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Vol. 302, Issue 3, 1023-1030, September 2002
Departments of Pharmacology (R.P.G.), Neurology (S.S., D.L.B., K.L.S., Y.D., D.R.L.), and Pediatrics (D.R.L.), University of Pennsylvania, School of Medicine, Children's Seashore House, Philadelphia, Pennsylvania
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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N-Methyl-D-aspartate (NMDA) receptors are calcium-permeable glutamate receptors that play putative roles in learning, memory, and excitotoxicity. NMDA receptor-mediated calcium entry can activate the calcium-dependent protease calpain, leading to substrate degradation. The major NMDA receptor 2 (NR2) subunits of the receptor are in vitro substrates for calpain at selected sites in the C-terminal region. In the present study, we assessed the ability of calpain-mediated proteolysis to modulate the NR1a/2A subtype in a heterologous expression system. Human embryonic kidney (HEK293t) cells, which endogenously express calpain, were cotransfected with NR1a/2A in addition to the calpain inhibitor calpastatin or empty vector as control. Receptor activation by glutamate and glycine as co-agonists led to calpain activation as measured by succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-aminomethyl coumarin (Suc-LLVY-AMC). Calpain activation also resulted in the degradation of NR2A and decreased binding of 125I-MK-801 (125I-dizocilpine) to NR1a/2A receptors. No stable N-terminal fragment of the NMDA receptor was formed after calpain activation, suggesting calpain regulation of NMDA receptor levels in ways distinct from that previously observed with in vitro cleavage. NR2 subunit constructs lacking the final 420 amino acids were not degraded by calpain. Agonist-stimulated NR1a/2A-transfected cells also had decreased calcium uptake and produced lower changes in agonist-stimulated intracellular calcium compared with cells cotransfected with calpastatin. Calpastatin had no effect on either calcium uptake or intracellular calcium levels when the NR2A subunit lacked the final 420 amino acids. These studies demonstrate that NR2A is a substrate for calpain in situ and that this proteolytic event can modulate NMDA receptor levels.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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NMDA
receptors are ionotropic glutamate receptors that play important roles
in learning and memory as well as many neurological disorders (Lynch
and Guttmann, 2001
). These receptors exist as heteromultimers composed
of subunits from two separate protein families (termed NR1 and NR2).
The NR1 subunit family consists of eight splice variants (NR1a-h),
whereas the NR2 family is composed of four members (NR2A-D) made from
separate genes (Nakanishi, 1992). Functional NMDA receptors usually
require members from each family and probably exist as a tetramer or a
pentamer in vivo (Lynch et al., 1995; Hawkins et al., 1999).
Each subunit of the NMDA receptor contains an extracellular N terminus
followed by four transmembrane domains. The second domain forms an
intramembrane loop, whereas the C terminus is intracellular and may
link the receptor to calcium-activated intracellular signaling pathway
systems (Niethammer et al., 1996). The interactions of the receptor
with signal transduction systems also may be modulated by the
association of the NMDA receptor C-terminal tail with anchoring proteins or other cytoskeletal elements (Bi et al., 1998a, Wechsler and
Teichberg, 1998). These interactions can lead to subtype-specific modulation of the receptor. For example, yotiao and protein kinase C
modulate NMDA receptors in subunit-specific manners based on the
properties of the C-terminal region (Grant et al., 1998; Lin et al.,
1998). The importance of the C-terminal region in proper NMDA receptor
function is further exemplified by the findings of Sprengel et al.
(1998), which demonstrated that gene-targeted mice lacking the
C-terminal tail of the NR2 subunit exhibited properties similar to mice
with a complete absence of an NR2 subunit. This occurs even though
receptors lacking the C-terminal region of NR2A or NR2C are
electrophysiologically similar to wild-type receptors (Sprengel et al.,
1998). This finding suggests that post-translational or
activity-dependent processing of the C-terminal tail plays a critical
role in the modulation of NMDA receptor activity, localization, or function.
