1Kinsmen Laboratory of Neurological Research, Department of Psychiatry, 2Department of Physiology, and 3Department of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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ABSTRACT |
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Chen, Nansheng, Timothy H. Murphy, and Lynn A. Raymond. Competitive Inhibition of NMDA Receptor-Mediated Currents by Extracellular Calcium Chelators. J. Neurophysiol. 84: 693-697, 2000. Calcium chelators have been widely used in electrophysiological recordings of N-methyl-D-aspartate (NMDA) receptor-mediated currents, as well as in studies of excitotoxicity. Intracellularly applied calcium chelators are known to inhibit, at least in part, such calcium-dependent processes as calmodulin-dependent inactivation, calcineurin-dependent desensitization, and rundown of NMDA receptors. On the other hand, the functional consequences and potential nonspecific effects of extracellularly applied chelators have not been extensively investigated. In whole-cell patch-clamp recordings from human embryonic kidney (HEK) 293 cells transiently transfected with recombinant NMDA receptors, we found that addition of calcium chelators such as EGTA shifted the glutamate dose-response curve to the right, from an EC50 for NR1A/NR2A of 8 µM in 1.8 mM Ca2+ to ~24 µM in a solution containing nominal 0 Ca2+/5 mM EGTA and further to ~80 µM in 20 mM EGTA. A similar shift in glutamate dose-response was observed for NR1A/NR2B currents. This dose-response shift was not due to a decrease in extracellular Ca2+ concentration because there was no change in the glutamate EC50 at Ca2+ concentrations ranging from 10 mM to nominal 0/200 µM EGTA. Moreover, addition of 5 mM EGTA fully chelated with 6.8 mM Ca2+ did not produce any shift in the glutamate dose-response curve. We propose that calcium chelators, containing four free carboxyl moieties, competitively inhibit glutamate binding to NMDA receptors.
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INTRODUCTION |
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N-methyl-D-aspartate
(NMDA)-type glutamate receptors play a key role in synaptogenesis,
synaptic plasticity, and some neuropathological conditions, largely due
to the receptor's high permeability to Ca2+
(Dingledine et al. 1999; Lipton and Rosenberg
1994
; McBain and Mayer 1994
). In the CNS, free
Ca2+ levels both inside and outside of cells are
highly regulated. The extracellular Ca2+
concentration under physiological conditions is approximately 2 mM
(Ghosh and Greenberg 1995
) but can drop as low as 0.5 mM during periods of intense neuronal activity (e.g., Heinemann et al. 1977
).
Calcium chelators have been widely used to investigate the role of free
Ca2+ on NMDA receptor function. For example,
buffering intracellular Ca2+ concentration by
perfusing 10 mM EGTA or 10 mM
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) via the recording pipette helped to reduce inactivation (Legendre et al. 1993), indicating inactivation is
Ca2+-dependent, a result further supported by
biochemical analysis and patch-clamp recordings (Ehlers et al.
1996
; Hisatsune et al. 1997
; Krupp et al.
1996
, 1999
; Wyszynski et al. 1997
; Zhang
et al. 1998
). This effect of Ca2+ on NMDA
receptor-mediated currents occurs rapidly since 10 mM BAPTA, a much
more rapid and selective Ca2+ chelator, was shown
to be more effective in preventing inactivation (Legendre et al.
1993
). Reducing extracellular Ca2+ could
also inhibit inactivation, suggesting that the effect of intracellular
Ca2+ chelators was most likely due to chelating
Ca2+ rather than directly acting on NMDA
receptors (e.g., Legendre et al. 1993
). Similarly,
experiments in which Ca2+ chelators were applied
intracellularly helped demonstrate that NMDA receptor-mediated rises
in intracellular Ca2+ cause the depolymerization
of the actin cytoskeleton and rundown of NMDA receptor-mediated peak
current (Rosenmund and Westbrook 1993a
,b
; for review,
McBain and Mayer 1994
). Therefore,
Ca2+ chelators are routinely included in
intracellular recording solutions to ensure stable NMDA
receptor-mediated current by inhibiting inactivation and rundown.
