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Next Article 
Volume 16, Number 11,
Issue of June 1, 1996
pp. 3549-3558
Copyright ©1996 Society for Neuroscience
Multiple Structural Elements Determine Subunit Specificity of
Mg2+ Block in NMDA Receptor Channels
Thomas Kuner1 and
Ralf Schoepfer1, 2
1 Zentrum für Molekulare Biologie der
Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany, and 2 University College London,
Laboratory for Molecular Pharmacology, London WC1E 6BT
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
In NMDA receptor channels, subtype-specific differences of
Mg2+ block are determined by the NR2 subunits.
Channels assembled from the NR1-NR2A or NR1-NR2B subunits are blocked
more strongly than channels formed by the NR1-NR2C or NR1-NR2D
subunits, predominantly reflecting a difference in voltage dependence.
A determinant of Mg2+ block common to the NR2
subunits is located in the M2 domain (N-site or
Q/R/N-site). However, subunit-specific differences of block
suggested that additional structural elements exist. Chimeric NR2
subunits were constructed by replacing segments of the least sensitive
NR2C subunit with homologous segments of the most sensitive NR2B
subunit. Mutant NR2 subunits were coexpressed with wild-type NR1 in
Xenopus oocytes, and Mg2+ block was
quantified. Replacement of the entire M1-M4 region resulted in a
chimera with a sensitivity of Mg2+ block similar
to that of the NR2B wild type. Replacing smaller segments or
introducing point mutations did not generate channels with
Mg2+ block characteristic of NR2B wild type.
However, combining in a single chimera three small segments (M1, M2-M3
linker, M4), each independently mediating an increase in
Mg2+ block, produced channels close to NR2B wild
type. Thus, differences in Mg2+ block as
controlled by the NR2 subunits cannot be explained by a single
structural determinant in addition to the N-site. Moreover,
three elements of the NR2 subunit are the major determinants of
subtype-specific differences of Mg2+ block in
heteromeric NMDA receptor channels.
Key words:
NMDA;
receptor;
channel;
structure;
recombinant;
Mg2+ block;
voltage dependence;
subunit-specific
INTRODUCTION
NMDA receptor (NMDAR) channels, composed from a
common NR1 subunit (Moriyoshi et al., 1991 ; Yamazaki et al., 1992) and
one or more of four different NR2 (A-D) subunits (Ikeda et al., 1992;
Kutsuwada et al., 1992; Meguro et al., 1992; Monyer et al., 1992; Ishii
et al., 1993), are thought to play a critical role in synaptic
plasticity (Collingridge and Singer, 1990; Bliss and Collingridge,
1993), development of functional neuronal networks (Constantine, 1990;
Shatz, 1990; Komuro and Rakic, 1993), and neuronal cell death (Choi,
1988). The NR1 subunit is expressed ubiquitously in the brain, whereas
the NR2 subunits show a distinct spatial and temporal expression
pattern (Watanabe et al., 1992; Akazawa et al., 1994; Monyer et al.,
1994), thereby generating functional diversity of the NMDAR channel.
For example, heterologously expressed NMDAR channels assembled from the
NR1 and any one of the NR2 subunits, show NR2-specific differences in
macroscopic kinetic properties (Monyer et al., 1992, 1994),
single-channel characteristics (Stern et al., 1992), fractional
Ca2+ currents (Burnashev et al., 1995), and
voltage-dependent Mg2+ block (Kutsuwada et al.,
1992; Monyer et al., 1992, 1994; Ishii et al., 1993).
The functional activation of NMDAR channels is linked to the membrane
potential by the voltage dependence of Mg2+ block
(Mayer et al., 1984; Nowak et al., 1984), allowing these channels to
sense coincidence of synaptic input (Bliss and Collingridge, 1993).
Differences in the extent of block by Mg2+ as
determined by the NR2 subunit could be an important mechanism to create
synapses with different characteristics for coincidence detection.
Indeed, regional differences of Mg2+ block and
changes during development have been reported (Kato et al., 1991;
Kleckner and Dingledine, 1991; Kato and Yoshimura, 1993; Nabekura et
al., 1994; Momiyama et al., 1995).
A determinant of Mg2+ block in NMDAR channels is
located in the M2 domain of the NR2 subunits (N-site or
Q/R/N-site) (Burnashev et al., 1992; Mori et al., 1992;
Sakurada et al., 1993). The N-site is part of an
octapeptide, which is conserved within the NR2 subunits. Despite having
this shared element, channels containing NR2A or NR2B are more
sensitive to Mg2+ block compared with NR2C- or
NR2D-containing channels (Monyer et al., 1994), suggesting that
additional elements exist that determine subunit specificity (Burnashev
et al., 1992; Hollmann and Heinemann, 1994; McBain and Mayer, 1994;
Sather et al., 1994; Seeburg et al., 1995).
