the cell surface in each experiment (see
B) was normalized for the difference in relative total
receptor expression of NR1A/NR2A and NR1A/NR2B (see
C).
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MK-801 block indicates peak Po is significantly larger
for NR1A/NR2A than NR1A/NR2B
MK-801 is a NMDA receptor open channel blocker that is
irreversible on a time scale of tens of minutes at hyperpolarized
potentials, making it a useful tool for estimating
Po (Huettner and Bean, 1988; Jahr,
1992; Rosenmund et al., 1995; Dzubay and Jahr, 1996). Therefore, we
recorded whole-cell NMDA receptor-mediated currents from transfected
HEK 293 cells in the absence and presence of MK-801. For these
experiments, the extracellular solution contained reduced calcium (0.2 mM) in order to minimize calcium-dependent inactivation and rundown (Jahr 1992; Legendre et al., 1993; Rosenmund and Westbrook, 1993; Ehlers et al., 1996; Krupp et al., 1998; Zhang et
al., 1998; Price et al., 1999). As mentioned earlier, a 20 msec agonist
pulse (1 mM glutamate and 50 µM glycine) evoked a fast inward current, with
a 10-90% rise-time < 10 msec, which deactivated rapidly
(although this decay phase was considerably slower for NR1A/NR2B; Fig.
1B,E). Such a protocol was applied every 20 sec until the peak current amplitude was stable, as defined by at least
five consecutive current responses with less than ± 2%
variability in peak amplitude. Then, following a protocol used by Jahr
(1992), 20 µM MK-801 was added to both the
control and agonist solutions, and a single agonist-evoked response was recorded [Fig.
3A2, trace
b, B2, trace b; peak amplitude of trace b
was normalized to the control response (trace a)] before
removing MK-801 from all solutions. For both NR1A/NR2A and NR1A/NR2B,
~60% of the peak current was blocked in the presence of an
equilibrium concentration (20 µM) of MK-801
(Fig. 3D), consistent with similar on-rates for MK-801 for
these two subtypes. Note also that for both subtypes, the time courses
of decay and charge transfer in the presence of MK-801 (Fig.
3A2,B2,
traces b, Sb, respectively) were markedly faster than in the
absence of MK-801 (Fig.
3A2,B2, traces a, Sa, respectively), indicating that
MK-801 blocked the currents not only during the peak but also during
deactivation. After extensive wash-out (2 min) of MK-801, subsequent
applications of agonist elicited currents with smaller amplitudes but
similar time courses, compared to the pre-MK-801 responses (Fig.
3A1, B1) as also
observed by Jahr (1992). After wash-out of 20 µM MK-801, both the peak and the charge
transfer in response to a single application of saturated agonist
showed a decrease of ~70% compared to the control response for both
subtypes (for NR1A/NR2A, 64±1%, n = 4; for NR1A/NR2B,
71±3%, n = 4, p = 0.25, unpaired
t test; Fig.
3A1,B1,C). These results are
similar to those found for neuronal NMDA receptors (Jahr, 1992) and
indicate that total Po for the peak
plus the deactivation phase of the current response is similar for the
two subtypes.
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Figure 3.
Peak current and decay phase attenuated in
continuous presence of MK-801. A1,
B1, Agonist-evoked current before
(Control) and after (Wash) the
application of 20 µM MK-801 in both agonist and control
solutions, recorded from an NR1A/NR2A (A1)-
or an NR1A/NR2B (B1)-transfected cell under
voltage-clamp configuration (60 mV). A2,
B2, Agonist-evoked current in the
absence (trace a) or in the presence (trace
b) of MK-801 in both solutions was recorded from an NR1A/NR2A
(A2)- or an NR1A/NR2B
(B2)-transfected cell. Peak current
in the presence of MK-801 (b) was normalized to
that in the absence of antagonist (a). Cumulative
charge transfer time course curves (trace Sa and
trace Sb) were obtained by integrating corresponding
traces (a and b, respectively) over time.
Fifty micromolar glycine was included in all solutions.
C, Bars show mean ± SEM for fraction of total charge
transfer remaining after wash-out of 20 µM MK801
(n = 5 for NR1A/NR2A and n = 6 for
NR1A/NR2B; p = 0.23, unpaired t test).
D, Mean ± SEM for peak current measured in the presence
of MK-801 normalized to that measured under control conditions
(n = 4 for both subtypes; p = 0.15 by
unpaired t test). The circles at the top
of the figure illustrate the tip of the piezo-driven tube (see
Materials and Methods) and the solutions used for recording:
C, Control; G, 1 mM
glutamate; M, MK-801. Extracellular solution contained
0.2 mM calcium.
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To estimate Po for the peak current,
we followed the analysis used by Jahr (1992). Since the peak current
response occurred during the 20 msec agonist application, after which
current decayed, we measured the fraction of total charge transfer
occurring within this 20 msec period. In the presence of 20 µM MK-801, ~55% of the charge transfer
occurred within 20 msec after the beginning of agonist application for
NR1A/NR2A, whereas only 10% of the charge transfer occurred in this
time period for NR1A/NR2B (Fig. 3A2,
traces b, Sb; B2, traces
b, Sb; n = 4). Increasing the MK-801 concentration above 20 µM did not result in any
increase in charge transfer during the 20 msec agonist application
(data not shown), as also reported by Jahr (1992). Thus, we estimate
that peak Po for NR1A/NR2A is 0.35 (=
0.64 * 0.55), whereas peak Po for
NR1A/NR2B is approximately fivefold smaller at 0.07 (= 0.71 *
0.10).
