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The Journal of Neuroscience, March 15, 2002, 22(6):2044-2053
The NMDA Receptor M3 Segment Is a Conserved Transduction Element
Coupling Ligand Binding to Channel Opening
Kevin S.
Jones,
Hendrika M. A.
VanDongen, and
Antonius M. J.
VanDongen
Department of Pharmacology and Cancer Biology, Duke University
Medical Center, Durham, North Carolina 27710
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ABSTRACT |
Ion channels alternate stochastically between two functional
states, open and closed. This gating behavior is controlled by membrane
potential or by the binding of neurotransmitters in voltage- and
ligand-gated channels, respectively. Although much progress has been
made in defining the structure and function of the ligand-binding cores
and the voltage sensors, how these domains couple to channel opening
remains poorly understood. Here we show that the M3 transmembrane segments of the NMDA receptor allosterically interact with both the
ligand-binding cores and the channel gate. It is proposed that M3
functions as a transduction element whose conformational change couples
ligand binding with channel opening. Furthermore, amino acid homology
between glutamate receptor M3 segments and the equivalent S6 or TM2
segments in K+ channels suggests that ion channel
activation and gating are both structurally and functionally conserved.
Key words:
ion channel gating; affinity; efficacy; receptor
structure; ligand binding; neurotransmitters; activation mechanism; NMDA receptor
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INTRODUCTION |
Cation channels consist of multiple
domains or subunits arranged around a central ion-conducting pore. The
pore-forming regions of K+ channels and
glutamate receptors (GluRs) display significant amino acid
sequence conservation (Wo and Oswald, 1995 ; Wood et al., 1995),
suggesting that they have evolved from a common ancestor (Wood et al.,
1995). Identification of GluR0, a prokaryotic ionotropic glutamate
receptor with a K+-selective pore (Chen et
al., 1999), confirmed this notion and further strengthened the idea
that K+ channels and glutamate receptors
are structurally related (Fig. 1A). The amino acid
sequence of the "missing link" GluR0 allowed us to extend this
homology to the transmembrane segment that follows the P region (Fig.
1B). In glutamate receptors, this M3 segment contains
a nine amino acid sequence (SYTANLAAF) that is highly conserved
throughout all members of the family. The alanine at position 8 of this
motif is mutated to threonine in the  2
glutamate receptor of the lurcher mutant mouse (Zhou et al.,
1997). Whereas wild-type 2 receptors are nonfunctional
in heterologous expression systems, the lurcher mutation
(A654T) results in constitutive activation of the channel and causes
neurodegeneration. Introducing the same mutation in the GluR1 AMPA
receptor increases agonist potency and converts a competitive
antagonist into an agonist (Taverna et al., 2000). Introducing the
lurcher mutation into AMPA, kainate, and NMDA receptors
reduced receptor desensitization and reduced the rate of receptor
deactivation (Kohda et al., 2000). Because the M3 segment is not part
of the ligand-binding core, these data suggest a role for M3 in channel
activation.
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Figure 1.
Topology and homology for K+
channels and glutamate receptors are shown. A, Ion
channels are modular proteins. The pore-forming region of the bacterial
KcsA K channel, formed by two transmembrane segments flanking a
re-entrant hairpin loop, represents a motif that is found in both
voltage-gated K+ channels and ionotropic glutamate
receptors. B, Amino acid sequence alignment of the
pore-forming regions and adjacent transmembrane segment for
voltage-gated K+ channels (P region
plus S6), the KcsA K+ channel
(P region plus TM2), and glutamate
receptors (P region plus M3). Conserved
residues are shown on a white background. The
prokaryotic glutamate-gated K+ channel GluR0 guides
the alignment in the S6-TM2-M3 region. C, Schematic
representation of four potential conformations of the ligand-binding
core of glutamate receptors: the agonist-free conformation
(R), the agonist-occupied-open conformation
(AR), the agonist-occupied-closed conformation
(AR*), and the agonist-free-closed conformation
(R*). Crystallographic data exist only for R and AR*.
D, Linear three-state model of agonist-induced receptor
activation. There are two coupled reactions. In the initial association
reaction, an agonist binds to lobe S1. In a second step, the
agonist-bound conformation can undergo domain closure. The equilibrium
constant KA reflects the affinity of the
agonist for the open conformation, whereas E is a
measure of the agonist efficacy (Colquhoun, 1998). E,
Allosteric, four-state model of agonist-induced receptor activation
(Colquhoun, 1998). The ligand-binding core is able to undergo domain
closure in both the absence and the presence of bound agonist. The
probability of being in the closed conformation is low in the absence
of agonist. Agonist occupancy stabilizes the closed conformation.
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The ligand-binding site in glutamate receptor subunits is formed by two
domains, S1 in the N terminal and S2 in the M3-M4 linker
(Stern-Bach et al., 1994) (Fig. 1A). The S1S2
ligand-binding core of GluR2 has been crystallized, with a partial
agonist molecule bound to the structure (Armstrong et al., 1998). More
recently, additional structures have been obtained for the GluR2
ligand-binding core, including an agonist-free (apo) conformation,
conformations with full and partial agonists, and an antagonist-bound
conformation (Armstrong and Gouaux, 2000). The S1-S2 structure reveals
two lobes connected by a hinge, forming a clamshell-like structure, similar to the bacterial periplasmic binding proteins (Oh et al., 1993). These novel GluR2 structures confirm and extend what was known
for the bacterial periplasmic binding proteins, namely that ligand
binding induces a conformational change in which the lobes rotate on
the hinge and collapse around the ligand. These crystallographic data
suggest a model for agonist activation of glutamate receptors (Armstrong and Gouaux, 2000), in which the binding of ligand stabilizes the closed form of the clamshell (Fig. 1C-E).