We investigated the presence of additional elements for blockade
by Mg2+ other than the N-site in the
NR2 subunits by constructing chimeras between the NR2B and NR2C
subunits. Homologous regions of the most sensitive NR2B subunit were
grafted into the least sensitive NR2C subunit. Receptor channels
containing any of the chimeric or wild-type NR2 subunits together with
the wild-type NR1 subunit were expressed in Xenopus oocytes
and functionally analyzed using a two-microelectrode voltage-clamp
system. Our results indicate that three structural elements are
required to convert an NR2 subunit with a low sensitivity to an NR2
subunit with a high sensitivity of Mg2+
block.
MATERIALS AND METHODS
Expression plasmid constructs. The coding regions of
the NR1 (1a splice form (Hollmann et al., 1993)) and the NR2 subunits
(Monyer et al., 1992, 1994) were cloned into a pSP64T-derived vector
optimized for expression in Xenopus oocytes (Kuner et al.,
1993). Noncoding regions were removed, and silent mutations were
introduced to facilitate mutagenesis. The resulting NR1-SP, NR2A-SP,
NR2B-SP, NR2C-SP, and NR2D-SP clones were used for all further
manipulations. All mutants were constructed in a truncated version of
the NR2C subunit as published by Monyer et al. (1992) (here denoted as
NR2C , 943 AA, see Fig. 5). Amino acids (AA) are shown in the
single-letter code. Mutants carrying one AA substitution are named as
``subunit(wild-type AA position in the mature proteinmutant
AA).'' Chimeras are named according to the putative transmembrane
domain(s) or loop(s) that were transplanted within a given fragment
(e.g., M14 contains M1-M4, L2 contains the loop
connecting M2 and M3; see Fig. 3 for detailed definition; Fig. 5 shows
a schematic diagram).
Fig. 5.
Schematic diagram of wild-type and chimeric
subunits. In all subunits, the core region containing M1-M4 is 281 amino acids long and is shown enlarged relative to the N and C termini.
Numbers without units denote number of amino acids. The
small numbers next to the schematic representation of the
chimeras indicate the number of differing amino acids that are replaced
by the given chimera. The middle panel shows
log(IC50, 100mV) values of
Mg2+ block;  is shown on the right
panel. Bars represent mean ± SEMs. Indication of
significance levels determined by the Tukey-Kramer test: c
or b = p < 0.05, cc or bb = p < 0.01 (comparison to NR2C or NR2B,
respectively).
[View Larger Version of this Image (24K GIF file)]
Fig. 3.
Alignment of the core region of rat NR2 subunit
amino acid sequences including the four hydrophobic domains (M1-M4).
An asterisk indicates the location of a determinant of
Mg2+ block in the M2 domain (N-site).
Residues printed in boldface were exchanged in a given
chimera, and underlined positions emphasize group-specific
differences ([NR2A = NR2B]  [NR2C = NR2D]) in amino acid
sequence. Numbers given on the right side
indicate the position of the rightmost amino acid in each row (72 AA)
within the given subunit. Fusion positions of chimeras are denoted by
capital letters in the order of their appearance:
M1 = A/B (only for this chimera, the sequence of
NR2A was inserted, printed in bold italic letters); M12 = A/D; L1 = B/C; M13 = A/E; M2a = SV..A
replaced by AI..G; L2 = D/E; M14 = A/I; M34 = E/I; M34a = F/I; M4 = G/I; M4a = G/H.
[View Larger Version of this Image (80K GIF file)]
Construction of chimeras. All mutants were constructed by
PCR-based methods (Ausubel et al., 1994). For the chimeras, a set of
primers was designed in a way to produce a PCR fragment of the desired
region of the donor subunit that additionally carried the required
restriction sites for subcloning into the recipient subunit. In most
cases, we used a four-primer strategy permitting the introduction of a
unique silent restriction site into the middle part of a fragment. This
newly introduced site could then be used to insert smaller fragments,
likewise carrying an additional site. Reiteration of this process
allowed the introduction of progressively smaller fragments. We chose a
distribution of restriction sites that allowed for reshuffling of most
chimeras. All constructs were sequenced over the entire length of the
replaced fragment.
Heterologous expression of NMDAR channels. NR2 wild-type and
chimeric subunits were expressed with the NR1 subunit at a ratio of
approximately 1:1 in Xenopus oocytes. Capped cRNA was
transcribed for each expression construct with SP6 RNA polymerase (80 U/µl, Promega, Madison, WI) and examined electrophoretically on a
denaturating agarose gel. To determine the concentration of the RNA
solution, the gel was stained with ethidium bromide, and the
intensities of the bands were compared with a length standard of
defined quantity (0.24-9.5 kb RNA Ladder, Gibco Life Technologies,
Eggenstein, Germany). Appropriate dilutions were prepared (0.01-1
µg/µl) to achieve expression levels in the range of 0.5-2 µA at
 50 mV. Xenopus laevis oocytes were manually dissected from
the ovary and placed in OR2 medium (NaCl, 82.5 mM; KCl, 2.5 mM;
Na2HPO4, 1 mM; HEPES, 5 mM;
MgCl2, 1 mM;
CaCl2, 1 mM; PVP, 0.5 gm/l,
pH adjusted to 7.2 with NaOH) supplemented with penicillin and
streptomycin (100 U/ml, Gibco). The oocytes were injected with 20-40
nl of RNA solution using a Nanoject injector (Drummond, Broomall, PA),
incubated at 19°C, and treated with collagenase type II (1 mg/ml,
Sigma, St. Louis, MO) after 12-24 hr of incubation. Whole-cell
recordings were made at room temperature 3-8 d after injection of the
oocytes.
