Vol.2, No.1, 8-15 (2010)
doi:10.4236/health.2010.21002
SciRes Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
Health
The role of intracellular sodium (Na+) in the regulation of
calcium (Ca2+)-mediated signaling and toxicity
Xian-Min Yu, Bradley R. Groveman, Xiao-Qian Fang, Shuang-Xiu Lin
Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, USA; [e-mail]
Received 15 October 2009; revised 1 December 2009; accepted 2 December 2009.
ABSTRACT
It is known that activated N-methyl-D-aspartate
receptors (NMDARs) are a major route of ex-
cessive calcium ion (Ca2+) entry in central neu-
rons, which may activate degradative processes
and thereby cause cell death. Therefore, NMD-
ARs are now recognized to play a key role in the
development of many diseases associated with
injuries to the central nervous system (CNS).
However, it remains a mystery how NMDAR ac-
tivity is recruited in the cellular processes
leading to excitotoxicity and how NMDAR activ-
ity can be controlled at a physiological level.
The sodium ion (Na+) is the major cation in ex-
tracellular space. With its entry into the cell, Na+
can act as a critical intracellular second mes-
senger that regulates many cellular functions.
Recent data have shown that intracellular Na+
can be an important signaling factor underlying
the up-regulation of NMDARs. While Ca2+ influx
during the activation of NMDARs down-regu-
lates NMDAR activity, Na+ influx provides an
essential positive feedback mechanism to over-
come Ca2+-induced inhibition and thereby po-
tentiate both NMDAR activity and inward Ca2+
flow. Extensive investigations have been con-
ducted to clarify mechanisms underlying Ca2+-
mediated signaling. This review focuses on the
roles of Na+ in the regulation of Ca2+-mediated
NMDAR signaling and toxicity.
Keywords: NMDA Receptors; Sodium and Calcium
Influx; Sodium and Calcium Signaling; Excitability;
Toxicity
1. INTRODUCTION
Cytoplasmic Ca2+ is the most common signaling factor in
all types of cells. Normal intracellular Ca2+ concentration
([Ca2+]i) is approximately 40,000-fold lower than ex-
tracellular [Ca2+], which ranges from 1 to 2 mM [1,2].
Ca2+ ions enter neurons via various pathways including
voltage-gated Ca2+ channels, ligand-gated Ca2+ channels
and the Ca2+ exchangers [2,3]. It is known that activated
NMDAR channels are a major route of excessive Ca2+
entry in neurons [4-10]. While excessive intracellular
Ca2+ may activate degradative processes and thereby
cause toxic effects [2,10-12], NMDAR channel activity
may be inhibited by intracellular Ca2+ through: 1) -
actinin/cytoskeleton dissociation from the NR1 subunit of
NMDARs [13]; 2) calmodulin activation [13-16], and 3)
activation of phosphatases, such as calcineurin which
dephosphorylates NMDARs [17-19]. The Ca2+-induced
down- regulation of NMDARs is considered an important
negative feedback mechanism to control NMDAR activ-
ity [20-23]. Based on these findings we questioned: How
do excessive amounts of Ca2+ get into neurons through
NMDARs if NMDARs are inhibited by Ca2+ influx?
Na+ is the major cation in the extracellular space, and it
can enter cells through a variety of routes including
permeation through ligand- (e.g., glutamate) and voltage-
gated cation channels, uptake via membrane exchangers
and gradient-driven co-transporters [24]. NMDAR chan-
nels are highly permeable to both Na+ and Ca2+. Short
burst or tetanic stimulation of afferents that induces
synaptic LTP increases [Na+] up to 40 or 100 mM in
spines and adjacent dendrites [25,26]. These increases
can essentially be prevented by the blockade of NMDARs,
indicating that they are mainly mediated by Na+ entry
through NMDARs [25,26].