Because glutamate receptors are tetramers (Laube et al., 1998;
Rosenmund et al., 1998), functional receptors contain four ligand-binding sites. NMDA receptors are a subtype of glutamate receptor, the activation of which requires binding of the co-agonist glycine in addition to glutamate (Johnson and Ascher, 1987). NMDA receptors (NRs) are heteromeric; they are composed of glycine-binding NR1 subunits (Kuryatov et al., 1994; Hirai et al., 1996) and
glutamate-binding NR2 subunits (Anson et al., 1998). An analysis of
agonist- and antagonist-binding rates suggested that each receptor
contains two glycine- and two L-glutamate-binding sites
(Benveniste and Mayer, 1991; Clements and Westbrook, 1991). Despite a
rather detailed understanding of ligand binding in glutamate receptors
and other ligand-gated ion channels, it is not clearly understood how
closure of the ligand-binding lobes leads to opening of the channel
gate (Colquhoun, 1998). The data presented here demonstrate that the M3
transmembrane segments of the NMDA receptor couple ligand binding to
channel gating.
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MATERIALS AND METHODS |
Mutations were generated using the megaprimer PCR method, as
described previously (Wood et al., 1995), and were confirmed by DNA
sequencing. cRNA for wild-type and mutant NMDA receptor subunits were
prepared by in vitro transcription, as described previously
(Wood et al., 1995).
Oocyte preparation and injection. Adult female Xenopus
laevis were anesthetized, and a few ovarian lobes containing stage V or VI oocytes were removed. The incision was sutured and the frog was
allowed to recover. Oocytes were dispersed by manually disrupting the
egg sac and were digested for 2 hr with collagenase type I (2 mg/ml)
(Invitrogen, Gaithersburg, MD) to further remove the follicular
layer. Injection of cRNA (75 nl of a 10-100 ng/µl solution) was
performed under a dissecting microscope with a micrometer-driven micropipetter. Oocytes were then transferred to SOS buffer (in mM: 100 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH 7.6) supplemented with gentamicin (100 µg/ml) and fungizone (100 µg/ml) and maintained at 19°C.
Two-electrode voltage clamp electrophysiology. Functional
expression was assessed 2-5 d after injection using a two-electrode voltage-clamp amplifier (OC-725; Warner Instrument, Hamden, CT) by
application of agonists during continuous perfusion of a buffer solution containing (in
mM): 100 NaCl, 5 KCl, 0.5 BaCl2, 10 HEPES, 10 µM EDTA, pH 7.3, at 25°C. Barium, rather than
calcium, was used as the divalent cation to minimize secondary
activation of calcium-activated Cl
currents (Leonard and Kelso, 1990). EDTA was used to chelate trace
amounts of the soft-metal divalent cations
Cd2+ and
Zn2+, which have been reported to
contaminate buffer solutions (Paoletti et al., 1997) and inhibit the
NMDA receptor by binding to a high-affinity site (Low et al., 2000;
Paoletti et al., 2000). EDTA also removes a zinc-dependent component of
desensitization (Zheng et al., 2001). Oocytes were placed in a
perfusion chamber (Warner Instrument) that was optimized for laminar
flow. Solution changes were accomplished using a gravity-fed,
computer-controlled perfusion system, which uses BIO-SIL silicone
rubber tubing (3.8 mm outer diameter; 1.5 mm inner diameter; Sil-Med,
Taunton, MA) and solenoid valves (General Valve, Fairfield, NJ). During
solution exchanges, the solution flow rate was ~15 ml/min. Solution
exchange kinetics was quantified as follows. Inward NMDA
currents were recorded at a holding potential of  60 mV in a buffer
containing (in mM) 100 NaCl and 0.5 BaCl2, after application of 10 µM glycine plus 100 µM
L-glutamate. When steady state was reached, all
of the Na+ ions were replaced with
impermeable
N-methyl-D-gluconate ions, resulting in an instantaneous change in the driving force. The inward
NMDA current decayed with a biexponential time course. The fast
component had a time constant of 180 msec and a relative amplitude of
89%. The remaining 11% decayed with a time constant of 2.3 sec. This
implies that ~90% of the solution exchange is complete within 200 msec, but the remaining 10% takes several seconds to complete.
Low-resistance glass microelectrodes (0.5-2.0 M ) were filled with 3 M KCl and 10 mM HEPES, pH
7.2, and used to impale the oocyte. Current traces were recorded from a
holding potential of 60 mV. Data acquisition and voltage control were accomplished with pClamp hardware and software (Axon Instruments, Burlingame, CA).
Curve fitting. The time courses of NMDA current deactivation
in Figure 3 were fitted with exponential models by minimizing the
residual sum of squares (RSS), using the Solver function in Microsoft
Excel (Microsoft, Redmond, WA). One- and two-exponential models
were compared using the asymptotic information criterion (AIC)
(Akaike, 1981; DiStephano and Landaw, 1984): AIC = N
log(RSS) + 2P, where N is the number of
data points and P is the number of parameters in the model.
The best model is the one that minimizes AIC. The remaining time
courses of deactivation and 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) modification were fitted with exponential models
using the Clampfit module in pClamp 6.0 (Axon Instruments).
