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Vol. 53, Issue 5, 933-941, May 1998
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania (K.W., A.J.P., N.D.N.), and Faculty of Pharmaceutical Sciences, Chiba University, Chiba, Japan (K.K., T.M., K.I.)
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Summary
Introduction
Materials & Methods
Results & Discussion
References
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A hallmark feature of N-methyl-D-aspartate
(NMDA) receptors is their voltage-dependent block by extracellular
Mg2+. The structural basis for Mg2+ block is
not fully understood. Although asparagine residues in the pore-forming
M2 regions of NR1 and NR2 subunits influence Mg2+ block, it
has been speculated that additional residues are likely to be involved.
Here, we report the unexpected finding that a tryptophan residue in the
M2 region of NR2 subunits controls Mg2+ block. An
NR2B(W607L) mutation abolished block and greatly increased permeation
of extracellular Mg2+. A similar effect was seen with a
mutation at the equivalent residue in NR2A but not with mutations at
the equivalent residue or adjacent residues in NR1. In NR2B, mutations
that changed NR2B(W607) to asparagine (W607N) or alanine (W607A) also
greatly reduced Mg2+ block, whereas mutations that changed
W607 to the aromatic residues tyrosine (W607Y) or phenylalanine (W607F)
had little or no effect on Mg2+ block. Furthermore, the
W607L, W607N, and W607A mutants, but not the W607Y and W607F mutants,
decreased Ba2+ permeability of NMDA channels. Thus, residue
NR2B(W607) may be involved in binding of divalent cations, in
particular Mg2+, through a cation-
interaction with the
electron-rich aromatic ring of the tryptophan. We previously suggested
that NR2B(W607) may contribute to the narrow constriction of the NMDA
channel. A model is now proposed in which the M2 loop of NR2B is folded in such a way that NR2B(W607) is positioned at the narrow constriction, at a level similar to NR2B(N616) and NR1(N616), with these three residues forming a binding site for Mg2+.
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Introduction |
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Summary
Introduction
Materials & Methods
Results & Discussion
References
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NMDA
receptors are glutamate-gated ion channels that are involved in
synaptic plasticity and ischemic neuronal cell death. A hallmark
feature of NMDA receptors is their voltage-dependent block by
extracellular Mg2+ (Mayer et al.,
1984
; Nowak et al., 1984). NMDA channels are blocked by
Mg2+ at resting membrane potentials, and the
block is relieved as neurons are depolarized, which allows the
receptors to function as synaptic coincidence detectors.
NMDA receptors are hetero-oligomers containing combinations of NR1 and
NR2 subunits in as-yet-undefined ratios and stoichiometries (Luo
et al., 1997; Monyer et al., 1992; Moriyoshi
et al., 1991; Sheng et al., 1994). The receptor
subunits are thought to contain three membrane-spanning regions (M1,
M3, and M4) and a reentrant loop (M2) that contributes to the
permeation pathway of the ion channel (Bennett and Dingledine, 1995; Wo
and Oswald, 1995; Wood et al., 1995). Asparagine
residues in the M2 segments of NR1 and NR2 subunits influence block by
Mg2+ and permeation of Ca2+
or Ba2+ (Burnashev et al., 1992; Mori
et al., 1992; Kawajiri and Dingledine, 1993; Sakurada
et al., 1993; Kupper et al., 1996). These
residues, which include N616 in NR1, N615 in NR2A, and N616 in NR2B,
seem to contribute to the narrowest constriction of the channel pore (Kuner et al., 1996; Wollmuth et al., 1996) and
may form part of a binding site for Mg2+. It is
conceivable that other residues in the channel pore may be involved in
block by Mg2+, but such
residues
have not yet been identified. Many studies of NMDA channel function
have focused initially on amino acid residues in the NR1 subunit,
followed by studies of the equivalent or adjacent residues in NR2
subunits. This strategy identified an important role of the asparagines
at NR1(N616) and NR2B(N616) in Mg2+ block, but
other residues that have been studied in the M2 region of NR1 have no
effect or only modest effects on Mg2+ block
(Burnashev et al., 1992; Mori et al., 1992;
Kawajiri and Dingledine, 1993; Sakurada et al., 1993; Kupper
et al., 1996). We recently found that tryptophan residues in
the M2 regions of NR2 subunits, including W607 in NR2B, influence
permeation of the novel polyamine channel blocker
N1-DnsSpm (Kashiwagi et al., 1997).