As an alternate approach for obtaining information on peak open
probability, we used a protocol in which MK-801 had access to open
channels only during the peak of the agonist-evoked current response.
As before, we recorded macroscopic current evoked by brief applications
(20 msec) of saturating agonist from HEK 293 cells transfected with
NR1A/NR2A or NR1A/NR2B. After establishing a stable peak current
response to glutamate alone (pulses applied at 20 sec intervals),
MK-801 was added to the agonist solution, and 20 msec pulses of agonist
and MK-801 were applied simultaneously. Figure
4 shows the results of this protocol
using a single application of 200 µM MK-801 and
illustrates three points. First, for both subtypes, 80-90% of the
peak current response was blocked, and the rise-time to peak was
attenuated significantly in the presence of 200 µM
MK-801; however, the time courses for decay of the current responses
with off-set of the MK-801/agonist pulse were identical to those
observed for the control response (Fig.
4A4,B4;
peak responses during and after MK-801 application were normalized to
that of the control response). These results indicate that currents
were affected by MK-801 only during the 20 msec pulse (peak response)
and not during the deactivation phase. Second, a "tail" current was
observed with removal of glutamate and MK-801 (Fig.
4A2,
B2; shown at higher gain in
A4, B4). The
rise-time for this tail current was similar to the rise-time of the
control current response to rapid application of glutamate alone,
consistent with opening of agonist-bound channels that had escaped
MK-801 block during the 20 msec application. Third, the agonist-evoked response after wash-out of MK-801 was markedly larger than that observed for the previous protocol (Fig.
4A3,B3),
consistent with the fact that only channels open during the peak
response were blocked, but those opening during the deactivation phase
were spared. Interestingly, when compared with the control response, the peak amplitude of the tail current with offset of the
MK-801/agonist pulse, as well as the residual agonist-evoked response
after wash-out of MK-801, were clearly larger for NR1A/NR2B than
NR1A/NR2A (Fig. 4A1-3,B1-3).
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Figure 4.
Brief pulse of 200 µM MK-801,
applied together with glutamate, attenuates only the peak NMDA
receptor-mediated current response. Recordings made from representative
cells expressing NR1A/NR2A (A) or NR1A/NR2B
(B). After establishing a stable current response to 20 msec application of 1 mM glutamate alone
(A1, B1,
Control), 200 µM MK-801 was added to
agonist solution, and a single response was recorded
(A2, B2,
MK-801). The glutamate-evoked current response recorded after
extensive wash-out of MK-801 is shown in
A3 and B3
(Wash). Normalized current traces shown on expanded time
scale in A4 and
B4 illustrate that the decay phases of
the control (thick line), MK-801, and wash (both shown
as thin lines) traces all follow the same time course;
open tip recordings for these experiments are shown above traces.
Extracellular solution contained 0.2 mM calcium.
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To quantitate the effects of MK-801 block on the peak response, we used
the protocol described for Figure 4 (see above) to measure progressive
attenuation of peak current amplitude with repeated applications of
agonist plus 20 µM MK-801 (Fig.
5). The peak amplitude of the tail
current response was taken as a measure of the residual peak response
to glutamate after each MK-801 application. As illustrated in Figure 5,
glutamate-evoked peak amplitude attenuation was only ~30% for both
subtypes (see insets), in contrast to the ~60% seen with glutamate
application in the presence of an equilibrium concentration of 20 µM MK-801 (Fig. 3D); the decreased block of peak current with nonequilibrium application of 20 µM MK-801 reflects the solution exchange time
over the whole cell. However, it is interesting to note that
progressive attenuation of peak NR1A/NR2A current proceeded at a
significantly faster rate than block of NR1A/NR2B peak current.
Progressive block over several episodes (20 sec intervals) was fit well
by a single exponential function (Fig.
6A,C),
with a mean decay constant (in episodes) of 2.4 ± 0.2 for NR1A/NR2A
and 8.1 ± 0.7 for NR1A/NR2B (n = 8 and 11 different cells, respectively). Consistent with a monoexponential
function, the ratio of the amplitude of each successive peak
(IN) to that of the previous peak
(IN 1) was stable (Figs. 5,
6B,D), and mean values for this
measure were significantly larger for NR1A/NR2B than NR1A/NR2A (Fig.
6E). In fact, the fraction of peak current blocked
with each successive pulse (1 [IN/IN1]) was approximately threefold higher for NR1A/NR2A than for NR1A/NR2B (0.28 ± 0.02, n = 8 vs 0.09 ± 0.02, n = 11, respectively; p < 0.01 by unpaired t
test; Fig. 6F). If a difference in MK-801 on-rate accounted for the difference in time course of progressive MK-801 block, then the time courses for the two subtypes should become identical at sufficiently high MK-801 concentrations. Therefore, we
compared progressive block of NR1A/NR2A and NR1A/NR2B currents by 10, 20, 50, 100, and 150 µM MK-801. As expected, we
observed a higher rate of block with increased MK-801 concentration,
but this reached a maximum for both NR1A/NR2A and NR1A/NR2B at MK-801 concentrations > 50 µM (Fig.