Our initial studies demonstrated that intracellular Na+
is an up-regulator of NMDARs, such that raising [Na+]i or
activating Na+ permeable channels may increase NMD-
AR-mediated currents [27-29]. We then identified that in
hippocampal neurons an increase of 5 ± 1 mM in [Na+]i
represents a threshold required to mask the down- regu-
lation of NMDARs induced by Ca2+ influx. Further in-
creases in Na+ influx not only significantly enhance Ca2+
influx induced by the activation of NMDARs, but also
overcome the Ca2+- dependent inhibition of NMDARs
[27,30]. This review focuses on the roles of Na+ in the
development of tissue injury and in the regulation of
Ca2+-mediated NMDAR signaling and toxicity.

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2. Na+ IN THE PROCESS OF
TISSUE INJURY
A significant increase in [Na+]i is a characteristic event
associated with tissue injury [31-37]. Application of
voltage-gated Na+ channel blockers reduce both Na+
entry and apoptotic neuronal death [32] whereas in-
creases of Na+ entry by application of the voltage-gated
Na+ channel activator, veratridine, induce neuronal apop-
tosis and caspase-3 activation [32,33]. There is a report
showing that during anoxia Na+ entry can occur through
either Gd3+-sensitive channels or via Na+/K+/2Cl- co-
transporters in cultured hippocampal neurons [38], im-
plying that multiple pathways for Na+ entry may be ac-
tivated during tissue injury.
It is known that Na+ influx into the cell is accompanied
by chloride ions (Cl-) and water, which can lead to acute
neuronal swelling and damage [4,39]. Previous studies
have shown that Na+ entry may cause an increase in cy-
tosolic Ca2+ through either Na+/Ca2+ exchangers or acti-
vation of voltage-gated Ca2+ channels [40,41], thereby
activating Ca2+-dependent signaling mechanisms. More-
over, Na+ entry via Na+/H+ exchange may cause changes
in intracellular pH, and thereby regulate many cellular
functions including enzyme activity, neuronal growth and
death [38,42-46]. A recent study showed that Na+ influx
plays an important role in the onset of anti-Fas-induced
apoptosis and that blocking Na+ influx may rescue pro-
grammed cell death in Jurkat cells [47]. Cox and col-
leagues reported that the binding of agonists to opioid
receptors on guinea pig cortical neuron membranes is
significantly reduced by increases in [Na+]i of 10-30 mM
[48]. Maximal inhibition of -, - and -opioid receptor
binding by Na+ is approximately 60%, 70% and 20%,
respectively [48]. Co-occurrence of Na+,K+-ATPase
dysfunction and Na+ influx causes α-amino-3-hydroxyl-
5-methyl-4-isoxa-zole-propionate receptor (AMPAR) pro-
teolysis and a rapid reduction of AMPAR cell-surface
expression [49]. Na+-mediated K+ channels such as Slo
gene-encoded K+ channels [50-54], are widely distributed
throughout the nervous system and are involved in both
the regulation of the after-potential following action po-
tentials [51,55], and the protection of neurons from hy-
poxic stimulation [51,52,54].
While the details of the mechanisms remain to be
clarified, significant pharmacological data have demon-
strated the protective effects of blocking Na+ influx dur-
ing injuries to the nervous tissue. The blockade of volt-
age-gated Na+ channels can prevent neurons from trau-
matic spinal cord injury [33,56-60] and the loss of white
matter [30,56,60,61], concurrently reducing the sensiti-
zation associated with pain [62-65] and preventing sei-
zures during kindling development [66]. The inhibition of
Na+/H+ exchange attenuates ischemia-induced cell death
[67,68]. As a result, a major focus of pharmaceutical
research has been on the search for effective therapeutic
approaches that target voltage-gated Na+ channels
[33,36].