One means of post-translational modification of the C terminus is
proteolytic processing by calpain. Calpain, most commonly activated in
brain by calcium entry through NMDA receptors (Adamec et al., 1998),
regulates numerous enzymes and membrane-associated proteins, including
cytoskeletal components, integral membrane proteins, and receptors
(Johnson and Guttmann, 1997). Calpain activity is inhibited by the
protein calpastatin, an endogenous and selective inhibitor of calpain
(Johnson and Guttmann, 1997).
The C-terminal region of the NR2 subunit is a substrate for calpain (Bi
et al., 1998b,c; Guttmann et al., 2001). Although NR2A appears to be a
substrate in vitro or with prolonged exposure to glutamatergic
agonists, it is not clear that physiological activation of calpain
results in NR2A cleavage or alters NMDA receptor activity. The
cleavages of the receptor subunit in vitro occur in the C-terminal
regions with all sites in the NR2A subunit on the C-terminal side of
amino acid 1051. Although two specific sites of cleavage in vitro occur
at amino acids 1279 and 1330 of NR2A, proteolysis at these sites does
not inherently alter NMDA receptor activity as receptor subunits
truncated to these exact sites retain basic electrophysiological
properties (Guttmann et al., 2001). In the present study we sought to
examine whether results from an in situ model system of transfected
cells would also demonstrate cleavage of the NMDA receptor by calpain
and whether such cleavage alters physiological properties of the NMDA receptor.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Materials
Glutamate, glycine, ketamine, aprotinin, pepstatin, phenylmethylsulfonyl fluoride, and anti-actin were from Sigma-Aldrich (St. Louis, MO); Dulbecco's modified Eagle's medium, horse serum, penicillin/streptomycin, and glutamine were from Invitrogen (Carlsbad, CA). Fetal bovine serum was from Hyclone Laboratories (Logan, UT). Suc-LLVY-AMC was from Bachem (Bubendorf, Switzerland); MK-801 was from Sigma/RBI (Natick, MA), 125I-MK-801 and 45CaCl2 were from PerkinElmer Life Sciences (Boston, MA), and HEK293t cells were from the American Type Culture Collection (Manassas, VA). Antibodies to the C-terminal portions of NR2A (AB1548) and NR1a (AB1516), with epitopes at amino acids 1445 to 1464 and 909 to 938, respectively, were from Chemicon International (Temecula, CA). An N-terminal antibody (amino acids 25-130 of NR2C) that cross-reacts with NR2A (A-6475) was from Molecular Probes (Eugene, OR). Porcine calpain I was purchased from Calbiochem. Anti-calpain I was a gift from Dr. John Elce (Queen's University, Kingston, ON, Canada), and rabbit calpastatin cDNA was a gift from Dr. Masatoshi Maki (Nagoya University, Nagoya, Japan).
Methods
Transfection of HEK293t Cells.
HEK293t cells were grown on
tissue culture-grade dishes (Corning brand; Corning Glassworks,
Corning, NY) in RPMI media containing 5% horse serum and 5% fetal
bovine serum supplemented with 2 mM glutamine and 100 units/ml
penicillin/streptomycin and placed in a 5% CO2
incubator at 37°C. Transfection of HEK293t cells with cDNA was
accomplished by calcium phosphate precipitation as previously described
(Grant et al., 1998). Twenty-four hours after transfection, the
medium was changed and treatments were added. Calcium uptake, calpain activation, and calcium imaging were routinely performed at
this point. Ketamine (500 µM) was added to the media during transfection to prevent NMDA receptor activation as previously described (Grant et al., 1997). Using HEK293t cells, the transfection efficiency is ~70%. As previously shown, individual HEK293t cells express all of the proteins that the cDNAs encode regardless of the
number of different cDNA plasmids that are transfected (Grant et al.,
1998).
Calpain Activity Assay with Suc-LLVY-AMC.