Ca2+ chelators are also included in extracellular
recording solutions for whole-cell patch-clamp recording (e.g.,
Zhang et al. 1998
) and especially for inside-out and
cell-attached single channel recordings (e.g., Gibb and
Colquhoun 1992
) to control Ca2+ level and
achieve long-lasting recordings. As well, extracellular Ca2+ chelators have been needed to help define
the role of Ca2+ in glutamate-induced
excitotoxicity (e.g., Murphy et al. 1988
).
NMDA receptors are most likely a tetrameric complex of two NR1 subunits
in combination with NR2A, NR2B, NR2C, and/or NR2D (reviewed by
Dingledine et al. 1999). Expression of these receptors in heterologous systems has been a fruitful approach to characterizing their structure, function, and modulation (Dingledine et al.
1999
). In this report, we have investigated the effects of
extracellular Ca2+ on NMDA receptor function. As
previously reported by others, we show large potentiation of NMDA
receptor-mediated currents with decreasing concentrations of added
extracellular Ca2+. Surprisingly, we have found
that Ca2+ chelators such as EGTA act as
competitive antagonists and shift the glutamate dose response curve to
the right. These results have important implications in interpreting
data from experiments in which external Ca2+
concentration is manipulated by chelators such as EGTA.
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METHODS |
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Cell culture and transfection
Human embryonic kidney 293 (HEK 293) cells (CRL 1573; ATCC,
Rockville, MD) were plated on 10-cm culture dishes (Falcon, Becton Dickson, Franklin Lakes, NJ) and maintained at 37°C in humidified 95% O2-5% CO2, as
described previously (Chen et al. 1997). Culture medium
was prepared from minimal essential medium (MEM; Life Technologies, Burlington, Ontario, Canada) and supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 1 mM sodium pyruvate (GIBCO, Canadian Life
Technologies, Burlington, Ontario, Canada), 100 U/ml
penicillin-streptomycin (GIBCO), and 1 mM glutamine. For transient
transfection using the calcium-phosphate method (Chen and
Okayama 1987
), the cDNA plasmid ratio for NR1A and NR2 subunits
was 1:1 with a total amount of 10 µg. Cells were transfected in
humidified 97% O2-3% CO2
at 37°C for ~8 h. After transfection, cells were transferred onto glass coverslips in 35-mm culture dishes (Falcon). The NMDA receptor antagonist (±)-2-amino-5-phosphonopentanoic acid (APV, 1 mM; Research Biochemicals, Natick, MA) and/or memantine (100 µM; Research
Biochemicals) were added to the medium to enhance cell survival
(Chen et al. 1997
; Raymond et al. 1996
).
Electrophysiology
Approximately 20-36 h after transfection, cells on coverslips
were transferred to the recording chamber on the stage of an inverted
microscope (Axiovert 100, Carl Zeiss, Thornburg, NY). Extracellular
solution, which contained 145 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl2, 11 mM glucose, and 10 mM HEPES and
titrated with NaOH to pH 7.4, flowed constantly through the chamber.
Intracellular solution contained 145 mM KCl, 5.5 mM BAPTA, 4 mM
adenosine 5'-triphosphate magnesium salt (MgATP), and 10 mM
HEPES, titrated to pH 7.25 with KOH. Recording pipettes were pulled
from borosilicate glass (Warner Instruments, Hamden, CT) with the
Narishige (Tokyo) PP-83 electrode puller. Pipettes with open tip
resistance of ~1-5 M were used.
Agonist-evoked currents were recorded using the patch-clamp technique
in the whole-cell voltage clamp configuration with a holding potential
of 60 mV. After forming the whole-cell recording configuration, the
cell was lifted from the chamber floor. Agonists were applied rapidly
by piezo-controlled switching of a
tube, which was positioned
~100 µm from the cell. Solutions were gravity fed through the two
sides of the
tube. The 10-90% rise time of solution exchange at
the open pipette tip was ~0.5 ms (Chen et al. 1999
).
Currents were filtered at 5 kHz and sampled at 2 kHz using pCLAMP6
software and the Axopatch 200A amplifier (Axon Instruments, Foster
City, CA). Analyses were conducted with Clampfit (Axon Instruments) and
Origin (Microcal Software, Northampton, MA) software.