Here, we show that mutations at NR2B(W607), but not at the equivalent
position in the NR1 subunit, greatly influence block by extracellular
Mg2+. This tryptophan residue may form part of
the selectivity filter and the Mg2+ binding site
of the NMDA channel, and the results of this study have implications
for understanding the structure of the M2 loop and the roles of
different NMDA receptor subunits in channel function.
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Materials & Methods
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cDNA clones and site-directed mutagenesis.
The wild-type NR1
clone (Moriyoshi et al., 1991) and the NR1(W608L) and
NR1(F609L) mutants (Sakurada et al., 1993) were gifts from
Dr. S. Nakanishi (Institute for Immunology, Kyoto University Faculty of
Medicine, Kyoto, Japan). The NR1(N616R) mutant (Kawajiri and
Dingledine, 1993) was a gift from Dr. R. J. Dingledine (Department of Pharmacology, Emory University, Atlanta, GA). The wild-type NR2A and
NR2B clones (Monyer et al., 1992) were gifts from Dr. P. H. Seeburg (Center for Molecular Biology, University of
Heidelberg, Germany). NR1 and NR2 mutants were prepared by
site-directed mutagenesis using the M13 phage system (Kunkel et
al., 1987; Sayers et al., 1992), and mutations were
confirmed by DNA sequencing. In some experiments, we used a rat brain
NR2B clone containing the W607L mutation (Kashiwagi et al.,
1997). In other experiments, we used a mouse brain NR2B clone, 
2
(Kutsuwada et al., 1992) (a gift from Dr. M. Mishina,
University of Tokyo, Tokyo, Japan), containing a 1.7-kb
HindIII/SphI fragment of the rat NR2B clone with
the W607L mutation. A similar construct was used to prepare the
NR2B(W607N), NR2B(W607A), NR2B(W607Y), and NR2B(W607F) mutants. In
control experiments, we found that an 2 clone containing the
wild-type HindIII/SphI fragment of rat NR2B had
properties indistinguishable from wild-type NR2B (data not shown).
Oligonucleotides (sense strands) used for preparation of NR2B mutants
were 5'-CTG GTG TTT AAC GGC TCC GTA CCT GT-3' for
NR2B(N616G), 5'-GGC AAA GCA ATT AAT TTA CTC TGG GGT C-3'
for NR2B(W607N), 5'-GGC AAA GCA ATT GCC TTA CTC TGG GGT-3'
for NR2B(W607A), 5'-GCA AAG CAA TTT ACT TAC TCT GGG GT-3'
for NR2B(W607Y), and 5'-GCA AAG CAA TTT TCT TAC TCT GGG
GT-3' for NR2B(W607F). The oligonucleotide for NR1(N616W) (antisense)
was 5'-CCC CAA TGC CGG ACC AGA GCA GGA CGC CC-3'. Underlined
nucleotides indicate the position of the mutations. The double mutant,
NR2B(W607L,N616G) was prepared by using the oligonucleotide for the
NR2B(N616G) mutation with a single-strand DNA fragment of NR2B that
contained the W607L mutation. Other NR1 and NR2 mutants were prepared
as described previously (Chao et al., 1997; Kashiwagi
et al., 1997). Most of the 5'-UTR was removed from the NR2B
(Williams, 1993) and 2 clones to improve expression in oocytes. The
5'-UTR of the 2 clone was truncated at the SalI site,
leaving a 5'-UTR of 50 nucleotides.
Expression in oocytes and voltage-clamp recording.