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RESULTS |
The role of the M3 segment in the activation pathway of glutamate
receptors was examined because (1) the lurcher phenotype suggested that an M3 residue (A654) had a role in receptor gating and
(2) the strict amino acid conservation in the C-terminal half of this
transmembrane domain among all of the members of the glutamate receptor
family suggested an important functional role. Amino acids in the
SYTANLAAF region of NMDA receptor subunits were individually substituted with cysteines. Because functional NMDA receptors are
heteromeric assemblies of NR1 and NR2 subunits, mutations were
introduced into each subunit separately and coexpressed in Xenopus oocytes with the complementary wild-type subunit.
NMDA receptors containing mutant subunits were then exposed to the thiol-modifying reagent MTSEA (Akabas et al., 1992) to explore the
function of these residues in activation of the receptor. Because both
the NR1 and NR2A subunits contain several extracellular cysteines, the
effect of MTSEA treatment was first characterized in the wild-type NMDA
NR1 plus NR2A receptor.
Effects of MTSEA on wild-type NMDA receptor function
MTSEA treatment of the wild-type NMDA receptors in the absence of
agonist had no significant effect on current amplitude (Fig. 2A). Simultaneous
application of MTSEA and agonists did cause a significant
inhibition of NMDA current, but this effect was reversed after washout
(Fig. 2B). Given its size and charge, it is likely
that MTSEA can act as an open channel blocker in NMDA receptors, as has
been shown for other channels (Liu and Siegelbaum, 2000). To test this
idea, MTSEA was applied at positive holding potentials, where it
exerted no effect on current amplitude (data not shown). Therefore, the
reversible inhibition by MTSEA in the presence of agonist likely
represents open channel block and not covalent modification of a thiol
group. Because MTSEA application had no lasting effect on the current
amplitude evoked from wild-type NMDA receptors, we conclude that either
the endogenous cysteines of the receptor were not available for
modification or their modification caused no discernible change in
current amplitude.
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Figure 2.
MTSEA modification of A7C-containing receptors is
agonist dependent. Representative current traces are shown from oocytes
expressing wild-type NR1 plus NR2A NMDA receptors and NMDA
receptors containing NR1-A7C or NR2A-A7C subunits, before and after a
60 sec exposure to 0.5 mM MTSEA in the absence (A,
C, E) or presence (B, D, F) of
supramaximal concentrations of agonists (100 µM
L-glutamate plus 10 µM glycine).
Discontinuous traces show breaks representing 90-180
sec washout periods. Dashed lines represent the
maximal response of the oocyte to agonist application before
MTSEA treatment. A, MTSEA application had no significant
effect on the maximal current evoked from wild-type NMDA receptors when
applied alone [change in maximal current, 11 ± 10% (mean ± SEM); n = 7; p = 0.27].
B, Coapplication of MTSEA and agonists transiently
inhibited current from wild-type receptors (change in maximal current,
43 ± 5%; n = 4), although there was no
lasting effect on the amplitude of subsequent activation. The change in
maximal current was 7 ± 5% (n = 4).
C, E, MTSEA application did not significantly affect the
maximal current evoked from NR2A-A7C or NR1-A7C mutant NMDA receptors
when applied in the absence of agonist: the change in maximal current
was 9 ± 6% (n = 9; p = 0.15) for NR1-A7C and 11 ± 5% (n = 10;
p = 0.06) for NR2A-A7C. ANOVA performed on the data
set described in A, C, and
E failed to identify a significant difference between
the wild-type and A7C-containing receptors
(p = 0.95). D, F,
Coapplication of MTSEA and agonists potentiated the agonist-evoked NMDA
current and slowed deactivation in A7C-containing receptors (change in
maximal current for NR1-A7C, 152 ± 11%; n = 8; change in maximal current for NR2A-A7C, 195 ± 13%;
n = 18).
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The SYTANLAAF region
Amino acids in the SYTANLAAF region of NMDA receptor subunits were
individually substituted with cysteines. Eight of the nine cysteine-substituted NR1 constructs were functional. However, NMDA
receptors containing the NR1-S646C mutation did not yield current. The
remaining receptors were exposed to the thiol-modifying reagent MTSEA,
both in the absence and in the presence of agonists. Four of the
cysteine-substituted NR1 receptors exhibited state-dependent responses
to MTSEA application. Simultaneous exposure to MTSEA and agonist
reduced the current amplitudes of receptors containing the NR1 mutants
T649C, N651C, or L652C by 75 ± 8%, 64 ± 9%, and 40 ± 8%, respectively (data not shown). However, exposure to MTSEA in
the absence of agonists did not reduce the NMDA currents in these
mutants (data not shown). MTSEA modification of NR1-A653C revealed a
critical role in receptor activation. This mutation targets the alanine
at position 7 of the SYTANLAAF region and will be referred to as A7C.
It should be noted that the A7C mutation immediately precedes the
lurcher mutation A8T. The remainder of this article
characterizes the unique properties conferred by MTSEA
modification of NMDA receptors containing the A7C mutation in either
the NR1 or NR2 subunit.
Access to residue A7C requires receptor activation
The amplitudes of currents evoked from NMDA receptors containing
the A7C mutation in the SYTANLAAF region of either NR1 or NR2A were
comparable with that of the wild-type receptor. The glutamate and
glycine concentration-response curves of the A7C mutants did not
indicate substantial changes in the apparent affinity (EC50) of either of the co-agonists (Table
1). This suggested that the overall
structure of the channel was not grossly disrupted by the mutation.
NMDA current evoked from receptors containing the A7C mutation in
either the NR1 or NR2A subunit was not affected by exposure to MTSEA in
the absence of agonist (Fig. 2C,E), although simultaneous application of MTSEA and agonists potentiated the current amplitude and markedly decreased the deactivation rate (Fig.