Experimental protocol: quantification of
Mg2+block. All recordings were
performed on a two-microelectrode voltage-clamp setup (TEC 01/2C, NPI
Electronic, Tamm, Germany) modified for computer-controlled, automatic
application of different solutions, voltage ramps, and data acquisition
(custom made using LabVIEW, National Instruments, Austin, TX).
Electrodes were pulled from borosilicate glass and filled with 3 M KCl. The resistance was kept in the range of
0.5-2 M . Oocytes were clamped at 50 mV, and voltage ramps were
applied (120 to +20 mV, within 2 sec). Current records were filtered
with a Bessel 4-pole low-pass filter at 30 Hz and digitized with a
sampling rate of 100 Hz. The recording chamber had a volume of 200 µl
and was continuously perfused at 1-2 ml/min. To minimize interactions
of Mg2+ and Ca2+ (Mayer and
Westbrook, 1987; Ascher and Nowak, 1988) and contamination by
Ca2+-activated Cl
currents (Leonard and Kelso, 1990), all experiments were made in low
Ca2+ Ringer's solution containing (in
mM): 0.18 CaCl2, 115 NaCl,
2.5 KCl, and 10 HEPES adjusted to pH 7.2 with NaOH.
The following protocol was used to obtain current-voltage
(I-V) curves in the presence of different
Mg2+ concentrations. First, the
Mg2+ concentration to be tested was applied in
the presence of glycine (10 µM) for 50-180 sec
to obtain the leakage I-V curve. Then, the same solution,
but in addition containing glutamate (100 µM),
was applied for 40 sec, and a test I-V curve was recorded.
Different Mg2+ concentrations were separated by a
3 min wash with low Ca2+ Ringer's. This type of
experiment was executed in the sequence 0 µM
(i.e., nominally Mg2+-free solution), 1 µM, 10 µM, 100 µM, 1 mM, 0 µM Mg2+. We refer to one
such sequence as a series, and a set consisted of two-to-three
consecutive series. Only sets with reasonably stable series were
selected for further analysis.
Data were analyzed using Igor (WaveMetrics). Leakage I-V
curves were subtracted from test I-V curves. Normalized
I-V curves were calculated relative to the value at 100
mV of the nominally Mg2+-free solution (Figs.
1, 4). For each Mg2+
concentration, the fractional block at 100 mV was determined. Data
derived from individual series of a set were averaged. The
IC50,100mV of a set was obtained by fitting the
averaged data points with the function:
![y=<FR><NU>100</NU><DE>1+<FENCE><FR><NU>[Mg<SUP>2+</SUP>]</NU><DE>IC<SUB>50</SUB></DE></FR></FENCE><SUP>n<SUB>H</SUB></SUP></DE></FR>,](/content/vol16/issue11/fulltext/3549/img001.gif)
|
(1)
|
where y is percentage of control current at a given
membrane potential, 100 is maximal current in nominally
Mg2+-free solution,
[Mg2+] is the concentration of extracellular
Mg2+, nH is the Hill
coefficient, and IC50 is half-maximal block by
Mg2+.
Fig. 1.
Differential Mg2+ block of
four NR1-NR2 subtypes expressed in Xenopus oocytes.
Representative I-V curves recorded in the presence of
different Mg2+ concentrations in low
Ca2+ Ringer's. I-V curves were
normalized to the current at 100 mV in nominally
Mg2+-free solution.
IC50,100mV values are shown in Table 1. The
control I-V curves of NR1-NR2A and NR1-NR2B were slightly
blocked by residual Mg2+ in the Ringer's.
However, the influence on calculation of the
IC50,100mV was negligible, and the values given
in Table 1 might be slight underestimates of the true
IC50,100mV. Variations of the reversal
potential in the range of 10 to 0 mV were observed between different
oocytes. In some instances, the outward current passed by NR1-NR2B
channels was potentiated in the presence of 1 mM
Mg2+.
[View Larger Version of this Image (23K GIF file)]
Fig. 4.
Selected I-V curves of four chimeras
representing different levels of Mg2+ block. See
also legend to Figure 1. IC50,100mV values are
shown in Table 1.
[View Larger Version of this Image (23K GIF file)]
Quantification of voltage dependence. Voltage
dependence of block (, fraction of the electric field that the
blocker experiences) and affinity of Mg2+ at 0 mV
[K0.5(0)] were calculated by determining
the IC50 (Eq. 1) at different potentials (25 to
115 mV, increments of 5 mV). The logarithm of the resulting
IC50 values was plotted against the holding
potential, and a straight line was fitted to the data points over a
voltage range from 80 to 30 mV. The apparent and
K0.5(0) were then calculated according to
Woodhull (1973):
|
(2)
|
where z is valence, V is membrane
potential, and R, T, and F have their
usual meaning.
