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Volume 17, Number 15,
Issue of August 1, 1997
pp. 5711-5725
Copyright ©1997 Society for Neuroscience
High-Affinity Zinc Inhibition of NMDA NR1-NR2A Receptors
Pierre Paoletti,
Philippe Ascher, and
Jacques Neyton
Laboratoire de Neurobiologie, Centre National de la Recherche
Scientifique Unité de Recherche Associée 1857, Ecole
Normale Supérieure, 75005 Paris, France
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Micromolar concentrations of extracellular Zn2+
are known to antagonize native NMDA receptors via a dual mechanism
involving both a voltage-independent and a voltage-dependent
inhibition. We have tried to evaluate the relative importance of these
two effects and their subunit specificity on recombinant NMDA receptors expressed in HEK 293 cells and Xenopus oocytes. The
comparison of NR1a-NR2A and NR1a-NR2B receptors shows that the
voltage-dependent inhibition is similar in both types of receptors but
that the voltage-independent inhibition occurs at much lower
Zn2+ concentrations in NR1a-NR2A receptors
(IC50 in the nanomolar range) than in NR1a-NR2B receptors
(IC50 in the micromolar range). The high affinity of the
effect observed with NR1a-NR2A receptors was found to be attributable
mostly to the slow dissociation of Zn2+ from its
binding site. By analyzing the effects of Zn2+ on
varied combinations of NR1 (NR1a or NR1b) and NR2 (NR2A, NR2B, NR2C),
we show that both the NR1 and the NR2 subunits contribute to the
voltage-independent Zn2+ inhibition. We have
observed further that under control conditions, i.e., in zero nominal
Zn2+ solutions, the addition of low concentrations
of heavy metal chelators markedly potentiates the responses of
NR1a-NR2A receptors, but not of NR1a-NR2B receptors. This result
suggests that traces of a heavy metal (probably
Zn2+) contaminate standard solutions and tonically
inhibit NR1a-NR2A receptors. Chelation of a contaminant metal also
could account for the rapid NR2A subunit-specific potentiations
produced by reducing compounds like DTT or glutathione.
Key words:
NMDA;
zinc;
DTT;
heavy metals;
recombinant receptors;
ionic channels
INTRODUCTION
Zn2+ ions are known to be
abundant in some nerve terminals and can be released in the synaptic
cleft at concentrations of nearly 1 µM (for review, see
Smart et al., 1994 ). In this context, the observation that
Zn2+ at micromolar concentrations inhibits NMDA
responses (Peters et al., 1987; Westbrook and Mayer, 1987) immediately
was given a major physiological significance. This significance was
reinforced by the observation of Zn2+ inhibitory
effects on the NMDA components of synaptic currents (Forsythe et al.,
1988; Mayer and Vyklicky, 1989) and by data indicating that
Zn2+ could play an important role in excitoxicity
(Koh and Choi, 1988; Koh et al., 1996).
The analysis of the mechanisms of the Zn2+
inhibition (Mayer et al., 1988, 1989; Christine and Choi, 1990;
Legendre and Westbrook, 1990) revealed that Zn2+
produces both a voltage-independent inhibition and a voltage-dependent block, the latter resembling that produced by Mg2+.
Inhibitory effects of Zn2+ on recombinant NMDA
receptors were observed first on receptors expressed from whole brain
RNA (Rassendren et al., 1990) and then, after the cloning of the main
NMDA receptor subunits, on heteromeric receptors associating NR1 and
NR2 subunits (Kutsuwada et al., 1992; Meguro et al., 1992) and on most
homomeric NR1 receptors (Hollmann et al., 1993; Zheng et al., 1994).
Mori et al. (1992) and Sakurada et al. (1993) then analyzed the effects
of mutations of a ring of asparagines found at the Q/R/N site, a
critical position of the M2 segment involved in the control of the
Mg2+ block of NMDA channels (see McBain and Mayer,
1994). They observed that a major reduction or even a complete
suppression of the Mg2+ block was associated with a
mild reduction of the Zn2+ block (see also Kawajiri
and Dingledine, 1993), probably because the mutations reduced or
abolished the Zn2+ voltage-dependent block but left
intact the Zn2+ voltage-independent inhibition.
The present study aimed at a better separation of the voltage-dependent
and the voltage-independent processes in recombinant receptors. In
attempting to measure the inhibitory effect of Zn2+
on NR1a-NR2A receptors expressed in Xenopus oocytes and
human embryonic kidney (HEK) cells, we observed inhibitions of very variable size at concentrations of a few tens of nanomolars. This was
found to be attributable to the variable degree of contamination of the
solutions by traces of heavy metal ions (possibly
Zn2+). By using chelators of these metals, we were
able to obtain a reliable estimate of the control response and to
demonstrate that the IC50 of the voltage-independent
Zn2+ inhibition is highly subunit-specific, ranging
from ~10 nM in the case of NR1a-NR2A receptors to 10 µM in NR1a-NR2C receptors. In contrast, the
voltage-dependent Zn2+ inhibition has an
IC50 in the micromolar range in NR1a-NR2A receptors and
can be suppressed selectively by a point mutation in the pore region
[NR2A(N595K)].
MATERIALS AND METHODS
Primary neuronal cultures
Cortical and diencephalic neurons taken from 15- to 16-d-old
mouse embryos were cultured for 2-5 weeks, as previously described by
Ascher et al. (1988).
NMDA receptor expression in HEK cells and
Xenopus oocytes
Plasmid constructions. All of the cDNAs used in this
study were subcloned in a modified (see Kupper et al., 1996) pcDNA3
vector (Invitrogen, Leek, Netherlands), allowing high-level expression of recombinant proteins in transfected mammalian cells as well as in
Xenopus oocytes after nuclear injection. The subunit cDNAs were subcloned from the following pBluescript-based plasmids: pN60
[Moriyoshi et al. (1991); gift from S. Nakanishi, Kyoto University, Japan] for NR1a, pNMDAR1-1b [Hollmann et al. (1993); gift from J. Boulter, Salk Institute, La Jolla, CA], NR2A and NR2C [Monyer et al.
(1992); gift from P. Seeburg, Center for Molecular Biology, Heidelberg,
Germany], and  2 [Kutsuwada et al. (1992); gift from M. Mishina,
Niigata University, Japan] for NR2B. The plasmid coding for the green
fluorescent protein (GFP) (Chalfie et al., 1994) was a gift from D. Pritchett.
Site-directed mutagenesis. Site-directed mutagenesis was
performed according to a method modified from Kunkel (1985). The presence of the mutation was verified by sequencing across the M2
region with the Sequenase Kit (Stratagene Cloning Systems, La Jolla,
CA).
Transfection of HEK cells. HEK cells were cultured in a DMEM
medium (with L-glutamine and 4.5 gm/l glucose added)
containing 10% heat-inactivated fetal calf serum and
penicillin-streptomycin (5000 U/ml). Low confluency cells were
transfected by the calcium phosphate precipitation method (Chen and
Okayama, 1987). Cells were cotransfected with a mixture containing NR1,
NR2, and GFP plasmids (0.3, 0.9, and 0.8 µg per 35 mm diameter dish,
respectively). After transfection, 100 µM
D-AP5 was added to the culture medium.
Expression of NMDA receptors in Xenopus oocytes.
Preparation of oocytes and nuclear injection of cDNAs coding for
wild-type and mutant NMDA receptors were performed as described by
Paoletti et al. (1995).
Chemicals
NMDA and D-2-amino-5-phosphopentanoic acid
(D-AP5) were obtained from Tocris Cookson (Bristol, UK).