Twenty-four hours
after transfection, cells were rinsed with serum-free medium and the
medium was replaced with serum-free media containing 100 µM
glutamate, 100 µM glycine in the presence or absence of 100 µM
MK-801 in addition to 80 µM Suc-LLVY-AMC. Cells were then replaced in
a 5% CO2 incubator at 37°C. After a 40-min
incubation, the plates were read in a Victor2
fluorescence plate reader (PerkinElmer Wallac, Turku, Finland) at
wavelength settings of 390 nm and 460 nm for excitation and emission,
respectively. Previous studies have demonstrated that this assay is
linear with cell number, and the activity measured is representative of
calpain activity observed with protein substrates (Johnson and Guttman,
1997; Guttmann and Johnson, 1998).
Analysis of NMDA Receptor Subunit Degradation by Calpain in
Situ.
Twenty-four hours after transfection, cells were rinsed with
serum-free media and the media were replaced with serum-free media
containing 100 µM glutamate, 100 µM glycine in the presence or
absence of 100 µM MK-801. Cells were then replaced in the incubator for 30 min. After incubation, the cells were rinsed once with PBS and
scraped into 1× Laemmli stop buffer without bromphenol blue, EGTA, or
dithiothreitol (DTT). Samples were heated to 100°C for 5 min and
briefly sonicated. Protein concentrations were determined using the
bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL).
Bromphenol blue and DTT were then added, and the samples were stored at
-20°C until used. For assessment of the blockade of calpain, cells
were cotransfected with calpastatin, a specific inhibitor of calpain.
This inhibitor shows fewer toxic actions than do synthetic calpain
inhibitors and their vehicles (reviewed in Johnson and Guttmann, 1997).
Western Blotting. For HEK293t cellular homogenates, 40 to 50 µg of total protein was loaded on a 7% polyacrylamide gel. After SDS gel electrophoresis, proteins were transferred to nitrocellulose, blocked with 3% bovine serum albumin, and incubated with primary antibodies to NR2A, NR1a, actin, or calpain. Blots were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies and developed with enhanced chemiluminescence (Pierce). Each blot was quantitated using imaging densitometry and analyzed using NIH Image software (National Institutes of Health, Bethesda, MD). Statistical differences were determined by analysis of variance.
Calcium Uptake Assay. Six-well plates of transfected HEK-293t cells were washed two times with HEPES-buffered saline solution (HBSS) without CaCl2, and then 2 × 106 cpm of 45Ca in HBSS were added to each well along with 100 µM glutamate and 100 µM glycine in the presence or absence of 100 µM MK-801. Plates were incubated at room temperature for 10 min and then washed once with HBSS containing 2 mM CaCl2. Cells were then harvested by addition of 500 µl of 0.05% trypsin for 5 min. Twenty milliliters of scintillation cocktail were then added, and the radioactivity was quantified using a Beckman (model LS 5000TD) scintillation counter (Beckman Coulter, Inc., Fullerton, CA). Statistical difference was determined by two-sample t test.
125I-MK-801 Binding.
Cell membranes were
prepared as previously described, using preparations that remove MK-801
and other NMDA receptor antagonists (Lynch et al., 1995). Briefly, to
remove MK-801, the membrane fractions were homogenized in assay buffer
(20 mM HEPES, pH 7.5, 100 µM glutamate, 100 µM glycine, and 300 µM MgCl2) and incubated at 32°C for 30 min.
Homogenates were then centrifuged, and the pellet was resuspended in
assay buffer. This process was repeated two more times. Membrane
suspensions were then assayed in saturating glycine (100 µM) and
glutamate (100 µM), spermidine (100 µM), 100 µM
MgCl2, and 300 pM
125I-MK-801 (Lynch et al., 1994). Membranes were
harvested (Brandel Harvester) onto polyethyleneimine-coated glass-fiber
filters (Schleicher & Schuell, Keene, NH), and the radioactivity was
quantified using a Beckman (model 5500B) gamma counter. Using this
protocol, binding to NR1a/2A combinations was observed, whereas no
binding to NR1a or NR2A was detected (Lynch et al., 1994). Statistical
differences were determined by two-sample t test.