Statistical analysis. The logarithm of the
IC50 values was used to calculate the mean, SEM,
and one-way ANOVA. Significance levels were assessed with the
Tukey-Kramer test using the GB-STAT software (Dynamic Microsystems,
Silver Spring, MD).
RESULTS
Mg2+ block of wild-type NMDAR channels expressed in
Xenopus oocytes
NMDAR channels assembled from the constitutive NR1 and any one of
the four NR2 subunits (A,B,C,D) were differently blocked by
Mg2+ (Fig. 1). Channels containing the NR2A or
NR2B subunit were more strongly blocked compared with channels
containing NR2C or NR2D. In the presence of 1 mM
Mg2+, the NR2A- or NR2B-containing channels were
almost completely blocked at 100 mV and passed maximal current at
approximately 25 mV. In contrast, NR2C- or NR2D-containing channels
were incompletely blocked at 100 mV and passed maximal current at a
less depolarized potential of approximately 35 mV (Fig. 1). In all
experiments reported here, current responses were nondesensitizing
given the time resolution of our system (not shown). To quantify
Mg2+ block, we determined the concentration of
Mg2+ for half-maximal block at 100 mV
(IC50,100mV, see Materials and Methods).
Dose-response curves for Mg2+ block of
individual records of NR1-NR2C and NR1-NR2B channels are illustrated in
Figure 2, A and B (shown as the
thick line). Averaged IC50,100mV
values are listed in Table 1 and shown
graphically in Figure 5.
Fig. 2.
Voltage dependence of Mg2+
block. A, Dose-response curves for
Mg2+ at different potentials (100, 80, 60,
40 mV) derived from the I-V curve of NR1-NR2C shown in
Figure 1. Values of the fractional block are shown as
squares. The fit used to determine the
IC50,100mV (in this example, 14.7 µM Mg2+) is shown as a
thick line. B, Same as in A for
NR1-NR2B; values of the fractional block derived from the
I-V curve shown in Figure 1 are shown as
triangles. The IC50,100mV is 2.4 µM Mg2+. C,
Difference in voltage dependence of the two representative
Mg2+ series shown in A and
B and Figure 1 (printed in black). The data were
fitted in the range from 80 to 30 mV, as indicated by the two
lines. Examples for the NR1-NR2A and NR1-NR2D are printed in
gray.
[View Larger Version of this Image (18K GIF file)]
The normalized I-V curves shown in Figure 1 suggest a
different voltage dependence of block between channels containing NR2A
or NR2B versus channels containing the NR2C or NR2D. Voltage dependence
was analyzed according to the Woodhull model (see Materials and
Methods). Figure 2, A and B, shows dose-response
curves at selected potentials derived from the records displayed in
Figure 1. The logarithm of IC50 values obtained
at different potentials was plotted against the holding potential, and
a straight line was fitted to determine and
K0.5(0). Individual examples for each
subtype are shown in Figure 2C. A deviation from the linear
relation predicted by the Woodhull expression was observed. The
deviation was most pronounced at very negative potentials and more
prominent in NR1-NR2A and NR1-NR2B channels compared with NR1-NR2C and
NR1-NR2D channels. This nonlinearity constituted the problem of finding
the appropriate voltage range to fit the data. At potentials more
negative than 90 mV, the deviation from linearity was too strong
(Fig. 2C), whereas at potentials more positive than 30 mV,
the error in calculating IC50 values was too
large. Within these borders, the most accurate fits were found in the
range of 80 to 30 mV, which was then taken to determine and
K0.5(0). NR2A- or NR2B-containing channels
displayed a higher voltage dependence ( ~ 1) compared with
channels containing NR2C or NR2D ( ~ 0.7). The four subtypes were
not significantly different regarding
K0.5(0) (Table 1). Because
K0.5(0) showed a considerable variability,
we applied two additional approaches (DiFrancesco et al., 1982;
Ruppersberg et al., 1994) to analyze voltage dependence of the four
subtypes. Both methods yielded similar results and a comparable degree
of variability (not shown).
Chimeric NR2 subunits: construction and functional analysis
Chimeric NR2 subunits were generated by replacing fragments of the
least sensitive NR2C subunit with homologous fragments of the most
sensitive NR2B subunit (Fig. 3; Fig. 5, left
panel). As the recipient NR2C subunit, a truncated version lacking
the C-terminal 275 amino acids (NR2C, see Materials and Methods) was
used. When coexpressed with the NR1 subunit, the resulting channel
showed no differences in Mg2+ block (Table 1,
Fig. 5) or single-channel characteristics (Schoepfer et al., 1994)
compared with channels containing the full-length NR2C subunit.
Comparison of the amino acid sequence between the core regions (M1-M4)
of the NR2 subunits shows a high overall sequence similarity (Fig. 3).