Glycine, L-glutamate, zinc chloride, HEPES,
N-tris(hydroxymethyl)methylglycine (tricine), diethylenetriaminepentaacetic acid (DTPA), EDTA,
N,N,N ,N-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN),
N-(2-acetamido)iminodiacetic acid (ADA), and
dithioerythritol (DTE) were obtained from Sigma (Saint Quentin
Fallavier, France). All other salts were obtained from Prolabo (Paris,
France) and were of "Normapur" grade. Zinc chloride stock solutions
(10 µM-100 mM) were prepared by progressive
dilutions with bidistilled water of a solution containing 100 mM ZnCl2 and 10 mM HCl. The stock solution of TPEN (50 mM) was prepared in ~100
mM HCl. Other stock solutions (EDTA, 250 mM;
DTE, 300 mM; tricine, 1 M; DTPA, 300 mM; ADA, 500 mM) were prepared in bidistilled
water, and their pH was adjusted to 7.4 with NaOH.
Buffered Zn2+ solutions
The marked effects of heavy metal chelators on the response of
NR1a-NR2A NMDA receptors (see Fig. 1, Table 1) and the apparent high
affinity of these receptors for Zn2+ (see Fig.
3B) required the use of buffered Zn2+
solutions to establish Zn2+ inhibition curves. The
heavy metal chelators tested in this study have a very high affinity
for Zn2+ (KD < 10 10 M; see Table 1), which precludes
their use to buffer Zn2+ in the 1-100
nM range. In this respect, tricine, with a
KD of 107.3 M
(Dawson et al., 1986), seemed more promising. We used MaxChel, a
program for buffer calculations [which takes into account the pH and
the ionic strength of the solutions (Bers et al., 1994)] with the
published binding constants of tricine, to prepare buffered solutions
for Zn2+ in the 10-100 nM range. We
expected that the responses recorded with NR1a-NR2A receptors with
such solutions would be smaller than the responses recorded in
Zn2+-free solutions but larger than the responses
recorded in nonbuffered solutions to which 100 nM
Zn2+ had been added. The actual responses were much
smaller than predicted, which suggested that we had used incorrect
binding constants for tricine. To estimate more accurate values for
these constants, we took advantage of the fact that at concentrations
of a few micromolars Zn2+ produces a
voltage-dependent inhibition. From the amount of
Zn2+ voltage-dependent block we estimated the free
Zn2+ concentration in our buffered solutions and
hence calculated an empirical KD of tricine for
Zn2+ in our experimental conditions of
105 M instead of
107.3 M. The buffered
Zn2+ solutions used to obtain the
Zn2+ dose-response curves shown in Figures
2A and 3A were prepared according to this
empirically established binding constant by adding to 10 mM
tricine the following concentrations of Zn2+ (in
µM): 0.26, 0.78, 2.6, 7.8, 26, 77.5, and 254. The
corresponding estimated concentrations of free Zn2+
were (in nM): 1, 3, 10, 30, 100, 300, and 1000, respectively. Note that in the calculations for the buffered solutions
the weak Zn2+ chelation properties of glycine and
glutamate (Dawson et al., 1986) have been neglected.
Fig. 1.
NR1a-NR2A responses are potentiated by the heavy
metal chelator TPEN. Recombinant NMDA receptors expressed in HEK 293 cells (A, B) or in Xenopus
oocytes (C, D) were activated by applying saturating concentrations of glutamate (100 µM) and
glycine (100 µM). The responses were compared before
(control) and after (TPEN) addition of 1 µM TPEN to the external solution.
A, B, HEK cells. Glutamate was applied on
a background of glycine for 2 sec every 10 sec on cells held at  50
mV. The external Ca2+ concentration was 1 mM. A, TPEN potentiates NR1a-NR2A
responses. Each trace is the average of five records.
B, NR1a-NR2B responses are not affected by 1 µM TPEN. Each trace is the average of
fifteen records. C, D,
Xenopus oocytes. Voltage ramps from 70 to +50 mV were
applied in the absence (control) or presence of 1 µM TPEN. Current-voltage curves
corresponding to the leak currents were substracted from those obtained
during steady applications of 100 µM glutamate and 100 µM glycine. The only external divalent cation was
Ba2+ (0.3 mM). C, TPEN
potentiates NR1a-NR2A responses over the whole voltage range.
D, NR1a-NR2B currents recorded in the absence or in the
presence of TPEN are superimposed.
[View Larger Version of this Image (17K GIF file)]
Fig. 3.
Concentration dependence of the
Zn2+ inhibition of NR1a-NR2A and NR1a-NR2B
receptors. Recombinant NMDA receptors were expressed in
Xenopus oocytes, and dose-response curves were
constructed from I-V curves obtained as in Figure 2. In
each experiment currents were expressed as a fraction of the current
recorded in the presence of a Zn2+ chelator
("0" Zn2+). The
curves in A and B
represent least-squares fits to the data points with the
two-binding-site isotherm y = 1 ((a/(1 + IC50(1)/[Zn2+])) + (b/(1 + IC50(2)/[Zn2+]))), in which
y is the relative current, and a and
b are the respective weights of each isotherm. The
curves in C and D
represent least-squares fits to the data points with the
single-binding-site isotherm y = 1 (a/(1 + (IC50/[Zn2+])n)),
in which y is the relative current, n is
the Hill coefficient, and a is a weight factor.
A, B, Dual antagonism by
Zn2+ of NR1a-NR2A responses recorded at negative
potentials (60 mV). A, Zn2+
concentrations correspond to free Zn2+
concentrations in solutions buffered with tricine (10 mM;
see Fig. 2 and Materials and Methods). Data are from seven cells, with
each point being the mean of three to four values. The
estimated IC50(1), IC50(2),
a, and b are 17 nM, 26 µM, 0.75, and 0.25, respectively. B,
Zn2+ concentrations correspond to added
Zn2+ concentrations corrected for an assumed 10 nM contaminating Zn2+. TPEN (1 µM; n = 13) or DTPA (2 µM; n = 4) were used for reference ("0" Zn2+ concentration). Data
are from 17 cells, each point being the value obtained
from 1 cell (30 and 300 nM added Zn2+)
or the mean value obtained from 2 cells (5 and 10 nM
and 3 µM), 3 cells (30 and 300 µM), 4 cells
(1 and 100 nM), 9 cells (10 and 100 µM), or
17 cells (1 µM). The estimated
IC50(1), IC50(2),
a, and b are 6 nM, 32 µM, 0.78, and 0.22, respectively. C, A
single-binding-site isotherm is sufficient to describe the
Zn2+ inhibition of NR1a-NR2B responses recorded at
negative potentials (60 mV). Zn2+ concentrations
correspond to added Zn2+ concentrations with no
correction for Zn2+ contamination. TPEN (1 µM) was used for the "0"
Zn2+ solution. Data are from five cells, with each
point being the mean of three to five values. The value of
a was fixed to 1. The estimated IC50 and
n are 490 nM and 0.78, respectively.
D, At positive potentials the low-affinity
Zn2+ inhibition of NR1a-NR2A responses is absent.
Zn2+ concentrations were corrected by assuming 10 nM contaminating Zn2+. TPEN (1 µM; n = 13) or DTPA (2 µM; n = 4) were used for the "0" Zn2+ concentration. Data are
from 17 cells, each point being the value for 1 cell (5, 30, and 300 nM added Zn2+) or the mean value for 2 cells (10 nM and 3 and 30 µM), 3 cells (1 nM and 300 µM), 4 cells (100 nM),
8 cells (10 and 100 µM), or 17 cells (1 µM). The value of n was fixed to 1. The
estimated values of the IC50 and of a are 5 nM and 0.79, respectively.
[View Larger Version of this Image (23K GIF file)]
Fig. 2.
Nanomolar external Zn2+
concentrations selectively inhibit NR1a-NR2A responses.
Leak-substracted NMDA currents were recorded at different
concentrations of external Zn2+ during voltage ramps
from 70 to +50 mV applied in Xenopus oocytes expressing NR1a-NR2A or NR1a-NR2B receptors. Glutamate and glycine were applied at saturating concentrations (100 µM each).
