From the Department of Neurobiology, University of Pittsburgh
School of Medicine, Pittsburgh, Pennsylvania 15261
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INTRODUCTION |
Cyanide is a mitochondrial poison that adversely affects cellular
respiration by inhibiting the reoxidation of cytochrome a3 by molecular oxygen, thereby obstructing the
electron transport chain and oxidative phosphorylation (1). Cytochrome
a3 and cytochrome a form the
cytochrome c oxidase complex, which is the terminal enzyme
in the electron transport chain. Transferring electrons to oxygen,
cytochrome c oxidase is the cellular respiratory component
responsible for the critical need for oxygen. Thus, cyanide
intoxication in cells is akin to oxygen starvation. In neuronal
systems, cyanide treatment has been a widely used model of hypoxia
(2-4), particularly in relation to excitotoxic processes. For example,
cyanide can raise extracellular glutamate levels (5, 6), increase
glutamate-triggered intracellular Ca2+ elevations in
neurons (7, 8), and potentiate glutamate toxicity (7, 9). Moreover,
specific inhibitors of N-methyl-D-aspartate (NMDA)1 receptors can inhibit
cyanide-induced Ca2+ influx in neurons (7, 10, 11) as well
as neurotoxicity (12-14).
Interestingly, a direct interaction between cyanide and the NMDA
receptor has recently been demonstrated. Cyanide treatment of cultured
rat hippocampal or cerebellar neurons potentiated NMDA-induced
physiological responses, including single channel activity in excised
outside-out membrane patches (15, 16). However, the precise site of
action of cyanide at the receptor remained to be elucidated. In the
present investigations we have evaluated the possible interaction of
KCN on the NMDA receptor redox modulatory sites (17). Via these sites,
disulfide-reducing agents such as dithiothreitol (DTT; see Refs. 17 and
18), dihydrolipoic acid (19), or tris(carboxyethyl)phosphine (20) enhance NMDA receptor-mediated physiological responses, whereas thiol-containing oxidants (21, 22), reactive quinones (23, 24), or
oxygen-derived free radicals (25, 26) can reverse the effects of
reductants or depress native responses. Cyanide has well established
properties as a disulfide reducing agent in many preparations (27-30),
and thus an effect on the NMDA thiol-sensitive sites would not be
surprising. Nonetheless, the experiments described here demonstrate
that cyanide can be used to distinguish between different NMDA receptor
subtypes by producing either a potentiation or a depression of the
physiological responses mediated by this ligand-gated ion channel.
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EXPERIMENTAL PROCEDURES |
Tissue Culture--
Tissue culture and all common reagents were
purchased from Sigma, excluding the following: iron-supplemented bovine
calf serum, HyClone Laboratories (Logan, UT), and minimal essential
medium, Life Technologies, Inc. Chinese hamster ovary cells (CHO-K1;
ATTC CCL61) were grown in Ham's F-12 nutrient medium with 10% fetal bovine serum, and 1 mM glutamine (CHO media) in 50- or
200-ml flasks at 37 °C in 5% CO2. Cells were passaged
at a 1:10 dilution at 80% confluency, approximately every 2 days, no
more than 30 times. Cerebral cortices were obtained from E-16
Sprague-Dawley C-D rats and dissociated according to methods described
previously (31). Briefly, cortices were incubated in minimal essential medium solution containing 0.03% trypsin for 2 h at 37 °C.
Dissociated cells were plated at a density of 3-5 × 105 cells/ml in growth medium (v/v 80% Dulbecco's
modified Eagle's minimum, 10% Ham's F12 nutrient mixture, 10%
heat-inactivated iron-supplemented bovine calf serum, 25 mM
HEPES, 24 units/ml penicillin, 24 µg/ml streptomycin, and 2 mM L-glutamine) into 35-mm tissue culture
dishes containing five 12-mm poly-L-lysine-coated glass
coverslips each, for electrophysiological recordings. Cells were
maintained at 37 °C in 5% CO2. Growth medium was
changed three times per week. After 15 days in culture, non-neuronal
cell proliferation was inhibited with 2 µM cytosine
arabinoside after which the growth medium contained 2% serum and no
F-12. Cells were mostly used between 22 and 29 days in vitro
(DIV), although some later experiments utilized cells at 8 DIV.