Chemicals and plasmid CDNAs
All chemicals were from Sigma (St. Louis, MO) unless otherwise
stated. NR1A (also known as NR1-1a; Hollmann et al.
1993) and NR2B cDNAs were gifts from Dr. S. Nakanishi, Kyoto
University, Kyoto, Japan. NR2A (from mouse brain, also known as
1)
was a gift from Dr. M. Mishina, University of Tokyo, Tokyo. These cDNAs were subcloned into eukaryotic expression vectors with the
cytomegalovirus promoter, as described (Chen et al.
1997
; Raymond et al. 1996
).
Statistical analysis and data presentation
Results are presented as mean ± SE. Significant differences were determined by paired or unpaired t-test. Figures were created with Origin software.
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RESULTS |
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Reducing extracellular Ca2+ concentration potentiates NMDA receptor-mediated current
At physiological extracellular Ca2+
concentrations, the Ca2+ influx resulting from
NMDA receptor activation induces partial pore block,
Ca2+-dependent inactivation, and peak current
rundown (Ascher and Nowak 1988; Ehlers et al.
1996
; Hisatsune et al. 1997
; Krupp et al.
1996
, 1999
; Legendre et al. 1993
;
Rosenmund and Westbrook 1993a
,b
; Wyllie et al.
1996
; Zhang et al. 1998
). We applied 1 mM
glutamate and 50 µM glycine for 2 s at intervals of 30 s
and a holding potential of
60 mV, while recording from NR1A/NR2A- or
NR1A/NR2B-transfected cells in the whole-cell mode. Within seconds of
reducing extracellular Ca2+ concentration by
switching from solutions containing 1.8 to 0.2 mM, then nominal zero,
then 5 mM EGTA, we observed a graded increase of the peak current
amplitudes mediated by both NR1A/NR2A (Fig. 1A) and NR1A/NR2B (data not
shown). In 5 mM EGTA, peak current amplitudes were approximately
sixfold larger than those recorded in control conditions (1.8 mM
Ca2+) for both subtypes [5.8 ± 2.1-fold,
n = 7 for NR1A/NR2A (Fig. 1A) and 6.3 ± 0.8-fold, n = 5 for NR1A/NR2B subtypes].
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Zn2+ has been shown to be a potent NMDA receptor
antagonist and Zn2+ contamination in the
nanomolar range in the recording solution can inhibit NR1A/NR2A-type
NMDA receptor-mediated current significantly (Chen et al.
1997; Paoletti et al. 1997
; Williams
1996
). Therefore, we investigated whether the potentiation of
NR1A/NR2A-mediated currents observed on switching solution to 5 mM EGTA
with no added Ca2+ was partly contributed by the
relief of Zn2+ block. Addition of the
Zn2+ chelator
N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) (1 µM) in the extracellular recording solution with
1.8 mM Ca2+ caused an increase in current
amplitude to 1.6 ± 0.2-fold of control (P < 0.001 by paired t-test; n = 5), consistent
with previously reported results (Paoletti et al. 1997
).
Thus, the effect of decreasing extracellular zinc accounted for only
~25% of the increase in peak amplitude for NR1A/NR2A-mediated
currents observed in recording solution containing
Ca2+ chelators and played no role in the
potentiation observed for NR1A/NR2B-mediated currents (since the
IC50 for zinc inhibition is in the micromolar
range; Chen et al. 1997
; Paoletti et al. 1997
; Williams 1996
).