The "0" Zn2+ concentration refers
to a solution containing a Zn2+ chelator with no
added Zn2+ (see Results). A,
NR1a-NR2A responses are inhibited by Zn2+
concentrations of a few nanomolars. The inhibition is
voltage-independent. At higher Zn2+ concentrations
the inhibition saturates at ~75% of the response in the
"0" solution. The indicated Zn2+
concentrations correspond to calculated free Zn2+
concentrations in solutions buffered with 10 mM tricine
(see Materials and Methods). The "0" solution
contained 10 mM tricine and no added
Zn2+. B, An additional
voltage-dependent inhibition is produced by micromolar concentrations
of Zn2+. The responses are from a different oocyte.
The indicated Zn2+ concentrations correspond to
nominal values. The "0" Zn2+
solution contained 1 µM TPEN. C,
Zn2+ antagonism of NR1a-NR2B responses is of lower
affinity, is total, and is mainly voltage-independent. The indicated
Zn2+ concentrations correspond to nominal values.
The "0" Zn2+ solution contained 1 µM TPEN. D, Expanding the current scale at
a high Zn2+ concentration (30 µM)
reveals a voltage-dependent component of the inhibition of NR1a-NR2B
responses. Shown is the same cell as in C. For clarity,
the data were fit with a third-order polynomial.
[View Larger Version of this Image (22K GIF file)]
Recording conditions
Neurons and HEK cells. Experiments on native neuronal
receptors were performed on nucleated patches (see Sather et al.,
1992). GFP-positive HEK cells were used for electrophysiological
recordings 16-48 hr after transfection in the whole-cell patch-clamp
configuration (Hamill et al., 1981). In all recordings HEK cells were
lifted off the chamber floor to permit rapid solution changes. Soft
glass patch pipettes of 3-5 M were filled with a solution
containing (in mM): 120 CsF, 10 CsCl, 10 EGTA, and 10 HEPES, pH-adjusted to 7.2 with CsOH.
The standard external solution (control solution) contained (in
mM): 140 NaCl, 2.8 KCl, 1 CaCl2, and 10 HEPES, pH-adjusted to 7.3 with NaOH. Drugs and agonists were applied to
the patch by means of an eight-barrel fast-perfusion system (Sather et
al., 1992). Solutions flowed continuously by gravity from all barrels. In all whole-cell and nucleated-patch experiments, glutamate (HEK cells) or NMDA (neurons) was applied on a continuous background of
glycine. NMDA was used at 200 µM. Glutamate and glycine
were each present at a saturating concentration (100 µM).
Xenopus oocytes. Two-electrode voltage-clamp recordings
were made 1-7 d after cDNA injection, using low resistance (0.5-1.5 M) electrodes filled with 3 M KCl. The standard solution
superfusing the oocytes contained (in mM): 100 NaCl, 2.8 KCl, 5 HEPES, and 0.3 BaCl2, pH-adjusted to 7.3 with
NaOH. The low external Ba2+ concentration was used
to minimize Ba2+ entry via NMDA channels and the
subsequent activation of Ca2+-dependent conductances
(Leonard and Kelso, 1990). The volume of the bath in the recording
chamber was ~100 µl. The rate of perfusion (~4 ml/min) allowed a
complete exchange of the solutions in 2-5 sec. Glycine (100 µM) and L-glutamate (100 µM)
were applied simultaneously, usually for 10 sec every 2 min, using
motor-driven valves. When the Zn2+ concentration was
varied, currents were recorded after 30-60 sec of preincubation at the
test Zn2+ concentration. The response at each test
Zn2+ concentration usually was compared with the
control responses (no added Zn2+ or "0"
Zn2+; see Results) before and after the change in
Zn2+ concentration.
All experiments were performed at room temperature (18-25°C).
Recording and data analysis
In neurons and HEK cells currents were recorded with a List
EPC-7 amplifier (Darmstadt, Germany) and a Racal FM tape recorder. The
voltage-clamp current usually was filtered (8-pole Bessel) with a
corner frequency of 250 Hz, sampled at twice this frequency, and later
analyzed by Strathclyde Electrophysiology Software (gift from John
Dempster, Strathclyde University, Glasgow, Scotland). For whole-cell
recordings the series resistance (3-8 M) was partially compensated
(50-90%) and monitored throughout the experiment.
Currents from oocytes were recorded with a Warner Instrument OC-725
amplifier (Hamden, CT). After being filtered at 500 Hz, traces were
acquired at 250 Hz with pClamp V6.0 (Axon Instruments, Foster City,
CA). Current-voltage curves were obtained with slow voltage ramps (2 sec duration) from 100 or 70 to +50 mV (the capacitive and leakage
currents were recorded before agonist application and substracted from
the glutamate-induced current traces).
For Zn2+ dose-response curves the response at each
tested Zn2+ concentration was bracketed by two
responses in a Zn2+-free solution (in the presence
of a Zn2+ chelator, usually 1 µM TPEN)
and then compared with the mean of these two
Zn2+-free responses. In the analysis of the
Zn2+ voltage-dependent block the "unblocked
fraction" curves (Fig. 5B) were obtained by dividing the
current-voltage curve obtained at each tested Zn2+
concentration by the current-voltage curve obtained in a
Zn2+-free solution (in these experiments TPEN, which
induces a moderate voltage-dependent block of the glutamate response at
potentials below 80 mV, was replaced by another
Zn2+ chelator, DTPA, which showed no detectable
voltage-dependent inhibitory effects in the range from 150 to +100
mV). We assumed that the Zn2+ voltage-dependent
block was relieved completely at +50 mV and therefore normalized all
unblocked fraction curves to 1 at +50 mV. The unblocked fraction values
at 0 mV could not be evaluated directly from the data (close to the
reversal potential, the ratios of the current traces take artifactual
infinite values). They were calculated by using polynomial fits to the
data collected outside the 10 to +10 mV range.
Fig. 5.
Low-affinity voltage-dependent
Zn2+ block of NMDA NR1a-NR2A responses.
A, Voltage ramps from 100 to +50 mV were applied in Xenopus oocytes expressing NR1a-NR2A receptors in the
absence of Zn2+ ("0"
Zn2+ solution containing 2 µM DTPA) or
after the addition of 1, 10, 30, 100, or 300 µM added
Zn2+. For clarity, leak-substracted
I-V curves are shown on an expanded current scale,
because the voltage-dependent block by Zn2+ appears
in a concentration range in which ~80% of the maximal response
already is eliminated by the high-affinity voltage-independent Zn2+ inhibition (see Figs. 2, 3). The
Zn2+ block increases with increasing
Zn2+ concentrations and with hyperpolarization.
However, even at the highest concentration of Zn2+
tested (300 µM) and at the most negative potentials, an
inward current can still be recorded. B, Voltage
dependence of the low-affinity Zn2+ inhibition.
Shown is the same cell as in A. The unblocked fraction was calculated by dividing, at each concentration of
Zn2+, the NMDA current by the NMDA current recorded
in "0" Zn2+ and by subsequently
normalizing to 1 for a membrane potential of +50 mV. Data points that
take artifactual values around the reversal potential have been
omitted. C, Concentration dependence of the
voltage-dependent block by Zn2+ at 100, 80,
60, 40, 20, 0, +20, and +40 mV. Data points were calculated from
curves similar to those shown in B, obtained in a series
of seven experiments. Each point corresponds to the mean
value obtained from three to seven measurements. The data points at 0 mV were obtained by interpolation with a polynomial fit (see Materials
and Methods). The lines drawn through the data points
are least-squares fits of the single binding isotherm: y = ymax · (1 (1/(1
+(IC50/[Zn2+])n))),
in which y is the relative voltage-dependent
Zn2+ inhibition and n is the Hill
coefficient. The weight factor ymax was
introduced to eliminate the residual voltage dependence seen at 1 µM Zn2+ (see Results). The estimated
IC50 and n are, respectively, 22 µM and 0.9 at 100 mV, 31 µM and 0.9 at
80 mV, 41 µM and 0.9 at 60 mV, 100 µM
and 0.9 at 40 mV, 346 µM and 0.9 at 20 mV, and 1162 µM and 0.8 at 0 mV. D, Voltage dependence
of the low-affinity Zn2+ inhibition. The
IC50 values are those estimated by the fits shown in
C. Note that, on this semi-log plot, a linear relation
does not fit the data. The slope of the relation between the
IC50 and the voltage increases with depolarization and
reaches a maximum between 40 and 0 mV. In this range the slope
(e-fold for ~16 mV, dotted line) is
consistent with an apparent electrical depth of the
Zn2+ binding site of 0.77. The dashed
line is drawn according to the equation used by Christine and
Choi (1990) for the fit of their single-channel data obtained in the
range between 70 and 20 mV (an IC50 at 0 mV of 909 µM and an electrical depth of 0.51).