The hydrophobic domains (especially M3) appear to be more conserved
than, for example, the L3 loop (for nomenclature, see Fig. 5,
left panel) or the region following M4. Also, the stretch of
amino acids preceding M1, a region involved in agonist binding
(Stern-Bach et al., 1994), exhibits almost 100% sequence identity. In
addition, the alignment reveals that group-specific differences (at
homologous positions: [NR2A = NR2B] [NR2C = NR2D],
underlined) occur more frequently within the hydrophobic
domains compared with other areas (e.g., L1 loop, L3 loop, or
C-terminal of M4).
All chimeras were coexpressed with the NR1 subunit and generated
whole-cell currents comparable to wild type (Table 1). Chimeras were
analyzed as described for the wild-type subunits; first we present
results based on the analysis of half-maximal block, then we describe
voltage dependence of Mg2+ block.
Influence of the M2 domain on subunit specificity of
Mg2+ block
In the close vicinity of the N-site within the M2
domain, few amino acid positions differ between the subunits (Fig. 3).
We created an M2 domain similar to NR2B by substituting three amino
acids (SV..A to AI..G, see Fig. 3) on the N-terminal side of the
N-site in the NR2C. The resulting NR2C-M2a chimera could not
be distinguished functionally from NR2C wild type (Fig. 5, Table 1). On
the C-terminal side of the N-site at the border of M2 (Fig.
3), two positions differ between NR2B and NR2C. When mutated
individually, the IC50,100mV of both
NR2C(I598V)- and NR2C(E599Q)-containing channels was close to wild-type
NR2C (Table 1).
Contribution of the M1-M4 segment
Because replacement of residues within M2 of NR2C was without
effect on Mg2+ block, we investigated regions
adjacent to the M2 domain. In a first attempt, the M1-M3 region was
transferred from the NR2B into the NR2C subunit to yield NR2C-M13
[Fig. 3A/E (the letters in superscript refer to
the segment exchanged between NR2B and NR2C)]. This chimera exhibited
a strongly increased sensitivity of Mg2+ block
compared with NR2C wild type (Fig. 4A, Table
1). However, Mg2+ block was not as sensitive as
wild-type NR2A or NR2B (Fig. 5). In a second attempt, we
transplanted the complete M1-M4 region of NR2B into NR2C, thereby
creating chimera NR2C-M14 (Fig. 3A/I).
Functional analysis of this chimera (Fig. 4B) revealed an
IC50,100mV indistinguishable from NR2A or NR2B
wild type (Table 1, Fig. 5).
Identification of individual elements within the
M1-M4 segment
In an attempt to detect single amino acid residues mediating the
increase of Mg2+ block found in NR2C-M14, the
M1-M4 segment was subdivided into smaller units (see Fig. 5,
left panel). The difference in sensitivity to
Mg2+ block between NR2C-M13 and NR2C-M14
suggested that the M3-M4 segment may contain a structural determinant,
in addition to elements present in the M1-M3 segment. To test this
idea, chimera NR2C-M34 was constructed (Fig.
3E/I). Functional analysis revealed a
significant increase in sensitivity of Mg2+ block
relative to NR2C wild type (Fig. 5, Table 1). However, this increase
was less pronounced than determined for its N-terminal counterpart,
NR2C-M13. Chimeras NR2C-M13 and NR2C-M34 were each subdivided to yield
an N-terminal and a C-terminal set of chimeras.
Chimera NR2C-M13 was split into NR2C-M12 and NR2C-L2, the former
containing M1 and M2 (Fig. 3A/D), the latter
carrying the L2 linker between M2 and M3 (Fig.
3D/E). Chimera NR2C-M12 was less sensitive
to Mg2+ block than NR2C-M13, but significantly
more sensitive than NR2C wild type (Fig. 5, Table 1). A slightly higher
sensitivity to Mg2+ block was found when chimera
NR2C-L2 was examined (Figs. 4C, 5). Interestingly, this
chimera introduced changes at only four amino acid positions, two of
which were already shown to have no effect on the
IC50,100mV [NR2C(I598V) and NR2C(E599Q), see
above]. The remaining two positions were mutated individually
[NR2C(R602K) and NR2C(L611S), see Fig. 3]. Unexpectedly, none of
these mutants differed significantly from wild-type NR2C with regard to
the IC50,100mV of Mg2+
block (Table 1). Chimera NR2C-M12 was divided into NR2C-M1, carrying M1
of NR2A (Fig. 3A/B) and NR2C-L1, containing
the small heterogeneous L1 linker between M1 and M2 of NR2B (Fig.
3B/C). Mg2+ block of
NR2C-M1 showed a similar sensitivity to that of NR2C-M12, but NR2C-L1
was indistinguishable from NR2C wild type (Fig. 5, Table 1).
Chimera NR2C-M34 was subdivided to yield NR2C-M34a, containing the
C-terminal half of L3, the M4 domain, and a short stretch of the C
terminus (Fig. 3F/I). NR2C-M34a exhibited a
similar sensitivity to Mg2+ block than NR2C-M34
(Fig. 5). In a further step, the C-terminal half of this chimera was
retained, thereby creating chimera NR2C-M4 (Fig.