6F). Importantly, the ratio of the mean value of
1 [IN/IN1] for
NR1A/NR2A to that of NR1A/NR2B remained significantly different at all
five concentrations of MK-801 (3.2, 3.1, 2.6, 2.2, and 2.1 for 10, 20, 50, 100, 150 µM, respectively). Together with
data shown in Figure 3D suggesting that MK-801 on-rates are
similar for the two subtypes, these data are consistent with the
conclusion that the difference in time course of progression of MK-801
block can be attributed mainly to a significant difference in peak
Po.
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Figure 5.
Representative current traces showing progressive
attenuation of peak glutamate-evoked currents by 20 µM
MK-801 present only in agonist solution. Recordings were made in the
continuous presence of 50 µM glycine from a cell
transfected with NR1A/NR2A (A) or NR1A/NR2B
(B). A0,
B0, Representative control responses
to 20 msec application of 1 mM glutamate alone.
A1-6,
B1-6, Successive current responses
to simultaneous application of 1 mM glutamate and 20 µM MK-801 for 20 msec at 20 sec intervals.
Insets show selected traces on an expanded time scale,
along with the open tip recording to indicate speed of solution
exchange in these experiments. Extracellular solution contained 0.2 mM calcium.
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Figure 6.
Progressive block by MK-801 of peak
glutamate-evoked current is more rapid for NR1A/NR2A than NR1A/NR2B.
A-D, Graphic representation of progressive MK-801 block
in recordings from cell transfected with NR1A/NR2A (A,
B) or NR1A/NR2B (C, D).
A, C, Peak current amplitude measured with each
successive pulse of glutamate plus MK-801 was normalized to the peak
current response to the second application of 1 mM
glutamate plus MK-801. Decay of peak current was fit by a single
exponential function. B, D, Peak current amplitude
recorded in response to each successive pulse of glutamate plus MK-801
(IN) was normalized to peak current
amplitude recorded in response to the previous glutamate plus MK-801
pulse (IN1). The mean ratio (± SEM) for
five pulses (the third through seventh pulse in MK-801) is shown at the
far right of each plot. E, Bars indicate
mean ± SEM for pooled data from n = 8 (NR1A/NR2A)
or n = 11 (NR1A/NR2B) different cells. Significant
difference by unpaired t test, **p < 0.01. F, The fraction of peak current blocked with
each successive pulse of MK-801 plus glutamate (1 [IN/IN1])
was calculated from recordings made from n = 5-11
cells, for each of five different MK-801 concentrations.
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9-Aminoacridine block demonstrates a larger fraction of channels
open after the peak for NR1A/NR2B than for NR1A/NR2A
Results of the experiments with MK-801 indicate that total
Po (for the peak and deactivation
phase of the macroscopic current response) is similar for NR1A/NR2A and
NR1A/NR2B, but that maximal (or peak)
Po is at least twofold to threefold
higher for NR1A/NR2A. These results suggest that the mean open time or
opening frequency is smaller, and/or the distribution of latencies to
first opening is shifted to longer times for NR1A/NR2B channels. We
used the use-dependent antagonist 9-aminoacridine (9-AA) to address
this issue more directly. As shown in Figure
7, A and B, brief
(20 msec) coapplication of 100 µM 9-AA and a
saturating concentration of agonist (1 mM
glutamate with 50 µM glycine) resulted in
80-90% block of peak current, followed by a tail current after
removal of agonist/9-AA. As for experiments with MK-801 (Fig. 4), the rise-time of the tail current was similar to that for current activation in response to agonist alone and likely represented opening
of agonist-bound channels that had escaped block (see also Benveniste
and Mayer, 1995). Also like the results with 200 µM MK-801, the amplitude of the tail current
relative to the control response was much larger for NR1A/NR2B than for
NR1A/NR2A, indicating that a larger proportion of channels opened for
the first time after the peak for NR1A/NR2B.
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Figure 7.
9-aminoacridine block reveals larger fraction of
channels open after the peak response for NR1A/NR2B than NR1A/NR2A.
A, B, Representative traces showing NR1A/NR2A
(A) or NR1A/NR2B (B)
mediated current evoked by 20 msec application of 1 mM
glutamate (Control, thick line) or 1 mM
glutamate plus 100 µM 9-AA (9-AA, thin
line). Holding potential was 60 mV. The top
trace in the inset is the open tip recording at
the end of the experiment. C-H, Representative traces
recorded from cell expressing NR1A/NR2A (C-E) or
NR1A/NR2B (F-H). Current elicited by voltage
switch from 100 to +60 mV 20 msec after a 500 msec application of 1 mM glutamate plus 100 µM 9-AA (C,
F, Tail current, thin lines).
Current evoked by 20 msec application of 1 mM glutamate at
a holding potential of +60 mV (D, G, Evoked, thick
lines). E and H show same
currents on expanded time scale and evoked current peaks are normalized
to those of tail currents. I-K, Bars represent mean ± SEM from n = 6 different cells for each subtype.
J (for NR1A/NR2A) and K (for NR1A/NR2B)
compare the time constant or constants of current decay for
voltage-activated currents immediately after 9-AA application
(filled bars) with those for glutamate-evoked
currents (open bars), both obtained at holding
potentials of +60 mV. **p < 0.01 by unpaired
t test. Extracellular solution contained 0.2 mM calcium.