Transfection Protocol--
The cDNAs for the NMDA subunits
and the positive transfection marker green fluorescent protein (32)
were previously subcloned into mammalian expression vectors (33-35).
Cells were seeded at 3 × 105 cells per well in 6-well
plates (35-mm wells) approximately 24 h prior to transfection with
1.3 µg of total DNA and 6 µl of LipofectAMINE (Life Technologies,
Inc.) in 1 ml of serum-free CHO media per well. Of the total DNA 0.3 µg were green fluorescent protein-containing plasmid, and the
remaining 1 µg was composed of a 1:3 ratio of NR1- to NR2-containing
plasmids (34). After a 5-h incubation at 37 °C in 5%
CO2 with the transfection solution, cells were refed with
CHO medium containing 300 µM ketamine or 1 mM
5,7-dichlorokynurenate to prevent the cell death that accompanies NMDA
receptor expression (34). Cells were used for recording approximately
40-50 h after the start of the transfection.
Site-directed Mutagenesis--
A construct containing the
cDNA for rat NR1 pN60 (36) was used as template for site-directed
mutagenesis to produce NR1(C744S). Mutagenic primers (36-mers) were
designed to mutate the codon for cysteine 744, TGC, to that of serine,
TCC, utilizing a commercially available PCR-based procedure
(Stratagene, La Jolla, CA). Cycling parameters were initial
denaturation at 94 °C for 1.25 min; 18 cycles of 94 °C for 0.75 min, 55 °C for 1 min, 68 °C for 15 min; and a final extension at
68 °C for 10 min. To remove methylated template DNA, the PCR
products were restriction-digested with DpnI (Stratagene).
Epicurian Coli XL-1 Blue supercompetent Escherichia coli
(Stratagene) were heat shock-transformed with the PCR products and
colonies selected; plasmid DNA was isolated and digested with HindIII and NotI (Life Technologies, Inc.) to
excise the mutated NR1 cDNA. This was subcloned into the equivalent
restriction sites of pRc/CMV. Sequencing of the restriction fragment
was performed to confirm the presence of the desired mutation and
absence of spontaneous unwanted sequence alterations. The
NR1(C744A,C798A) double mutant was the kind gift of S. Traynelis, Emory
University, Atlanta, GA.
Whole Cell Recordings--
Electrophysiological measurements
were obtained at a membrane voltage of
60 mV using the whole cell
patch-clamp configuration. Methods of data acquisition and analysis
have been described previously (18). The external recording solution
contained 150 mM NaCl, 2.8 mM KCl, 1.0 mM CaCl2, 10 mM HEPES, and 10 µM glycine (pH 7.2). For recordings from cortical neurons
0.25 µM tetrodotoxin (Calbiochem) was added to this
solution. Patch electrodes (2-4 M
) were filled with 140 mM CsF, 10 mM EGTA (Sigma), 1 mM
CaCl2, and 10 mM HEPES (pH 7.2). NMDA,
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), DTT, potassium cyanide
(KCN) or
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) were dissolved in extracellular solution and applied onto cells
by a multi-barrel fast perfusion system (Warner Instrument Corp.,
Hamden, CT). All results are expressed as the mean ± S.D. For
statistical comparisons a parametric test (t test) was used, unless the standard deviations of the two groups analyzed were significantly different from each other, in which case a non-parametric test was utilized (Mann Whitney two sample test).