2D,F). This indicates a pronounced
activation-state dependence for MTSEA modification of the
A7C-containing receptors. Similar results were obtained when MTSEA was
substituted with the membrane-impermeable analog
[2-(trimethylammonium)ethyl] methane thiosulfanate (data not shown), indicating that access to residue A7C occurs from the
extracellular space. Taken together, these data suggest that MTSEA
gains access to the A7C residue only when the receptor is activated.
MTSEA modification slows deactivation of A7C NMDA receptors
The current evoked during activation of ligand-gated ion channels
is rapidly abolished after removal of the agonist. This deactivation
occurs as the agonist dissociates from its binding site and the ion
channel returns to its resting state. Deactivation of wild-type
NR1/NR2A NMDA receptors proceeded with a biexponential time course
(Fig. 3). This time course may in part be
determined by the relatively slow perfusion system (see Materials and
Methods). The deactivation kinetics of A7C-containing NMDA receptors
was also biexponential, and the time constants obtained were
statistically indistinguishable from the wild-type receptor (Fig. 3).
MTSEA application in the absence of agonists did not affect the
deactivation kinetics of the wild-type or A7C-containing NMDA
receptors. Concurrent application of MTSEA and agonist had no effect on
the wild-type NMDA receptor, but it dramatically slowed deactivation of
NR1-A7C and NR2A-A7C receptors. Figure 3 summarizes the effects of
MTSEA modification on the deactivation kinetics of A7C-containing NMDA receptors, as well as the requirement for the presence of agonists. MTSEA modification also potentiated the amplitude of currents evoked
from the A7C NMDA receptors. Interestingly, the potentiation of NMDA
current occurred despite activation of the A7C NMDA receptors with
supramaximal concentrations of agonists (Fig. 2D).
The effect that MTSEA exerts on the deactivation kinetics and current
amplitudes of the A7C NMDA receptors was examined more thoroughly.
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Figure 3.
MTSEA modification slows deactivation of A7C
receptors. Left panels,
Representative current traces showing deactivation time course of
wild-type (WT) and A7C-containing NMDA receptors before treatment
(A), after MTSEA treatment
(B), and after MTSEA treatment in the presence of
10 µM glycine and 100 µM
L-glutamate (C). To facilitate
comparison, the deactivation traces are offset on the time axis and
normalized. Exponential fits (solid lines) are
superimposed on the experimental data. The NR1-A7C trace after
MTSEA plus agonists was best described by a single exponential
component, whereas all of the other traces required a sum of two
exponential components, according to the AIC (see Materials and
Methods). The curves in C did not return to baseline,
and they required an additional offset amplitude. The constitutive
activity as a percentage of the maximum MTSEA-modified current was 19%
for NR1-A7C and 54% for NR2A-A7C receptors, respectively.
Right panels, Bar graphs
illustrating time constants (means ± SEM) of the exponential
models that best describe the deactivation time courses for the three
experimental conditions. Black bars represent the
dominant time constant (that with the largest relative
amplitude). The white bars represent the time
constant with the smallest relative amplitude. The width of each column
indicates the relative amplitude of each exponential component; the
total width of the black bar plus the white bar represents 100%.
Numbers in parentheses represent the number of oocytes.
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Modification of A7C requires closure of both glutamate- and
glycine-binding cores
The experiments described above suggested that activation
of the NMDA receptor is required for MTSEA to gain access to the A7C
residue. Agonist binding induces receptor activation by a series of
conformational changes that culminate in channel opening. This process
is initiated by closure of the bilobate binding cores (Armstrong and
Gouaux, 2000). In the NMDA receptor, activation requires binding of
both glycine and glutamate at the NR1 and NR2A subunits, respectively.
Therefore, we investigated whether the application of either co-agonist
alone was sufficient to allow MTSEA access to the A7C residue.
Coapplication of MTSEA and L-glutamate failed to evoke
current from either NR1-A7C- or NR2A-A7C-containing receptors and did
not result in their covalent modification (data not shown). However,
coapplication of MTSEA and glycine did produce a very slowly developing
current in A7C-containing NMDA receptors that required several minutes
to reach steady state (Fig. 4). Interestingly, glycine by itself was found to partially activate the
NMDA receptor to a level between 1 and 5% of the maximal activation obtained with both co-agonists (Fig. 4). These data suggest that our
glycine solutions may have been contaminated by trace amounts of
L-glutamate, or alternatively, that glycine may act as a
partial agonist at the glutamate-binding site. In both scenarios, the glutamate-binding site is partially activated by an agonist. Therefore, we investigated whether the glutamate-binding site plays a critical role in the glycine-only NMDA current, which supports the modification by MTSEA. Coapplication of the competitive L-glutamate
antagonist APV reduced the glycine-only currents evoked from the A7C
NMDA receptors in a dose-dependent manner (Fig. 4). Concomitant with the reduction in receptor activation, APV also reduced the rate and
extent of MTSEA modification observed during glycine application to the
A7C NMDA receptors (Fig. 4). Although these results do not fully
resolve the nature of the glycine-only currents evoked from
A7C-containing NMDA receptors, they indicate that MTSEA modification of
A7C requires that both the glycine- and glutamate-binding sites be
occupied by effective agonists. The absence of glycine from its binding
site or the presence of an antagonist in the glutamate site is
sufficient to prevent MTSEA modification.
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Figure 4.
APV prevents MTSEA modification by glycine alone.
Representative current traces show modification of NR1-A7C by 0.5 mM MTSEA and 10 µM glycine, in the absence
and the presence of 100 or 500 µM APV. Similar results
were obtained in NR2A-A7C-containing receptors (data not shown). Traces
are normalized to the current amplitude evoked by the supramaximal
agonist application represented by the dashed
line.