Defolliculated oocytes were prepared and maintained as described
previously (Williams et al., 1993). Oocytes were injected with NR1 plus NR2 cRNAs in a ratio of 1:5 (0.2-4 ng of NR1 plus 1-20
ng of NR2). Macroscopic currents were recorded with a two-electrode voltage-clamp using a GeneClamp 500 amplifier (Axon Instruments, Foster
City, CA) or an OC-725 amplifier (Warner Instruments, Hamden, CT) as
described previously (Williams, 1993, 1994; Williams et al.,
1993). Oocytes were continuously superfused with a saline solution (96 mM NaCl, 2 mM KCl, 1.8 mM
BaCl2, 10 mM HEPES, pH 7.5)
(Na+ saline). In most experiments, oocytes were
injected with K+-BAPTA (100 nl of 40 mM, pH 7.0-7.4) on the day of recording (Williams, 1993).
50 mV and at +50 mV were
measured by linear regression of data from 45 to 55 mV and from +45
to +55 mV. In experiments using Mg2+ as the
extracellular charge carrier, the Na+ saline was
replaced with a solution that contained 64 mM
MgC12, 2 mM KCl, and 10 mM HEPES, pH 7.5 (Mg2+ saline). In
most experiments, the pH of this solution was adjusted to pH 7.5 using
NaOH, and the Mg2+ saline therefore contained
~6 mM Na+. In some experiments, the
pH of the solution was adjusted using Mg(OH)2,
and the solution did not contain KCl; thus Mg2+
was the only extracellular cation in those experiments. No differences were observed between the reversal potentials measured in
Mg2+ saline in which pH was adjusted with NaOH or
with Mg(OH)2. In experiments using
Ba2+ as the extracellular charge carrier, the
Na+ saline was replaced with a solution that
contained 64 mM BaC12, 2 mM KCl, and 10 mM HEPES, pH 7.5 (Ba2+ saline). There were small liquid junction
potentials (+3 to +8 mV) when changing from Na+
saline to Mg2+ or Ba2+
saline. The reported values of Vrev have been
corrected for these junction potentials.
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Results and Discussion |
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Summary
Introduction
Materials & Methods
Results & Discussion
References
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NR2B(W607) controls Mg2+ block.
We initially
studied W-to-L mutations at several positions in the M2 loop of the NR1
and NR2B subunits (Fig. 1A). Wild-type NR1/NR2B receptors were
inhibited by Mg2+ with an
IC50 value of 19 µM at 70 mV
(Fig. 1, B and C; Table 1). Mutations in the NR1 subunit at W608L,
W611L, or an adjacent aromatic residue, F609L, had no effect on
Mg2+ block. In contrast, mutation NR2B(W607L)
almost abolished block by extracellular Mg2+,
which had little or no effect at concentrations up to 300 µM (Fig. 1C; Table 1). At higher concentrations (1-3
mM), Mg2+ potentiated
glutamate-induced currents at NR1/NR2B(W607L) receptors, presumably due
to an effect of Mg2+ at the stimulatory polyamine
site (Paoletti et al., 1995). Stimulation by 1-3
mM Mg2+ was also seen at wild-type
NR1/NR2B receptors at depolarized potentials (+40 to +60 mV) (data not
shown). This form of Mg2+ stimulation, like that
of spermine, seems to be voltage-independent and is presumably unmasked
at negative membrane potentials at NR1/NR2B(W607L) receptors because of
the lack of Mg2+ block at these receptors. A
W-to-L mutation at residue NR2B(W610L), three residues downstream of
W607 (Fig. 1A), also had a small effect on Mg2+
block, increasing the IC50 by ~3-fold (Table
1). Block by Mg2+ was also reduced in receptors
containing a W-to-L mutation in the NR2A subunit at position
NR2A(W606), although the effect of this mutation on the
IC50 for Mg2+ was smaller
than that of the equivalent mutation (W607L) in NR2B (Table 1).