[View Larger Version of this Image (22K GIF file)]
Unless otherwise specified, error bars represent SD.
RESULTS
Heavy metal chelators potentiate NMDA NR1a-NR2A
receptor activity
Recombinant NMDA receptors were expressed both in HEK cells and
Xenopus oocytes. Responses were elicited by applying
saturating concentrations of both glycine (100 µM) and
glutamate (100 µM). As shown in Figure
1A, NR1a-NR2A whole-cell currents
recorded at 50 mV in HEK cells were enhanced markedly (~threefold
increase in the peak current) by the addition of a low concentration (1 µM) of TPEN, a highly specific heavy metal chelating
agent (see Table 1; Arslan et al., 1985). This
potentiation was rapid and fully reversible. When glutamate pulses were
applied at 10 sec intervals, the potentiation was already maximal for
the response to the first pulse applied after the addition of TPEN.
Similarly, a complete recovery was observed for the response to the
first pulse applied in the TPEN-free solution (data not shown). Unlike NR1a-NR2A responses, NR1a-NR2B responses recorded in the same conditions were not affected by TPEN (Fig. 1B).
Similar results were obtained for NMDA receptors expressed in
Xenopus oocytes. Figure 1, C and D,
shows leak-substracted NMDA currents recorded during voltage ramps
applied in oocytes expressing either NR1a-NR2A receptors (Fig.
1C) or NR1a-NR2B receptors (Fig. 1D).
Whereas TPEN (1 µM) had no effect on the NR1a-NR2B
current, it induced an approximately twofold potentiation of the
NR1a-NR2A current. Moreover, as illustrated in Figure 1C,
the potentiation of NR1a-NR2A currents produced by the addition of
TPEN was voltage-independent.
The ratio of the peak current recorded at 50 mV in the presence of
TPEN over the peak current recorded in the absence of TPEN was close to
1 for NR1a-NR2B receptors expressed in HEK cells (1.07 ± 0.04;
n = 6) or oocytes (1.03 ± 0.02; n = 10), whereas for NR1a-NR2A receptors the mean ratio was 2.9 ± 0.3 (n = 6) in HEK cells and slightly lower in oocytes
(2.05 ± 0.6; n = 17).
These results suggested that TPEN removes from the external medium a
metal that tonically inhibits NR1a-NR2A, but not NR1a-NR2B, receptors. Because the effect is seen with a concentration of chelator as low as 1 µM, one can exclude effects
involving the divalent cations Ca2+ or
Ba2+ present in the extracellular solutions at
millimolar concentrations, and one might suspect the involvement of a
heavy metal such as Zn2+ or
Cu2+.
In both expression systems the magnitude of the potentiation of the
NR1a-NR2A responses was quite variable, ranging in oocytes from 1.5 to
3.7 and from 2.5 to 3.3 in HEK cells (for HEK cells, see Fig.
7A). A likely explanation of this variability is that the
putative heavy metal removed by TPEN is present in the external solutions at concentrations varying from one experiment to the other.
Fig. 7.
Most native NMDA receptors are potentiated by
TPEN. A, Comparison of the effects of TPEN (1 µM) on the peak amplitude of NMDA currents recorded in
neurons and in HEK 293 cells expressing either NR1a-NR2A or NR1a-NR2B
receptor subtypes. Native NMDA responses were elicited by a 2 sec pulse
of NMDA (200 µM) on a background of glycine (10 µM). Recombinant NMDA responses were recorded with protocols identical to those shown in Figure 1. The holding potential was 50 mV. Each circle corresponds to the peak ratio
(TPEN/control) obtained from one experiment. The
filled and hatched circles correspond to
the two separate experiments, which are illustrated in
B. The potentiation of native NMDA responses by TPEN was
highly variable, with peak ratios ranging from 1.0 (no potentiation) to
a maximum of 1.6. The mean value was 1.25 ± 0.17 (n = 12). The mean peak ratios for NR1a-NR2A and
NR1a-NR2B receptors were 2.9 ± 0.3 (n = 6)
and 1.07 ± 0.04 (n = 6), respectively.
B, Variability of the effect of TPEN on neuronal NMDA
responses. Shown are superimposed traces recorded in the control
solution and in the presence of 1 µM TPEN. Each
trace is the average of three individual responses. The
top panel shows an example with a marked potentiation by
TPEN (the peak ratio TPEN/control of 1.41 corresponds to the
filled circle in A), whereas the
bottom panel shows an example in which TPEN was
ineffective (the peak ratio of 1.0 corresponds to the hatched
circle in A).
[View Larger Version of this Image (14K GIF file)]
TPEN was chosen initially for its powerful heavy metal chelating
properties and its poor Ca2+ affinity (Arslan et
al., 1985). However, it has been shown that TPEN permeates readily
biological membranes (Arslan et al., 1985), and the possibility had to
be considered that external TPEN enters the cell and potentiates
NR1a-NR2A responses via an intracellular mechanism. To evaluate this
hypothesis, we tested the effects of three membrane-impermeant heavy
metal chelators: EDTA, DTPA, and ADA (see Table 1; Dawson et al.,
1986). The experiments were performed in oocytes in the presence of 0.3 mM Ba2+. As shown in Table 1, each of
the three compounds potentiated NR1a-NR2A responses recorded at
negative potentials. Moreover, the potentiations produced by each
chelator were very similar to the potentiation produced by TPEN. This
result argues against an intracellular mechanism of action of TPEN and
suggests that the four compounds act by complexing a contaminant heavy
metal present in the external solution at a concentration high enough to inhibit NR1a-NR2A responses. Candidates for trace impurity are
numerous, including Zn2+, Cd2+,
Mn2+, Cu2+,
Fe2+, and Al3+.
Zn2+ inhibits NR1a-NR2A responses in the
nanomolar range
The effects of various heavy metals on NMDA receptors have been
studied in many neuronal preparations (Mayer et al., 1989; Eimerl and
Schramm, 1993; Trombley and Shepherd, 1996; Vlachova et al., 1996).
These studies have shown that Zn2+,
Cd2+, Fe2+, and
Cu2+ are NMDA antagonists, among which
Zn2+ is the most potent, inhibiting native NMDA
receptor activity at concentrations of a few micromolars. However, in
all of these experiments the possible presence in the external
solutions of endogenous Zn2+ (and of other heavy
metals) was not taken into account, and the solutions with no added
Zn2+ were assumed to be
Zn2+-free. The observation that heavy metal
chelating agents potentiate NMDA NR1a-NR2A responses, by suggesting
that Zn2+ could be present as an endogenous metal in
the external solutions at a submicromolar concentration (necessarily
lower than that of the chelator producing a large potentiation),
imposed a reevaluation of the inhibitory constants deduced from early
work.
To evaluate the potency of Zn2+ as a high-affinity
NMDA receptor antagonist, we used three different protocols. The first
two involved the construction in oocytes of dose-response curves
either with or without buffered Zn2+ solutions. The
third approach was based on the measurement in HEK cells of the current
relaxations after step changes in Zn2+
concentrations.
To use buffered Zn2+ solutions, we looked for a
chelating agent capable of producing buffered free
Zn2+ concentrations in the nanomolar to micromolar
range. For this purpose the binding constants of TPEN, EDTA, DTPA, and
ADA with Zn2+ were much too high (absolute
KD  109.7 M;
Table 1), so we selected tricine, which binds Zn2+
with a low affinity (we estimated the absolute
KD as 105 M;
see Materials and Methods), but has an even lower affinity for
Ca2+ or Ba2+ (absolute
KD ~100 mM) present in the
external solutions at millimolar concentrations. We first compared, on
NR1a-NR2A receptors expressed in oocytes, the effect of tricine with
that of the other chelating agents used previously. Tricine (10 mM) potentiated NR1a-NR2A responses by the same amount as
TPEN, EDTA, DTPA, and ADA (see Table 1). In what follows, "0"
Zn2+ solution refers to a solution with no added
Zn2+ but containing one of the five chelating
agents.