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From the data described thus far, it could be concluded that lobe
closure of both the glutamate- and glycine-binding sites is required to
allow the conformational change in M3 that makes residue A7C accessible
to MTSEA. However, lobe closure at the ligand-binding sites induces a
cascade of conformational rearrangements that result in channel
opening, Therefore, it is not clear which of the several steps involved
directly causes MTSEA accessibility to residue A7C. The conformational
change in M3 could result directly from ligand binding, or
alternatively, it could be a consequence of channel gating. This point
was further elaborated by investigating the correlation between the
rate of MTSEA modification with ligand binding and gating
parameters. Because thiol modification by MTSEA is extremely rapid, up
to 105 M1
sec1 (Stauffer and Karlin, 1994), the chemical
reaction is not the rate-limiting step of modification. Rather, the
rate of MTSEA modification depends critically on the relative amount of
time that the cysteine at position A7 is exposed to the aqueous
environment of the receptor.
MTSEA modification rate depends on agonist concentration
The relationship between MTSEA modification rate and agonist
concentration was investigated to correlate the activation state of the
A7C NMDA receptors with MTSEA accessibility to residue A7C. When lower
concentrations of agonists were used, MTSEA was found to potentiate the
NMDA currents substantially (Fig.
5A), suggesting that modified
receptors are much more sensitive to agonists. The rate of MTSEA
modification was much slower when lower concentrations of agonists were
used (Fig. 5A). The time course of current potentiation was
best fit by a single exponential function, and the time constant was
dependent on the concentration of both
L-glutamate and glycine. However, the
MTSEA-induced potentiation of current evoked from the NR2A-A7C receptor
became biexponential at the highest agonist concentrations. In all of
the cases, the NMDA current potentiation became faster with increasing
agonist concentration and the kinetics saturated at higher
concentrations. Modification rate constants were plotted as a function
of the agonist concentration and fitted with the Hill equation. The
resulting half-maximal concentrations (EC50) and
Hill coefficients are summarized in Table 1. Normalization of the rate
constants allowed for direct comparison between the modification
kinetics and the activation state of the NMDA receptor, as reflected by
the concentration-response curves (Fig. 5B,C). The
kinetics of current amplitude potentiation paralleled the
concentration-dependent activation of the receptors. The two curves,
steady-state activation and MTSEA modification rate, overlapped when
plotted as a function of glycine concentration for the NR1-A7C
receptors (Fig. 5B). However, the MTSEA modification rate
curve of the NR2A-A7C receptors was significantly left-shifted from the
activation curve when plotted as a function of glutamate concentration
(Fig. 5C). This discrepancy suggests that A7C accessibility is not directly determined by the activation state (the open
probability) of the receptor, as reflected by the normalized
concentration-response curve. Instead, the conformational changes in
M3 display a differential sensitivity to occupancy at the glycine- and
glutamate-binding sites that cannot be derived from channel gating
alone.
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Figure 5.
MTSEA modification rate depends on agonist
concentration. A, Representative NR1-A7C NMDA currents
showing the effect of lowering the agonist concentration on the MTSEA
modification rate. Supramaximal agonist concentrations (100 µM glutamate plus 10 µM glycine) were first
applied to establish the maximal current (indicated by the dotted
line). A submaximal concentration of glycine (100 µM
glutamate plus 100 nM glycine) was then applied, resulting
in a fractional response. Addition of 0.5 mM MTSEA resulted
in a slowly developing potentiation to a level that exceeded the
maximal response before treatment. B, Glycine
concentration-response curves for NR1-A7C in the presence of a
saturating concentration of L-glutamate (100 µM). Graphs are shown for normalized NMDA currents
(open circles) and normalized MTSEA modification rates
(closed squares). C, Glutamate
concentration-response curves for NR2A-A7C in the presence of a
saturating concentration of glycine (100 µM). Graphs are
shown for normalized NMDA currents (open circles) and
normalized MTSEA modification rates (closed
squares). All of the data were fitted with the Hill
equation: R/Rmax = 1/[1 + (EC50/[agonist])n], where
R is the response (current or rate) for the given
agonist concentration, Rmax is the maximal
response, n is the Hill coefficient, and
EC50 is the concentration midpoint. The values for the Hill
coefficient and EC50 are listed in Table 1.
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MTSEA modification of residue A7C depends on agonist efficacy
The recent crystallographic structures of the GluR2 ligand-binding
core suggest a model in which interaction of ligands with their binding
sites is characterized by two distinct processes, agonist binding and
domain closure (Fig. 1C-E). These molecular reactions can
be described by either a linear three-state model or a more general
four-state allosteric model (Colquhoun, 1998). In the linear model, the
agonist interacts with the open conformation of the ligand-binding core
in an initial binding reaction. Once bound, the agonist stabilizes a
closed conformation of the ligand-binding clamshell (Fig.
1D). This closed conformation of the ligand-binding core promotes receptor activation and ultimately results in channel opening. In the allosteric model, the ligand-binding core is able to
visit the closed conformation spontaneously, even in the absence of
bound agonist, although the probability is very low (Fig.
1E). However, agonist occupancy facilitates domain
closure by stabilizing the closed conformation.
The concentration dependence of the MTSEA modification rate (Fig.
5B,C) correlates A7C accessibility with agonist occupancy. However, there are two distinct conformations in which the agonist occupies the ligand-binding core: the agonist-bound-open (A·R) state
and the agonist-bound-closed (A·R*) state (Abele et al., 2000).
