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Cardiac Naⴙ-Ca2ⴙ Exchange
Molecular and Pharmacological Aspects
Munekazu Shigekawa, Takahiro Iwamoto
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Abstract—The Na⫹-Ca2⫹ exchanger (NCX) is one of the essential regulators of Ca2⫹ homeostasis in cardiomyocytes and
thus an important modulator of the cardiac contractile function. The purpose of this review is to survey recent advances
in cardiac NCX research, with particular emphasis on molecular and pharmacological aspects. The NCX function is
thought to be regulated by a variety of cellular factors. However, data obtained by use of different experimental systems
often appear to be in conflict. Where possible, we endeavor to provide a rational interpretation of such data. We also
provide a summary of current work relating to the structure and function of the cardiac NCX. Recent molecular studies
of the NCX protein are beginning to shed light on structural features of the ion translocation pathway in the NCX
membrane domain, which seems likely to be formed, at least partly, by the phylogenetically conserved ␣-1 and ␣-2
repeat structures and their neighboring membrane-spanning segments. Finally, we discuss new classes of NCX
inhibitors with improved selectivity. One of these, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate
(KB-R7943), appears to exhibit unique selectivity for Ca2⫹-influx–mode NCX activity. Data obtained with these
inhibitors should provide a basis for designing more selective and clinically useful drugs targeting NCX. (Circ Res.
2001;88:864-876.)
Key Words: Na⫹-Ca2⫹ exchange 䡲 Ca2⫹ transport 䡲 cardiac muscle 䡲 excitation-contraction coupling
he Na⫹-Ca2⫹ exchanger (NCX) catalyzes electrogenic
exchange of Na⫹ and Ca2⫹ across the plasma membrane
in either the Ca2⫹-efflux or Ca2⫹-influx mode, depending on
the electrochemical gradients of the substrate ions. Thus,
NCX is modulated by electrical activity of cardiac myocytes.
On a beat-to-beat basis, the primary function of NCX in the
heart is extrusion of Ca2⫹ from myocytes during relaxation
and diastole, which balances Ca2⫹ entry via L-type Ca2⫹
channels during cardiac excitation.1 The contributions of
different ion transporters to Ca2⫹ removal from myocytes
during twitch relaxation have been summarized in a recent
review by Bers.2 NCX extrudes ⬇30% of the Ca2⫹ required to
activate the myofilaments in rabbit, guinea pig, and human
ventricles but a much smaller portion (⬇7%) in rat and mouse
ventricles. Sarcoplasmic reticulum (SR) Ca2⫹-ATPase removes most of the remaining Ca2⫹. In failing rabbit or human
heart, NCX and SR Ca2⫹-ATPase contribute nearly equally to
Ca2⫹ removal from the cytoplasm.2 Such differences in the
NCX contribution mostly reflect differences in the expression
levels of NCX activity in the sarcolemma,3,4, Ca2⫹-ATPase
activity in the SR,4,5 and possibly [Na⫹]i6 in cardiomyocytes
from these animal species in normal and disease conditions.
The physiological significance of Ca2⫹ influx via NCX in
the excitation-contraction coupling of cardiac muscle is
controversial. Triggering the release of Ca2⫹ from the SR via
NCX during membrane depolarization has been shown to be
much less efficient than via L-type Ca2⫹ channels.7–9 However, when both L-type Ca2⫹ channels and NCX are at work,
Ca2⫹ entry via NCX appears able to synergistically amplify
the effect of triggering SR Ca2⫹ release via the L-type
current.10 In heart failure, on the other hand, enhanced Ca2⫹
entry due to increased NCX expression may provide inotropic
support for failing myocytes, in which the SR function is
often defective.4,11 Under other pathological conditions, such
as cardiac ischemia/reperfusion12 or digitalis intoxication,13
the NCX-mediated increase in Ca2⫹ entry or decrease in Ca2⫹
exit due to a rise in [Na⫹]i results in Ca2⫹ overloading of the
SR, leading to mechanical and electrical dysfunction of
myocytes.
Because the SR Ca2⫹ load, which is a predominant determinant of cardiac contractility,2 is determined by the competition between SR Ca2⫹-ATPase and NCX for the cytosolic
Ca2⫹, modulation of NCX activity by physiological regulatory factors as well as by alterations in cytosolic ion concentrations and the action potential duration in disease states
exerts profound influences on the overall contractile function
of the heart. In the present review, we describe recent
advances in molecular and pharmacological studies of the
cardiac NCX function. The mechanism and further physiological and pathological aspects of NCX function have been
discussed in several recent reviews 14 –16 and short
comments.17,18
T
Original received February 22, 2001; revision received March 15, 2001; accepted March 16, 2001.
From the Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Correspondence to Munekazu Shigekawa, MD, PhD, Department of Molecular Physiology, National Cardiovascular Center Research Institute,
Fujishiro-dai 5-7, Suita, Osaka 565-8565, Japan. E-mail [email protected]
© 2001 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
864
Shigekawa and Iwamoto
Naⴙ-Ca2ⴙ Exchange
865
Figure 1. Minimum model for transport
cycle of cardiac NCX1 (A) and outward
exchange currents in excised giant membrane patch (B). A, Exchanger assumes
conformations with inward-facing (E1) and
outward-facing (E2) ion transport sites (K1/2
values for substrate ions are indicated).
Exchange activity is regulated by intracellular Ca2⫹ (Ca2⫹i) and intracellular Na⫹ (Na⫹i).
B, Time-dependent changes in outward
exchange currents initiated by application of
100 mmol/L Na⫹i at 1 ␮mol/L Ca2⫹i (bold
line) are shown. Removal of Ca2⫹i inactivates the current, whereas intracellular
application of modifiers such as ATP, PIP2,
or Ca2⫹i decelerates the current inactivation.
Cardiac NCX
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The mammalian NCX forms a multigene family of homologous proteins comprising 3 isoforms: NCX1,19 NCX2,20 and
NCX3.21 These isoforms share ⬇70% amino acid identity in
the overall sequences and thus presumably have a very
similar molecular structure. NCX1 is the first NCX cloned
and is highly expressed in cardiac muscle and brain and to a
lesser extent in many other tissues. NCX2 and NCX3 are not
expressed in adult rat heart at the protein level, but an
NCX2-specific transcript can be detected faintly by using
reverse transcriptase–polymerase chain reaction.22 NCX2 and
NCX3 are expressed in a few limited tissues, such as brain,
and their molecular properties and functions remain unclear.
Besides these isoforms, splice variants with variation in a
small region of the large central loop of the exchanger
molecule are generated from NCX1 and NCX3 genes in a
tissue-specific manner.22,23 However, the physiological significance of such diversity is not clear, although some splice
variants of NCX1 seem to exhibit the regulatory property
differences.24,25 In heart, a splice variant of NCX1, designated
NCX1.1, is predominantly expressed.22
NCX1 gene expression occurs early in cardiogenesis in the
mouse embryo, before the onset of ventricular myosin light
chain 2 expression and before the occurrence of the first heart
beat.26 It is expressed in a heart-restricted pattern through the
critical early stages of heart development until at least 11 days
postcoitus (dpc). Recent targeted disruption of the NCX1
gene revealed that NCX1-deficient (Ncx1⫺/⫺) embryonic
mice died between 9 and 10 dpc.27 In the case of Ncx1⫺/⫺
embryos at 9.5 dpc, ⬇70% of them showed no heartbeat,
whereas the remainder exhibited only very slow and arrhythmic heart contraction. The ventricular wall was very thin and
contained few myocytes in Ncx1⫺/⫺ mice compared with
Ncx1⫹/⫹ mice, although there was apparently no defect in the
expression of heart-specific genes in the ventricles of Ncx1⫺/⫺
mice. Myocytes displayed morphological signs of apoptosis.