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|
Unlike MK-801, block of open channels by 9-AA is reversible on a time
scale of seconds (at hyperpolarized membrane potentials) to
milliseconds (at depolarized potentials) and traps agonist in the bound
state (Benveniste and Mayer, 1995), as evidenced by the slow
decay of tail currents seen in Figure 7, A and B. These properties can be exploited to obtain a more quantitative estimate of the proportion of channels opening after the peak for the
two subtypes. As previously reported (Benveniste and Mayer, 1995), a 500 msec pulse of 9-AA together with agonist applied at
hyperpolarized potentials will result in accumulation of channels in
the blocked agonist-bound state. Because the time constant for 9-AA
unblock is <3 msec at +60 mV, all of the agonist-bound channels that
had accumulated in the blocked state will open nearly simultaneously
after depolarization (Fig.
7C,E,F,H).
Thus, the ratio of the peak amplitude of the agonist-evoked current
response at +60 mV (Fig. 7D,G) to
that of the tail current evoked by depolarization to +60 mV after 9-AA
block (Fig. 7C,F) provides a rough
estimate of the fraction of channels open at the peak of the current
response compared with the Po(total)
for the 500 msec agonist application (Benveniste and Mayer,
1995). This ratio is approximately twofold larger for NR1A/NR2A
than NR1A/NR2B (0.72 ± 0.04, n = 6 and 0.35 ± 0.02, n = 4, respectively; p < 0.01 by unpaired t test; Fig. 7I), indicating
that a larger fraction of channels open after the peak for NR1A/NR2B
(65%) than for NR1A/NR2A (28%). Comparison of the decay phase of the
outward current evoked by the +60 mV depolarization after 9-AA block to
that of the agonist-evoked response revealed a similar time course for
NR1A/NR2A (Fig. 7E,J). In
contrast, the voltage-activated current (after 9-AA block) mediated by
NR1A/NR2B exhibited a fast component, representing ~68% of the total
peak with a time constant  = 29 ± 2 msec
(n = 6), that was not apparent in the decay of the
agonist-evoked response; the remainder of the decay followed a similar
time course to that exhibited by the agonist-evoked response (Fig.
7H,K). Together, these results suggest that peak
Po for NR1A/NR2B is significantly
smaller than that for NR1A/NR2A at least in part because of a longer
latency to first opening after saturation of receptors with agonist.
| |
DISCUSSION |
Our results demonstrate that the maximal channel open probability
of NMDA receptors is dependent on subunit composition. According to
differences in macroscopic peak current density and block by MK-801,
peak Po for NR1A/NR2A is twofold to
fivefold higher than that for NR1A/NR2B. Previous studies using 10-20
µM MK-801 to measure peak
Po for neuronal NMDA receptors have
yielded results in the range of 0.04 to 0.3 (Jahr, 1992; Rosenmund et
al., 1995; Dzubay and Jahr, 1996), and it has been suggested that peak
Po for NMDA receptor-mediated current
responses may differ for recordings made in the whole-cell versus
outside-out patch mode (Benveniste and Mayer, 1995; Rosenmund et
al., 1995). Interestingly, our estimate of peak
Po, using a protocol and analysis from
Jahr (1992), was 0.35 for NR1A/NR2A and 0.07 for NR1A/NR2B, values
similar to the high and low estimates reported for neuronal NMDA
receptors. Moreover, the charge transfer time course for
NR1A/NR2A-mediated whole-cell current that we observed in the presence
of 20 µM MK-801 is in excellent agreement with
that found by Jahr (1992) for NMDA receptors in outside-out patches
from hippocampal neurons. In addition, our estimate of peak
Po for NR1A/NR2A matches the value
reported for open probability within NR1A/NR2A superclusters in
single-channel recordings from outside-out patches (Wyllie et al.,
1998). Therefore, our results suggest that at least some of the
variability in measurements of peak Po
for neuronal NMDA receptors may be caused by differences in receptor
subunit composition.
In experiments in which 20 µM MK-801 was present in both
control and agonist solutions, the percentage block of peak current and
charge transfer after wash-out of MK-801 was similar for currents mediated by NR1A/NR2A and NR1A/NR2B. These results indicate that Po(total) (~0.7) for the peak and
deactivation phase in response to brief agonist stimulation is similar
for the two subtypes. On the other hand, results of experiments in
which MK-801 had access to channels only during the peak of the current
response (Figs. 4-6) revealed distinct differences between NR1A/NR2A
and NR1A/NR2B, consistent with significant differences in peak
Po. Assuming some forebrain neurons or
synapses express predominantly NR2A or NR2B subunits, this latter
protocol may prove a useful tool for determining neuronal NMDA receptor
subunit composition.