3. ROLES OF Na+ IN THE REGULATION OF
Ca2+-MEDIATED NMDAR SIGNALING
AND TOXICITY
3.1. Calcium Influx through Activated
NMDARs is Regulated by Na+ Influx
Activated NMDARs are highly permeable to both Na+
and Ca2+ [20,22,23]. Prolonged increases of intracellular
Ca2+ during NMDAR activation may act as a negative
feedback mechanism controlling NMDAR activity
[20,22,23]. In light of our findings demonstrating that: 1)
intracellular Na+ up-regulates NMDAR channel gating
and 2) multiple types of receptor/channels such as AM-
PARs, voltage-gated Na+ channels, non-selective cation
channels and remote NMDARs may regulate NMDAR
activity through a Na+-dependent mechanism [27,28], we
investigated how NMDARs are regulated when both Ca2+
and Na+ flow into neurons during the same time period
through activated NMDARs [27,30]. Recordings were
conducted in the cell-attached single-channel configura-
tion. In this recording model, recorded surface NMDARs
are isolated by a recording electrode from the bath envi-
ronment and therefore cannot be directly stimulated by
bath-applied agents. We recorded the activity of surface
NMDARs before and after activation of remote
NMDARs (outside the patch) induced by bath application
of NMDA or L-aspartate [30]. To prevent toxic effects
which may be induced by application of NMDA or L-as-
partate, a standard extracellular solution in which NaCl
and KCl were replaced by Na2SO4 and Cs2SO4, was
utilized [30]. Consistent with previous findings [17,28,39,
69,70], no damage of neurons bathed with this standard
solution was observed following NMDA or aspartate
application. NMDAR single-channel activity was evoked
with 10 µM NMDA and 3 µM glycine included in the
standard extracellular solution filling the recording elec-
trodes.
We found that bath application of NMDAR agonists
may change NMDAR channel activity recorded in
cell-attached patches in a concentration-dependent man-
ner. While a significant increase in NMDAR channel
gating occurred during L-aspartate (>100 µM) applica-
tion to neurons bathed with the standard extracellular
solution, the activity of NMDARs was inhibited in neu-
rons when Na+ influx was blocked by replacing ex-
tracellular Na+ with Cs+ or N-methyl-D-glutamine
(NMDG) [28,30].
We measured the ratio of fluorescence at 346 nm ver-
sus 380nm for the Na+-sensitive dye, sodium-binding
benzofuran isophthalate (SBFI), and the Ca2+-sensitive

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10
dye, Fura-2, in the soma region of neurons. When the Na+
gradient across the cell membrane was decreased by
reducing extracellular Na+ concentration ([Na+]e) to 20
mM and the Na+ ionophore, monensin (10 M) was in-
cluded in the extracellular solution, basal [Ca2+]i and
[Na+]i of neurons were approximately 84 nM and 16 mM,
respectively. Under this condition bath application of
L-aspartate increased [Ca2+]i by 66 nM, decreased [Na+]i
by 5.8 mM and inhibited NMDAR activity [30]. On av-
erage, the overall channel open probability and mean
open time were reduced to 64% and 77% of controls. The
burst and cluster lengths were also significantly reduced.
These inhibitory effects produced by the bath application
of L-aspartate were prevented by either application of
APV or removal of Ca2+ from extracellular solution,
indicating that the activation of remote NMDARs may
also down-regulate recorded NMDAR activity through
Ca2+ influx [30]. Thus, it is demonstrated that NMDARs
can be up- and down-regulated by influxes of Na+ and
Ca2+, respectively.
We then measured changes of [Na+]i and [Ca2+]i in
neurons bathed with extracellular solution containing a
[Na+] of 10, 20 or 145 mM before and during the activa-
tion of NMDARs induced by bath application of
L-aspartate. We found that with an increase in [Na+]e, the
activation NMDARs produced increases in [Na+]i as ex-
pected, but also increased [Ca2+]i. Excluding the effect of
Ca2+ influx-induced Ca2+ release (CICR) from intracel-
lular stores, the increase in [Ca2+]i of neurons bathed with
extracellular solution containing 145 mM Na+ was still
significantly higher than that found in neurons bathed
with extracellular solution containing 10 mM Na+ [28,30 ].