We then recorded currents from NR1A/NR2A-transfected cells evoked by a subsaturating concentration of glutamate (10 µM, with 50 µM glycine). Under these conditions, we observed the same trend of increasing current amplitude with decreasing concentration of added extracellular calcium (Fig. 1B). However, with the addition of 5 mM EGTA (with no added Ca2+) to the extracellular solution, we made two surprising observations. The first was that the current amplitude decreased significantly compared with the no added calcium condition (Fig. 1B). The second was that the current activation time course was significantly slowed (Fig. 1B). The 10-90% rise time was 34.5 ± 1.5 ms in 1.8 mM Ca2+ and 81.4 ± 9.5 ms in 5 mM EGTA with no added Ca2+ (P < 0.01, paired t-test; n = 6). Recordings from NR1A/NR2B-transfected cells also revealed significant slowing of the activation time course in 5 mM EGTA/no added Ca2+, to 126 ± 13 ms from 65.4 ± 7.2 ms in 1.8 mM Ca2+ (P < 0.01, paired t-test; n = 5). As well, apparent desensitization was abolished (Fig. 1B). The slowed rise time, decreased peak amplitude, and lack of apparent desensitization of glutamate-evoked current observed on addition of EGTA suggested to us that NMDA receptors might exhibit decreased sensitivity to agonist under these conditions.
Glutamate dose-response curve is shifted to the right in extracellular solution containing 5 mm EGTA/no added Ca2+
To test the possibility that extracellular Ca2+ modulates the glutamate EC50, we generated glutamate dose-response curves for both NR1A/NR2A and NR1A/NR2B in the presence of 1.8 mM extracellular Ca2+ or no added Ca2+ plus 5 mM EGTA (Fig. 2). Consistent with our prediction, glutamate dose-response curves of both subtypes were shifted significantly to the right for 5 mM EGTA/no added Ca2+ compared with 1.8 mM extracellular Ca2+ (Fig. 2). The glutamate EC50 for NR1A/NR2A was 8.1 ± 0.6 and 24.0 ± 0.7 µM for extracellular Ca2+ of 1.8 mM versus 5 mM EGTA with no added Ca2+, respectively. For NR1A/NR2B, the glutamate EC50 was 1.6 ± 0.1 and 3.6 ± 0.6 µM for the same two conditions. Since the glutamate dose-response curve shift was observed under conditions of very low extracellular Ca2+ concentration and in the presence of the Ca2+ chelator EGTA, we next addressed whether the shift was an effect of extracellular Ca2+ or EGTA.
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Extracellular Ca+ concentration does not modulate the glutamate dose-response using cells transfected with NR1/NR2A
We constructed a series of glutamate dose-response curves under
conditions in which we varied either extracellular
Ca2+ or EGTA concentration (Fig.
3). In the absence of added
Ca2+, contamination of standard extracellular
recording solutions by this ion has been estimated in the range of 20 µM (Hoth 1995; Xiong et al. 1997
).
Therefore, if the rightward shift of the glutamate dose response curve
were due to elimination of extracellular calcium, we would predict a
similar shift with a lower but effective concentration of EGTA (200 µM with no added Ca2+), as we observed for 5 mM
EGTA/no added Ca2+. Instead, the glutamate
dose-response curve in the presence of 200 µM EGTA/no added
Ca2+ was not significantly different from that
generated in normal (1.8 mM) extracellular Ca2+
(Fig. 3A). This result indicates that the glutamate
EC50 of NR1A/NR2A remains unchanged with a shift
in extracellular Ca2+ concentration from a
millimolar to sub-micromolar level, or over an ~1000-fold range,
suggesting extracellular Ca2+ concentration does
not modulate the glutamate dose-response. On the other hand, increasing
extracellular EGTA concentration from 5 to 20 mM (with no added
Ca2+) further shifted the glutamate dose-response
curve to the right with an EC50 of 86.4 ± 2.4 µM (n = 4; Fig. 3B). Moreover, 5 mM BAPTA (which also contains four carboxyl groups) produced a similar shift in the glutamate dose-response for NR1A/NR2A as did 5 mM EGTA
(not shown). Notably, the addition of 5 mM EGTA fully chelated with 6.8 mM Ca2+ had no effect on the
glutamate EC50. Taken together, our results suggest that Ca2+ chelators containing free
carboxyl groups compete for binding at the glutamate site of NMDA
receptors.