In the normal mammalian heart, NCX1 expression reaches a
maximum near birth and then decreases postnatally to a
significantly lower level in the adult stages.28 This is in
contrast to SR Ca2⫹-ATPase, which is increasingly expressed
postnatally. In rat heart, thyroid hormones play an important
role in the reciprocal control of the expression of these 2 Ca2⫹
transporters.29 Thus, the contribution of NCX to the control of
[Ca2⫹]i is greater in the immature heart than in the mature
heart.
NCX is a high-capacity and low–Ca2⫹-affinity transporter
exchanging 3 Na⫹ and 1 Ca2⫹ across the plasma membrane7,14,30 (however, see a recent study by Fujioka et al31
reporting coupling ratios ⬎3 to 1). The maximum turnover
numbers (up to 5000 per second)32,33 for NCX1 and its Km for
intracellular Ca2⫹ (⬇6 ␮mol/L)34 –36 are much greater than
those for SR Ca2⫹-ATPase (100 to 150 per second for Ca2⫹
transport calculated from the activity of purified ATPase at
37°C37 and ⬇0.3 ␮mol/L for Ca2⫹ affinity5,38). The density of
NCX1 in the sarcolemma has been estimated to be 250 to 400
NCX/␮m2 in the guinea pig ventricular myocyte by electrical
measurement of NCX currents from whole-cell and excised
giant patches.32,33 The density of NCX1 in the sarcolemma
appears to differ significantly in some species. In one report,3
the rate of Ca2⫹ extrusion via NCX1 per unit membrane
capacitance is 2- to 4-fold larger in guinea pig and hamster
myocytes than in rat myocytes. Immunocytochemically,
NCX1 is localized in the T-tubule membrane as well as in the
peripheral sarcolemma and the intercalated disks in rat and
guinea pig ventricular myocytes, which does not appear to
support the predominant proximity of NCX1s to the ryanodine receptors in the dyadic junction.14,39 Whether NCX1
with a relatively low Ca2⫹ affinity is capable of reducing
[Ca2⫹]i to a low resting level is an interesting question. NCX1
has been shown to be the predominant Ca2⫹ extrusion
mechanism in resting rabbit or guinea pig cardiomyocytes,7,40
although the [Ca2⫹] underneath the sarcolemma could be
significantly higher than the bulk resting [Ca2⫹]i. When
cloned cardiac NCX1 is expressed in heterologous CCL39
cells with little endogenous NCX, the exchanger is capable of
completely suppressing Ca2⫹-regulated plasma membrane
processes, such as Ca2⫹/calmodulin-dependent activation of
the Na⫹-H⫹ exchanger NHE1 by a physiological agonist,
␣-thrombin.41
Figure 1A shows a simplified model for Na⫹-Ca2⫹ exchange by NCX1 that is supported by many previous studies.33,34,42– 44 The model represents a consecutive or ping-pong
mechanism in which only 1 substrate ion is translocated at a
time, with the indicated values for the apparent transport and
regulatory site affinities for extracellular and intracellular
Na⫹ or Ca2⫹.34 –36,45 Interaction of NCX1 with these ions is
intrinsically asymmetric in that the apparent affinity for
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Circulation Research
May 11, 2001
Factors Influencing Naⴙ-Ca2ⴙ Exchange
Cardiac Muscle
Activation
2⫹
Ca
i
(regulatory site K1/2: 0.022–0.4 ␮mol/L)
Squid Axon
Ca i (regulatory site K1/2: ⫺ATP, 4–8 ␮mol/L;
⫹ATP, 0.4–0.8 ␮mol/L)
2⫹
Protein phosphorylation
Protein phosphorylation
PIP2 (giant cardiac or oocyte patches, sarcolemmal
vesicles)
PIP2 (giant oocyte patches expressing NCX-SQ1)
䡠䡠䡠
Phosphoarginine
PKC activators
Monovalent cations
䡠䡠䡠
Monovalent cations
Redox reagents (free radicals)
Proteolysis
Inhibition
Na⫹ (Na⫹i-dependent inactivation)
䡠䡠䡠
䡠䡠䡠
Na⫹ (Na⫹i-dependent inactivation)
Protons
Protons
Ni2⫹, La3⫹, Cd2⫹
Ni2⫹, La3⫹, Cd2⫹
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XIP
XIP
FMRF, FRCRCFa
FMRF
KB-R7943
KB-R7943
intracellular Ca2⫹ is several hundred times higher than that for
extracellular Ca2⫹, although the affinities for intracellular Na⫹
and extracellular Na⫹ differ little, and these 2 transport
substrates exert regulatory influences from only the inside
(see below). The effects of alkali metal cations and protons on
Na⫹-Ca2⫹ exchange are also highly asymmetric (also see
below). In addition to Na⫹-Ca2⫹ exchange, NCX also catalyzes coupled exchanges of internal Ca2⫹ for external Ca2⫹
and internal Na⫹ for external Na⫹.14 The voltage dependence
of Na⫹-Ca2⫹ exchange by NCX1 is attributed mostly to a
voltage dependence of the Na⫹ translocation step or Na⫹
binding, which is rate limiting in overall reaction.33,34,44
However, the net negative charge is also moved by cardiac
NCX1 during outward Ca2⫹ translocation from the cytoplasmic side, suggesting that the ion-binding site in unliganded
NCX protein has a negative charge slightly more than
⫺2.32,44
Regulation
The NCX1 function is regulated by a variety of extracellular
and intracellular factors. The Table lists factors involved in
acute regulation of both the mammalian cardiac NCX1 and
the invertebrate squid exchanger (NCX-SQ1), which are the
most intensively studied NCXs. Exchange activity of cardiac
NCX1 is stimulated by phospholipase C–activating agonists,
such as phenylephrine, endothelin 1, angiotensin II, and some
growth factors, in rat adult or neonatal cardiomyocytes and
cells transfected with cloned dog cardiac NCX146 – 48 as well
as in sarcolemmal vesicles isolated from rat heart.49 The
effects of agonists are mimicked by phorbol ester46,48,50 or the
phosphatase inhibitor okadaic acid.46 Furthermore, all these
stimulatory effects of agonists are blocked by selective
inhibitors of protein kinase C (PKC). Therefore, extracellular
signals activate NCX activity by a mechanism involving PKC
activation. It should be noted that although the observed
effects of PKC activators on NCX1 activity are modest (30%
to 40% stimulation), the basal NCX activity might have
already been elevated in neonatal cardiomyocytes or NCX1transfected cells, because PKC inhibitors or PKC downregulation by prior 24-hour exposure to phorbol ester decreases
basal NCX activity by 30% to 40%.46,50 Thus, the overall
effects of PKC on NCX1 could be substantial, at least in in
vitro cell systems. Under equivalent conditions, NCX activity
in cells expressing cloned NCX2 is not affected by phorbol
ester or PKC inhibition.48
In neonatal cardiomyocytes and NCX1-transfected cells,
the NCX1 protein is phosphorylated at specific serine residues under basal and stimulated conditions, but the phosphorylation is abolished by PKC inhibitors.46,48 However, phosphorylation of the NCX1 protein is not required for PKCdependent NCX activation, because cells expressing an
NCX1 mutant with its phosphorylatable residues mutated to
alanines exhibit a normal response to PKC activation.48
Interestingly, the phorbol ester– dependent stimulation of
NCX activity is abolished in cells expressing an NCX1
mutant lacking most (amino acids 246 to 672) of the central
hydrophilic loop (see Figure 2A) or in a loss-of-function
mutant of the XIP segment in the same loop48,51 (see below).