When [Na+]e was reduced to 10 mM, the activation of
NMDARs produced increases in [Na]i and [Ca2+]i by
around 0.8 mM and 35 nM, respectively. Under this
condition, the activation of remote NMDARs inhibited
NMDAR activity recorded in cell-attached patches [30].
When [Na+]e was increased to 20 mM, NMDAR activa-
tion produced a 5 mM increase in [Na+]i and a 50 nM
increase in [Ca2+]i, but no change in the activity of re-
corded NMDARs [30]. Similarly, increasing [K+]e by 30
mM in an extracellular solution containing 170 mM Na+
and 1 M TTX produced increases in [Na+]i and [Ca2+]i
by around 7 mM and 48 nM, respectively, but again
showed no change in the activity of NMDARs recorded
in cell-attached patches either [28,30 ]. Thus, an increase
in [Na+]i of approximately 5 mM appeared to be a critical
concentration for masking the inhibitory effects induced
by Ca2+ influx on NMDARs in cultured hippocampal
neurons [30]. Since a modest increase of [Ca2+]i by ap-
proximately 35 nM inhibited NMDAR activity when
[Na+]e was reduced to 10 mM [30], it was possible that
Na+ influx not only enhanced Ca2+ influx but also masked
the inhibitory effects of Ca2+.
To confirm this hypothesis, we recorded NMDAR
single-channel activity before and during the activation of
remote NMDARs in cell-attached patches with pipettes
filled with a Ca2+-free extracellular solution containing
200 mM Na+ from neurons that had been pre-treated with
BAPTA-AM (10 M for 4 hrs) and bathed with the same
Ca2+-free extracellular solution, or with pipettes filled
with extracellular solution containing 0.3 or 1.2 mM Ca2+
from neurons bathed with the extracellular solution con-
taining the same amount of Ca2+, respectively. We found
that the activation of remote NMDARs produced a simi-
lar up-regulation of NMDAR channel activity when local
and bath [Ca2+] was set at 0, 0.3 and 1.2 mM, implying
again that the effects of Ca2+ influx in the regulation of
NMDARs by remote NMDARs are overcome by Na+
under normal condition [30]. Furthermore, removal of
extracellular Ca2+ did not produce any effect on the
up-regulation of NMDARs by remote NMDARs in neu-
rons bathed with the standard extracellular solution con-
taining 200 mM Na+ [28,30]. Thus, we conclude that Ca2+
influx through activated NMDARs is regulated by Na+
influx, and that the effect of Na+, which overcomes
Ca2+-induced inhibition, provides an essential positive
feedback mechanism enhancing both the NMDAR activ-
ity and the inward flow of Ca2+.
3.2. Depletion of Extracellular Ca2+
Enhances Na+ Influx and Thereby
Causes NMDAR-Mediated Toxicity
Based on the findings that glutamate concentration may
increase in both humans [71,72] and animals after nervous
system injury [73], and that application of NMDAR an-
tagonists may protect neurons from excitotoxic injuries in
both humans [74,75] and animal [39,74,76], it has been
believed that NMDAR-mediated excitoxicity plays a ke- y
role in the development of neuronal death associated with
stroke/traumatic CNS injury. However, it remained unclear
how NMDARs were recruited to cause neurotoxicity.