The fact that Po(total) is similar but
peak Po is approximately twofold to
fivefold higher for NR1A/NR2A than for NR1A/NR2B suggests that
NR1A/NR2B channels have longer latencies to first opening, lower
opening frequency, shorter mean open times, or some combination of
these. Experiments with 9-aminoacridine suggest that some of the
difference in peak Po is attributable
to a smaller fraction of NR1A/NR2B channels opening within the first 20 msec after a jump into a saturating concentration of agonist compared with NR1A/NR2A. Because the 10-90% rise-time for the macroscopic current response is slightly, but significantly, longer for NR1A/NR2B, the peak of the distribution of latencies to first opening may be
slightly shifted to longer times, but may also be broader than that of
NR1A/NR2A. On the other hand, Colquhoun et al. (Edmonds and Colquhoun,
1992; Wyllie et al., 1998) have suggested that NMDA
receptor-mediated macroscopic current responses to jumps into a
saturating concentration of agonist can be approximated by the ensemble
average of aligned superclusters (recorded as single-channel openings
during steady-state exposure to low agonist concentrations), as long as
opening latency is ignored. If so, our results from MK-801 and 9-AA
experiments could also be explained if NR1A/NR2B channels exhibited
longer duration superclusters and smaller intracluster channel open
probability compared with NR1A/NR2A, similar to the comparison between
NR1A/NR2D and NR1A/NR2A reported by Wyllie et al. (1998). Because some
NR1A/NR2A and NR1A/NR2B channels may escape block on first opening
(because of open times less than ~1 msec; see Wyllie et al., 1998),
the subtype with lower opening frequency within the supercluster would
show less peak current attenuation. As well, this line of reasoning
could explain why increasing the on-rate of MK-801 (by increasing
its concentration) had a larger effect on block of NR1A/NR2B peak current than that of NR1A/NR2A; as the channel blocking rate increased from ~250 sec1 at 10 µM to ~2500 sec
1 at 100 µM MK-801 (Heuttner and
Bean, 1988; Jahr, 1992), the fraction of first openings that escaped
block would have markedly decreased. Clearly, a comparison of NR1A/NR2A
and NR1A/NR2B superclusters in single-channel recordings from
transfected mammalian cell lines would help resolve these issues.
Results of experiments with the agonist-trapping, use-dependent
antagonist 9-aminoacridine indicate that the fraction of channels open
at the peak of the macroscopic current response is more than twofold
higher for NR1A/NR2A than for NR1A/NR2B. The estimated fractions, 0.72 and 0.35, respectively, are much higher than the values calculated from
the MK-801 experiments (0.35 and 0.07, respectively) and are likely to
be overestimates of the actual peak open probability for several
reasons (see also Benveniste and Mayer, 1995). First, 9-AA
unblock is not instantaneous, so some of the blocked channels will not
contribute to the peak of the tail current. Second, because 9-AA
unblock occurs (slowly) even at hyperpolarized potentials, 9-AA will
have already dissociated from some of the channels during the interval
between removal of agonist/9-AA and switching voltage from 100 to +60
mV (Fig. 7C,F). Thus, the tail current
peak amplitude underrepresents
Po(total) for the 500 msec application
of agonist/9-AA. It is worth noting that agonist-trapping properties
and dissociation rates of 9-AA have not been compared for NR1A/NR2A and
NR1A/NR2B, and any differences between the subtypes in these parameters
would alter the relative values of estimated peak
Po. However, by applying depolarizing voltage within 20 msec after offset of 9-AA, we have attempted to
minimize the effect that differences in 9-AA dissociation rates might
have on our results. Third, since a fraction of available channels may
have opened very briefly or not at all during the 500 msec application
of agonist/9-AA, Po(total), as
represented experimentally by the tail current amplitude, may be <1.
It is interesting to note that not only the peak amplitude but also the
time course of current decay at +60 mV is markedly different for the
tail compared with evoked currents mediated by NR1A/NR2B. The large
proportion (~68%) of tail current decaying by the fast (29 msec)
time constant, which is absent in the agonist-evoked response, is
consistent with synchronized channel opening after 9-AA unblock and a
low opening frequency of NR1A/NR2B agonist-bound channels.
In concluding that numbers of surface receptors were similar for
NR1A/NR2A and NR1A/NR2B from data analyzing expression of NR1A, we
assumed that: (1) Every surface receptor containing NR1A also contained
NR2 subunit or subunits; (2) Complexes of NR1A with NR2A shared the
same stoichiometry as those of NR1A with NR2B; and (3) All surface
receptors were functional. The first assumption is supported by data
indicating that NR1A subunits expressed heterologously in the absence
of NR2 subunits show little or no surface expression (McIlhinney et
al., 1996). Although we have not directly tested the second and third
assumptions, the ratio of peak Po for
NR1A/NR2A to that of NR1A/NR2B measured from MK-801 block experiments
was similar to that predicted by the difference in peak macroscopic
current density for the two subtypes. Therefore, it is likely that our
assumptions were valid and that expression of functional receptors at
the surface of transfected HEK 293 cells was similar for NR1A/NR2A and
NR1A/NR2B under our experimental conditions.
A variety of data indicate that at typical CNS synapses, the
presynaptic release of a single quantum of glutamate results in
saturation of binding to postsynaptic NMDA receptors (Clements et al.,
1992; Tang et al., 1994; Tong and Jahr, 1994). Therefore, the NMDA
receptor-mediated component of the EPSC amplitude is directly
proportional to the density of postsynaptic receptors and their open
probability. Our results suggest that when expressed at equal density,
postsynaptic NMDA receptors composed predominantly of NR1A/NR2A will
mediate peak currents approximately fourfold larger than those mediated
by receptors composed of NR1A/NR2B. In support of this conclusion, a
recent study in rat brain frontal cortex showed that, after repeated
electroconvulsive seizures, upregulation of NR2B
expression (in the absence of any change in NR2A expression) was
associated with an approximately threefold decrease in peak amplitude
of NMDA-evoked currents recorded from pyramidal neurons (Hiroi et al.,
1998). Another study in cultured rat cerebellar granule cells
demonstrated a significant decrease in amplitude of NMDA-evoked calcium
transients that correlated with BDNF-induced downregulation of NR2A
without a change in NR1A and NR2B expression levels (Brandoli et al.,
1998). Interestingly, the alteration in NMDA receptor subunit
composition resulting from BDNF treatment was associated with decreased
vulnerability to excitotoxic neuronal death.