Calcium Imaging.
Twenty-four hours after transfection, cells
were rinsed twice with HBSS. The medium was then replaced with HBSS
containing 2 µM Fura-2 acetoxymethyl ester and returned to the
incubator. After a 30-min incubation, cells were rinsed twice with HBSS
and placed on the stage of a Nikon Eclipse TE300 microscope (Nikon, Melville, NY). Images of cells were obtained and analyzed using the
Metafluor imaging system (Universal Imaging, Downingtown, PA).
Calibrations were done as previously described (Guttmann and Johnson,
1998; Lynch et al., 2001). Prior to agonist application, images
were obtained for several minutes to establish a stable baseline
calcium measurement. Agonists (glutamate and glycine) were then
applied, and images were obtained at 1-s intervals. Peak calcium
concentrations were typically observed in less than 30 s.
Cell Toxicity Assay.
Toxicity assays were performed by
modifications of our previously described techniques using
cotransfection with GFP as a surrogate marker of cell viability
(Anegawa et al., 2000). HEK293t cells were cotransfected with NMDA
receptor combinations, calpastatin or vector control, and GFP.
Twenty-four hours later, cells were rinsed twice with HBSS and the
media were replaced with HBSS containing 100 µM glutamate and
100 µM glycine. Selected plates had 100 µM MK-801 included
throughout the transfection to provide a control for
non-receptor-mediated cell death. Zero, 4, or 8 h after agonist addition, the medium was aspirated and the cells were collected in 1×
Laemmli stop buffer without bromphenol blue, EGTA, or DTT. Samples were
heated to 100°C for 5 min and briefly sonicated. Protein
concentrations were determined using the bicinchoninic acid assay
(Pierce Chemical Co., Rockford, IL). Bromophenol blue and DTT were then
added, and the samples were stored at -20°C until use. To quantitate
cell death, samples were separated by SDS-PAGE and immunoblotted for
the presence of intact GFP. Assays were performed in triplicate, and
results were normalized to the mean cell viability at the beginning of
agonist application. Statistical differences were determined by
analysis of variance.
cDNA Constructs.
The shortened NR2A subunit NR2A1051 was
constructed as previously described (Grant et al., 1998). This
construct does maintain the epitope tag required for immunoreactivity
to antibody AB1548, consisting of the last six amino acids of the NR2A
subunit, and has normal electrophysiological and ligand-binding
properties (Grant et al., 1998; Guttmann et al., 2001).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Identification of Calpain in HEK293t Cells and Selective Activation
of Calpain by NMDA Receptors.
To determine whether calpain I was
present within HEK293t cells, 40 µg of total cellular homogenate was
separated by SDS-PAGE and immunoblotted with a monoclonal antibody
directed at calpain I (a generous gift of Dr. John Elce). An 80-kDa
band was detected, indicating that calpain is present in this cell line
(Fig. 1A).
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). Agonist stimulation of
NMDA receptors in transfected HEK293t cells activated calpain as
measured by the increase in fluorescence resulting from substrate cleavage. Calpain was not activated in cells cotransfected with NR1a/2A
and the specific calpain inhibitor calpastatin since the increase in
fluorescence was not significantly different from that observed in
untransfected cells treated with agonists. Some increase in
fluorescence was observed in both untransfected cells and cells
cotransfected with calpastatin, likely due to the ability of other
proteases to cleave Suc-LLVY-AMC (Guttmann et al., 1997). However,
because the increase in fluorescence in NR1a/2A-transfected cells was
inhibited by calpastatin, the proteolysis stimulated by agonist must
result from calpain activation. In contrast, activation of endogenous
adenosine receptors with ATP, which activates a variety of
calcium-dependent processes (Short and Taylor, 2000), did not stimulate
calpain activity as measured by increased Suc-LLVY-AMC fluorescence
(Fig. 1B, inset) in ATP-treated cells. This selective activation of
calpain by NMDA receptors matches the patterns observed in neurons
(Adamec et al., 1998).