3G/I). Sensitivity to
Mg2+ block of NR2C-M4 was comparable to the
effect found for NR2C-M34 (Fig. 5). To investigate whether the M4
domain or the heterogeneous part C-terminal to M4 contains the
responsible determinant, we constructed chimera NR2C-M4a (Fig.
3G/H). This chimera exhibited the same
sensitivity to Mg2+ block as NR2C-M4 (Fig.
5).
To summarize, three small elements within the M1-M4 segment
independently mediated an increase in sensitivity to
Mg2+ block. These elements are contained on
chimeras NR2C-M1, NR2C-L2, and NR2C-M4a. Chimera NR2C-L2 was further
analyzed for single amino acid determinants, but no attempt was made to
identify single amino acid residues within the NR2C-M1 and NR2C-M4a
chimeras.
Combination of the three elements M1, L2, and M4
Insertion of the M1-M4 region of the NR2B into the NR2C resulted
in a channel with an NR2B-like phenotype of Mg2+
block. However, no single determinant could be detected within the
M1-M4 region. We combined the three smallest elements that showed a
significant effect by themselves in one single chimera, NR2C-M1L2M4a
(Fig. 3A/B,D/E,G/H).
Mg2+ block of the resulting channel was enhanced
more than observed for any of the elements alone and was close, but not
identical, to that of wild-type NR1-NR2B (Figs. 4D, 5, Table
1).
Voltage dependence of chimeric NR2 subunits
Analysis of the chimeras for voltage dependence of
Mg2+ block revealed a pattern as expected from
the IC50,100mV values described above.
Consistent with the observation that NMDAR wild-type channels differ in
their but exhibit a similar K0.5(0), we
found that the chimeras also differed in but were not statistically
different with regard to the affinity of Mg2+ at
0 mV. Most of the chimeras displayed intermediate values and were
not significantly different from either NR2B or NR2C wild types (Fig.
5, Table 1). Only the N-terminal mutants NR2C-M1, NR2C-M12, NR2C-L2,
and NR2C(L611S) exhibited significantly increased voltage dependence
compared with NR2C wild type. The NR2C-M1L2M4a chimera showed the
strongest increase in voltage dependence and could not be distinguished
from wild type NR2B (Fig. 5). The chimeras with the largest decrease in
IC50,100mV, NR2C-M13 and NR2C-M14, exhibited an
increase of , albeit not statistically significant. In contrast to
the N-terminal chimeras, C-terminal chimeras showed no significant
change in voltage dependence. IC50,100mV values
of all mutants could be calculated from pairs of averaged
K0.5(0) and values given in Table 1 and
presented with the same pattern as found for the measured
IC50,100mV values (not shown).
DISCUSSION
The present study revealed structural determinants for NR2
subunit-specific Mg2+ block in heteromeric NMDAR
channels. As indicated by the work of Monyer et al. (1994), channels
containing NR1 and any one of four NR2 subunits fall into two groups
with regard to their sensitivity to external Mg2+
block. Receptor channels containing the NR2A or NR2B subunits are
characterized by a higher sensitivity
(IC50,100mV ~ 2 µM),
whereas channels containing NR2C or NR2D exhibit a lower sensitivity of
Mg2+ block (IC50,100mV ~ 10-15 µM). Differences of
IC50,100mV values shown here compared with
IC50 values reported in other studies arise in
part from calculation of the fractional block at potentials less
negative than 100 mV (Kawajiri and Dingledine, 1993). In addition,
differences may be attributable to the presence of millimolar
concentrations of Ca2+ or
Ba2+ (Ishii et al., 1993; Kawajiri and
Dingledine, 1993) in the external solution. To reduce interaction
between Ca2+ and Mg2+
(Mayer and Westbrook, 1987; Ascher and Nowak, 1988), Ringer's solution
containing 0.18 mM Ca2+ was
used in our study.
Analysis of voltage dependence in wild-type NMDAR channels
The Woodhull model of voltage-dependent block predicts a linear
relationship between the ln(IC50,V) of the
blocker and the membrane potential. In contrast, we found a deviation
from linearity toward a lower voltage dependence of
Mg2+ block, consistent with the observations of
Kleckner and Dingledine (1991). This deviation was most prominent at
potentials more negative than 80 mV in all four subunits, but the
more sensitive NR1-NR2A and NR1-NR2B channels deviated more strongly
compared with the less sensitive NR1-NR2C and NR1-NR2D channels. The
best approximation of a linear Woodhull relation was found in the range
of 80 to 30 mV; therefore, this range was used to determine and
K0.5(0). Two explanations for the
inconsistencies between data and model may be relevant (Ascher and
Nowak, 1988; Kleckner and Dingledine, 1991). First,
Mg2+ might be a permeant blocker (Mayer and
Westbrook, 1987). Second, Ca2+ attenuates
Mg2+ block at hyperpolarized potentials (Mayer
and Westbrook, 1987). These two actions may be additive and
subunit-specific.
NMDAR channels containing the more sensitive NR2A or NR2B subunits show
a higher voltage dependence ( ~ 1) compared with channels
containing NR2C or NR2D ( ~ 0.7). However, both groups show a
similar affinity for Mg2+ at 0 mV
(K0.5(0) ~ 3.5 mM).