We examined the effects of extracellular Ca2+ depletion
and reperfusion, which may occur in stroke patients, on
cultured hippocampal neurons [27,77]. Neurons were
bathed initially with an extracellular solution containing:
140 mM NaCl, 5 mM CsCl, 1.8 mM CaCl2, 33 mM
glucose, 25 mM HEPES; pH: 7.35; osmolarity: 310-320
mOsm. The reduction of [Ca2+]e from 1.8 mM to 0.5 or 0
mM caused a significant increase in Caspase-3 activity
and morphological changes in neurons such as swelling,
beading, and/or process disintegration. Significantly less
formazan was observed in 3-(4,5-dimethylthiazol-2-yl)
2,5-diphenyl-tetrazolium bromide (MTT) assays in which
neurons were treated with the extracellular solution con-
taining 0.5 or 0 mM Ca2+, indicating a change in mito-
chondrial function associated with neuronal injury
[78-81]. Unexpectedly, application of NMDAR antago-
nists APV (100 M) and MK801 (2 M) significantly
prevented the above mentioned changes in neurons only

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11
when the drugs were applied concomitant to the reduction
of [Ca2+]e from 1.8 to 0 mM. No protective effects of the
drugs could be found when they were applied during Ca2+
reperfusion or when [Ca2+]e was reduced from 1.8 to 0.5
mM [77]. These findings suggest that the depletion of
extracellular Ca2+ may evoke NMDAR-mediated neuro-
toxicity [77], and also raised the questions of how and
when NMDAR activity is recruited to induce neuronal
injury following the removal of extracellular Ca2+.
To address this question, we recorded NMDAR sin-
gle-channel activity before and during a depletion of
extracellular Ca2+ from 1.8 to 1.3, 0.5 or 0 mM in cell-
attached patches from cultured hippocampal neurons. To
prevent cell damage during the reduction of extracellular
Ca2+, the Cl- in the standard extracellular solution was
replaced by SO4
2- [28,30,39,77]. Bath application of a
low [Ca2+] solution to neurons caused a parallel shift of
the current-voltage (I/V) relationship in NMDAR sin-
gle-channels recorded in cell-attached patches, which
indicates that there is a cell-depolarization, but no change
in single-channel conductance [30,77]. In order to ac-
count for this, the holding potential was re-adjusted to
maintain a 70 mV patch-potential from the reversal po-
tential of recorded channels. We found that a depletion of
extracellular Ca2+ from 1.8 to 1.3 or 0.5 mM did not in-
duce any significant change in the activity of recorded
channels until [Ca2+]e was reduced from 1.8 to 0 mM
[28,30,77]. The channel activity could be subsequently
abolished with application of the NMDAR antagonist,
MK801, confirming that a [Ca2+]e reduction from 1.8 to 0
mM produces increases in NMDAR activity [28,30,77].
Since the concomitant blockade of NMDARs to the re-
duction of [Ca2+]e from 1.8 to 0.5 mM may actually in-
crease the number of injured neurons [77], the up-regu-
lation of NMDARs appears to be essential in triggering of
toxicity mediated by NMDARs and the application of
NMDAR antagonists in the Ca2+ reperfusion model may
be protective only when NMDARs are recruited.
To identify the mechanisms by which the removal of
extracellular Ca2+ results in the up-regulation of NMD-
ARs, we measured [Ca2+]i and [Na+]i in cultured hippo-
campal neurons before and during reductions of ex-
tracellular Ca2+. A [Ca2+]e reduction-dependent decrease
in [Ca2+]i and increase in [Na+]i were observed [77]. A
depletion of extracellular Ca2+ from 1.8 to 0 mM pro-
duced sufficient increases in [Na+]i capable of enhancing
NMDAR activity [28,30,77]. Furthermore, we found that
the up-regulation of NMDAR activity induced by ex-
tracellular Ca2+ depletion was prevented by the blockade
of Na+ influx [77].
Previous studies showed that the removal of extracel-
lular Ca2+ to 0 mM may increase NMDAR single-channel
conductance [20,23,82], and that reducing intracellular
Ca2+ may reduce the Ca2+-dependent inhibition of
NMDARs and thereby enhance NMDAR channel activity
[13-20,23]. Therefore, it is possible that NMDAR gating
may be enhanced by the removal of extracellular Ca2+
through Na+ and/or Ca2+-dependent mechanisms.