Therefore, we wanted to determine which of these
conformations support the accessibility change of residue
A7C. This important issue was investigated by activating A7C NMDA
receptors with partial agonists before exposure to MTSEA. The models
describing the interaction between the agonist and its binding site
(Fig. 1C,D) are reduced to a two-state scheme at saturating
agonist concentrations, in which the probability of observing the
unoccupied receptor state(s) becomes vanishingly small. Under these
conditions, the ligand-binding cores alternate stochastically between
the closed (A·R*) and open (A·R) conformations. The degree of
domain closure induced by agonists can vary substantially (Armstrong
and Gouaux, 2000) and is an important determinant of the efficacy of
the agonist (E in Fig. 1D,E).
Experimentally, agonist efficacy is estimated by its intrinsic activity,  (Ariens, 1954).
By activating A7C NMDA receptors with saturating concentrations of
agonists with different intrinsic activities, it is possible to
correlate the accessibility of residue A7C with the position of the
equilibrium between the A·R and A·R* conformations of the ligand-binding core. MTSEA modification of A7C NMDA receptors proceeded
more slowly when partial agonists were substituted for L-glutamate or glycine (Fig.
6A). Therefore,
accessibility of residue A7C, as measured by the rate of MTSEA
modification, depends critically on the stability of the
agonist-bound-closed conformation (A·R*) of the ligand-binding core.
The MTSEA modification rate of the NR1-A7C receptor correlated linearly
with the intrinsic activity of the glycine-site agonists, and the
modification rate of the NR2A-A7C receptor correlated linearly with the
intrinsic activity of the glutamate-site agonists (Fig.
6B,C). This could be expected because closure of the
NR1 ligand-binding core is induced by glycine and glycine-like
compounds, and closure of the NR2 ligand-binding domains are induced by
L-glutamate and glutamate-like compounds.
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|
Figure 6.
MTSEA modification rate depends on intrinsic
activity. A, Representative traces show modification of
NR2A-A7C in the presence of saturating concentrations of various full
and partial agonists. Left trace, The full agonists
L-glutamate and glycine (100 µM glutamate
plus 10 µM glycine) allow rapid modification of the NMDA
receptors by MTSEA. Middle trace, The partial agonist QA
replaced glutamate (5 mM; EC50 of 1200 µM and 600 µM for NR1-A7C and NR2A-A7C,
respectively). Right trace, The partial agonist
HA-966 (500 µM) replaced glycine. (Note: the
EC50 of HA-966 could not be accurately determined, but
modification rates were identical at 500 µM and 5 mM, implying saturation of the binding site in NR1-A7C and
NR2A-A7C.) B, C, Plots of MTSEA modification rate versus
agonist intrinsic activity. Squares represent
modification rates obtained with the full agonists
L-glutamate and glycine. Open circles
represent rates obtained with the partial glycine-site agonists HA-966
(500 µM) or D-cycloserine (100 µM; EC50 of 14 µM and 32 µM in NR1-A7C and NR2A-A7C, respectively). Closed
circles represent rates obtained with the partial
L-glutamate agonists QA, NMDA, or PDA. The
modification rate for NR1-A7C correlates with the intrinsic
activity of the glycine site agonists
(R2 = 0.926), but does not
correlate linearly with the glutamate site agonists. Modification of
NR2A-A7C correlates with both glycine and glutamate site agonists
(R2 = 0.975 and 0.982, respectively). Intrinsic activity () was calculated as the fraction
of maximal current evoked by application of saturating concentrations
of partial agonist: = Ipartial
agonists/Iglutamate + glycine.
D, E, Bar graphs indicating the fraction of maximal
current evoked by each partial agonist before and after MTSEA
modification.
|
|
Surprisingly, MTSEA modification of NR2A-A7C receptors also correlated
linearly with the intrinsic activity of the agonist bound to the NR1
subunit (Fig. 6C). However, modification of the NR1-A7C
receptor did not correlate linearly with the intrinsic activity of
NR2-bound agonists (Fig. 6B). For example,
substituting L-glutamate with agonists that have
intermediate intrinsic activities did not reduce the rate of MTSEA
modification, although substitution with
(±)-cis-piperidine-2,3-dicarboxylic acid (PDA), a ligand of
particularly low efficacy ( = 0.07), did result in a
significantly slower modification. These data suggest that the access
of MTSEA to residue A7C is promoted when the binding sites are in the
agonist-bound-closed (A·R*) state. Furthermore, the data revealed a
distinct asymmetry in the intersubunit communication between the NR1
and NR2A NMDA receptor subunits, in which the agonist bound at the
glycine site exerts a dominant influence over the M3 conformation in
its own NR1 subunit as well as the neighboring NR2 subunits.
MTSEA modification increases the efficacy of partial agonists
MTSEA modification of the A7C NMDA receptors increased the current
amplitude to levels that were equal to or greater than the responses
evoked by full agonists. This occurred in 9 of 10 cases (Fig.
6D,E). The current amplitude evoked by the
lowest-efficacy glycine agonist, (±)-3-amino-1-hydroxy-2-pyrrolidone
(HA-966), was potentiated 70-fold by MTSEA modification. Whereas before modification the relative current amplitudes evoked by partial agonists
varied widely, reflecting large differences in intrinsic activity,
MTSEA modification potentiated current amplitudes to levels that
are much more similar (Fig. 6D,E). Therefore, MTSEA modification of A7C NMDA receptors appears to increase the intrinsic activity of partial agonists, converting the majority to full agonists.
| |
DISCUSSION |
The data shown here implicate the M3 segment as a critical element
in the activation pathway of NMDA receptors. Cysteine substitution of
the alanine at position 7 of the SYTANLAAF motif (A7C) revealed agonist-induced accessibility changes. A previous report that studied
the same cysteine substitutions (Beck et al., 1999) concluded that none
of the positions displayed agonist-dependent accessibility to MTSEA.