In Situ Proteolysis of NR2A in NR1a/2A-Transfected HEK293t
Cells.
Since endogenous calpain was activated by NMDA receptor
agonists, we tested the ability of calpain to cleave NMDA receptor subunits. HEK293t cells were cotransfected with NR1a/2A in the presence
or absence of calpastatin and analyzed by SDS-PAGE and immunoblotting
for NR1a, NR2A, and actin (Fig. 2A).
Agonist stimulation of NR1a/2A-transfected cells for 30 min decreased
NR2A immunoreactivity to a C-terminal antibody by 45% (Fig. 2B)
compared with cells that were treated with the NMDA receptor antagonist
MK-801 in addition to glutamate and glycine. There was no significant
decrease in NR2A immunoreactivity in agonist-stimulated cells that were cotransfected with calpastatin, identifying the role of calpain activity in the cleavage of NR2A. NR1a subunit levels were not altered
following NMDA receptor activation, and no change in the amount of the
poor calpain substrate actin was observed. Interestingly, the
immunoreactivity at the beginning of agonist addition was slightly
increased in NR1/2A-transfected cells that were cotransfected with
calpastatin, compared with vector controls (although statistical significance was not reached), suggesting that basal calpain activity may play a role in turnover of this NMDA receptor combination (see
inset Fig. 2B). These data indicate that activation of calpain by NMDA
receptor stimulation leads to selective cleavage of the NR2A subunit.
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; Lynch et al.,
1995). 125I-MK-801 binding of agonist-treated
NR1a/2A/vector-transfected cells was decreased by 20% compared with
cells that were protected with MK-801 during agonist exposure (Fig.
2C). This decrease following agonist treatment was not observed in
NR1a/2A-transfected cells that were cotransfected with calpastatin,
demonstrating that calpain activity leads to decreased levels of
125I-MK-801 binding to assembled receptors, and
further suggesting that calpain may play a role in NMDA receptor
turnover and processing.
Since the epitope of NR2A necessary for immunoreactivity is located in
the last 20 amino acids of the C terminus, the absence of a relatively
large immunoreactive fragment (Fig. 2D) suggests that the calpain
cleavage sites are located near this epitope in the C-terminal region
of NR2A, consistent with previous data from in vitro experiments
(Guttmann et al., 2001) and the intracellular localization of calpain.
We therefore sought to identify whether a stable N-terminal product of
calpain was produced using an antibody to the N terminus of NR2A.
Activation of NR1a/2A-transfected cells with glutamate and glycine
decreased immunoreactivity using this antibody, but no stable
N-terminal product was detectable (see Fig. 2D). This strongly suggests
that in situ activation of calpain by the NMDA receptor leads directly
or indirectly to more complete degradation of the NMDA receptor.
These results contrast with previous demonstrations of the stability of
the N-terminal two-thirds of the NMDA receptor to in vitro digestion by
calpain (Bi et al., 1998a; Guttmann et al., 2001), and under postmortem
conditions (Wang et al., 2000). This could be explained if the initial
cleavages by calpain allow complete receptor degradation by other
cellular processes. To assess this, we investigated whether a truncated
form of NR2A (NR2A1051), which is not a substrate for calpain in vitro
(Guttmann et al., 2001), was also degraded in a receptor-activated
manner. Whereas activation of the mutant NR1a/2A1051 receptor resulted
in activation of calpain as measured by increased Suc-LLVY-AMC
fluorescence (Fig. 3A), activation of the
receptor did not decrease immunoreactivity for this NR2A1051 mutant
(Fig. 3B), and inclusion of calpastatin had no effect on NR2A1051
protein levels (data not shown). This shows that the crucial structural
determinants for in situ degradation of the NR2 subunit by calpain
localize to the C-terminal region of the receptor and that other
processing systems are likely involved subsequent to calpain-mediated
proteolysis.