These findings, together with an absence of effect on cloned
NCX2, suggest that enhanced NCX activity is not secondary
to the PKC-dependent modulation of other ion transporters.
One likely mechanism for the PKC-dependent regulation of
NCX1 is involvement of a phosphorylatable cytosolic ancillary protein(s) that interacts with the exchanger. In this
context, it is important to note that a novel 13-kDa cytosolic
protein recently isolated from the axoplasm and brain of
squid has been shown to be involved in ATP-dependent
regulation of NCX-SQ1.52
Protein kinase A activation by forskolin and/or other
reagents has been reported to stimulate activities of cardiac
NCX1 expressed in BHK cells50 and Xenopus oocytes,24
although the same clone expressed in CCL39 cells is not
affected by 8-bromo-cAMP or 8-bromo-cGMP.48 In frog
heart, however, ␤-receptor– dependent or cAMP-dependent
Shigekawa and Iwamoto
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stimulation results in the inhibition of NCX activity, in which
a novel 9 –amino acid exon of the frog exchanger seems to
play a role.53 However, the part played by protein kinase A in
this inhibition is not clear. On the other hand, Condrescu et
al54 found that protein phosphatase inhibitors, such as calyculin A, caused substantial inhibition of intracellular Na⫹–
dependent Ca2⫹ influx into CHO cells expressing bovine
cardiac NCX1. Because use of the PKC inhibitor and deletion
of the central hydrophilic loop of NCX1 did not block such
inhibition, protein phosphorylation by a kinase(s) other than
PKC might be involved.
To date, studies of NCX currents using excised giant
cardiac or oocyte membrane patches have failed to provide
evidence for the involvement of protein kinases, although
high ATP concentrations (K1/2 ⬎4 mmol/L) have been shown
to stimulate NCX1 activity in these patches.55,56 Such failure
could be due to loss of diffusible factors from excised
patches. Using dialyzed squid axons, DiPolo and Beaugé57,58
have accumulated a large body of data supporting the
involvement of protein phosphorylation in the ATPdependent regulation of squid NCX-SQ1, although known
protein kinase inhibitors are reportedly unable to abolish such
an ATP effect. In the squid axon, ATP markedly increases the
affinities of NCX-SQ1 for the transport substrates (intracellular Ca2⫹ and extracellular Na⫹) without a change in Vmax and
also increases the affinity for the regulatory intracellular
Ca2⫹58,59 (see below). However, comparable data have not yet
been obtained for the cardiac NCX1.
Over a longer time range, upregulation of NCX1 activity
occurs due to increased gene expression at the transcription
level in adult cardiac myocytes in the pressure-overloaded
heart, in the infarcted heart (peri-infarcted area), and in the
chronically failed heart.4,60,61 In isolated adult and neonatal
cardiomyocytes, NCX1 gene expression is increased significantly in response to ␣-adrenergic (with phenylephrine)62 or
␤-receptor/cAMP– dependent stimulation.63 Furthermore,
long exposure to high external Ca2⫹ and enhanced Na⫹ influx,
procedures that raise [Ca2⫹]i or [Na⫹]i, result in a significant
increase in the NCX1 mRNA level in adult and neonatal
cardiomyocytes.60,63 In neonatal cardiomyocytes, transforming growth factor-␤1 and interleukin-1␤ stimulate and inhibit
NCX1 gene expression, respectively, and PKC inhibitors
significantly reduce both the basal and transforming growth
factor-␤1–stimulated levels of NCX1 mRNA in these cells.64
The NCX1 gene contains 3 tissue-specific promoters and
multiple 5⬘-untranslated region exons upstream from the
coding region that undergo alternative splicing.65– 67 From an
analysis of the cardiac-specific promoter, cis-acting elements
important for the expression of NCX1 in neonatal cardiomyocytes as well as for its induction by ␣1-adrenergic stimulation
have been identified.67 However, the mechanisms for upregulation or downregulation of the NCX1 gene by many of the
above signals remain unclear.
Cardiac NCX activity is intrinsically regulated by ATPdependent mechanisms as well as by physiologically important cations (Table). When ATP levels in adult rat cardiomyocytes are depleted ⬎90% by treatment with metabolic
inhibitors, NCX activity measured as intracellular Na⫹–
dependent Ca2⫹ uptake is inhibited by ⬇80%.68 ATP deple-
Naⴙ-Ca2ⴙ Exchange
867
tion also reduces exchange activity in cells expressing cloned
cardiac NCX1.46,69 ATP depletion affects many aspects of
cell metabolism, causing reduced phosphorylation of proteins
and active metabolites, such as phosphatidylinositol 4,5bisphosphate (PIP2), alteration of the cytoskeleton structure,
and induction of various stress responses.70 In CHO cells
expressing cloned NCX1, the effect of ATP depletion can be
partially mimicked by the action of cytochalasin D, an agent
that enhances depolymerization of actin microfilaments.69
Furthermore, NCX1 protein has previously been shown to
bind to the cytoskeletal protein ankyrin.71 Therefore, ATP
depletion may inhibit NCX activity by changing its membrane anchorage because of disruption of the submembrane
cytoskeleton. However, in rat adult cardiomyocytes, cytochalasin D was reported not to affect the NCX activity significantly.72 The effect of ATP depletion is absent or greatly
reduced in cells expressing an NCX1 mutant lacking a large
portion of the central hydrophilic loop48,69 or a loss-offunction mutant of the XIP or Ca2⫹-regulatory segment in the
same loop51 (see below), suggesting an important role for this
loop in regulation by ATP depletion.
Collins et al,55 Hilgemann et al,56 and He et al73 observed
that millimolar ATP markedly activates NCX currents in
giant membrane patches excised from guinea pig ventricular
myocyte blebs or oocytes expressing cloned NCX1. This
activating effect of ATP is mimicked by exogenous PIP2
applied to the cytoplasmic surface of the patch, but it is
attenuated by procedures that reduce the effective PIP2 level
in the patch, such as treatment with anti-PIP2 antibody.73,74
Thus, the ATP-dependent NCX activation is most likely due
to the generation of PIP2 from its precursors in excised
patches. This situation resembles that of ATP-sensitive K⫹
(KATP) channels, in which ATP usually suppresses channel
activity with a Ki of ⬇1 mmol/L in intact cells.75 In giant
patches excised from oocytes expressing cloned KATP channels, the Ki for ATP is as low as ⬇10 ␮mol/L, which
increases to a normal value when PIP2 is added to the
cytoplasmic surface of the patch. Therefore, the PIP2 level is
low in excised giant patches, and the resting endogenous level
of PIP2 is likely to be required to maintain the physiological
ATP sensitivity of KATP channels in intact cells.75 It is
possible that endogenous PIP2 plays a similar constitutive role
in cardiomyocytes, thus maintaining NCX activity at a high
level. However, because PIP2 exerts a strong regulatory
influence on NCX activity in excised patches, changes in
levels of PIP2 or in other acidic phospholipids in myocytes in
response to stimuli may contribute to the regulation of NCX
activity in intact cells. This requires further clarification.