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RESULTS |
Potentiation of NMDA-induced Whole Cell Responses by KCN in
Cortical Neurons--
Cyanide as a disulfide-reducing agent is used in
biochemical experiments at concentrations in the range of 1-40
mM (27-29). Since the NMDA receptor reduction process is
both time- and concentration-dependent (18), we opted to
use a concentration roughly 2-20 times higher than the concentrations
utilized by Isom and co-workers (100 µM to 1 mM; see Refs. 15 and 16) to accelerate and maximize the reduction process but without damaging the cells. Cortical neurons were
therefore incubated for several minutes in extracellular recording
solution containing 2 mM KCN. Whole cell measurements of
NMDA (30 µM) receptor-mediated currents were performed
under control conditions and at 1-min intervals during the KCN
treatment (Fig. 1A). During
the KCN incubation itself, we noted a rapid, small, variable increase
in inward leak current, which always decreased in amplitude and
eventually disappeared a few seconds later, even while the toxin was
still present in the bath. We observed that the NMDA-induced responses
increased in amplitude in a time-dependent fashion during
the cyanide exposure. After a 3-min treatment with the mitochondrial
inhibitor, the NMDA-generated currents had increased 1.6 ± 0.8-fold (x-fold = x2/x1; n = 10) over control responses; by 5 min, responses were potentiated
1.8 ± 0.4-fold (n = 3). This action of KCN in
cortical neurons is similar to that reported earlier for NMDA-induced
responses in cerebellar granule cells (16). The oxidizing agent DTNB
(0.5 mM) quickly (<1 min) and effectively reversed the
actions of KCN and further depressed the NMDA-induced current
amplitudes to 81.1 ± 17.3% (n = 3) of the
initial control responses (Fig. 1A). Hence, KCN produced
effects on the NMDA receptor resembling those previously reported for
the sulfhydryl-reducing agent DTT (17, 18).

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Fig. 1.
Potentiation of NMDA-induced whole cell
currents by KCN in mature neurons. A, KCN (2 mM) applied via rapid perfusion produced a
time-dependent enhancement of currents elicited by 30 µM NMDA applied to a cortical neuron from a second
perfusion barrel (in the absence of KCN). The oxidizing agent DTNB (0.5 mM) reversed this effect of KCN within 1 min. Currents were
recorded at 60 mV under the whole cell voltage clamp configuration in
this and subsequent figures. For all figures, each treatment was rinsed
off the cells with control solution prior to the next application of a
redox substance. B, NMDA (30 µM)-induced
currents are not substantially altered when KCN (2 mM) is
rapidly co-applied from a second perfusion barrel. KCN, when applied
alone, produces a small, irregular increase in inward current.
Bars above the traces show application intervals of the
indicated drugs. C, currents elicited by 30 µM
NMDA are potentiated by a 9-min incubation with 4 mM DTT
but are not further enhanced by a subsequent treatment with DTT plus 2 mM KCN together (6 min).
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We also monitored whole cell responses to brief (1-1.5 s) applications
of either NMDA (30 µM) or KCN (2 mM) alone or
both these drugs together (Fig. 1B). Currents induced by
NMDA + KCN were slightly (6.0 ± 5.3%; n = 6) but
significantly (p < 0.001, paired t test)
smaller than those induced by NMDA. Moreover, KCN alone was able to
elicit inward currents but with maximal amplitudes that were only
8.0 ± 4.2% (n = 6) of the NMDA-elicited
responses. These KCN-induced currents are thus likely responsible for
the aforementioned transient increase in current during the first few
seconds of the prolonged incubations with the toxin. The potentiating actions of KCN on the NMDA-induced currents thus required relatively longer incubation periods (>3 min) with the toxin, again reminiscent of the actions of DTT in this preparation (18).
To test whether DTT and KCN altered NMDA receptor-mediated currents via
a similar mechanism, we obtained NMDA-induced responses during a
prolonged treatment with DTT (4 mM, approximately 10 min)
and following a subsequent exposure to DTT together with 2 mM KCN (6 min; Fig. 1C). Currents were
potentiated during both these treatments 2.6 ± 1.3- and 2.9 ± 1.6-fold, respectively (n = 9). The ratio of DTT + KCN to DTT potentiation for all cells tested was 1.1 ± 0.2. Therefore, KCN was unable to potentiate further NMDA responses after
the DTT treatment, suggesting that both these agents act via a common
site.