The ensemble currents produced by the summation of
consecutive super-clusters were compared before (1.8
mM) and after reducing [Ca2+]e to 0 mM. We found that
the removal of extracellular Ca2+ may significantly in-
crease the decay time of ensemble currents and that this
effect can be abolished by blocking Na+ influx. This
suggests that the removal of extracellular Ca2+ may affect
NMDAR-mediated whole-cell responses through the
action of Na+ [77].
Large reductions in [Ca2+]e have been found during
instances of high neuronal activity [83-85], the develop-
ment of seizures [86], hypoglycemic coma [87], and
periods of hypoxia and ischemia [88,89]. Ca2+-depletion
has also been reported to induce cell injury and death [90].
Thus, the Na+-dependent enhancement of NMDAR activ-
ity induced by depletion of extracellular Ca2+ may be an
important mechanism underlying the development of neu-
rotoxicity in the CNS.
3.3. Na+ Regulation of Ca2+ Homeostasis
Under resting conditions [Ca2+]i in neurons is normally
maintained at 10–100 nM, and is tightly regulated by both
Ca2+ influx and efflux across the membrane. [Ca2+]i can
be increased by Ca2+ entry through Ca2+ channels (in-
cluding ligand- and voltage-gated Ca2+ channels and
non-selective cation channels) located on the plasma
membrane and by CICR from the endoplasmic reticulum
(ER) upon binding of inositol trisphosphate (IP3) to the
inositol trisphosphate receptor (IP3R). Ca2+-mediated
injury is usually acute and rapid [91]. Disturbances of
Ca2+ homeostasis in the cytoplasm, ER, or mitochondria
can be harmful to cells [3]. Since Ca2+ stores are closely
connected within the cells and interact with each other,
dysregulation of one compartment is usually followed by
responses from the others. Together, they may overwhelm
the cell’s capacity to maintain overall homeostasis and
kill the cell [3].
In the plasma membrane Ca2+-ATPase and the Na+/
Ca2+ exchanger act to transport cytosolic Ca2+ to the ex-
tracellular space. The Na+/Ca2+ exchanger has a low af-
finity for Ca2+ but a high velocity; as such, it removes
Ca2+ only when cytosolic concentrations are high. The
Ca2+-ATPase, has a high affinity for Ca2+ and pumps out
Ca2+ even at low cytosolic concentrations [3,92,93]. In
resting cells, [Ca2+] in the mitochondrial matrix is around
100 nM. When cytosolic [Ca2+] rises, Ca2+ can enter the
mitochondria through a uniporter and thereby regulate
Ca2+ signals [94]. In mitochondria the Na+/Ca2+ ex-
changer extrudes Ca2+ [3,94,95]. However, the activity of
the Na+/Ca2+ exchanger may be reversed on the influx of
Na+ [96]. This reversal in Na+/Ca2 exchange is observed
under pathological conditions [3]. If the mitochondrial

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Na+/Ca2+ exchanger is overwhelmed by Ca2+ entry, the
Ca2+ levels in the mitochondrial matrix may increase
enough to trigger a mitochondrial permeability transition.
The sustained transitions may cause mitochondrial de-
polarization, inhibition of ATP production, and cell death
[97-99]. In the nucleus Ca2+ is involved in the gene
transcription and DNA metabolism [100-102]. Unlike in
the mitochondria, nuclear Ca2+ is found to be rapidly
equilibrated with cytosolic Ca2+. This may occur by dif-
fusion across nuclear pores [103] and/or Ca2+ channels in
the nuclear envelope [104].