Besides being transport substrates, intracellular Ca2⫹ and
Na⫹ exert important modulatory effects on NCX activity.56,58,76,77 Both the Ca2⫹-influx and Ca2⫹-efflux modes of
NCX1 are activated only when regulatory intracellular Ca2⫹
binds to a high-affinity site located in the central hydrophilic
loop of NCX135,78 (Figure 2A). For this intracellular Ca2⫹–
dependent activation, K1/2 values of 0.1 to 0.4 ␮mol/L have
been obtained by measuring the peak of the outward exchange current in excised membrane patches without ATP
(giant patches excised from myocyte blebs,56 oocytes expressing cloned NCX1,35,79 and large inside-out “macro-
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patches” excised from intact myocytes36). In contrast, a much
smaller K1/2 of 22 nmol/L was obtained by Miura and
Kimura45 for activation of the whole-cell outward exchange
current in guinea pig myocytes. It is possible that the excised
patch experiments without ATP could have produced overestimates of K1/2, because high ATP concentrations have been
shown to reduce K1/2 values in excised bleb patches55 or in
dialyzed squid axons58 (see Table). In a recent study, Weber
et al80 examined the effect of changing [Ca2⫹]i on NCX
activity in voltage-clamped intact cardiomyocytes while
monitoring the bulk [Ca2⫹]i. They reported that NCX activity
in ferret myocytes is regulated by a physiological range of
[Ca2⫹]i with a K1/2 of 0.125 ␮mol/L, although similar regulation is not detected in mouse myocytes at [Ca2⫹]i ⬎0.1
␮mol/L. It has previously been observed that NCX1 is
capable of extruding Ca2⫹ from resting rabbit7 or guinea pig40
cardiomyocytes. On the other hand, Haworth and Goknur,81
who analyzed the beat-dependent activation of 22Na flux in
isolated adult rat cardiomyocytes, have suggested that NCX1
is activated predominantly at a [Ca2⫹]i above the resting level.
Overall, NCX appears to exhibit relatively low activity in
intact myocytes at rest, with further activation occurring at a
higher physiological [Ca2⫹]i. In addition, there might be some
species-specific differences in this regulation.80
In the presence of extracellular Ca2⫹ and regulatory intracellular Ca2⫹, a high [Na⫹] applied to the cytoplasmic surface
of inside-out excised patches rapidly activates the exchanger,
followed by an inactivation process in which the exchange
current slowly decays to a steady state77 (see Figure 1B). This
process, called intracellular Na⫹– dependent inactivation, is
suggested to occur when the transport sites in NCX1 are fully
loaded with Na⫹ from the cytoplasmic side. An analysis of
exchange current noise suggests that this process is a manifestation of the relatively slow (t1/2 1 to 5 seconds) conformational transition between the active and inactive states of
the exchanger that occurs during Na⫹-Ca2⫹ exchange.82 Intracellular Na⫹– dependent inactivation is influenced by a
variety of factors; it is enhanced by high intracellular protons
(at low pHi)77,83 but attenuated by micromolar intracellular
Ca2⫹ (K1/2 ⬇2 ␮mol/L), millimolar ATP, or PIP2.56,73,74
Furthermore, this inactivation process and its modulation by
the above factors are all abolished when the intracellular
surface of the NCX protein is partially digested with a
proteolytic enzyme,84 suggesting that the large cytoplasmic
loop is involved in the exchanger inactivation. Likewise,
treatment of the NCX1 protein with redox reagents (dithiothreitol and FeSO3) results in marked stimulation of NCX
activity.85 A recent study has provided evidence that redox
reagents enhance NCX activity mainly by attenuating the
intracellular Na⫹– dependent inactivation.86
In whole-cell patch-clamp measurements, NCX activity
can be modulated by an inactivation process similar to the
intracellular Na⫹– dependent inactivation observed in excised
giant patches.87 Therefore, an intriguing question is to what
extent the activity of NCX1 is modulated by regulatory
intracellular Ca2⫹ and Na⫹ in beating cardiomyocytes, in
which [Ca2⫹]i oscillates between ⬇0.1 and ⬇1.0 ␮mol/L with
some variation in [Na⫹]i around ⬇10 mmol/L.2,6 The activation and deactivation of NCX activity by regulatory intracel-
lular Ca2⫹ per se appear to be very fast, because the inward
NCX currents can be turned on or off by the addition or
removal of intracellular Ca2⫹ within the time needed for fast
solution change.35,36 On the other hand, the intracellular
Ca2⫹– dependent modulation of the steady-state outward
NCX current evoked under a high [Na⫹]i (see Figure 1B) in
giant excised cardiac bleb or oocyte patches exhibits much
slower kinetics (t1/2 5 to 10 seconds).35,56 However, recent
experiments using macropatches excised from intact cardiomyocytes36 have shown that deactivation and activation of the
steady-state outward NCX currents in 50 mmol/L intracellular Na⫹ by removal and readdition (to 5 ␮mol/L) of intracellular Ca2⫹ occur in fast and slow phases, with an overall t1/2 of
⬇0.5 and ⬇0.4 seconds, respectively. These kinetics are
much faster than those obtained from giant excised patches.35,56 Therefore, at the physiological [Na⫹]i, NCX activity is
likely to be modulated on a beat-to-beat basis by intracellular
Ca2⫹– dependent modulation. This interpretation is consistent
with a recent demonstration of very fast induction of outward
NCX current in the intact ferret myocytes when the latter are
exposed to caffeine.80 However, it is not clear to what extent
a normal level of intracellular Na⫹ causes intracellular Na⫹–
dependent inactivation and how fast physiological levels of
intracellular Ca2⫹ deactivate this intracellular Na⫹ effect in
beating myocytes.
The steady-state exchange activity of NCX1 exhibits a
pronounced pHi dependence, increasing monotonically from
a near-zero activity at pHi 6 to a high activity at pHi 9.83,88 In
myocardial ischemia/reperfusion, the intracellular Na⫹–
dependent inactivation of NCX activity due to a prevailing
high intracellular Na⫹, low ATP, and/or low pHi level may be
beneficial, because the resulting decrease of NCX-mediated
Ca2⫹ entry would reduce Ca2⫹ overloading and myocyte
damage.12 Consistent with this idea, cells expressing an
NCX1 mutant exhibiting no intracellular Na⫹– dependent
inactivation are highly sensitive to cell damage caused by
intracellular Na⫹– dependent Ca2⫹ overloading.51 In contrast,
reperfusion after a prolonged period of ischemia is known to
be associated with a burst of oxygen-derived free radical
production.89 Goldhaber90 has provided evidence that
oxygen-derived free radicals enhance Na⫹-Ca2⫹ exchange in
intact rabbit ventricular myocytes. Because redox reagents
activate NCX activity primarily by attenuating the intracellular Na⫹-dependent inactivation (see above), free radicals
generated during reperfusion after prolonged myocardial
ischemia may enhance NCX activity, promoting intracellular
Ca2⫹ overload and triggering arrhythmia. The molecular
mechanism of free radical–induced activation of NCX is
currently unclear.