This apparent conflict can be reconciled by considering the different
experimental approaches used. In the present experiments, glutamate and
glycine were always coapplied and the buffer used between agonist
applications was agonist-free. In the studies performed by Beck et al.
(1999), glycine was present throughout the experiment and receptors
were activated by the addition of L-glutamate.
Consequently, when A7C was treated with MTSEA, it was found that the
baseline current slowly increased, which made additional testing
difficult. As we have shown, glycine by itself will minimally activate
NMDA receptors and permit MTSEA modification, which very slowly
increases the size of the current. This modification is prevented by
occupying the glutamate-binding site with an antagonist, suggesting
that agonist occupancy of both binding sites is required to allow MTSEA
access to A7C. Alternatively, it is possible that occupancy of the
glycine-binding site is sufficient to activate the receptor partially.
In this scenario, the inhibitory effect of APV suggests that glycine
binding at the NR1 subunit allows the glutamate-binding core to close
in the absence of ligand.
The activation-dependent accessibility of residue A7C does not by
itself place M3 in the chain of events that links ligand binding to
channel opening. M3 could undergo a conformational change concomitant
with channel opening without having a causative role. However, the
additional data shown here strongly support the idea that M3 is a
transduction element critically involved in coupling ligand binding to
channel opening (Fig. 7).
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|
Figure 7.
A model for activation of K channels and glutamate
receptors and a proposed schematic model of the role of the M3-S6-TM2
segments in channel activation are shown. M3, S6, and TM2 are shown as
transduction elements coupling voltage-sensor movement or
ligand-binding core closure to the channel gate. Open
arrows represent allosteric interactions that M3 has with both
the ligand-binding core and the gate. Experimental support for three of
the four allosteric interactions (a,
b, and c) is described in the
Discussion.
|
|
M3 is functionally distinct from the gate
The NMDA receptor is unique among receptors in its dual agonist
requirement. Binding reactions at all four subunits allosterically project to a single gate, which "integrates" the information and generates a single output, the open probability. If the accessibility of A7C is completely determined by open probability, then MTSEA modification rate should depend only on the state of activation. However, the modification rate shows a concentration dependence that is
distinct (in both its Hill coefficient and its midpoint) from
the normalized response curves (Fig. 5, Table 1). Furthermore, the
MTSEA modification rate of NR1-A7C is the same for the partial agonist
quinolinic acid (QA) and the full agonist L-glutamate, despite that fact that QA activates the NMDA receptor by only 50%
(Fig. 6B). Therefore, A7C accessibility is not
determined strictly by the open probability of the channel. It appears
that M3 receives information on events taking place at the glycine- and
glutamate-binding sites that cannot be extracted from channel gating alone.
M3 interacts allosterically with the glutamate- and
glycine-binding sites
The MTSEA modification rate of A7C-containing NMDA receptors was
determined by both agonist concentration and efficacy. Supramaximal concentrations of the full agonists glycine and glutamate, which promote efficient closure of the ligand-binding lobes, permitted rapid
modification. Likewise, low concentrations of full agonists or high
concentrations of partial agonists, both of which induce infrequent or
incomplete domain closure, significantly slowed the rate of
modification. These results imply that the activation state of the
ligand-binding cores modulates conformational changes in M3 (Fig. 7,
arrow a). Conversely, M3 also affects the behavior of the
ligand-binding lobes, as MTSEA modification of A7C increased the
efficacy of the partial agonists. This suggests that MTSEA modification
of position A7 in the SYTANLAAF region of the M3 domain alters the
interaction between the ligand-binding core and its agonist (Fig. 7,
arrow b). Introducing a bulky side chain at A7C
allosterically shifts the equilibrium between the open and closed cleft
conformations. From this it is concluded that the M3 segments and the
ligand-binding cores of NMDA receptors interact through a reciprocal,
allosteric mechanism that determines agonist efficacy.
M3 interacts allosterically with the gate
The conformation of M3 affects the behavior of the gate, which is
the pore structure directly responsible for opening the channel. MTSEA
modification of A7C dramatically affects NMDA receptor gating by
decreasing the rate at which the receptor deactivates after agonist
removal (Fig. 3C). MTSEA modification also results in
activation of the NMDA receptor to a level not obtainable by agonists
alone (Figs. 2F, 6E). Taken
together, these results suggest that activation of the NMDA receptor
evokes conformational changes in the M3 domain, which are then
allosterically transduced to affect the behavior of the gate (Fig. 7,
arrow c). An allosteric interaction between M3 and the gate
must necessarily be bidirectional, such that alterations in gating
behavior (caused by a mutation or drug) should affect conformational
changes in M3 (Fig. 7, arrow d). We have not presented data
that support this idea.