We then tested the effects of various Zn2+
concentrations in tricine-buffered solutions on NR1a-NR2A receptors
and found that they revealed two inhibitory effects differing markedly
in their IC50 values and voltage dependence. The first is
illustrated in Figure 2A, which shows
the inhibition produced by Zn2+ at concentrations
from 3 nM to 1 µM. The reduction of the
conductance is very similar over the whole potential range tested. An
estimated concentration of 3 nM free
Zn2+ is sufficient to produce a clear inhibition;
the response is reduced by more than one-half at an estimated
concentration of 30 nM free Zn2+ and by
nearly 80% at 1 µM Zn2+. Figure
2B shows that for concentrations of
Zn2+ above 1 µM (at these
concentrations there is no noticeable difference between buffered and
nonbuffered solutions) the pattern of inhibition changes. There is
nearly no additional inhibition in the positive potential range,
suggesting that the voltage-independent inhibition has reached its
maximum; on the other hand, in the negative potential range the
inhibition continues to increase with the Zn2+
concentration and the more so at more negative potentials.
These results indicate that Zn2+ exerts a dual block
on NMDA NR1a-NR2A receptors: one is voltage-independent and seen with
nanomolar concentrations of Zn2+; the other is
voltage-dependent and requires much higher Zn2+
concentrations (in the micromolar range). At 60 mV the dose-response curve obtained with tricine-buffered solutions was well fit with a
two-binding-site isotherm, using IC50 values of 17 nM and 26 µM having relative weights of 75 and 25%, respectively (Fig. 3A). Qualitatively, these results are reminiscent of the actions of Zn2+ described on native NMDA receptors (Christine
and Choi, 1990; Legendre and Westbrook, 1990), but quantitatively the
affinity of the site involved in the voltage-independent process
appears to be three orders of magnitude higher than reported on native NMDA receptors.
Dose-response curves obtained by applying Zn2+ in
nonbuffered solutions (nominal Zn2+ concentrations)
resembled those obtained with buffered solutions and showed two well
separated regions of inhibition at negative potentials. However, as
expected from the presence of contaminating traces of
Zn2+ (or another compound) in the control solutions,
the points obtained at low Zn2+ concentrations were
poorly fit by a standard isotherm (data not shown). The fit was
improved greatly by adding to the nominal Zn2+
concentrations an assumed contaminating Zn2+
concentration. As shown in Figure 3B, at 60 mV a
satisfactory fit could be obtained by assuming a contaminating level of
Zn2+ of 10 nM. This correction led to
IC50 values (6 nM and 32 µM) and
to the relative weight of the two effects (78 and 22%), which were
very similar to those found with tricine-buffered solutions (see
above). Moreover, at positive potentials (e.g., at +50 mV), only the
high-affinity Zn2+ inhibition was present. Its
IC50 (5 nM) was identical to that calculated at
60 mV, and it saturated at ~20% of the maximal current (Fig.
3D).
In contrast to NR1a-NR2A responses, NR1a-NR2B responses were not
affected by heavy metal chelators. This suggested that, if Zn2+ inhibits NR1a-NR2B receptors, the inhibition
must be of lower affinity than that of NR1a-NR2A receptors. Indeed, as
illustrated in Figure 2B, NR1a-NR2B responses
recorded in oocytes were sensitive to Zn2+
concentrations (nonbuffered solutions) in the hundreds of nanomolars range and could be inhibited fully, at all potentials, by
Zn2+ concentrations of a few tens of micromolars.
Interestingly, in this latter concentration range careful observation
of the (already highly inhibited) currents revealed a voltage-dependent
component of the inhibition (Fig. 2D). This
voltage-dependent inhibition was similar to that observed for
NR1a-NR2A receptors (see Fig. 5A). Figure 3C
shows the dose-response curve obtained at 60 mV from five cells
recorded in the same conditions as those used in Figure
2B. In contrast to the case of NR1a-NR2A receptors, fitting the data obtained on NR1a-NR2B receptors did not require correction of the Zn2+ concentration for
contaminating Zn2+. At 60 mV the inhibition of
NR1a-NR2B responses by Zn2+ could be described by a
single-binding-site isotherm with an IC50 of 490 nM ranging from 0 to 100% of the maximal response. Moreover, the IC50 for Zn2+ inhibition
is in large part voltage-independent. The relative current amplitudes
measured at +50 mV for NR1a-NR2B receptors were fit correctly by a
single isotherm with an IC50 of 530 nM (n = 3; data not shown). Thus, Zn2+
behaves as a full antagonist of NR1a-NR2B receptors acting mainly via
a voltage-independent mechanism.
In summary, Zn2+ exerts a dual block on both NMDA
NR1a-NR2A and NR1a-NR2B receptors: the first, which is
voltage-independent, is seen at lower Zn2+
concentrations than the second, which is voltage-dependent.
Quantitatively, there are two major differences between the
voltage-independent Zn2+ inhibition of NR1a-NR2A
and NR1a-NR2B receptors. The IC50 of the
voltage-independent inhibition is, under the recording conditions used
here, 50-fold lower for NR1a-NR2A receptors than for NR1a-NR2B receptors. Moreover, the voltage-independent inhibition can block only
80% of NR1a-NR2A responses, whereas it can block entirely NR1a-NR2B
responses.
Slow dissociation of Zn2+ from
NR1a-NR2A receptors
Further evidence for a high-affinity voltage-independent
Zn2+ inhibition of NR1a-NR2A responses was obtained
by analyzing the relaxations produced by Zn2+
concentration jumps applied during a NMDA response. These experiments were performed on transfected HEK cells lifted from the bottom of the
dish to allow rapid solution exchange. Tricine (10 mM) was
present both in the "0" Zn2+ solution and in the
test solution. The presence of free tricine at a nearly constant level
throughout the experiment minimized the chance of repetitive binding of
Zn2+ in a putative region of restricted diffusion
that artifactually could slow the relaxation kinetics. The free
Zn2+ concentration was a few tens of nanomolars, a
range in which Zn2+ produces only the high-affinity
voltage-independent inhibition (see Fig. 3A). Representative
traces of concentration jump experiments are shown in Figure
4. The current relaxations observed at the onset and at
the offset of the Zn2+ application were well fit by
single exponentials. In the example illustrated, the on and off time
constants were 390 msec and 1.7 sec, respectively, for a free
Zn2+ concentration of 20 nM (Fig.
4A). The corresponding values were 116 msec and 1.9 sec with 100 nM free Zn2+ (Fig.
4B). Similar values were obtained in a series of four
experiments performed at 50 mV with a free Zn2+
concentration of 20 nM:  on = 370 ± 80 msec; off = 1.7 ± 0.2 sec.
Fig. 4.
Slow dissociation of Zn2+ from
NR1a-NR2A receptors. Current relaxations that follow a
Zn2+ concentration jump were analyzed in HEK 293 cells expressing NR1a-NR2A receptors. Each trace
represents an individual response to a 14.5 sec pulse of glutamate (100 µM) on a background of glycine (100 µM).
Tricine (10 mM) was present throughout the experiment. Zn2+ was applied for 3 sec during the pulse of
glutamate once the response had reached a steady level. The
Zn2+ concentrations as indicated in the figure
correspond to the calculated free Zn2+ concentration
in the tricine-buffered solutions (see Materials and Methods). The
onset and offset of the inhibition by Zn2+ were fit
by single exponentials (superimposed on the current traces) with time
constants on and off, as indicated in the figure. Data were filtered at 100 Hz and sampled at 140 Hz. The holding
potential was 50 mV.