External alkali metal ions, such as Na⫹, K⫹, or Li⫹,
increase the Vmax of NCX activity up to 2- to 3-fold with low
affinity91–94 (K1/2 several tens of mmol/L for NCX1 in the case
of Li⫹). These cations bind to a site(s) that is distinct from the
transport sites, and they are not transported by the exchanger.91,94 Under physiological conditions, Na⫹ functions as both
the transport substrate and an activator of NCX activity. In
squid axons, intracellular alkali metal cations also stimulate
NCX activity, but this seems to require the simultaneous
presence of an external alkali metal cation.91 How these
Shigekawa and Iwamoto
Naⴙ-Ca2ⴙ Exchange
869
cations regulate the transport properties of the exchanger is
not clear. On the other hand, Egger and Niggli95 have
reported an interesting effect of extracellular protons on the
NCX transport property in guinea pig cardiomyocytes; at pHo
5 or 6, the inward NCX current is strongly inhibited, whereas
the corresponding rate of extracellular Na⫹– dependent Ca2⫹
extrusion is decreased only weakly. Thus, extracellular protons appear to modify the electrogenicity or stoichiometry of
Na⫹-Ca2⫹ exchange.
Structure and Function
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The cardiac NCX1 consists of 970 amino acids with a
molecular mass of 110 kDa.19 During its biosynthesis, a
signal sequence of 32 amino acids in the N-terminus is
cleaved off to yield a mature protein.96 On SDS-PAGE under
a reducing condition, the mature NCX1 protein often appears
as 2 bands with molecular masses of 120 and 70 kDa (and a
faint 140- to 160-kDa band). The 70-kDa component is
generally considered to be a proteolytic fragment of the
120-kDa protein.97 Approximately half of the NCX1 protein
constitutes a transmembrane domain, whereas the remaining
half (⬇550 amino acids) forms a large domain exposed on the
cytoplasm. The latter domain does not appear to be required
for the transport function of NCX1, because a mutant lacking
most of it (⌬240-679) still retains exchange activity.84 Recent
topological studies98 –100 suggest that the mature NCX1 protein comprises 9 transmembrane segments (TMs) and a large
hydrophilic loop between TMs 5 and 6, with N- and
C-termini located on the external and internal sides, respectively (see the model in Figure 2A). The N-terminal and
C-terminal halves of the membrane domain contain 2 internal
repeat sequences of ⬇40 amino acids, which are designated
the ␣-1 and ␣-2 repeats.101 These repeat sequences are
conserved in all members of the NCX family as well as in
related cation exchangers,16,101 suggesting the functional
importance of these segments.
In the NCX1 protein, the ␣-1 repeat consists of portions of
putative TM2 and TM3 and a loop connecting these TMs,
whereas the ␣-2 repeat consists of a portion of putative TM7
and the C-terminal non-TM segment. Recent substituted
cysteine scanning analyses98,102 have provided evidence suggesting that the loop of the ␣-1 repeat and the non-TM
segment of the ␣-2 repeat together with its C-terminal
neighboring region form reentrant membrane loops originating from the opposite sides of the membrane, respectively
(Figure 2A). Cysteine substitutions at many residues in these
loop regions render the exchanger sensitive to inhibition by
externally or internally applied membrane-impermeable sulfhydryl reagents, suggesting that these residues might be
exposed on the ion pathway.98,102 On the other hand, Qiu et
al,103 using cysteine mutagenesis with disulfide cross-linking,
have shown that the same interface of TM7 is close to TM2
on the extracellular side but is adjacent to TM3 near the
intracellular side of the membrane and that TM2 adjoins
TM8. Thus, the ␣-1 and ␣-2 repeats and their loop regions are
most likely in proximity within the membrane (Figure 2B).
Site-directed mutagenesis studies have permitted the identification of a number of amino acid residues in the ␣ repeats
whose mutations significantly alter the transport properties of
Figure 2. Topological models for cardiac Na⫹-Ca2⫹ exchanger.
A, Putative transmembrane helices are indicated by cylinders
with arabic numbers. N and C indicate N- and C-terminals,
respectively. B, Model for helix packing of TMs 2, 3, 7, and 8
suggested by cross-linking data103 is shown.
cardiac NCX1. When 45Ca2⫹ uptake activity was measured in
Xenopus oocytes expressing mutants of carboxyl- or
hydroxyl-containing amino acid residues within putative TMs
2, 3, and 7, many of them exhibited no or only low NCX
activity.104 Furthermore, mutation of conserved glycines
(Gly138 and Gly837) alters the slope of the current-voltage
relationship of NCX1. Mutation of Thr103 at the cytoplasmic
portion of TM2 increases the apparent affinity of NCX1 for
the substrate intracellular Na⫹ and also seems to produce Li⫹
transport capacity, suggesting alteration in the ionic selectivity of the exchanger.105 On the other hand, the putative TMs
4 and 5 contain regions of similarity to the Na⫹,K⫹-ATPase
and SR Ca2⫹-ATPase, and mutation of Glu199 or Thr203 in
TM5 results in the loss of NCX activity.104 Glu199 of NCX1
corresponds to Glu309 of SR Ca2⫹-ATPase, which, as revealed in the high-resolution 3D structure of SR Ca2⫹ATPase, is one of the residues directly liganding the transported Ca2⫹ in the membrane.106
In the putative loop regions of the ␣ repeats in cardiac
NCX1, mutations of 3 conserved aspartic acids (Asp130,
Asp825, and Asp829) result in up to 6-fold reduction in the
apparent affinity for the substrate, extracellular Ca2⫹.102
Furthermore, mutations of other residues in the ␣ repeat loop
regions (Asn125, Thr127, and Val820) render the exchanger
up to 8-fold less sensitive to inhibition by external Ni2⫹,107 a
competitive inhibitor for the transport substrate, extracellular
Ca2⫹.93 Similar studies have resulted in the identification of
Val820, Gln826, and Gly833 in the ␣-2 repeat loop, whose
mutations alter the apparent affinity for 2-[2-[4-(4nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate
870
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May 11, 2001
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(KB-R7943)108 (see below). Mutation at Gly833 renders
NCX1 almost insensitive to inhibition by KB-R7943. Furthermore, simultaneous mutations of Val820 and Gln826 alter
the extent of stimulation of NCX activity by external Li⫹.107
Thus, interactions of NCX1 with the transport substrate
(extracellular Ca2⫹), inhibitors (Ni2⫹ and KB-R7943), and an
activator (Li⫹) are significantly influenced by the mutation of
residues in the ␣ repeat loops, although some of the residues
identified do not appear to interact directly with ions and
modifiers, because the observed effects of mutations are mild.
Taken together, these data suggest that both the TMs and loop
regions of the ␣-1 and ␣-2 repeats participate in the formation
of the ion translocation pathway in NCX1.