70 mV (Table 1). Thus, the presence of an amino acid with
an aromatic side chain (Y or F) at position NR2B(W607) can restore
block by extracellular Mg2+.
Block by Mg2+ is voltage dependent, and we
therefore studied I-V relationships of glutamate-induced currents in
the absence and presence of Mg2+. At wild-type
NR1/NR2B receptors, block by Mg2+ (1-1000
µM) was strongly concentration- and voltage-dependent (Fig. 1D). In contrast, at concentrations below 100 µM,
Mg2+ had no effect on the I-V relationship at
NR1/NR2B(W607L) receptors (data not shown), and at concentrations of
100-1000 µM Mg2+ produced a very
small block at hyperpolarized membrane potentials (Fig. 1E). The
NR2B(W607Y) mutation does not affect the potency of
Mg2+ measured at 70 mV (Table 1). However,
voltage-dependent block by Mg2+ at
NR1/NR2B(W607Y) receptors was more shallow than at wild-type receptors.
At wild-type receptors, there was a clear region of negative slope
conductance with 10-1000 µM Mg2+
and a complete block at extreme negative potentials with 1000 µM Mg2+. In contrast, at
NR1/NR2B(W607Y) receptors, the slope conductance with 10-1000
µM Mg2+ was shallow, and there was
an incomplete block with 1000 µM
Mg2+ (Fig. 1F) The difference between wild-type
NR1/NR2B and NR1/NR2B(W607Y) receptors is illustrated in Fig. 1G. These
data suggest that Mg2+ is able to permeate
NR1/NR2B(W607Y) channels more easily than wild-type channels, manifest
as a partial relief from block at very negative potentials, although
Mg2+ block at 70 mV is unaffected by W607Y
(Table 1). In experiments using Mg2+ as the main
extracellular charge carrier, we subsequently found that this is indeed
the case (see below). The profile seen with NR2B(W607F) was similar to
that of the wild-type channels, with a steep region of negative slope
conductance in the presence of extracellular Mg2+
(data not shown).
Some mutations in the M2 region of NR1, including NR1(N616Q), reduce
the sensitivity of NMDA receptors to proton inhibition and increase the
potencies of glutamate and glycine (Kashiwagi et al., 1997).
These effects presumably reflect long range allosteric changes or
disruptions to channel gating because NR1(N616) lies in the ion
permeation pathway and is not directly involved in binding of protons
or agonists. We determined the effects of NR2B(W607) mutations on
sensitivity to glutamate, glycine, and pH. These parameters were
unaffected by mutations at NR2B(W607) (Table 2).
Permeation of Mg2+ and Ba2+ through NMDA
channels.
Although the NMDA channel is a relatively nonselective
cation channel, the pore must contain a selectivity filter that allows passage of Ca2+ and Ba2+,
which are highly permeable, but not of Mg2+,
which permeates native NMDA channels very poorly (Mayer and Westbrook,
1987; Stout et al., 1996). To determine whether NR2B(W607) mutations alter Mg2+ permeability and to study
the relationship between block and permeation of
Mg2+, we measured currents through wild-type and
mutant channels with Na+ or
Mg2+ as the major extracellular cation. In the
absence of Mg2+, and with
Na+ as the main extracellular cation
(Na+ saline), currents through NR1/NR2B and
NR1/NR2B(W607L) receptors had similar reversal potentials, close to 0 mV (Fig. 2, B and C). Under conditions where extracellular
Na+ was replaced by Mg2+,
outward currents were observed at wild-type NR1/NR2B receptors and
these currents asymptotically approached zero as the oocytes were
hyperpolarized (Fig. 2B). This suggests that Mg2+
does not permeate these channels or that the reversal potential (Vrev) for Mg2+ is more
negative than 100 mV. In contrast, large inward currents were seen in
Mg2+ saline at NR1/NR2B(W607L) receptors, and
Vrev in Mg2+ saline was
25 ± 1 mV (16 oocytes) (Fig. 2). A similar increase in
Mg2+ permeability was seen with a mutation at
NR2A(W606L), a position equivalent to NR2B(W607L), and
Vrev in Mg2+ saline at
NR1/NR2A(W606L) receptors was 28 ± 2 mV (nine oocytes).