NMDA Receptor Subunit-specific Effects of KCN--
To facilitate
the examination of the putative actions of KCN on the redox-sensitive
sites of the NMDA receptor, we first investigated the actions of DTT
and DTNB on recombinant receptors expressed in CHO cells (33-35). We
used site-directed mutagenesis to substitute cysteine 744 of the NR1
subunit with a serine residue (NR1(C744S)). This mutation abolishes DTT
sensitivity in recombinant NMDA receptors composed of NR1 together with
either the NR2B, NR2C, or NR2D subunit but not NR2A (37), as this
latter subunit may contain a separate DTT-sensitive site (38, 39). NR1
or NR1(C744S) subunits were transiently transfected into CHO cells
along with either NR2A or NR2B to yield four separate groups of cells,
each expressing one of the following subunit combinations: NR1/NR2A,
NR1(C744S)/NR2A, NR1/NR2B, and NR1(C744S)/NR2B. NMDA (30 µM)-induced responses were recorded from these cells
(Fig. 2) during control conditions and
following treatments with DTT (4 mM, 3 min) and DTNB (0.5 mM, 1 min). NMDA-evoked currents in NR1/NR2A-,
NR1(C744S)/NR2A-, and NR1/NR2B-expressing cells were similarly
potentiated by DTT (Table I). As
expected, NMDA-induced responses in cells transfected with
NR1(C744S)/NR2B were unaltered by the DTT treatment (Table I). In
contrast, DTNB depressed the currents in all subunit configurations (Table I). Interestingly, the effects of DTNB on NR2B-containing receptors, but not on NR2A, were significantly greater for the mutated
form of NR1, when compared with the wild-type (p < 0.05, Mann-Whitney two sample test). In all cases, the inhibitory
effects of DTNB were fully reversible by a subsequent incubation with 4 mM DTT. The depressive actions of DTNB on the
NR1(C744S)/NR2B subunit configuration could thus be due to the
formation of a mixed disulfide (between one-half of the DTNB molecule
and a sulfhydryl group in the protein) with a second critical cysteine
present on NR1. This amino acid, Cys-798, is also important for the
formation of the redox-sensitive site on this subunit (37), perhaps by forming a disulfide bond with Cys-744 (40, 41). A mixed disulfide should be subject to reduction by DTT, and this appears to be the
case.

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Fig. 2.
Redox sensitivity of recombinant NMDA
receptors expressed in CHO cells. NMDA (30 µM)-evoked responses were recorded under
control conditions, following a 3-min incubation with 4 mM
DTT, and after a 1-min treatment with 0.5 mM DTNB, in CHO
cells transfected with the NMDA receptor subunits
indicated above the traces. Note the failure of
DTT to potentiate the NMDA-induced current in cells expressing the
NR1(C744S)/NR2B receptor configuration.
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Table I
Effects of redox agents on recombinant NMDA receptor-mediated currents
Currents elicited by a 30 µM NMDA application to CHO
cells transfected with the NMDA receptor subunits indicated were
recorded under control conditions, following a 3-min incubation with 4 mM DTT and after a 1-min treatment with 0.5 mM
DTNB. The values given are means ± S.D. from 4 to 7 experiments.
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A separate group of CHO cells transfected with the four aforementioned
subunit configurations was also incubated with KCN (2 mM,
3-6 min). We observed that cyanide produced a 1.4 ± 0.1-fold potentiation of 30 µM NMDA-elicited responses in cells
expressing NR1/NR2A receptors (n = 6), with DTNB (0.5 mM) rapidly (<1 min) reversing this effect (to 82.1 ± 14.1% of control, n = 5; Fig. 3A). Moreover, KCN had a very
similar effect to 4 mM DTT in CHO cells transfected with
NR1(C744S)/NR2A receptors (1.8 ± 0.3-fold increase,
n = 4; Fig. 3B). The potentiating action of
KCN was spontaneously reversible upon washout, albeit slowly, similar to what has been observed following DTT treatment in native receptors (18). A concentration-response curve for the potentiating action of KCN
on wild-type NR1/NR2A receptors after 3-min incubations was then
generated (Fig. 4). The data were fitted
to a logistic function; the calculated EC50 of KCN
potentiating NMDA-induced responses in this preparation was 0.5 mM. Surprisingly, 2 mM KCN did not potentiate
but, in fact, somewhat depressed NMDA-induced currents in cells
transfected with NR1/NR2B (to 83.2 ± 14.4% of control;
n = 4), even though 4 mM DTT produced its
expected response augmentation in these cells (Fig.