[View Larger Version of this Image (14K GIF file)]
Assuming that there is only one high-affinity voltage-independent
Zn2+ binding site and that Zn2+
interaction with the receptor follows a simple bimolecular reaction (as
suggested by the Hill coefficient, close to 1; see Fig. 3), the onset
time constant, on, should depend on the
Zn2+ concentration [on = 1/([Zn2+]kon + koff), in which kon is
the Zn2+ association rate constant and
koff the Zn2+ dissociation
rate constant]. The offset time constant, off, should be independent of the Zn2+ concentration
(off = 1/koff). These
simple predictions were, indeed, fulfilled. kon
was found to be close from 108
M1 · sec1.
koff was estimated to be 0.6 sec-1. The KD derived from
these values (KD = koff/kon)
was 6 nM, a value in very good agreement with the values
estimated independently from dose-response curves. It is worth noting
that such a result does not depend on the expression system, because
concentration jumps were performed on HEK cells, whereas dose-response
curves were obtained with Xenopus oocytes.
We also performed concentration jump experiments on NR1a-NR2B
receptors. Two series of experiments were performed, one under conditions similar to those used for NR1a-NR2A receptors (1 mM extracellular Ca2+ ions) and the
other in which extracellular Ca2+ ions were replaced
by 0.3 mM Ba2+ to mimic the conditions
used for obtaining Zn2+ dose-response curves in
oocytes. Because of the low affinity of the NR1a-NR2B receptors for
Zn2+ (see Fig. 3C), these experiments
required relatively high Zn2+ concentrations (1-5
µM). As a consequence, the
Zn2+-induced on-relaxations were very fast ( 50 msec), and the evaluation of their time constant was made uncertain
by the relatively slow speed of our whole-cell perfusion system (time
constant, ~20 msec). This limitation did not apply to the evaluation
of the off-relaxations. In the two sets of experiments the
off-relaxations after Zn2+ withdrawal were very
similar [off = 63 ± 17 msec (n = 6) with 1 mM Ca2+ and off = 70 ± 14 msec (n = 3) with 0.3 mM
Ba2+] and much faster than those observed with
NR1a-NR2A receptors (see above). The rate of Zn2+
dissociation calculated from these relaxations was on the order of 15 sec1, i.e., ~25-fold larger than that calculated
for NR1a-NR2A receptors.
Using this dissociation rate value and the amount of steady-state
Zn2+ inhibition measured at the end of the
Zn2+ application [53 ± 7% (n = 4) with 1 mM Ca2+ and 5 µM Zn2+; 49 ± 2% with 0.3 mM Ba2+ and 1 µM
Zn2+], we calculated Zn2+
association rates of 3.2 · 106
M1 · sec1 and 1.4 · 107 M1 · sec1, respectively. The sensitivity of these rates
to the external divalent ions Ca2+ and
Ba2+ suggests that these ions compete with
Zn2+ for occupancy of the voltage-independent
inhibition site in NR1a-NR2B. This would explain why the steady-state
inhibition observed with these receptors in the presence of 1 mM Ca2+ and 5 µM
Zn2+ was much lower than that predicted by the
apparent KD of 0.5 µM measured in
oocytes. Replacing external Ca2+ by
Ba2+ had no significant effect on
Zn2+ inhibition of NR1a-NR2A receptors (data not
shown). The difference in Zn2+ on-rates between
NR1a-NR2A and NR1a-NR2B receptors thus could, at least partly, result
from a lowering of the NR1a-NR2B on-rate by the competition among
Zn2+, Ca2+, and
Ba2+.
Overall, the results of the concentration jump experiments indicate
that the difference of Zn2+ affinity between
NR1a-NR2A and NR1a-NR2B receptors is attributable mainly to a
difference in the dissociation rate of Zn2+.
The low-affinity voltage-dependent
Zn2+ block
As shown in Figures 2 and 3, at negative potentials and at high
enough Zn2+ concentrations (1-100 µM)
a voltage-dependent blocking action of Zn2+ is
superimposed on the voltage-independent one. This voltage-dependent inhibition is seen with both NR1a-NR2A and NR1a-NR2B responses, but
in the second case it is masked by the fact that it overlaps with the
voltage-independent block (see Fig. 2C,D). Consequently, the
voltage-dependent Zn2+ inhibition was characterized
more easily on NR1a-NR2A receptors.
Figure 5A shows representative
current-voltage relations of NR1a-NR2A currents recorded in
Xenopus oocytes at different Zn2+
concentrations in the micromolar range (1, 10, 30, 100, and 300 µM from 100 to +50 mV). The traces are shown on an
expanded current scale to restrict the display to the component of the
maximal response that remains after addition of 1 µM
Zn2+ has saturated the site responsible for the
high-affinity voltage-independent inhibition. Increasing the
concentration of Zn2+ above 1 µM
produced little change in the outward currents but markedly reduced the
inward currents. The fraction of the channels that were not blocked by
the voltage-dependent process (the unblocked fraction) was evaluated as
the ratio (normalized to 1 at +50 mV) of the glutamate-induced current
measured during a voltage ramp in the presence of
Zn2+ over the current in "0"
Zn2+ solution. Figure 5B shows that the
block increased as the potential was made more negative. The voltage
dependence of the Zn2+ block differs from that seen
in the same system with Mg2+ (see Kuner and
Schoepfer, 1996): in the presence of Zn2+, below
60 mV the inward current becomes nearly independent of the potential,
in contrast to the case of Mg2+, in which at very
negative potentials the current tends to zero, leading to the
"bell-shaped" current-voltage relations described by Nowak et al.
(1984) and by Mayer et al. (1984). Even at the highest
Zn2+ concentration tested (300 µM) and
at the most negative potentials applied (100 mV), the response is not
blocked fully. The absence of a parallel shift between the different
unblocked fraction curves obtained with increasing
Zn2+ concentrations further suggests that the
voltage-dependent Zn2+ inhibition is different from
the Mg2+ block of NMDA responses, possibly because
Zn2+ ions can permeate (escape) more readily through
the channel (see Discussion).
The affinity for Zn2+ of the site accounting for the
voltage-dependent block of the NR1a-NR2A receptor was determined at
different membrane potentials, using values obtained from normalized
unblocked fraction plots such as the one shown in Figure 5B
(see Materials and Methods). The block by Zn2+ was
well fit by a single-binding-site isotherm over the range from 100 to
0 mV (Fig. 5C), assuming that the slight voltage dependence
observed at 1 µM Zn2+ is attributable
to an unrelated effect (e.g., binding to surface charges or to a
superficial divalent binding site in the external vestibule of the
pore; see Ascher and Nowak, 1988; Premkumar and Auerbach, 1996). The
calculated apparent KD values were 22 µM at 100 mV, 31 µM at 80 mV, 41 µM at 60 mV, 100 µM at 40 mV, 346 µM at 20 mV, and 1162 µM at 0 mV (Fig.
5D). The last value (at 0 mV) is five orders of magnitude
higher than that of the voltage-independent antagonism (~10
nM; see Fig. 3). The fact that the apparent
KD is not a simple exponential function of the membrane potential is expected from a model in which the blocking ion
is permeant (see Discussion).
A pore mutation in NR2A selectively suppresses the
voltage-dependent Zn2+ inhibition
Our data are in agreement with the idea that
Zn2+ acts at two different sites on NR1a-NR2A
receptors: one extracellular, outside the membrane field and accounting
for the high-affinity voltage-independent inhibition, and the other
inside the channel, accounting for the voltage-dependent block
(Christine and Choi, 1990). To try to dissociate the two sites, we used
mutated NMDA receptors and took advantage of the fact that the
voltage-dependent Zn2+ inhibition presents some
similarities with the Mg2+ block of NMDA channels
and that molecular determinants involved in Mg2+
block have been identified. The strongest effects on
Mg2+ block were obtained by mutating the residue
occupying the Q/R/N site of the M2 region, the putative channel-forming
segment of the protein. In particular, Burnashev et al. (1992),
Sakurada et al. (1993), and Kawajiri and Dingledine (1993) showed that the substitution of asparagine N598 of the NR1 subunit by an arginine results in a total suppression of the external Mg2+
block. We found that a similar effect could be obtained by replacing asparagine N595 of the NR2A subunit by a lysine [NR2A(N595K)] (unpublished results). Consequently, we expressed NR1a-NR2A(N595K) receptors in Xenopus oocytes and compared their sensitivity
to Zn2+ and to heavy metal chelators with that of
wild-type NR1a-NR2A receptors.