At present, however, little is known about the detailed
structure of the NCX1 molecule, in particular, the ion-binding
sites and the shape and dimensions of the ion transport
pathway, the requirement of the oligomeric protein structure
for the function, and changes in the conformation of the
exchanger associated with ion transport. Recently, the lowresolution 3D structure of the Na⫹-H⫹ exchanger NhaA from
Escherichia coli has been solved, which reveals that it has a
highly asymmetric molecular organization comprising 12
TMs and exists as dimer.109 Therefore, the structure of NCX1
does not seem to be similar to NhaA. The unique topology
and functional importance of the ␣-1 and ␣-2 repeats of
NCX1 are rather reminiscent of a somewhat similar structural
feature reported for the water channel aquaporin-1, in which
2 prominent loop regions with functionally important residues penetrate the membrane from opposite sides, thereby
forming part of the pore.110
As noted above, mild proteolysis of the internal surface of
the exchanger greatly stimulates its activity and eliminates
regulation by intracellular Ca2⫹, intracellular Na⫹, protons,
PIP2, and ATP. Similarly, an NCX1 mutant with deletion of
a large portion of the central cytoplasmic loop (⌬240-679) is
not regulated by intracellular Ca2⫹, intracellular Na⫹,84 or
PKC.48 Thus, the large cytoplasmic loop is most likely to be
involved in the regulation of NCX activity. At the N-terminal
end of this loop near the membrane-lipid interface, there is a
20 –amino acid segment, designated the XIP region, whose
sequence is rich in both basic and hydrophobic residues,19 as
in the calmodulin-binding domain (see Figure 2A). This
region, to which calmodulin does not seem to bind strongly,111 is considered to play a pivotal role in the regulation of
NCX activity (see below). On the other hand, C-terminal to
the XIP region, there is a region of ⬇135 amino acids (amino
acids 371 to 508) containing 2 conserved clusters of acidic
amino acids. This 135–amino acid region, when expressed as
a fusion protein and assayed directly, binds 45Ca2⫹ with high
affinity.78 NCX1 mutants carrying mutations within the acidic
clusters exhibit markedly lowered affinity for regulatory
intracellular Ca2⫹, suggesting that Ca2⫹ binding to this region
is responsible for intracellular Ca2⫹– dependent regulation of
NCX activity.35
In the large cytoplasmic loop of the NCX1 protein, there
are two ⬇70 –amino acid internal repeat motifs, designated
the ␤ repeats, which are conserved in the NCX family.101
Although the functions of these sequences are not clear, the
␤-1 repeat almost overlaps the N-terminal portion of the
Ca2⫹-regulatory site, which is reportedly required for highaffinity 45Ca2⫹ binding.78 The ␤-2 repeat is located on the
C-terminal side of the Ca2⫹-regulatory site. A recent study of
tryptic digestion of scallop membranes has shown that limited
proteolysis occurs at both ends of the Ca2⫹-regulatory site,
with the C-terminal end cleaved only in the absence of
Ca2⫹.112 Thus, the Ca2⫹-regulatory site and its C-terminal
neighboring region containing the ␤-2 repeat may form a
folded structure, and Ca2⫹ removal from the regulatory site
appears to induce a large conformational change in these
regions. A substantial conformational change associated with
Ca2⫹ binding to a fusion protein containing the Ca2⫹regulatory site was also detected as a large mobility shift
during SDS-PAGE.78 Finally, there is a region near the
C-terminal end of the cytoplasmic domain in which alternative splicing involving 6 small exons occurs in a tissuespecific manner.22,23
As described above, NCX1 is inactivated when regulatory
intracellular Ca2⫹ is removed. Similarly, intracellular Na⫹–
dependent inactivation at a high [Na⫹]i generates an almost
fully inactive exchanger state in the absence of ATP or at low
pHi. Although the underlying mechanism(s) that gives rise to
such an inactivated state is not clear, the endogenous XIP
region may play a critical role in the process. First, a synthetic
peptide having the same sequence as the endogenous XIP
region completely inactivates NCX activity (Ki ⬇0.1 ␮mol/L)
when applied from the intracellular side of an excised giant
patch.111 This peptide is also an effective inhibitor of the
whole-cell outward exchange current in ventricular myocytes.113 Second, mutations of the XIP region eliminate or
accelerate the intracellular Na⫹– dependent inactivation of the
exchange activity as measured by using excised oocyte giant
patches.79 Mutations in the XIP region that abolish intracellular Na⫹– dependent inactivation cause loss of responsiveness to modulation by ATP, PIP2, or PIP2 antibody.114
Furthermore, cells expressing an XIP mutant exhibiting no
intracellular Na⫹– dependent inactivation do not respond to
inhibition by ATP depletion or to activation of PKC by
phorbol ester.51 All of these results are consistent with the
hypothesis that the XIP region functions as an autoinhibitory
domain that plays a central role in the activation and
inactivation of NCX activity. The receptor site interacting
with XIP in NCX1 has not yet been identified.
Modulations of NCX activity by intracellular Ca2⫹ and Na⫹
appear to be complex processes involving multiple regions of
the exchanger molecule. The regulatory effects of both ions
are abolished by deletion of a small segment (⌬680-685) of
NCX1 near the C-terminal end of the large cytoplasmic
loop.115 Furthermore, wild-type NCX1s with different splicing patterns in the region located upstream from the abovedeleted residues (Figure 2A) exhibit altered kinetics of
regulation by intracellular Na⫹ or Ca2⫹.24,25 In addition, some
mutations within the XIP region, which primarily affect
intracellular Na⫹– dependent inactivation, significantly reduce the apparent affinity for regulatory intracellular Ca2⫹.79
Conversely, intracellular Ca2⫹-regulatory site mutations attenuate intracellular Na⫹– dependent inactivation.35 Therefore, it appears that structural integrity of the large cytosolic
loop is required for the transduction of ion-binding signals.
Shigekawa and Iwamoto
On the other hand, cysteine substitution of Asn101, modeled
to be localized near the cytosolic interface between TM2 and
the first intracellular loop, renders the exchanger insensitive
to regulation by intracellular Ca2⫹ or Na⫹.105 Thus, a region(s)
of the NCX1 protein other than the large cytoplasmic loop
may also be involved in the transduction of ion-binding
signals, which transmit regulatory information on transport
by influencing the TMs that catalyze ion translocation. In this
sense, CALX1, an NCX from Drosophila melanogaster, is
interesting in that activity of the wild-type exchanger is
inhibited by intracellular Ca2⫹, which is the opposite pattern
of intracellular Ca2⫹– dependent regulation.116 In CALX1,
Ca2⫹ binding probably occurs normally, but the binding
signal appears to be transduced differently.