5A). A more pronounced current depression by KCN was observed in cells expressing the DTT-insensitive NR1(C744S)/NR2B receptor configuration (to 49.3 ± 15% of
control; n = 3; Fig. 5B). The depressive
actions of KCN on the mutant receptor were not reversible by a
subsequent treatment with 4 mM DTT. Similar to the effects
of DTNB on NR2B-containing receptors, KCN produced a significantly
greater depression of the NMDA-induced currents in cells expressing
NR1(C744S)/NR2B subunits, when compared with NR1/NR2B
(p < 0.05, Mann-Whitney two sample test).

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Fig. 3.
Effects of KCN on NR2A-containing
receptors. A, KCN (2 mM, 3 min) potentiates
30 µM NMDA-elicited whole cell responses in CHO cells
expressing NR1/NR2A. This effect of KCN was readily reversed by 0.5 mM DTNB (1 min). B, DTT (4 mM) and
KCN (2 mM) treatments (3 min) independently enhanced 30 µM NMDA-induced currents in CHO cells expressing
NR1(C744S)/NR2A receptors.
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Fig. 4.
Concentration-response curve for the
potentiating actions of KCN on NR1/NR2A receptors. From data such
as those shown in Fig. 3A, the fold increase in 30 µM NMDA-mediated current amplitude was plotted
versus the KCN concentration, after a 3-min incubation with
the metabolic inhibitor. Symbols represent the mean ± S.E. for 3-6 cells tested at each concentration of KCN. The data were
fitted to a logistic function; the calculated EC50 was 0.5 mM.
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Fig. 5.
Effects of KCN on NR2B-containing
receptors. A, DTT (4 mM, 3 min) enhanced
but KCN (2 mM, 3 min) depressed the NMDA responses in cells
transfected with NR1/NR2B subunits. B, in cells expressing
DTT-insensitive NR1(C744S)/NR2B receptors, KCN still depressed the
NMDA-evoked currents.
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The depressing actions of KCN on NR1/NR2B or NR1(C744A)/NR2B receptors
could be due to reaction of these substances with either Cys-744 or
Cys-798 in the wild-type subunit or with the remaining Cys-798 in the
case of the mutant. These interactions would lead to the formation of a
DTT-insensitive thiocyanate adduct. In order to test this hypothesis we
obtained a series of recordings with a double NR1(C744A,C798A)/NR2B
mutant. Interestingly, the inhibitory actions of 2 mM KCN
(2 min) were significantly decreased but still present in the double
mutant, when compared with the single mutant (p < 0.05, unpaired t test; Fig.
6). This effect was not reversible by 4 mM DTT. Responses obtained from NR1(C744A,C798A)/NR2B
receptors after cyanide treatment were 84.2 ± 19.9%
(n = 6) of control currents, a value very similar to
the inhibitory effect of KCN on wild-type NR1/NR2B receptors.
Interestingly, 0.5 mM DTNB had a similar inhibitory effect
on NR1(C744A,C798)/NR2B receptors (84.2 ± 6.9% of control, n = 5) as in the wild-type NR1/NR2B subunit
configuration and significantly different from its effect on the single
mutant NR1(C744S)/NR2B (p < 0.0001; unpaired
t test). These results support the hypothesis that DTNB and
KCN interact with Cys-798 in the single NR1 mutant and suggest a novel
effect of these substances with a different site in either the
wild-type or double mutant NR1/NR2B receptor. Comparative measurements
were obtained on the NR1(C744A,C798A)/NR2A subunit combination. A 3-min
incubation with either 2 mM KCN or 4 mM DTT
produced a 1.7 ± 0.5-fold (n = 4) or a 2.2 ± 0.8-fold (n = 4) potentiation, respectively, of
these receptors.