CICR during NMDAR activation has been reported
[20,105,106]. We observed that when extracellular solu-
tion contained more Na+, NMDAR activation produced
greater increases in both [Na+]i and [Ca2+]i [30]. Fur-
thermore, in neurons bathed with extracellular solution
containing 145 mM Na+, NMDAR activation-induced
increases in [Ca2+]i were significantly reduced from 100 
30 nM (n = 5) to 62  8 nM (n = 8) with thapsigargin (0.1
µM) treatment [30], which depletes intracellular stores of
Ca2+ by blocking Ca2+ re-uptake. In the absence of thap-
sigargin, NMDAR activation only produced a 35  8 nM
(n=8) increase in [Ca2+]i in neurons bathed with ex-
tracellular solution containing 10 mM Na+ [30]. The
blockade of Ca2+ influx by removal of extracellular Ca2+
abolished the NMDAR activation-induced increase in
[Ca2+]i, (data not shown) [2,107]. The increase in [Ca2+]i
induced by Ca2+ release from intracellular stores during
NMDAR activation in neurons bathed with extracellular
solution containing 10 mM Na+ was significantly reduced
when compared with that in neurons bathed with ex-
tracellular solution containing 145 mM Na+ [30]. These
data suggest that CICR from intracellular stores during
NMDAR activation may be regulated by intracellular Na+.
Stys and colleagues provided direct evidence showing
that intra-axonal Ca2+ release during ischemia in rat optic
nerves is mainly dependent on Na+ influx. This Na+ ac-
cumulation stimulates three distinct intra-axonal sources
of Ca2+: 1) the mitochondrial Na+/Ca2+ exchanger driven
in the Na+ import/Ca2+ export mode; 2) positive modula-
tion of ryanodine receptors; and 3) promotion of IP3
generation by phospholipase C [108].
4. QUESTIONS AND FUTURE STUDIES
Na+ entry is a key factor that initiates fast action poten-
tials and shapes sub-threshold electrical properties to
thereby regulate neuronal excitability and neuronal dis-
charge activity [109-113]. Present data have shown that: 1)
intracellular Na+ up-regulates NMDARs; 2) via increas-
ing intracellular Na+, multiple types of receptor/channels
such as AMPA receptors, voltage-gated Na+ channels and
non-selective cation channels, may regulate NMDAR
activity; 3) Na+ influx may enhance Ca2+ influx, mask the
Ca2+-dependent inhibition of NMDARs and significantly
alter Ca2+ homeostasis.
Based on combined investigations of protein crystal
structures in-vitro and functions in cells, Na+ binding
motifs have been characterized in a number of proteins
such as thrombin, Na+/K+-ATPase and various neuro-
transmitter transporters. Thrombin is a serine protease,
the activity of which is regulated by Na+ binding. The
sequence, CDRDGKYG, in the Na+ binding loop is
highly conserved in thrombin from 11 different species
[114]. Investigations into the crystal structure of a bacte-
rial homologue of the Na+/Cl- dependent transporters
from Aquifex aeolicus revealed that there are two Na+
binding sites, named Na1 and Na2 [115]. Na+/ K+-ATPase
is found to have three Na+ biding sites. Na1 is formed
entirely by the side chain oxygen atoms of residues on
three helices in the transmembrane regions (TM) 5, 6 and
8. Na2 is formed almost ‘‘on’’ the TM4 helix with three
main chain carbonyls plus four side chain oxygen atoms
(Asp 811 and Asp 815 on TM6 and Glu 334 on TM4).
The Na3 binding site is contiguous to Na1. The carbonyls
of Gly 813 and Thr 814 (TM6), the hydroxyl of Tyr 778
(TM5), and the carboxyl of Glu 961 (TM9) contribute to
the Na3 binding site [116,117].
To date there is no evidence of a similar amino acid
sequence corresponding to a Na+ binding site, as seen in
these Na+ binding proteins, present in NMDAR subunit
proteins (Yu, unpublished data). Molecular mechanisms
underlying the regulation of NMDARs and Ca2+ signaling
by intracellular Na+ remain unclear. Investigations aiming
to identify critical Na+ targeting site(s) in the regulation of
NMDARs and Ca2+ homeostasis are essentially needed
for understanding activity-dependent neuroplasticity in
the CNS.
5. ACKNOWLEDGMENTS
This work was supported by grant NIH (1R01 NS053567) to X.-M.Y.
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