Our analysis of recombinant NMDAR channels revealed values that are in
the range of previously published data obtained from native NMDAR
channels. For example, and K0.5(0)
values of 1 and 8.8 mM have been reported by
Ascher and Nowak (1988), ~0.8 and 1.8 mM by
Jahr and Stevens (1990), 0.73 and 1.1 mM by Chen
and Huang (1992). Inspection of the data presented here, as well as
comparison to data published by others, reveals that the values
reported for are in good agreement, whereas the
K0.5(0) values show a considerable
variability. This might be related to a systematic error in
quantification of K0.5(0), which in most
methods is determined by extrapolation of a fit applied to a voltage
range distant from 0 mV. Thus, small errors in fitting the data can
create a large variability of K0.5(0). In
conclusion, the four subtypes appear to be similar in binding given the
sensitivity of our system, but small differences cannot be ruled out.
Nevertheless, subunit-specific differences in
Mg2+ block predominantly reflect a difference in
voltage dependence ().
Structural elements mediating subunit specificity of
Mg2+ block
Our data demonstrate that an NR2 subunit with a low
sensitivity of Mg2+ block can be transformed into
one with a high sensitivity by exchanging the M1-M4 region. Hence,
determinants for subunit specificity of Mg2+
block are located within the M1-M4 segment.
Subdividing the M1-M4 region into smaller units resulted in a
progressive loss of Mg2+ sensitivity. However,
three small elements within this region mediated an increase in
sensitivity to Mg2+ block. These elements are the
M1 domain, the small L2 linker connecting M2 and M3, and the M4 domain
(see Fig. 3 for definition). Interestingly, NR1-NR2C-M1 and NR1-NR2C-L2
channels exhibited a significant increase in voltage dependence. The
NR2C-L2 chimera differs in only four amino acid positions from wild
type. Unexpectedly, mutation of any individual amino acid did not
result in a change of the IC50,100mV,
indicating that a specific set of residues is required to generate this
phenotype. Combination of the three elements in chimera NR2C-M1L2M4a
resulted in a channel with identical voltage dependence () of block
but a slightly different IC50,100mV compared
with NR2B wild type. On the other hand, channels containing NR2C-M14
exhibited an IC50,100mV identical with NR2B
wild type and a slightly, but not significantly, decreased value of
. This discrepancy may reflect limitations of our experimental
approach to resolve differences as small as encountered in this case.
The three elements contain most of the group-specific differences in
amino acid sequence within the M1-M4 segment (see Fig. 3), suggesting
that they mediate a group-specific function. We conclude that the M1,
L2, and M4 domains are the major structural constituents that mediate
subunit-specific differences of Mg2+ block in
heteromeric NMDAR channels.
Implications for structure and mechanism
Initially, the subunit-specific difference in voltage
dependence () suggested that a single site, in addition to the
N-site, might be sufficient to create subunit specificity of
Mg2+ block. This can be clearly ruled out by the
results shown in this study. Alternatively, four models might be
considered for the interpretation of our results in structural terms.
Subunit specificity of Mg2+ block might be
generated by the presence of multiple amino acids at different sites:
(1) affecting global properties of the channel (e.g., gating); (2)
affecting the positioning of a functionally critical amino acid in the
channel (e.g., N-site); (3) affecting the interaction of
Mg2+ with other ions (e.g.,
Ca2+); (4) interacting directly with
Mg2+ in the channel (e.g., local changes in
surface potential).
Elucidation of the mechanism underlying subunit specificity of
Mg2+ block may provide evidence favoring one of
the models, but it is also conceivable that several mechanisms act in
parallel. For example, subunit-specific differences in single-channel
properties (Stern et al., 1992) are consistent with model (1).
Interpretation of the difference in as a difference in location of
a site relative to the vertical axis of the channel seems compatible
with model (2). Interaction of Mg2+ and
Ca2+ in the channel, which also may account for
the deviation from the Woodhull model, sustains model (3). The
subunit-specific difference in voltage dependence, interpreted as a
difference in local surface potential, favors model (4).
Considering models (3) and (4), it appears relevant that the three
elements are putative transmembrane (M1,M4) or membrane-associated (L2)
regions, and thus may be located close to the conduction pathway.
Functional data (Burnashev et al., 1992), as well as cysteine
substitutions (Kuner et al., 1995), indicate that the M2 segment forms
at least part of the channel lining. Little is known for the other
putative transmembrane segments. Changes in Mg2+
block mediated by the M1 segment and RNA editing in the M1 segment of
GluR6 (Köhler et al., 1993), in combination with the recently
proposed three transmembrane domain (3TM) topology model of glutamate
receptors (Hollmann et al., 1994; Wo and Oswald, 1994; Bennett and
Dingledine, 1995), indicate that M1 may contribute to the outer
vestibule of the channel. Similarly, the L2 loop and parts of M3 might
be positioned close to the channel, consistent with the observation
that M3 has a modulatory effect on pore structure (Ferrer-Montiel et
al., 1995). The 3TM model and results obtained from cysteine scanning
analysis of the M2 segment (Kuner et al., 1995) place the L2 loop on
the cytoplasmic side of the membrane. Differences in interaction with
ions present at the cytoplasmic channel opening may affect external
Mg2+ block (Ruppersberg et al., 1994). To date,
nothing is known about the positioning of M4 relative to the conduction
pathway. It is intriguing to speculate that M4 might contribute to the
lining of the channel in conjunction with other transmembrane
domains.