Pharmacology
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Potent and selective blockers of NCX activity would be
extremely useful for clarifying the precise functions of NCX1
and other isoforms at the cell, organ, and whole-animal
levels, as well as for studying the molecular mechanism of
Na⫹-Ca2⫹ exchange. Currently, an NCX blocker is not clinically used. However, because NCX moves ions and charge in
either direction, mode-specific inhibitors of NCX, if available, may have high therapeutic potential. For example,
specific blocking of excessive Ca2⫹ influx via reverse-mode
NCX activity may reduce Ca2⫹ overload due to cardiac
glycoside toxicity or to ischemia and reperfusion in the heart
and other tissues. Increased Ca2⫹ influx via NCX is also
implicated in some forms of hypertension, particularly in
relation to an endogenous ouabain-like compound.14,117 In
heart failure, enhanced Ca2⫹ efflux due to upregulation of
NCX activity may impair the systolic function by reducing
the SR Ca2⫹ loading and is also directly implicated as a cause
of arrhythmia.17,18,118 On the other hand, excess Ca2⫹ influx
via NCX during the terminal phase of the action potential
plateau could contribute to abnormally slow relaxation in
failing myocytes.119 However, to what extent mode-specific
blockers of NCX may be beneficial in therapeutic control of
the overall function of a failing heart and/or other diseased
organs remains to be determined.
As blockers of NCX activity, many divalent and trivalent
cations, such as La3⫹, Ni2⫹, and Cd2⫹ (Table), and a variety of
organic compounds, including amiloride derivatives (eg,
dichlorobenzamil) or the substituted pyrrolidine ethanamine
(eg, bepridil), have long been known (see previous reviews14,120 and references therein). These cations and organic
agents also exert blocking effects on other cell systems
sometimes showing cardiovascular activities, which limits
their use as specific blockers of NCX activity. However, Ni2⫹
at up to 5 mmol/L is being used as an NCX blocker under
conditions in which other membrane currents sensitive to
Ni2⫹ are already suppressed by different agents. More recently, a few new NCX inhibitors, such as an isothiourea
derivative (KB-R7943, formerly No. 7943) and peptides and
their analogues with much improved selectivity for NCX,
have been developed.
KB-R7943 has been reported to be a potent and selective
inhibitor of NCX at low micromolar concentrations.121,122 Its
effect on NCX1 or NCX2 is 3-fold less potent than its effect
Naⴙ-Ca2ⴙ Exchange
871
on NCX3.93 It is an amphiphilic molecule with a positively
charged isothiourea group at neutral pH and is soluble at up
to 100 ␮mol/L in an aqueous buffer. Its inhibitory action is
relatively fast and washable. However, when cells are preincubated with KB-R7943 for a longer period (⬎2 to 3
minutes), more time is needed for its washout. Similarly, the
inhibitory potency of the drug seems to increase as the
preincubation time is lengthened.
Intriguingly, KB-R7943 exerts a preferential effect on
reverse-mode (Ca2⫹-influx mode) NCX activity,121–123 although such an effect disappeared under a certain condition.124 It inhibits intracellular Na⫹– dependent Ca2⫹ influx
into rat cardiomyocytes or some other NCX1-expressing cells
with an IC50 of 1.2 to 2.4 ␮mol/L, whereas it inhibits
extracellular Na⫹– dependent Ca2⫹ efflux from these cells
only weakly (IC50 ⬇30 ␮mol/L).121 On the other hand,
KB-R7943 inhibits whole-cell outward NCX currents from
ventricular myocytes or NCX1-transfected cells with an IC50
of 0.3 to 0.9 ␮mol/L,108,122,123 but it is much less potently
inhibitory to whole-cell inward NCX current (IC50 17 ␮mol/
L).122 Inhibition by KB-R7943 is reportedly noncompetitive
with extracellular Ca2⫹ or Na⫹121 of a mixed-type (competitive and noncompetitive with extracellular Ca2⫹)93 or competitive with extracellular Ca2⫹,122 suggesting that the mode
selectivity may be partly due to competition with extracellular Ca2⫹. However, the selective blocking of Ca2⫹ influx via
NCX is observed irrespective of the presence or absence of
extracellular Ca2⫹.121,123 Interestingly, the same mode selectivity as seen in KB-R7943 is seen in the inhibition of NCX
activity by Ni2⫹107 or some protein phosphatase inhibitors54
(see above), whereas the opposite selectivity has been reported for 3⬘,4⬘-dichlorobenzamil.122 Similarly, in cardiac
sarcolemmal vesicles, the XIP peptide111 and a Ca2⫹ channel
blocker, nicaldipine (0.1 to 10 ␮mol/L),125 depressed the rate
of Na⫹-dependent Ca2⫹ uptake much more potently than that
of Na⫹-dependent Ca2⫹ efflux. Although the data suggest a
significant difference in the properties of NCX1 in the
forward and reverse modes, the underlying molecular mechanism for this peculiar directional specificity is not clear.
Some progress has been made in elucidating the mechanism by which KB-R7943 acts. First, current evidence
strongly suggests that KB-R7943 inhibits NCX activity from
the external side in intact cells.108,122 When whole-cell outward NCX currents are measured, inhibition is not observed
if the drug is applied internally through a pipette solution.
Furthermore, rapid (⬍5-second) inhibition of the outward
NCX current is induced when the latter is evoked by external
application of both Ca2⫹ and KB-R7943.108 However, KBR7943 may be capable of inhibiting NCX activity from the
cytoplasmic side also, inasmuch as it inhibits NCX activity in
inside-out giant patches excised from oocytes expressing
NCX1.126 Second, structural determinants of KB-R7943 sensitivity in the exchanger have been sought by analyzing the
functions of chimeras between NCX1 and NCX3 and sitedirected mutants of NCX1108 (see above). The results suggest
that the highly conserved ␣-2 repeat of the exchanger is
exclusively responsible for the drug response and that mutation at Val820, Gln826, or Gly833 in the portion of the ␣-2
repeat forming the putative reentrant membrane loop (Figure
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2B) alters drug sensitivity. Mutation at Gly833 causes a
particularly large (ⱖ30-fold) reduction in KB-R7943 sensitivity. Considering the external side of the drug action in the
intact cell, the simplest interpretation of these data would be
that mutations of above residues alter the conformation of the
external drug-binding site, thereby influencing its affinity for
the drug. However, the possibility remains that Gly833 is part
of the KB-R7943 receptor.108
KB-R7943 at up to 30 ␮mol/L has little effect on the
Na⫹-Ca2⫹-K⫹ exchanger,108 the Na⫹-H⫹ exchanger, sarcolemmal Ca2⫹-ATPase, SR Ca2⫹-ATPase, or Na⫹,K⫹-ATPase.121
On the other hand, the drug has been reported to inhibit
voltage-sensitive Na⫹ currents, L-type Ca2⫹ currents, and
inward rectifier K⫹ currents with IC50s of 14, 8, and ⬇7
␮mol/L, respectively, in guinea pig cardiomyocytes.122 However, the ramp pulse protocol used in these latter measurements appears to have overestimated the effect of KB-R7943
(see pertinent study122). In rat ventricular myocytes, 5 ␮mol/L
KB-R7943 does not alter steady-state twitches, Ca2⫹ transients, Ca2⫹ load in the SR, or rest potentiation, but it
prolongs the late low plateau of the action potential, suggesting modest inhibition of K⫹ currents.123 In guinea pig
papillary muscle, however, KB-R7943 at up to 10 ␮mol/L
does not significantly affect the resting membrane potential
or various action potential parameters.121,127 Similarly, the
spontaneous beating rate and developed tension are not
affected by 10 or 30 ␮mol/L KB-R7943 in isolated guinea pig
atria.128 Thus, KB-R7943 at ⬍5 ␮mol/L could be used as a
fairly selective blocker for reverse-mode NCX activity in
isolated cardiomyocytes. However, in other cell types, such
as bovine adrenal chromaffin cells129 and rat hippocampal
neurons,130 KB-R7943 has recently been reported to inhibit
neuronal nicotinic acetylcholine receptors (IC50 0.3 to 6.5
␮mol/L) and N-methyl-D-aspartate receptor channels (2 IC50s
0.8 and 11 ␮mol/L), respectively, although the latter is in
contradiction with another report.131 Furthermore, storeoperated Ca2⫹ entry into cultured neurons and astrocytes is
significantly inhibited by 10 ␮mol/L KB-R7943.132 All these
possible side effects need to be taken into account if KBR7943 is to be used as an NCX blocker.