50 mV and +50 mV
is plotted for each mutant. Thus, although the W607Y and W607F mutants
are more permeable to Mg2+ than wild-type
channels, the Mg2+ block at these mutants is much
more profound than at the W607L, W607N, or W607A mutants. The values of
Vrev for Mg2+ observed with
the different NR2B(W607) mutants were very similar (Fig. 2C) even
though the W-to-Y and W-to-F mutants produced very small currents (Fig.
2D). This suggests that residue NR2B(W607) predominantly affects block
by Mg2+ per se rather than affecting the
rate of permeation of the ion after the block is relieved. Thus, the
strength of the block at wild-type channels that contain a tryptophan
at NR2B(W607) presumably accounts for the lack of permeation of
Mg2+ through those channels.
To determine whether the NR2B(W607L) mutations alter
Ba2+ permeability, we compared
Vrev in Na+ saline and in a
solution containing 64 mM Ba2+
(Ba2+ saline). Because wild-type NMDA channels
are highly permeable to Ba2+, there is a large
positive shift in Vrev when switching from Na+ saline to Ba2+ saline
(Hume et al., 1991). Even with Ba2+ in
the extracellular solution it is possible to activate secondary Cl
conductances that would influence the
measured reversal potential. We attempted to eliminate these
Cl currents by injecting oocytes with BAPTA, by
limiting the duration of agonist application, and, in some experiments
with Ba2+ saline, by reducing the concentration
of glutamate from 10 to 1 µM. At wild-type NR1/NR2B
receptors, the shift in Vrev was +22 ± 1 mV
(Fig. 2E). At receptors containing the NR2B mutants W607L, W607N, and
W607A, but not W607Y or W607F, Ba2+ permeability
was markedly reduced (Fig. 2E). Thus, residue NR2B(W607) influences
Ba2+ permeability as well as
Mg2+ block. It may be that transient binding of
Ba2+ to NR2B(W607), and subsequent unbinding and
passage of Ba2+ through the channel, is involved
in Ba2+ flux through wild-type channels. These
data are consistent with the reported interactions between
Ba2+ (or Ca2+) and
Mg2+ within NMDA channels (Mayer and Westbrook,
1987).
Reversal potentials in extracellular Na+,
Mg2+, and Ba2+ were
measured for receptors containing various combinations of NR1 and NR2
mutants (Figs. 3 and 4). A W-to-L mutation at NR1(W608L) had no effect
on Mg2+ permeation and produced a small decrease
in Ba2+ permeability (Figs. 3A and 4A). Mutations
at the second tryptophan residue in the M2 loop of NR2 subunits,
NR2B(W610L) and NR2A(W609L), but not at the equivalent position in NR1,
increased permeation of Mg2+ (Fig. 4, A and D),
although inward currents carried by Mg2+ through
these channels were very small (Fig. 3C) and reversal potentials were
more negative than that seen with NR2B(W607L). When the NR2B(W610L)
mutant was studied in combination with the NR1(W608L) or NR1(W611L)
mutants, there was no further increase in Mg2+
permeability and only a small change in Ba2+
permeability. Similarly, the combination of NR2B(W607L) with NR1(W608L)
or NR1(W611L) did not produce any further change in Mg2+ permeability compared with NR1/NR2B(W607L)
receptors (Fig. 4A). Thus, of the tryptophan residues present in this
region of the pore, only the first tryptophan in NR2 subunits is a
major determinant of Mg2+ block. GluR subunits of
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate
receptors contain a leucine residue at the position equivalent to
NR1(W611) and NR2B(W610). Mutation of this leucine to tryptophan in the
GluR1 subunit has been reported to increase divalent cation
permeability of homomeric GluR1 channels (Ferrer-Montiel et
al., 1996). Our results show that mutations NR1(W611L) and NR2B(W610L), either alone or in combination, have little or no effect
on Ba2+ permeability (Fig. 4A), suggesting that
the tryptophan at this position in NMDA channels plays only in minor
role in controlling Ba2+ permeability.