In summary, our experiments show that at least three amino
acid clusters of the NR2 subunit, likely to be positioned close to the
conduction pathway, are required to determine sensitivity of
Mg2+ block in heteromeric NMDAR channels. The
mechanism underlying subunit specificity of Mg2+
block remains to be established; however, the models that we provide
may guide future studies.
FOOTNOTES
Received Nov. 8, 1995; revised March 20, 1996; accepted March 26, 1996.
This work was supported by SFB Grant 317/B9 to Peter H. Seeburg. R.S.
is a Senior Wellcome Trust Fellow. We thank Dr. Peter H. Seeburg for
his interest and support and Dr. Lonnie P. Wollmuth for critical
discussions and technical advice. We are grateful to Drs. David
Colquhoun, Dirk Feldmeyer, and Georg Köhr for commenting on this
manuscript. We thank Annette Herold for DNA sequencing, Ulla Amtmann
for technical assistance, and Christine Beck and Nicole Bender for help
with mutagenesis.
Correspondence should be addressed to Thomas Kuner at the above
address.
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H.-H. Chen, C.-T. Wei, Y.-R. Lin, T.-H. Chien, and M.-H. Chan
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Z. Fu, S. M Logan, and S. Vicini
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A. Qian, A. L. Buller, and J. W. Johnson
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V. L. Arvanian, W. J. Bowers, J. C. Petruska, V. Motin, H. Manuzon, W. C. Narrow, H. J. Federoff, and L. M. Mendell
Viral Delivery of NR2D Subunits Reduces Mg2+ Block of NMDA Receptor and Restores NT-3-Induced Potentiation of AMPA-Kainate Responses in Maturing Rat Motoneurons
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P. E. Chen, A. R. Johnston, M. H. S. Mok, R. Schoepfer, and D. J. A. Wyllie
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V. Gauck and D. Jaeger
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E. Harvey-Girard and R. J. Dunn
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L. J Gentet and D. Ulrich
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P. Rossi, E. Sola, V. Taglietti, T. Borchardt, F. Steigerwald, J. K. Utvik, O. P. Ottersen, G. Kohr, and E. D'Angelo
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D. Billups, Y.-B. Liu, S. Birnstiel, and N. T. Slater
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C.-F. Hsiao, N. Wu, M. S. Levine, and S. H. Chandler
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D. Chollet, P. Franken, Y. Raffin, A. Malafosse, J. Widmer, and M. Tafti
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L. Cathala, C. Misra, and S. Cull-Candy
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E. D. Kirson and Y. Yaari
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C. Misra, S. G Brickley, D. J A Wyllie, and S. G Cull-Candy
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S. M. Antonov and J. W. Johnson
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E. D Kirson, C. Schirra, A. Konnerth, and Y. Yaari
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G. Martin, S. H. Ahmed, T. Blank, J. Spiess, G. F. Koob, and G. R. Siggins
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R. Dingledine, K. Borges, D. Bowie, and S. F. Traynelis
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B. Saul, T. Kuner, D. Sobetzko, W. Brune, F. Hanefeld, H.-M. Meinck, and C.-M. Becker
Novel GLRA1 Missense Mutation (P250T) in Dominant Hyperekplexia Defines an Intracellular Determinant of Glycine Receptor Channel Gating
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J. C. Brimecombe, M. J. Gallagher, D. R. Lynch, and E. Aizenman
An NR2B Point Mutation Affecting Haloperidol and CP101,606 Sensitivity of Single Recombinant N-Methyl-D-Aspartate Receptors
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D. J A Wyllie, P. Behe, and D. Colquhoun
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L. C. Anson, P. E. Chen, D. J. A. Wyllie, D. Colquhoun, and R. Schoepfer
Identification of Amino Acid Residues of the NR2A Subunit That Control Glutamate Potency in Recombinant NR1/NR2A NMDA Receptors
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C. T. Smothers, J. J. Mrotek, and D. M. Lovinger
Chronic Ethanol Exposure Leads to a Selective Enhancement of N-Methyl-D-aspartate Receptor Function in Cultured Hippocampal Neurons
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J. C. Brimecombe, F. A. Boeckman, and E. Aizenman
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P. Paoletti, P. Ascher, and J. Neyton
High-Affinity Zinc Inhibition of NMDA NR1-NR2A Receptors
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J. Chao, N. Seiler, J. Renault, K. Kashiwagi, T. Masuko, K. Igarashi, and K. Williams
N1-Dansyl-Spermine and N1-(n-Octanesulfonyl)-Spermine, Novel Glutamate Receptor Antagonists: Block and Permeation of N-Methyl-D-Aspartate Receptors
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