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Fig. 6.
Mutation affecting KCN sensitivity of
NR2B-containing receptors. A, as noted earlier, KCN (2 mM, 3 min) depressed 30 µM NMDA-induced
currents in CHO cells expressing NR1C744S/NR2B receptors. B,
this effect of KCN is substantially decreased in cells expressing
double mutant NR1(C744A,C798A)/NR2B receptors.
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Effects of KCN on NMDA-induced Responses in Neurons at Early
Developmental Stages--
Cortical neurons in culture have been shown
to express primarily, but not solely, NR1 and NR2B early in development
(42, 43). As neurons mature in vitro, expression of NR2A
becomes more prominent. Hence, KCN was tested for its ability to alter NMDA (30 µM)-induced responses in young neurons (8 DIV).
The metabolic inhibitor produced a modest potentiation of these
responses after a 5-min incubation (to 1.2 ± 0.1-fold of control,
n = 5). It is possible that the relatively small
amounts of NR2A expression at this developmental stage are sufficient
to prevent a current depression by KCN in these neurons, such as that
seen for the NR1/NR2B combination. Nonetheless, the aforementioned
potentiating actions of KCN observed in mature neurons were more
prominent than in the younger cells (p < 0.05, Mann-Whitney two sample test). Interestingly, 2 mM KCN
applied for 3-5 min to CHO cells transfected with NR1, NR2A, and NR2B
produced neither a potentiation nor a depression of NMDA-elicited
currents (n = 4). One must interpret this result with
caution, however, since the proportion of functional receptors that
co-assemble all three subunits in this situation is not known.
Actions of KCN on NR1/NR2A Are Not Due to Zinc Chelation--
A
recent report (39) has suggested that the previously proposed
redox-sensitive site on the amino-terminal of NR2A (38) may actually
represent a high affinity zinc recognition site. Through this site, low
background zinc levels present in extracellular solutions may
constitutively inhibit NR1/NR2A channel activity. Since DTT has good
metal chelating properties (44), Paoletti and co-workers (39) proposed
that the rapid onset and quickly reversing component of the effects of
the reducing agent on NR1/NR2A-mediated currents actually represents
the removal and re-introduction of endogenous zinc block. We therefore
evaluated whether the actions of KCN on NR1/NR2A may, in fact, be
mediated by a similar mechanism as this substance can also chelate
metals to some extent (45). We initially tested the actions of 1 µM TPEN, a high affinity zinc chelator (46), on
NMDA-elicited currents in mature (25-29 DIV) cortical neurons (Fig.
7A) and in
NR1/NR2A-transfected CHO cells (Fig. 7B). TPEN did not alter
the peak currents elicited by the agonist in both these preparations
(92.3 ± 13.5% of control, n = 8 for neurons;
94.2 ± 11.4% of control, n = 5 for NR1/NR2A), although a small effect on NR1/NR2A current desensitization was noted.
This suggests that the levels of zinc in our external recording solution may be below the binding affinity of this metal for its site
on NR2A (approximately 5 nM; see Refs. 39 and 47). We investigated the integrity of our TPEN solution by evaluating its
effect on chelating exogenously added zinc on NMDA receptor-mediated currents in cortical neurons. One µM TPEN significantly
(p < 0.001, paired t test) reversed the
blocking actions of 1 µM zinc on these currents, that is
the steady-state responses to 30 µM NMDA in the presence
of zinc were 64.1 ± 10.1% of control, whereas in zinc and TPEN
they were 86.7 ± 10.6% of the currents produced by NMDA alone
(n = 6). Finally, and most importantly, the inclusion of 1 µM TPEN in all external solutions did not affect the
potentiating actions of 2 mM KCN on responses mediated by
the activation of NR1/NR2A receptors (1.5 ± 0.3-fold increase,
n = 4; Fig. 7C). This suggest that the
actions of KCN on this receptor configuration are not due to removal of
zinc.