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DISCUSSION |
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Our major finding is that Ca2+ chelators are
competitive antagonists at the glutamate-binding site of NMDA
receptors. This conclusion is based on three observations. First, the
decrease in current amplitude evoked by submaximal glutamate
concentrations observed in the presence of 5 mM EGTA was associated
with a slowing in rise-time to peak and lack of glutamate-induced
desensitization, typical of responses to lower concentrations of
glutamate (e.g., see Fig. 1). Second, the glutamate dose-response curve
was shifted to the right by EGTA in a dose-dependent manner, without a
decrease in the maximal response to saturating concentrations (i.e., 1 mM) of glutamate. Third, the effect of calcium chelators on the glutamate dose-response was not a result of altering extracellular Ca2+ concentration, since
Ca2+ concentrations in the range of ~20 µM up
to 1.8 mM had no effect on glutamate potency. Furthermore, the amount
of chelator necessary to produce a change in the glutamate affinity is
several hundred-fold in excess of what is required to effectively
chelate any residual extracellular Ca2+ (~20
µM Ca2+ and 5 mM EGTA). Consistent with our
findings, Gu and Huang (1994) also reported no effect of
extracellular Ca2+ on trigeminal NMDA receptor
dose-response to NMDA.
Xiong et al. (1997) demonstrated that a transient
decrease in extracellular Ca2+ concentration
alone could evoke currents in patch-clamp recordings from cells ranging
from neurons to HEK 293 cells. These currents were mediated by a cation
selective, Ca2+ sensing channel. Our results
could not be complicated by that effect because extracellular
Ca2+ was maintained at a constant level when
switching between control and agonist-containing solutions to evoke
NMDA receptor-mediated currents. In addition, although a decrease in
extracellular Ca2+ concentration would alter
charge-shielding effects on the membrane surface, this effect would be
predicted to decrease, rather than increase, current amplitude through
the cation-selective NMDA receptor channel.
Recent studies suggest that there may be differences in mechanisms of
modulation of recombinant versus native neuronal NMDA receptors, since
tyrosine phosphorylation by src affects zinc inhibition of
recombinant (NR1A/NR2A- and NR1A/NR2B-type) but not native neuronal
NMDA receptors (Xiong et al. 1999). However, this
interaction depends on alterations made to receptor structure by
intracellular proteins or second messengers, which may differ between
neurons and the nonneuronal cells used to analyze recombinant receptors. On the other hand, results of studies restricted to correlations between structure (e.g., subunit composition) and binding/gating properties of recombinant NMDA receptors have been remarkably informative with regard to native neuronal NMDA receptors (for review, see Dingledine et al. 1999
). Therefore, we
predict that our finding of competitive inhibition of glutamate binding by EGTA for recombinant NMDA receptors can be extended to native neuronal NMDA receptors composed of NR1A, NR2A, and/or NR2B subunits.
The fact that EGTA acts as a competitive antagonist for glutamate binding is not a complete surprise, since EGTA has two pairs of carboxyl residues, whereas glutamate has one pair. Recordings made in the presence of other extracellular Ca2+ chelators that contain free carboxyl groups, including EDTA and BAPTA, showed similar shifts in the NMDA receptor glutamate dose-response curve (Chen, unpublished data). Moreover, effects consistent with competitive inhibition by calcium chelators were also observed for glutamate-evoked currents mediated by the non-NMDA receptors GluR2 and GluR6 (Chen, unpublished data). These results suggest that inclusion of Ca2+ chelators in the extracellular solution during patch-clamp recording or excitotoxicity experiments will have an effect other than lowering Ca2+ concentration, complicating the interpretation of data.
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ACKNOWLEDGMENTS |
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T. H. Murphy is a Medical Research Council Scientist.
This project was supported by operating grants from the Medical Research Council (MRC) Canada to L. A. Raymond and T. H. Murphy. N. Chen is supported by a postdoctoral fellowship from the Hereditary Disease Foundation.
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FOOTNOTES |
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Address for reprint requests: L. A. Raymond, Kinsmen Laboratory of Neurological Research, Dept. of Psychiatry, University of British Columbia, 4N3-2255 Wesbrook Mall, Vancouver, British Columbia V6T 1Z3, Canada (E-mail: LYNNR{at}interchange.ubc.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 February 2000; accepted in final form 19 April 2000.
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REFERENCES |
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