We also carried out experiments to compare the effects of the
NR2B(W607L) mutant with those of mutations at NR1(N616) (Figs. 3B and
4B) and NR2B(N616) (Figs. 3D and 4C), in some cases combining the NR1
and NR2 mutants to determine whether the effect of one mutant
predominates. N-to-G, N-to-R, and N-to-W mutations at NR1(N616) all
increased Mg2+ permeability and decreased
Ba2+ permeability (Fig. 4B), although inward
currents carried by Mg2+ through these mutants
were very small (Fig. 3B). With the exception of NR1(N616R), the
NR2B(W607L) mutant seemed to predominantly influence
Mg2+ permeability (Fig. 4B). In contrast, the
NR1(N616) mutants predominantly influenced Ba2+
permeability in NR1(N616X)/NR2B(W607L) channels (Fig. 4B).
The NR2B(N616G) mutant greatly increased Mg2+
permeability and reduced Ba2+ permeability,
similar to the NR2B(W607L) mutant (Figs. 3D and 4C). A double mutant,
NR2B(W607L,N616G), did not have a greater effect than either mutant
alone, nor did the combination of NR2B(N616G) with NR1(N616G) (Fig.
4C). Similar results were seen with an N615G mutant in NR2A, which
increased Mg2+ permeability comparable with the
NR2A(W606L) mutant (Fig. 4D). The lack of additivity of the mutations
at NR2B(W607) and NR2B(N616) suggests that these residues may make a
similar or functionally overlapping contribution to the
Mg2+ binding site and they may be closely
positioned in the pore (Fig. 5).
Implications for understanding Mg2+ block and the
structure of NMDA channels.
A tryptophan residue is present in all
glutamate receptor subunits, with the exception of NR-L, and in some
K+ channel subunits at a position equivalent to
NR2B(W607). In NMDA channels (Kuner et al., 1996) and in
Shaker K+ channels (Lü and
Miller, 1995), the tryptophan at this position has been reported to
face the lumen of the channel pore. This would be consistent with a
direct interaction of Mg2+ with NR2B(W607) in the
NMDA channel lumen, possibly involving a cation- interaction of
Mg2+ with the aromatic ring of W607 (Kumpf and
Dougherty, 1993). In support of this idea, block by
Mg2+ was drastically reduced in mutants
containing leucine, asparagine, or alanine but was largely unaffected
in mutants containing tyrosine or phenylalanine. Based on the kinetics
of block by external and internal Mg2+, a
barrier-well energy profile has been proposed for
Mg2+ block of the NMDA channel (Li-Smerin and
Johnson, 1996). That profile contains a very high energy barrier that
prevents crossover and permeation of internal
Mg2+ (Li-Smerin and Johnson, 1996). It is
tempting to speculate that the tryptophan residue at position
NR2B(W607) is the actual physical determinant of that energy barrier,
and it will be of interest to study the influence of NR2B(W607) on
block by intracellular Mg2+.