Previous studies have demonstrated that NR1A/NR2B is the predominant
NMDA receptor subtype expressed in cortex and hippocampus during early
developmental stages (Monyer et al., 1994; Sheng et al., 1994)
and that this receptor subtype mediates the markedly slower time course
of EPSC decay found in immature versus mature neurons (Carmignoto and
Vicini, 1992; Hestrin, 1992; Flint et al., 1997). Although these slow
EPSCs would be optimized for temporal integration of nonsynchronous
synaptic inputs, our results suggest that they would be smaller in
amplitude than those typically found in mature cortical and hippocampal
neurons. Moreover, in mature forebrain neurons that express both NR2A
and NR2B, stimuli that induce a switch in the relative expression
levels of these subunits would be expected to alter EPSC amplitude and
trigger a long-lasting change in synaptic efficacy, perhaps mediating
synaptic plasticity and altered vulnerability to excitotoxicity.
| |
FOOTNOTES |
Received Jan. 19, 1999; revised May 26, 1999; accepted June 1, 1999.
This work was supported by a grant from the Medical Research Council
(MRC) (L.A.R.). N.C. was supported by a fellowship from the
Huntington's Disease Society of America. L.A.R. is an MRC (Canada)
Scholar. We thank Drs. T. H. Murphy and C. J. Price for useful discussions and suggestions, and Moira Thejomayen for assistance in manuscript preparation.
Correspondence should be addressed to Dr. Lynn A. Raymond, Division of
Neuroscience, Department of Psychiatry, University of British Columbia,
4N3-2255 Wesbrook Mall Vancouver, BC, Canada V6T 1Z3.
| |
REFERENCES |
-
Benveniste M,
Mayer ML
(1995)
Trapping of glutamate and glycine during open channel block of rat hippocampal neuron NMDA receptors by 9-aminoacridine.
J Physiol (Lond)
483:367-384[Abstract/Free Full Text].
-
Brandoli C,
Sanna A,
De Bernardi MA,
Follesa P,
Brooker G,
Mocchetti I
(1998)
Brain-derived neurotrophic factor and basic fibroblast growth factor downregulate NMDA receptor function in cerebellar granule cells.
J Neurosci
18:7953-7961[Abstract/Free Full Text].
-
Brimecombe JC,
Boeckman FA,
Aizenman E
(1997)
Functional consequences of NR2 subunit composition in single recombinant N-methyl-D-aspartate receptors.
Proc Natl Acad Sci USA
94:11019-11024[Abstract/Free Full Text].
-
Carmignoto G,
Vicini S
(1992)
Activity-dependent decrease in NMDA receptor responses during development of the visual cortex.
Science
258:1007-1011[Abstract/Free Full Text].
-
Chen C,
Okayama H
(1987)
High efficiency transformation of mammalian cells by plasmid DNA.
Mol Cell Biol
7:2745-2752[Abstract/Free Full Text].
-
Chen N,
Moshaver A,
Raymond LA
(1997)
Differential sensitivity of recombinant N-methyl-D-aspartate receptor subtypes to Zinc inhibition.
Mol Pharmacol
51:1015-1023[Abstract/Free Full Text].
-
Chen N,
Luo T,
Wellington C,
Metzler M,
McCutcheon K,
Hayden MR,
Raymond LA
(1999)
Subtype-dependent modulation of NMDA receptor mediated currents by mutant huntingtin.
J Neurochem
72:1890-1898[Web of Science][Medline].
-
Clements JD,
Lester RA,
Tong G,
Jahr CE,
Westbrook GL
(1992)
The time course of glutamate in the synaptic cleft.
Science
258:1498-1501[Abstract/Free Full Text].
-
Dingledine R,
Borges K,
Bowie D,
Traynelis SF
(1999)
The glutamate receptor ion channels.
Pharmacol Rev
51:7-61[Abstract/Free Full Text].
-
Dzubay J,
Jahr CE
(1996)
Kinetics of NMDA channel opening.
J Neurosci
16:4129-4134[Abstract/Free Full Text].
-
Edmonds B,
Colquhoun D
(1992)
Rapid decay of average single-channel NMDA receptor activations recorded at low agonist concentration.
Proc R Soc Lond B Biol Sci
250:279-286[Medline].
-
Ehlers MD,
Zhang S,
Bernhardt JP,
Huganir RL
(1996)
Inactivation ofNMDA receptors by direct interaction of calmodulin with the NR1 subunit.
Cell
84:745-755[Web of Science][Medline].
-
Flint AC,
Maisch US,
Weishaupt JH,
Kriegstein AR,
Monyer H
(1997)
NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex.
J Neurosci
17:2469-2476[Abstract/Free Full Text].
-
Hestrin S
(1992)
Developmental regulation of NMDA receptor-mediated synaptic currents at a central synapse.
Nature
357:686-689[Medline].
-
Hessler NA,
Shirke AM,
Malinow R
(1993)
The probability of transmitter release at a mammalian central synapse.
Nature
366:569-572[Medline].
-
Hiroi N,
Marek GJ,
Brown JR,
Ye H,
Saudou F,
Vaidya VA,
Duman RS,
Greenberg ME,
Nestler EJ
(1998)
Essential role of the fos B gene in molecular, cellular, and behavioral actions of chronic electroconvulsive seizures.
J Neurosci
18:6952-6962[Abstract/Free Full Text].
-
Hollmann M,
Boulter J,
Maron C,
Beasley L,
Sullivan J,
Pecht G,
Heinemann SF
(1993)
Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor.