Cooperativity and asymmetry
The model in Figure 7 schematically illustrates how the M3 segment
allosterically mediates the coupling between the ligand-binding core
and channel opening. However, the model is incomplete because it shows
the ligand-binding site and M3 segment for a single subunit, whereas
functional NMDA receptors are most likely tetrameric. Moreover, our
results have uncovered a high degree of cooperativity between the
movement of the M3 segments in NMDA receptor subunits that needs to be
taken into account. Closure of just the glutamate- or glycine-binding
lobes is not sufficient to induce the conformational changes in M3 that
make residue A7C accessible to MTSEA. However, when both types of
binding lobes are closed, MTSEA gains access to residue A7C. This was
required whether the cysteine substitution was introduced in the NR1 or
NR2A subunit. The rate of modification of a NR2A-A7C-containing NMDA
receptor depended critically on the activation status of both its
"own" glutamate-binding core as well as the glycine-binding core
localized to the neighboring NR1 subunit. The MTSEA modification of the
NR1-A7C receptor was similarly dependent on the conformation of the
ligand-binding cores, although the relationship between the
modification rate and efficacy for partial glutamate agonists was
distinctly nonlinear. This suggests that domain closure of the
individual glycine and glutamate bilobate binding cores does not
provide enough free energy to induce a conformational change in the M3
segments. Closure of the ligand-binding lobes at both the NR1 and NR2A
subunits is required to produce the change in accessibility.
This high degree of cooperativity in the behavior of the M3 segments
can be explained by their interaction with a common, single gate
(Colquhoun, 1998). Alternatively, the subunits may interact with each
other, potentially through the M3 or M1 segments, to produce the
cooperativity that was observed. This could also make it easier to
explain the asymmetry that has been observed in the interaction between
the glutamate- and glycine-binding sites (Benveniste et al., 1990;
Lester et al., 1993) (Fig. 6B,C).
A conserved transduction element in glutamate receptors and
K+ channels
The M3 domains allosterically interact with both the gate and the
ligand-binding sites in the NMDA receptor and mediate their coupling.
Ligand binding alters the conformation of M3, which in turn changes the
probability that the channel will open. What is the nature of this
conformational change that occurs in M3? Data obtained recently for K
channels may provide a clue. Pore-forming regions are structurally
conserved between ionotropic glutamate receptors and
K+ channels (Fig. 1A,B).
The glutamate receptor M3 segment corresponds to TM2 in the bacterial
KcsA K channel, which has been crystallized recently (Doyle et al.,
1998). Electron paramagnetic resonance spectroscopy measurements in
KcsA have shown that the -helical TM2 segment displays rotational
and translational movements after activation of the channel (Perozo et
al., 1998, 1999). The data presented here are consistent with a similar
rotation/translation of the M3 segment underlying NMDA receptor activation.
Furthermore, introduction of a cysteine in the C-terminal half of S6,
the equivalent transmembrane segment of the voltage-gated Shaker
K+ channel, results in a phenotype very
similar to that of A7C in the NMDA receptor. Like A7C-containing NMDA
receptors, modification of the S6 cysteine requires channel activation.
In addition, deactivation of the Shaker
K+ channel was slowed by the presence of
soft-metal divalent cations, which can form intersubunit metal bridges
(Holmgren and Yellen, 1998). Therefore, we propose that the M3, TM2,
and S6 segments are structurally and functionally conserved. They
represent universal transduction elements whose translocation couples
ligand binding or voltage-sensor movement to channel opening. It
therefore appears that glutamate receptors and K channels not only
contain structurally highly conserved pore-forming regions but also
share a common gating mechanism.
| |
FOOTNOTES |
Received May 30, 2001; revised Dec. 19, 2001; accepted Dec. 20, 2001.
This work was supported by National Institutes of Health Grants NS31557
and MH61506 to A.M.J.V. and by a Minority Predoctoral Fellowship in
Neuroscience from the American Psychological Association to K.S.J. We
thank Dr. John York for his critical reading of the manuscript.
Correspondence should be addressed to Antonius M. J. VanDongen,
Department of Pharmacology and Cancer Biology, Duke University Medical
Center, P.O. Box 3813, Durham, NC 27710. E-mail: vando005{at}mc.duke.edu.
| |
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[Abstract]
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M. L. Chapman and A. M.J. VanDongen
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[Abstract]
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B. Hu and F. Zheng
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[Abstract]
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P. E. Chen, M. T. Geballe, P. J. Stansfeld, A. R. Johnston, H. Yuan, A. L. Jacob, J. P. Snyder, S. F. Traynelis, and D. J. A. Wyllie
Structural Features of the Glutamate Binding Site in Recombinant NR1/NR2A N-Methyl-D-aspartate Receptors Determined by Site-Directed Mutagenesis and Molecular Modeling
Mol. Pharmacol.,
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[Abstract]
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B. Hu and F. Zheng
Differential Effects on Current Kinetics by Point Mutations in the lurcher Motif of NR1/NR2A Receptors
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[Abstract]
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T. G. Banke, S. M. Dravid, and S. F. Traynelis
Protons Trap NR1/NR2B NMDA Receptors in a Nonconducting State
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[Abstract]
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T. L. Kalbaugh, H. M. A. VanDongen, and A. M. J. VanDongen
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[Abstract]
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M. V. Yelshansky, A. I. Sobolevsky, C. Jatzke, and L. P. Wollmuth
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A. M. J. VanDongen
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PNAS,
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[Abstract]
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R. L. McFEETERS and R. E. OSWALD
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FASEB J,
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[Abstract]
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N. Chen, B. Li, T. H. Murphy, and L. A. Raymond
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[Abstract]
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A. I. Sobolevsky, M. V. Yelshansky, and L. P. Wollmuth
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C. Jatzke, M. Hernandez, and L. P Wollmuth
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K. Williams, M. Dattilo, T. N. Sabado, K. Kashiwagi, and K. Igarashi.
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N. Strutz, C. Villmann, H.-G. Breitinger, M. Werner, R. J. Wenthold, P. Kizelsztein, V. I. Teichberg, and M. Hollmann
Kainate-binding Proteins Are Rendered Functional Ion Channels upon Transplantation of Two Short Pore-flanking Domains from a Kainate Receptor
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TOP
ABSTRACT
INTRODUCTION