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Effects of Calpastatin Cotransfection on 45Ca Uptake,
Intracellular Free Calcium, and Cell Death.
To determine whether
calpain-mediated proteolysis alters NMDA receptor function, the effects
of calpain inhibition on NMDA receptor-mediated calcium influx and
agonist-induced intracellular calcium transients were studied. Like
125I-MK-801 binding, these assays serve as
markers of physiologically active receptors, because channel activity
in this heterologous expression system is only present when a receptor
contains both NR1 and NR2 subunits (Grant et al., 1997). Calcium uptake
from the media in NR1a/2A cells cotransfected with calpastatin
was increased over NR1a/2A/vector-transfected cells by 50% (Fig.
4A) during agonist stimulation for 10 min. Similarly, agonist-induced intracellular calcium responses of
NR1a/2A-transfected cells were significantly potentiated by
cotransfection with calpastatin, suggesting that calpain activation
functionally limits NMDA receptor activity. To verify that the
physiological effects of calpastatin are directed at the same
structural region of the receptor as calpain cleavage, the effects of
calpastatin on agonist-induced calcium uptake and intracellular calcium
level changes in NR1a/2A1051- transfected cells were examined. The
presence of calpastatin did not significantly increase either calcium
uptake (see Fig. 4A) or intracellular calcium changes (see Fig. 4B) in
NR1a/NR2A1051 receptors. Although these truncated receptors may lack
regulatory sites of NMDA receptor control, the correlation of preserved
receptor levels with physiological properties of these truncated
constructs further supports the possibility that the effects of calpain
on calcium uptake and intracellular calcium levels occur through the
structural region regulating calpain-mediated degradation of the
receptor, the last 420 amino acids of NR2A (Grant et al., 2001).
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, 2000). Cells cotransfected with
calpastatin in addition to NR1a/2A/GFP died at an accelerated rate
compared with those transfected with NR1/2A/GFP and vector control
(Fig. 5) as determined by the amount of
GFP immunoreactivity remaining after either 4 or 8 h of agonist
stimulation. This is consistent with the increased intracellular
calcium rise observed in calpastatin-cotransfected cells (see Fig. 4, A
and B) and prior results linking increased extent of NMDA
receptor-mediated cell death with increasing rises in intracellular
calcium (Anegawa et al., 1995, 2000). These results suggest that
calpain can slow the rate of NMDA receptor-mediated cell death in
HEK293t cells.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The present study demonstrates that the NR2A subunit is a
selective in situ substrate for calpain in a cell culture expression system and that calpain-mediated proteolysis in the C terminus results
in NMDA receptor degradation and reduced activity. The effects of
calpain are mediated by the final 420 amino acids of the NR2A subunit
inasmuch as receptor constructs truncated to amino acid 1051 are not
cleaved by calpain, consistent with in vitro results (Guttmann et al.,
2001). However, in contrast to in vitro studies, the N-terminal portion
of the receptor is not stable following calpain activation, suggesting
that in this in situ system, the N-terminal products of calpain
cleavage are further degraded by other mechanisms. Taken together,
these data suggest that calpain-mediated cleavage can be the
controlling event for stability of the NMDA receptor and that calpain
processing in the C-terminal region of the NMDA receptor may modulate
NMDA receptor function in vivo.