As shown in Figure 6A, at 60 mV the
strong inhibition produced by 1 µM
Zn2+ was comparable for the mutant and wild-type
receptors. The ratio of the current recorded in 1 µM
added Zn2+ over the current recorded in "0"
Zn2+ was 0.19 ± 0.02 (n = 5)
for the mutant receptors and 0.22 ± 0.04 (n = 17)
for the wild-type receptors (see Fig. 3B). In contrast, the
additional voltage-dependent inhibition of NR1a-NR2A responses produced by 100 µM Zn2+ was not
observed with NR1a-NR2A(N595K) responses (n = 7).
Fig. 6.
A pore mutation selectively eliminates the
low-affinity voltage-dependent Zn2+ inhibition.
A, Comparison of the inhibitory effects of
Zn2+ on NMDA responses recorded at 60 mV in
oocytes expressing wild-type NR1a-NR2A receptors or mutant
NR1a-NR2A(N595K) receptors. Shown are superimposed individual
responses to a 20 sec pulse of glutamate and glycine (100 µM each) recorded in the presence of a
Zn2+ chelator ("0"
Zn2+ concentration; 1 µM TPEN for
wild-type receptors and 2 µM DTPA for mutant receptors)
or in the presence of 1 or 100 µM added Zn2+. Both types of receptors are strongly inhibited
by 1 µM Zn2+, but the presence of 100 µM Zn2+ fails to produce an additional
inhibition on the mutant receptors. B, Leak-substracted
I-V curves from Xenopus oocytes
expressing the mutant NR1a-NR2A(N595K) receptors in the absence
("0" Zn2+; 2 µM
DTPA) or in the presence of added Zn2+ (1 and 100 µM). The traces obtained in 1 and 100 µM Zn2+ are superimposed almost
perfectly over the whole voltage range, indicating that the
voltage-dependent inhibition by Zn2+ has been
suppressed by the mutation.
[View Larger Version of this Image (15K GIF file)]
Voltage ramps applied to oocytes expressing NR1a-NR2A(N595K) receptors
confirmed that the currents recorded in the presence of 1 and 100 µM Zn2+ were identical over the whole
voltage range tested (from 100 to +50 mV), indicating the
disappearance of the voltage-dependent block. The voltage-independent
inhibition was not affected by the mutation and, as in the wild-type
receptors, affected only ~80% of the maximal response at saturating
Zn2+ concentrations (Fig. 6B,
reproduced in seven cells). Moreover, we found that, like NR1a-NR2A
responses, NR1a-NR2A(N595K) responses were potentiated by the addition
of 1 µM TPEN (current ratio TPEN/control = 2.1 ± 0.1; n = 2).
Heavy metal chelators potentiate native NMDA responses
The effect of TPEN was investigated on native NMDA
receptors in nucleated patches excised from embryonic mouse cortical
and diencephalic neurons (Sather et al., 1992). The experimental
conditions were identical to those used for the HEK cells, except that
glutamate was replaced by NMDA to activate specifically the NMDA type
of glutamate receptors. Most neuronal NMDA responses were potentiated by the presence of 1 µM TPEN (11 cells of 12), but the
potentiating factor was highly variable from one neuron to the other,
ranging from 1.1-fold to a maximum of 1.6-fold (Fig.
7A; mean potentiation 1.25 ± 0.17;
n = 12). Figure 7B illustrates two examples
representative of the range of the effect of TPEN on neuronal NMDA
currents: one with a clear potentiation (top panel, peak
current ratio TPEN/control = 1.4) and the other with virtually no
potentiation (bottom panel, peak current ratio
TPEN/control = 1.0).
The potentiations observed in neuronal receptors were never larger than
twofold, whereas a factor of three was common with NR1a-NR2A
receptors. This could indicate that, in our cultures, there are none or
very few neurons endowed exclusively with NR1a-NR2A receptors,
consistent with some previous findings (see Discussion and Paoletti et
al., 1995). On the other hand, the results show that the large majority
of neurons does express NMDA receptors that are potentiated by the
addition of micromolar concentrations of TPEN and therefore are
inhibited substantially by a contaminant heavy metal present at a
submicromolar concentration. This suggests that most of the tested
neurons contained a fraction of NMDA receptors highly sensitive to
Zn2+.
The large variability of the magnitude of the potentiation by TPEN of
native receptors probably reflects the subunit composition heterogeneity known to occur in primary cultures of neurons (Williams et al., 1993; Audinat et al., 1994; Paoletti et al., 1995). This suggestion was reinforced by expanding the study of the effects of the
addition of TPEN and by determining the Zn2+ binding
affinities of recombinant NMDA receptors (expressed in Xenopus oocytes) other than NR1a-NR2A and NR1a-NR2B. The
absence of an effect of TPEN on NR1a-NR2B receptors was found also for NR1b-NR2B receptors (current ratio TPEN/control = 0.98 ± 0.02; n = 4). The potentiating effect of TPEN seen on
NR1a-NR2A receptors also was seen with NR1b-NR2A receptors, but it
was smaller (current ratio TPEN/control = 1.21 ± 0.02;
n = 3). As shown in Table 2, the
voltage-independent Zn2+ antagonism has a unique
pharmacological profile on each recombinant receptor tested. The
comparison of these profiles shows that the voltage-independent
Zn2+ inhibition extends over a range of three orders
of magnitude (NR1a-NR2A receptors being the most sensitive and
NR1a-NR2C receptors being the least sensitive) and depends on the
nature of both NR2 and NR1 subunits. NR1b subunits contain exon 5, a
21-amino-acid N-terminal exon that is positively charged and known to
affect the interaction of NMDA receptors with positively charged
molecules and ions (Hollmann et al., 1993; Williams, 1994; Williams et
al., 1994; Zhang et al., 1994; Zheng et al., 1994; Paoletti et al., 1995). It is interesting to note that it also appears to modify the
interaction of recombinant heteromeric NMDA receptors with Zn2+.
Table 2.
Subunit-specific voltage-independent
Zn2+ inhibition
|
1a/2A |
1b/2A |
1a/2B |
1b/2B |
1a/2C |
|
| IC50
(nM) |
~10 |
70 |
490 |
2500 |
14,000
|
|
(see Fig. 3) |
(n = 4) |
(see Fig.
3) |
(n = 5) |
(n = 4) |
|
|
Recombinant NMDA receptors were expressed in Xenopus
oocytes. The IC50 values were determined as described in
Figure 3B for NR1b-NR2A receptors and Figure 3C
for NR1b-N2B and NR1a-NR2C receptors. As for NR1a-NR2A receptors,
the voltage-independent Zn2+ inhibition of NR1b-NR2A
receptors is partial, saturating at ~70% of the maximal current. In
contrast, as for NR1a-NR2B receptors, NR1b-NR2B receptors and
NR1a-NR2C receptors are fully inhibited by Zn2+, mainly
via a voltage-independent mechanism. The external
Ba2+ concentration was 0.3 mM,
and the external pH was 7.3.
|
|
Zn2+ chelation is probably responsible for the
NR2A-specific fast potentiation of NMDA responses by reducing
agents
Experiments on recombinant NMDA receptors expressed in HEK cells
have shown that sulfhydryl reducing agents such as dithiothreitol (DTT)
or reduced glutathione potentiate recombinant NMDA channel activity via
a dual mechanism. All of the receptors tested (heteromers composed of
NR1 subunits and of one type of the NR2 subunit family) show a slowly
developing potentiation (time scale of minutes) that does not reverse
after washout of the reducing agent (Köhr et al., 1994).