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Fig. 7.
Actions of KCN on NR1/NR2A are not due to
zinc chelation. A, whole cell currents from a mature
cortical neuron in culture in response to 30 µM NMDA
under control conditions or in the continuous presence of 1 µM TPEN. B, similar responses obtained from a
CHO cell expressing NR1/NR2A receptors. Note that in both A
and B, TPEN does not increase the peak response to the
agonist, although a small effect on current desensitization can be
noted in the CHO cell response. C, NMDA (30 µM)-induced currents in a CHO cell expressing NR1/NR2A
receptors recorded in the continuous presence of 1 µM
TPEN before and after a 3-min treatment with 2 mM
KCN.
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DISCUSSION |
Cyanide has been used extensively to characterize and identify
critical thiols in a variety of enzymes and other proteins (28-30, 48,
49). The present study not only confirms the previously proposed direct
interaction of the toxin with the NMDA receptor (15, 16) but suggests a
possible effect on the functional thiol groups of the protein. Our data
indicate that the modifications of these sites by KCN can produce
different, subunit-dependent physiological outcomes as
follows: either an enhancement or a depression of receptor channel
function. Hence, mature cortical neurons in culture, which have been
shown to express comparatively high levels of NR2A, in conjunction with
NR1 and NR2B (42, 43), show a more pronounced sensitivity to
KCN-mediated NMDA current potentiation than younger neurons, which
express primarily NR1/NR2B receptors and less NR2A. This is in stark
contrast to the potentiating actions of DTT on native NMDA receptors,
which we have recently reported to be substantially larger in immature
cultured neurons than in older cells (50).
Studies by Köhr et al. (38) and Sullivan et
al. (37) suggested that the NMDA receptor contained two separate
redox agent-sensitive centers. Köhr and co-workers (38), by using
chimeric NR subunits, showed that the extracellular amino-terminal of
NR2A contained a DTT-sensitive site(s) that influenced NMDA-induced
currents. Investigations by Sullivan et al. (37) employed
site-directed mutagenesis to demonstrate that Cys-744 and Cys-798 of
NR1, located in the extracellular loop between the putative
transmembrane domains III and IV, were critically important for
rendering redox sensitivity to NR1/NR2B, NR1/NR2C, and NR1/NR2D
receptors but, as expected, not to the NR1/NR2A subunit
combination.
As mentioned earlier, a recent study has questioned the existence of a
redox modulatory site on NR2A. Paoletti et al. (39) have
suggested that the observed rapid effects of DTT on NR1/NR2A receptors
represent zinc chelation and removal from solution with the consequent
relief of a high affinity block by the metal. We believe that not all
of the effects of DTT on NR1/NR2A receptors can be explained by zinc
chelation, however. First, DTT (and KCN) produces a slow potentiation
of the currents in our experiments, which is different from the rapid
onset (<10 s) of the DTT and TPEN effects seen by Paoletti et
al. (39) in their investigations. Second, the strong metal
chelator TPEN, in our hands, had practically no effect on NMDA
receptor-mediated currents in either mature neurons or in CHO cells
expressing NR1/NR2A receptors, suggesting that background zinc levels
in our preparation are very low. These observations may lead one to
conclude that the slow effects produced by DTT or KCN on native
receptors on mature neurons or on NR1/NR2A recombinant receptors may
then be due to the interactions of these substances with the redox site
on NR1(Cys-744 and Cys-798; see Ref. 37). However, these substances
work well in potentiating currents mediated by NR1(C744S)/NR2A or
NR1(C744A,C798A)/NR2A receptors, which would suggest that a separate
redox site exists on either NR1 or NR2A. The fact that KCN depresses
slightly the currents mediated by NR1/NR2B receptors, which contain
Cys-744 and Cys-798 on NR1, potentially as the only DTT-sensitive
site(s), would seem to indicate that a KCN/DTT-sensitive site does lie within NR2A in NR1/NR2A receptors.