; Kawajiri and
Dingledine, 1993; Kupper et al., 1996). Another possibility
is that the structure, folding, or positioning of the M2 loop in NR2
subunits is different from that in subunits of non-NMDA receptors and,
perhaps, different from that in NR1, and that this accounts for the
role of NR2B(W607) in Mg2+ block
We previously found that the NR2B(W607L) but not the NR1(W608L)
mutation reduced block and increased the apparent permeation of the
polyamine-derivative N1-DnsSpm (Kashiwagi
et al., 1997). Those results led us to propose that residue
NR2B(W607) may contribute to the narrowest constriction of the channel
pore (Kashiwagi et al., 1997) together with the asparagines
at positions NR1(N616) and NR2B(N616) (Kuner et al., 1996;
Wollmuth et al., 1996), although this is not consistent with
current models of the proposed structure of the M2 loop region in which
NR2B(W607) lies some distance below NR2B(N616) (Kuner et
al., 1996; Sutcliffe et al., 1996). The finding that
W607 in NR2B but not W608 in NR1 affects Mg2+
block reinforces the idea that the M2 regions of NR1 and NR2 make
nonsymmetrical contributions to channel structure. It may be that the
secondary structure of at least part of the M2 loop in NR1 and NR2
subunits is different, leading to placement of NR2B(W607) at a position
in the channel pore such that it can interact with
Mg2+ whereas NR1(W608) cannot (Fig. 5). If
residue NR2B(W607) is located at a level in the channel similar to
NR1(N616) and NR2B(N616) (Fig. 5A), it is possible that some or all of
the M2 region extends in a plane parallel, rather than perpendicular,
to the plane of the membrane, as has been proposed for the pore-forming
loop of a cyclic nucleotide-gated channel (Sun et al.,
1996). A limitation of models such as those shown in Fig. 5 is that
they are "static," and it is possible that the M2 loops are very
flexible, as has been proposed for pore-forming loops of voltage-gated
Na+ and K+ channels
(Bénitah et al., 1997; Liu et al., 1996;
Tsushima et al., 1997) and that under some conditions they
can arrange themselves such that NR2B(W607) is very close to or forms
part of the narrow constriction and the selectivity filter in the
channel.
We found that a W-to-L mutation at the second tryptophan in NR2B,
NR2B(W610), also had a small effect on Mg2+
block. In the study by Kuner et al. (1996), the equivalent
tryptophan residues in NR1 and NR2C were not accessible to thiol
reagents after mutagenesis to cysteine, suggesting that this tryptophan is not solvent accessible in the channel. If NR2B(W610) does not face
the channel lumen, it is possible that the W-to-L mutation causes a
nonspecific disruption of channel structure, perhaps influencing the
position of the NR2B(W607) or NR2B(N616) residues. In this context, it
is notable that mutations at NR1(S617) have a small effect on
Mg2+ block (Kawajiri and Dingledine, 1993),
although this position is also not accessible to thiol reagents after
cysteine mutagenesis (Kuner et al., 1996). Another
possibility is that residues such as NR2B(W610) and NR1(S617) do face
the channel lumen but their accessibility is limited because of
shielding by other residues or by ions passing through the channel.
In conclusion, we propose that there are at least three residues that
make critical contributions to the narrow constriction and selectivity
filter of the NMDA channel. These include N616 in NR1 and N616 and W607
in NR2B (Fig. 5). The N and W residues in NR2 subunits predominantly
influence block and permeation of extracellular
Mg2+ and probably form part of a
Mg2+ binding site. Although the schematics shown
in Fig. 5 are undoubtedly a gross oversimplification, future models of
the M2 region will need to take into account a critical role for the
conserved tryptophan in NR2 subunits in block by
Mg2+, as well as the potential positioning of
this residue at the narrow constriction of the channel (Kashiwagi
et al., 1997).
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Footnotes |
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Received October 14, 1997; Accepted January 29, 1998
This work was supported by United States Public Health Service Grant NS35047 from the National Institute of Neurological Disorders and Stroke, a Grant-in-Aid from the American Heart Association, and grants from the Japan Health Science Foundation and the Yamanouchi Foundation for Research on Metabolic Disorders.
Send reprint requests to: Dr. Keith Williams, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6084. E-mail: williams{at}pharma.med.upenn.edu
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Abbreviations |
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NMDA, N-methyl-D-aspartate; N1-DnsSpm, N1-dansyl-spermine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; GluR, glutamate receptor; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; I-V, current-voltage.
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References |
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Summary
Introduction
Materials & Methods
Results & Discussion
References
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), NR1(W608L)/NR2B (
), and
NR1/NR2B(W607L) (
) at 
) by using voltage ramps. At NR1/NR2B
receptors, inward currents could not be detected in Mg2+
saline and a symbol with an arrow is drawn below 