Despite its side effects, several pharmacological actions of
KB-R7943 provide information on the role of reverse-mode
NCX activity. First, the fact that, as noted above, 5 ␮mol/L
KB-R7943 has no effect on normal Ca2⫹ transients and
contractions123 suggests that Ca2⫹ influx via NCX is not
important for physiological excitation-contraction coupling,
at least in rat cardiomyocytes. Second, blocking the Na⫹-K⫹
pump by cardiac glycosides increases [Na⫹]i, thereby inducing positive inotropy as well as toxic myocyte Ca2⫹ overload.13 In rat ventricular myocytes treated with 50 ␮mol/L
strophanthidin, 5 ␮mol/L KB-R7943 suppressed glycoside
toxicity but preserved positive inotropy.123 Similar effects by
30 ␮mol/L KB-R7943 were observed in spontaneously beating isolated guinea pig atria pretreated with 3 ␮mol/L
ouabain.128 These data are interpreted by Satoh et al123 as
suggesting that the reduction of Ca2⫹ efflux via NCX due to
competition with elevated [Na⫹]i causes the inotropic effect,
whereas the net Ca2⫹ entry via NCX is responsible for the
generation of glycoside toxicity. Third, reverse-mode NCX
activity has been implicated as the cause of Ca2⫹ overload
associated with cardiac ischemia/reperfusion.12 Recent studies have provided evidence that KB-R7943 at 3 to 20 ␮mol/L
is effective in reducing cytosolic Ca2⫹ and Na⫹ overload, cell
injury, and arrhythmias that are associated with ischemia/
reperfusion, the Ca2⫹ paradox, and substrate-free hypoxia/
reoxygenation in different types of cardiac preparations (rat
cardiomyocytes,121,133 guinea pig papillary muscle,127 and
Langendorff-perfused rat hearts133,134). KB-R7943 has also
been reported to be significantly protective against anoxia/
reoxygenation or ischemia/reperfusion damage in brain135
and kidney.136
Some synthetic peptides are also effective NCX inhibitors.
The XIP peptide derived from the primary sequence of
cardiac NCX1 (see above) decreases the Vmax of NCX
activity.111 As noted earlier, it is significantly less potent in
inhibiting Na⫹-dependent Ca2⫹ efflux from sarcolemmal vesicles than in inhibiting the reverse reaction. XIP has little
effect on Na⫹,K⫹-ATPase, SR Ca2⫹-ATPase, or L-type Ca2⫹
currents, and it does not increase membrane conductance
when applied to the intracellular surface by use of the
excised-patch technique.111,113 However, it may bind calmodulin and could thus interfere with the function of calmodulin-binding proteins. Furthermore, its usefulness as an NCX
inhibitor is limited because it acts only from the cytoplasmic
side.
Other peptides, such as the molluscan cardioexcitatory
tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFa) and its analogues and the cyclic hexapeptide Phe-Arg-Cys-Arg-CysPhe-CONH2 (FRCRCFa), which are much smaller than XIP,
have been reported to inhibit NCX activity.137,138 FMRFa and
its related peptides inhibit the NCX activity of cardiac
sarcolemmal vesicles, with IC50s ranging from 1 to 1000
␮mol/L. On the basis of structure/activity studies of these
peptides, a new cyclic peptide, FRCRCFa, with an intramolecular disulfide bond has been synthesized. FRCRCFa exhibits improved inhibitory potency and resistance to proteolytic degradation, and in sarcolemmal vesicles it inhibits
NCX activity completely, with an IC50 of 2 to 10 ␮mol/L
without competing with extravesicular Ca2⫹ and Na⫹.138 In
the rabbit ventricular myocyte, FRCRCFa inhibits whole-cell
NCX currents much more potently, with an IC50 of 0.023
␮mol/L, and exhibits a rapid onset of action.139 Furthermore,
it reportedly has no effect on L-type Ca2⫹ channels or delayed
rectifier and inward rectifier K⫹ channels. Thus, FRCRCFa
appears to have several advantages over XIP. This peptide
acts from the intracellular side, but its binding site has not
been identified.
Concluding Remarks
NCX is essential for maintaining Ca2⫹ homeostasis in cardiomyocytes. Despite the great advances that have been made, it
is evident from this survey that we are still far from a detailed
understanding of the functions of cardiac NCX1, and the roles
and activities of other NCX isoforms remain totally unknown.
NCX may be regulated in situ by multiple fast and slow
signaling mechanisms. Features requiring further exploration
are as follows: the parts played by protein kinases, a possible
regulatory protein factor(s), and acidic phospholipids, such as
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PIP2; the roles of intracellular Ca2⫹ and Na⫹ in the beat-tobeat regulation of NCX activity; and the functional significance of altered expression of NCX activity in heart diseases.
At the molecular level, high-resolution structural analysis and
biophysical and biochemical studies of structural changes in
the NCX molecule associated with ion transport must be
actively pursued. Genetically modified animal models expressing excess or reduced levels of NCX activity (eg, Ncx⫹/⫺
mice) or expressing NCX mutants are particularly useful in
elucidating the consequences of modification of the NCX
function in physiological, pathological, and pharmacological
contexts, although differences among species may confuse
the data interpretation. Cardiac overexpression of normal
NCX1 (up to ⬇200%) or of an NCX1 mutant devoid of
intracellular Ca2⫹– and intracellular Na⫹– dependent regulation in transgenic mice has been reported to produce a
relatively normal myocyte function, except that Ca2⫹ fluxes
via NCX and the SR Ca2⫹ content are increased in the former
mice, and rest potentiation is substantially enhanced in
papillary muscle from the latter.16,115 Finally, highly potent
and selective new drugs targeting NCXs are being developed.
The extent to which these can be of clinical benefit remains
to be seen.
12.
13.
14.
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16.
17.
18.
19.
20.
21.
22.
23.
Acknowledgments
This study was supported by grants-in-aid for scientific research
(Nos. 10470013 and 12670102) from the Ministry of Education,
Culture, Sports, Science, and Technology, a Research Grant for
Cardiovascular Diseases (11-C) from the Ministry of Health, Labor,
and Welfare, and grants from the Uehara Memorial Foundation and
the Cardiovascular Research Foundation. We thank Drs Satoshi
Matsuoka and Shigeo Wakabayashi for helpful discussions and
critically reviewing the manuscript.
24.
25.
26.
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Cardiac Na+-Ca2+ Exchange : Molecular and Pharmacological Aspects
Munekazu Shigekawa and Takahiro Iwamoto
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Circ Res. 2001;88:864-876
doi: 10.1161/hh0901.090298
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