This interpretation provides an understanding of paradoxical components
of previous studies examining calpain-mediated cleavage of the NMDA
receptor. Although selective cleavage of the NR2A subunit in the
C-terminal region (an intracellular domain which is not required for
activity) (Sprengel et al., 1998) has been shown, stably cleaved forms
of the NMDA receptor have not been readily found in neurons (Dingledine
et al., 1999). In addition, some studies suggest that a loss of NMDA
receptor activity is produced by calpain cleavage in situ (Bi et al.,
2000), even though cleavage occurred in a region that is nonessential
for activity. The present results suggest that calpain cleavage may act
as a trigger for NMDA receptor processing by other intracellular
proteases. This hypothesis is consistent with the findings of Wang et
al. (2000), who demonstrated that NR2 subunits were rapidly degraded in
post-mortem brain to fragments similar in size to those observed previously in vitro (Guttmann et al., 2001) and also retained ligand
binding, although a direct role for calpain was not determined.
Protease-mediated regulation of glutamate receptors has been proposed
previously in tissue plasminogen activator regulation of NMDA receptors
(Nicole et al., 2001) and AMPA receptor cleavage by calpains or
caspases (Glazner et al., 2000). The proposed calpain cleavage of the
NMDA receptor may be important in controlling secondary modulatory
systems similar to those in AMPA receptor regulation, where
caspase-mediated degradation of the AMPA receptor specifically shifts
cell death from necrosis to apoptosis (Glazner et al., 2000). However,
the putative mechanisms that lead to further NMDA receptor degradation
beyond calpain are not yet clear. Recent studies have demonstrated
regulation of NMDA receptor levels by binding of interacting proteins
such as F-actin and specific internalization motifs in the C terminus
(Lan et al., 2001; Lei et al., 2001; Roche et al., 2001; Scott et al.,
2001). Cleavage by calpain could alter interactions with such binding
proteins, perhaps leading to degradation by other proteases or
revealing internalization motifs that cause lysosomal degradation (a
subject of future study).
Since calpain in this system is stimulated directly by NMDA receptor
activation, the present results suggest that calpain cleavage of the
NR2A subunit can be a selective pathway for feedback inhibition of NMDA
receptor activity, perhaps by decreasing the number of functional
receptors. Prolonged stimulation of NMDA receptors results in a
phenomenon known as calcium-dependent inactivation (Legendre et al.,
1993) for which multiple mechanisms have been proposed. Several studies
have shown that calcium-dependent inactivation does not involve
calpain, because inactivation was not altered by calpain inhibitors but
required a specific calmodulin-binding component of the NR1 subunit
(Krupp et al., 1996; Zhang et al., 1998) and involved dissociation of
the NMDA receptor from actin. Another proposed calpain-independent
mechanism of NMDA receptor inactivation involves the dissociation of
NMDA receptors from actin by the actin-cleaving protease gelsolin
(Furukawa et al., 1997). The physiological effects observed in the
present study, however, suggest that in addition to these mechanisms,
there is a calpain-mediated pathway that can result in decreased NMDA
receptor activity. Additionally, in contrast to other proposed
mechanisms, neither the NR1 subunit nor actin is significantly degraded
in the present paradigm.
The present results link calpain cleavage of NMDA receptors to models of excitotoxicity using modest-duration exposure to glutamate in which biochemical assays and physiological approaches were performed in parallel with similar results. Although synaptic modifications of NMDA receptors may occur over a shorter time period than those used in the present study, the results of calcium imaging studies suggest that calpain may modulate NMDA receptor function either at a basal level of activity or rapidly after agonist-induced activation of the receptor. In addition, the degree of NMDA receptor modification necessary for significant modification of synaptic architecture and the exact properties of calpain activation in single dendritic spines make quantitative comparison of the events in our model system with synaptic events difficult. However, direct application of the results of the present study to neuronal paradigms may help to better define the role of calpain cleavage of the NMDA receptor in synaptic modification.
Besides the NMDA receptor, other synaptic proteins are physiological
substrates of calpain including neuronal nitric-oxide synthase,
calmodulin kinase, and other calcium channels (Dosemeci and Reese,
1995; Hell et al., 1996). Class L-type calcium channels have increased
calcium permeability following calpain cleavage (Hell et al., 1996),
whereas ryanodine receptor channel activity is decreased by calpain
proteolysis (Shevchenko et al., 1998). In the case of NR2A, calpain
cleavage appears to result primarily in decreased NMDA receptor amount.