Neuron
10:943-954[Web of Science][Medline].
-
Huettner JE,
Bean BP
(1988)
Block of N-methyl-D-aspartate-activated current by the anticonvulsant MK-801: selective binding to open channels.
Proc Natl Acad Sci USA
85:1307-1311[Abstract/Free Full Text].
-
Jahr CE
(1992)
High probability opening of NMDA receptor channels by L-glutamate.
Science
255:470-472[Abstract/Free Full Text].
-
Kohr G,
Seeburg PH
(1996)
Subtype-specific regulation of recombinant NMDA receptor-channels by protein tyrosine kinases of the src family.
J Physiol (Lond)
492:445-452[Abstract/Free Full Text].
-
Krupp JJ,
Vissel B,
Heinemann SF,
Westbrook GL
(1996)
Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific.
Mol Pharmacol
50:1680-1688[Abstract].
-
Krupp JJ,
Vissel B,
Heinemann SF,
Westbrook GL
(1998)
N-terminal domains in the NR2 subunit control desensitization of NMDA receptors.
Neuron
20:317-327[Web of Science][Medline].
-
Krupp JJ,
Vissel B,
Thomas CG,
Heinemann SF,
Westbrook GL
(1999)
Interactions of calmodulin and alpha-actinin with the NR1 subunit modulate Ca2+-dependent inactivation of NMDA receptors.
J Neurosci
19:1165-1178[Abstract/Free Full Text].
-
Legendre P,
Rosenmund C,
Westbrook GL
(1993)
Inactivation of NMDA channels in cultured hippocampal neurons by intracellular calcium.
J Neurosci
13:674-684[Abstract].
-
Lu YM,
Roder JC,
Davidow J,
Salter MW
(1998)
Src activation in the induction of long-term potentiation in CA1 hippocampal neurons.
Science
279:1363-1367[Abstract/Free Full Text].
-
McBain CJ,
Mayer ML
(1994)
NMDA receptor structure and function.
Physiol Rev
75:723-760.
-
McIlhinney RAJ,
Molnar E,
Atack JR,
Whiting PJ
(1996)
Cell surface expression of the human N-methyl-D-aspartate receptor subunit 1a requires the co-expression of the NR2A subunit in transfected cells.
Neuroscience
70:989-997[Web of Science][Medline].
-
Monyer H,
Sprengel R,
Schoepfer R,
Herb A,
Higuchi M,
Lomeli H,
Burnashev N,
Sakmann B,
Seeburg PH
(1992)
Heteromeric NMDA receptors: molecular and functional distinction of subtypes.
Science
256:1217-1221[Abstract/Free Full Text].
-
Monyer H,
Burnashev N,
Laurie DJ,
Sakmann B,
Seeburg PH
(1994)
Developmental and regional expression in the rat brain and functional properties of four NMDA receptors.
Neuron
12:529-540[Web of Science][Medline].
-
Price CJ,
Rintoul GL,
Baimbridge KG,
Raymond LA
(1999)
Inhibition of calcium-dependent NMDA receptor current rundown by calbindin-D28k. J.
Neurochem
72:634-642.
-
Raymond LA,
Moshaver A,
Tingley WG,
Shalaby I,
Huganir RL
(1996)
Glutamate receptor ion channel properties predict vulnerability to cytotoxicity in a transfected non-neuronal cell line.
Mol Cell Neurosci
7:102-115[Web of Science][Medline].
-
Rosenmund C,
Westbrook GL
(1993)
Calcium-induced actin depolymerization reduces NMDA channel activity.
Neuron
10:805-814[Web of Science][Medline].
-
Rosenmund C,
Feltz A,
Westbrook GL
(1995)
Synaptic NMDA receptor channels have a low open probability.
J Neurosci
15:2788-2795[Abstract].
-
Sheng M,
Cummings J,
Roldan LA,
Jan YN,
Jan LY
(1994)
Changing subunit composition of heteromeric NMDA receptors during development of rat cortex.
Nature
368:144-147[Medline].
-
Stern P,
Béhé P,
Schoepfer R,
Colquhoun D
(1992)
Single-channel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors.
Proc R Soc Lond B Biol Sci
250:271-277[Medline].
-
Stern P,
Cik M,
Colquhoun D,
Stephenson FA
(1995)
Single channel properties of cloned NMDA receptors in a human cell line: comparison with results from Xenopus oocytes.
J Physiol (Lond)
476:391-297[Abstract/Free Full Text].
-
Sucher NJ,
Awobuluyi M,
Choi YB,
Lipton SA
(1996)
NMDA receptors: from genes to channels.
Trends Pharmacol Sci
17:348-355[Medline].
-
Sugihara H,
Moriyoshi K,
Ishii T,
Masu M,
Nakanishi S
(1992)
Structures and properties of seven isoforms of the NMDA receptor generated by alternative splicing.
Biochem Biophys Res Commun
185:826-832[Web of Science][Medline].
-
Tang CM,
Margulis M,
Shi QY,
Fielding A
(1994)
Saturation of postsynaptic glutamate receptors after quantal release of transmitter.
Neuron
13:1385-1393[Web of Science][Medline].
-
Tong G,
Jahr CE
(1994)
Block of glutamate transporters potentiates postsynaptic excitation.
Neuron
13:195-203.