NR1-NR2A receptors show an additional marked potentiation ( threefold) with a very rapid onset (maximal from the first pulse in
DTT; see Köhr et al., 1994). This fast potentiation is reversed by the removal of the reducing agent and is associated with a slowing
of desensitization and an acceleration of deactivation of
glutamate-activated currents (Köhr et al., 1994). Both types of
potentiation have been assumed to involve sulfhydryl-containing redox
sites (Köhr et al., 1994). However, the striking similarities (in
magnitude, in kinetics, in subunit specificity) between the NR2A
subunit-specific potentiation by DTT and the NR2A subunit-specific potentiation by heavy metal chelators could be accounted for by the
strong Zn2+ binding properties of DTT (absolute
KD of 1010.3 M
at pH 9.2; Cornell and Crivaro, 1972), suggesting that the subunit-specific action of DTT is attributable to chelation of a
contaminant heavy metal.
Experimental evidence for this hypothesis was obtained by testing the
additivity of the potentiations produced by TPEN and DTE, the
erythroisomer of DTT. In HEK cells expressing NR1a-NR2A receptors,
glutamate-activated currents were recorded in control conditions, in
the presence of TPEN (1 µM) or DTE (3 mM),
and in the presence of both TPEN and DTE. The concentration of DTE was
similar to the concentration of DTT used by Köhr et al. (1994). As shown in Figure 8A, the
potentiation produced by TPEN, by DTE, and by DTE plus TPEN were
similar (current ratios: TPEN/control = 2.8 ± 0.01, n = 2; DTE/control = 2.9 ± 0.1, n = 2; TPEN+DTE/control = 2.9 ± 0.1, n = 2).
All three potentiations were immediate (maximal from the first pulse
applied 10 sec after the change in condition) and fully reversible
(data not shown). Moreover, all three potentiations were associated
with a similar acceleration of the dissociation of glutamate as
measured by the decrease in the time constant of the glutamate
off-relaxation (Fig. 8B). The mean off
value was 139 ± 7 msec (n = 7) in control
conditions, 44 msec ± 12 msec (n = 4) in TPEN,
48 ± 16 msec (n = 2) in DTE, and 41 msec
(n = 1) in DTE plus TPEN. Similar current amplitudes of
the responses recorded at 60 mV in the presence of DTE (3 mM) alone and TPEN (1 µM) plus DTE (3 mM) also were observed in Xenopus oocytes
expressing NR1a-NR2A receptors (n = 6). Moreover, we
observed that DTE (3 mM) fails to induce fast potentiation
effects when applied in the presence of other
Zn2+-chelating agents (100 µM EDTA; 1 mM ADA; 10 mM tricine; 1 µM DTPA)
(data not shown).
Fig. 8.
The NR2A subunit-specific potentiation produced by
reducing agents is probably the consequence of Zn2+
chelation. Shown are glutamate-evoked currents recorded in an HEK 293 cell expressing NR1a-NR2A receptors. The cell was exposed either to a
control solution or to TPEN (1 µM) and DTE (3 mM) applied separately or simultaneously. The holding
potential was 50 mV. A, Each trace is
an individual response to a 2 sec pulse of glutamate (100 µM) applied on a background of glycine (100 µM). The potentiations produced by DTE and
TPEN are not additive. B, Glutamate
off-relaxations are shown on an expanded time scale and after
normalization to the steady-state response amplitude. The decay of the
current was fit by a single exponential function (solid
lines superimposed on the current traces), with a time constant
off indicated in the figure. TPEN and
DTE applied separately or simultaneously induce similar
accelerations of the off response.
[View Larger Version of this Image (12K GIF file)]
Therefore, the fast potentiations of NR1a-NR2A responses by DTE (or
DTT) and by TPEN involve a similar mechanism. We propose that the NR2A
subunit-specific potentiation of NMDA receptor activity by sulfhydryl
reducing agents is caused by chelation of contaminant heavy metal
traces, resulting in the relief of a tonic high-affinity inhibition. In
the light of the data presented in this work, it seems most likely that
Zn2+ is the contaminant metal.
DISCUSSION
Our observations show that the two inhibitory effects of
Zn2+ (voltage-independent and voltage-dependent)
previously described on native NMDA receptors by Christine and Choi
(1990) and by Legendre and Westbrook (1990) also are observed in
recombinant NMDA receptors. However, in one class of recombinant
receptors (NR1a-NR2A or NR1b-NR2A), the voltage-independent
inhibition presents two atypical properties. First, the
IC50 of the inhibition is in the nanomolar range (~10 and
70 nM in NR1a-NR2A and NR1b-NR2A receptors,
respectively), i.e., much lower than what previously had been measured
in native receptors. Second, the inhibition is never complete, and
saturation of the voltage-independent Zn2+ site only
reduces the response by 70-80%. While this work was in progress,
qualitatively similar conclusions were reported by two other groups
(Moshaver and Raymond, 1996; Williams, 1996).
Consequences of the presence of a high-affinity and partial
voltage-independent Zn2+ inhibition in NR1-NR2A
receptors
The effects of heavy metal chelators described in the present
study suggest that contaminating traces of Zn2+ (or
of another metal) may be sufficient to strongly inhibit NR1-NR2A receptors through the high-affinity Zn2+ binding
site. This may account for some of the discrepancies concerning
Zn2+ inhibition of NMDA receptors found in the
literature. For example, Hori et al. (1987) were able to block NMDA
responses with Zn2+ at micromolar concentrations in
slices of rat brain cortex, whereas the same Zn2+
concentrations were ineffective in experiments of Hegstad et al.
(1989). This could mean either that in the second case the NMDA
receptors are insensitive to Zn2+ (as the authors
believed) or, on the contrary, that the receptors are so sensitive to
Zn2+ that the voltage-independent inhibition already
was saturated by contaminating Zn2+ levels. The
partial nature of this inhibition in NR1-NR2A receptors may reinforce
such misleading conclusions: on a background of Zn2+
contamination, an exogenous Zn2+ application will
bring the receptors from an already strongly inhibited state to a
maximally inhibited state in which ~20% of the maximal response
persists. The small effect observed may be recognized falsely as
indicating a very low-affinity Zn2+ inhibition (see
Grimwood et al., 1996). This illustrates the need for a systematic use
of metal chelators in evaluating the "control" responses. The
presence of contaminant Zn2+ under control
conditions in the work of Williams (1996) also may explain the
eightfold difference in affinities for Zn2+ of
NR1a-NR2A receptors reported in that study and this paper.
A second consequence of the high-affinity Zn2+
inhibition is that it leads to a reevaluation of the potentiations
produced by DTT and glutathione on recombinant NMDA receptors
(Köhr et al., 1994). These authors described two potentiations
produced by reducing agents: one slow and irreversible, observed in all
of the subtypes tested, and one fast, reversible, and specific to the
NR1-NR2A combination. The similarity and the mutual occlusion between
this fast potentiation and that produced by metal chelators suggest that the rapid and subunit-specific effects of the reducing agents are
better explained by chelation of Zn2+ (or of another
metal) contaminating extracellular solutions than by their reducing
properties. This reinterpretation further suggests that the "slow"
redox modulation of the NMDA receptor, which is sensitive to the
mutation of two cysteines identified in the NR1 subunit by Sullivan et
al. (1994), is the sole modulation involving redox processes and is not
subunit-specific. After suppression of this modulation by mutation of
the two cysteines, the residual potentiation observed in NR1-NR2A
receptors corresponds to the effects of complexing the contaminant
metal.
Using a chimera approach, Köhr et al. (1994) identified a domain
in the N-terminal region of the NR2A subunit (between residues 250 and
400) responsible for the "fast redox modulation." This domain most
likely contains some of the molecular determinants of the high-affinity
voltage-independent Zn2+ binding site. The point
mutagenesis experiments of Köhr et al. (1994) indicate that the
cysteine residues contained in this domain are not involved in the
subunit-specific potentiation, but the region contains other residues
often involved in protein Zn2+ binding sites, like
histidines, glutamates, or aspart |