Although the C-terminal region of NR2A where calpain cleaves is not
required for electrophysiological activation of the receptor, it may
affect other properties to a modest degree (Sprengel et al., 1998).
Thus, if neuronal stabilizing mechanisms exist, calpain cleavage might
lead to intermediate NMDA receptors with novel properties as previously
suggested (Guttmann et al., 2001). In addition, because previous
studies have shown that NR2A phosphorylation by protein kinase C
inhibits calpain cleavage of the NR2 subunit (Bi et al., 1998a),
protein kinase C phosphorylation may be involved in blocking
calpain-mediated turnover of NMDA receptors. Thus, some of these second
messenger systems themselves may be regulated by calpain, and further
amplification of the direct calpain-mediated NMDA receptor modulation
may also occur.
Because both NMDA receptors and calpain have been linked to
neuropathological conditions such as excitotoxicity (Lynch and Guttmann, 2001), the present findings suggest new possibilities for understanding the mechanisms of neurodegeneration and
neuroprotection. Calpain inhibitors have been proposed as
neuroprotective agents. The present data suggest that although calpain
inhibition may block the cellular degradation in response to
excitotoxic stimuli, it may indirectly potentiate cell death. Based on
the current findings, antagonists to NMDA receptors, while preventing
the normal activation of calpain, would decrease NMDA receptor
turnover. The presence of additional NMDA receptors may lead to a
delayed and more dramatic increase in intracellular calcium once the
pharmacological inhibitor is removed or metabolized. Alternatively, the
low molecular weight products generated by the selective calpain
cleavage of the NR2A subunit may have specific roles in neuronal
function or pathology, as similar calpain-generated fragments from
troponin have been shown to have in cardiac disease (Murphy et al.,
2000). This is consistent with our previous finding that NMDA receptors lacking the final 420 amino acids are less toxic when transfected into
cells, despite having similar physiologic properties (Anegawa et al.,
2000; Guttmann et al., 2001).
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Acknowledgments |
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We thank Drs. Michael Robinson and Amy Brooks-Kayal for helpful comments, Dr. Masatoshi Maki for the calpastatin cDNA, Dr. John Elce for the calpain I antibody, and the Mental Retardation and Research Center for DNA sequencing.
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Footnotes |
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Accepted for publication May 3, 2002.
Received for publication April 4, 2002.
This work was supported by Grants DA07130, NS01789, NS39126, MH14654, and NS1084 from the National Institutes of Health; a Beeson Fellowship from the American Federation for Aging Research; and Grant 9920365U from the American Heart Association (R.P.G.). Support for DNA sequencing was provided through the Molecular Genetics Core of the Mental Retardation Research Center Grant HD26979.
DOI: 10.1124/jpet.102.036962
Address correspondence to: Dr. David R. Lynch, Division of Neuroscience Research, Children's Hospital of Philadelphia, Philadelphia, PA 19104-4318. E-mail: lynch{at}pharm.med.upenn.edu
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Abbreviations |
|---|
NMDA, N-methyl-D-aspartate;
NR1, NMDA receptor
subunit 1;
HEK, human embryonic kidney;
MK-801, dizocilpine;
DTT, dithiothreitol;
HBSS, HEPES-buffered saline solution;
GFP, green
fluorescence protein;
PAGE, polyacrylamide gel electrophoresis;
Suc-LLVY-AMC, succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-aminomethyl
coumarin;
AMPA, 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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,
p < 0.05, n = 3-5). No
significant difference in fluorescence was observed in calpastatin- or
vector control-transfected cells incubated in the presence of 100 µM
ATP (B, inset).
). Arrowheads indicate
position of molecular weight markers in kilodaltons.