-
Vicini S,
Wang JF,
Li JH,
Zhu WJ,
Wang YH,
Luo JH,
Wolfe BB,
Grayson DR
(1998)
Functional and pharmacological differences between recombinant N-methyl-D-aspartate receptors.
J Neurophysiol
79:555-566[Abstract/Free Full Text].
-
Wang YT,
Yu X-M,
Salter MW
(1996)
Ca2+-independent reduction of N-methyl-D-aspartate channel activity by protein tyrosine posphatase.
Proc Natl Acad Sci USA
93:1721-1725[Abstract/Free Full Text].
-
Wyllie DJA,
Behe P,
Colquhoun D
(1998)
Single-channel activations and concentration jumps: comparison of recombinant NR1a/NR2A and NR1a/NR2D NMDA receptors.
J Physiol (Lond)
510:1-18[Abstract/Free Full Text].
-
Zhang S,
Ehlers MD,
Bernhardt JP,
Su CT,
Huganir RL
(1998)
Calmodulin mediates calcium-dependent inactivation of N-methyl-D-aspartate receptors.
Neuron
21:443-453[Web of Science][Medline].
Copyright © 1999 Society for Neuroscience 0270-6474/99/19166844-11$05.00/0
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|
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|
|
|
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[Full Text]
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|
|
|
|
|
|
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[Full Text]
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|
|
|
|
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|
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|
|
|
|
|
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|
|
|
|
|
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|
|
|
|
|
|
|
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|
|
|
|
|
|
|
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|
|
|
|
|
|
|
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|
|
|
|
|
|
|
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|
|
|
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|
|
|
|
|
|
|
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|
|
|
|
|
|
|
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[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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[Full Text]
[PDF]
|
|
|
|
|
|
|
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278(36):
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[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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23(17):
6876 - 6883.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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3364 - 3372.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Szinyei, O. Stork, and H.-C. Pape
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J. Neurosci.,
April 1, 2003;
23(7):
2549 - 2556.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Ren, Y. Honse, B. J. Karp, R. H. Lipsky, and R. W. Peoples
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J. Biol. Chem.,
January 3, 2003;
278(1):
276 - 283.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. E. Chapman, K. A. Keefe, and K. S. Wilcox
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J Neurophysiol,
January 1, 2003;
89(1):
69 - 80.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Rossi, E. Sola, V. Taglietti, T. Borchardt, F. Steigerwald, J. K. Utvik, O. P. Ottersen, G. Kohr, and E. D'Angelo
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J. Neurosci.,
November 15, 2002;
22(22):
9687 - 9697.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Prybylowski, Z. Fu, G. Losi, L. M. Hawkins, J. Luo, K. Chang, R. J. Wenthold, and S. Vicini
Relationship between Availability of NMDA Receptor Subunits and Their Expression at the Synapse
J. Neurosci.,
October 15, 2002;
22(20):
8902 - 8910.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C Pina-Crespo and A. J Gibb
Subtypes of NMDA receptors in new-born rat hippocampal granule cells
J. Physiol.,
May 15, 2002;
541(1):
41 - 64.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. K. Abrams, M. V. L. Bennett, V. K. Verselis, and T. A. Bargiello
Voltage opens unopposed gap junction hemichannels formed by a connexin 32 mutant associated with X-linked Charcot-Marie-Tooth disease
PNAS,
March 19, 2002;
99(6):
3980 - 3984.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Rodrigues, G. E. Schafe, and J. E. LeDoux
Intra-Amygdala Blockade of the NR2B Subunit of the NMDA Receptor Disrupts the Acquisition But Not the Expression of Fear Conditioning
J. Neurosci.,
September 1, 2001;
21(17):
6889 - 6896.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-y. Lan, V. A. Skeberdis, T. Jover, X. Zheng, M. V. L. Bennett, and R. S. Zukin
Activation of Metabotropic Glutamate Receptor 1 Accelerates NMDA Receptor Trafficking
J. Neurosci.,
August 15, 2001;
21(16):
6058 - 6068.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. A. Skeberdis, J.-y. Lan, X. Zheng, R. S. Zukin, and M. V. L. Bennett
Insulin promotes rapid delivery of N-methyl-D- aspartate receptors to the cell surface by exocytosis
PNAS,
March 13, 2001;
98(6):
3561 - 3566.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Chen, J. Ren, L. A. Raymond, and T. H. Murphy
Changes in Agonist Concentration Dependence That Are a Function of Duration of Exposure Suggest N-Methyl-D-aspartate Receptor Nonsaturation during Synaptic Stimulation
Mol. Pharmacol.,
February 1, 2001;
59(2):
212 - 219.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
M. Umemiya, N. Chen, L. A. Raymond, and T. H. Murphy
A Calcium-Dependent Feedback Mechanism Participates in Shaping Single NMDA Miniature EPSCs
J. Neurosci.,
January 1, 2001;
21(1):
1 - 9.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Chen, T. H. Murphy, and L. A. Raymond
Competitive Inhibition of NMDA Receptor-Mediated Currents by Extracellular Calcium Chelators
J Neurophysiol,
August 1, 2000;
84(2):
693 - 697.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Steigerwald, T. W. Schulz, L. T. Schenker, M. B. Kennedy, P. H. Seeburg, and G. Kohr
C-Terminal Truncation of NR2A Subunits Impairs Synaptic But Not Extrasynaptic Localization of NMDA Receptors
J. Neurosci.,
June 15, 2000;
20(12):
4573 - 4581.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
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ABSTRACT
INTRODUCTION