A recent study from our laboratory (35) demonstrated that two
DTT-sensitive sites could be electrophysiologically distinguished in
recombinant NMDA receptors, at least at the single channel level. Redox
agents were shown to affect both open channel frequency and open time
in NR1/NR2A receptors but only frequency in NR1/NR2B channels. The
behavior of receptors putatively composed of NR1/NR2A/NR2B subunits was
more akin to the latter configuration. The present investigations show
that two putative redox-active sites can be chemically distinguished by
cyanide. This toxin mimics the actions of DTT for the NR1/NR2A subunit
configuration, potentiating NMDA-induced currents, but behaves more
like DTNB at the NR1/NR2B receptor by depressing the responses. As NR2A
is expressed in our cultures (43) and in similar preparations (42), at
the time we perform most of our recordings the effects of KCN on NR2A
appear to override, or somehow occlude, the actions of the toxin on any
NR2B-containing receptors that are present in the neurons (NR1/NR2B or
NR1/NR2A/NR2B). This complex subunit interaction is complemented by our
recent single channel studies (35) that revealed a masking of a
subunit-specific redox property when both NR2A and NR2B co-assembled in
the same receptor with NR1. Therefore, detection of the modification of the NR1 redox site by KCN may not be possible, at least
macroscopically, when NR2A is present in the final receptor and
regardless of whether or not there is co-assembly with NR2B. This
possibility is strengthened by the fact that the redox site on NR1 can
only be alkylated with N-ethylmaleimide (18) when this
subunit combines with NR2B but not with NR2A (38).
The inhibitory actions of KCN on NR1(C744S)/NR2B receptors could be due
to the cyanolation (formation of a thiocyanate adduct) of Cys-798 of
NR1. It is unlikely, however, that such a chemical modification can
produce equivalent allosteric changes on the receptor as those induced
by the hypothetical formation of a disulfide bond between Cys-744 and
Cys-798 after DTNB-induced oxidation (40, 41). Therefore, it remains to
be seen what effects cyanide has, if any, on NR1(C798S)/NR2B receptors.
Experiments are currently underway to address these issues.
Nonetheless, the effect of DTNB on NR1(C744S)/NR2B receptors are
similar to that produced by KCN on these channels and significantly
larger than its effect on NR1/NR2B channels. Since KCN also depresses
NMDA-stimulated currents in cells expressing wild-type NR1/NR2B
subunits, it is attractive to speculate that the preferred effect of
the toxin on this receptor configuration is cyanolation of the same
site where DTNB oxidizes. In addition, this site may be neither Cys-744
or Cys-798 on NR1, as the actions of both DTNB and KCN are similar in
wild-type or double mutant NR1(C744A,C798A)/NR2B receptors. Although
this possibility can only be definitively tested by further studies, it
may pave the way for future experiments aimed at identifying additional sites susceptible to thiol modification on all NMDA receptor subunits, which could also be susceptible to cyanolation.
In summary, cyanide represents the first described molecule able to
distinguish chemically between two putative redox sites on the NMDA
receptor. Furthermore, the chemical reduction of the receptor by
cyanide, leading to the potentiation of NMDA-induced currents in
neurons, may be sufficient to account for the enhancement in
excitotoxicity produced by the metabolic inhibitor, similar to what has
been seen for DTT (51). The ability of cyanide to produce different
effects on NMDA receptor function, depending on the subunit
composition, further supports the notion that two or more putative
redox sites modify NMDA receptor function by different allosteric
mechanisms (35). Finally, the use of cyanide and cyanide adducts may
help to characterize biochemically the mechanisms whereby the redox
modulatory sites influence NMDA receptor channel activity.
We thank J. Brimecombe and K. Hartnett for
helpful discussions. We also thank Drs. S. Nakanishi, P. Seeburg, D. Lynch, S. Traynelis, and M. Chalfie for the NR and green fluorescent
protein plasmids.