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(Circulation Research. 2001;88:864.)
© 2001 American Heart Association, Inc.

Review

Cardiac Na⁺-Ca²⁺ Exchange

Molecular and Pharmacological Aspects

Munekazu Shigekawa, Takahiro Iwamoto

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 shigekaw{at}ri.ncvc.go.jp

	Abstract

Top Abstract Introduction Cardiac NCX Regulation Structure and Function Pharmacology Concluding Remarks References

 
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.

Key Words: Na⁺-Ca²⁺ exchange • Ca²⁺ transport • cardiac muscle • excitation-contraction coupling

	Introduction

Top Abstract Introduction Cardiac NCX Regulation Structure and Function Pharmacology Concluding Remarks References

 
The 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 8 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 15 16} and short comments.^{17 18}

	Cardiac NCX

Top Abstract Introduction Cardiac NCX Regulation Structure and Function Pharmacology Concluding Remarks References

 
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 membrane^{7 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 K_m for intracellular Ca²⁺ (6 µmol/L)^{34 35 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 µmol/L for Ca²⁺ affinity^{5 38} ). The density of NCX1 in the sarcolemma has been estimated to be 250 to 400 NCX/µm² 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 43 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 35 36 45} Interaction of NCX1 with these ions is intrinsically asymmetric in that the apparent affinity for 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}

View larger version (21K): [in this window] [in a new window]   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 µmol/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.

	Regulation

Top Abstract Introduction Cardiac NCX Regulation Structure and Function Pharmacology Concluding Remarks References

 
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 NCX1^{46 47 48} as well as in sarcolemmal vesicles isolated from rat heart.⁴⁹ The effects of agonists are mimicked by phorbol ester^{46 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 NCX1-transfected 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

View this table: [in this window] [in a new window]   Table 1. Factors Influencing Na+-Ca2+ Exchange

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 PKC-dependent 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 loop^{48 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

View larger version (25K): [in this window] [in a new window]   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.

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, ß-receptor–dependent or cAMP-dependent 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 (K_1/2 >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 Beaugé^{57 58} have accumulated a large body of data supporting the involvement of protein phosphorylation in the ATP-dependent 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 V_max and also increases the affinity for the regulatory intracellular Ca^{2+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 ß-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-ß₁ and interleukin-1ß stimulate and inhibit NCX1 gene expression, respectively, and PKC inhibitors significantly reduce both the basal and transforming growth factor-ß₁–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 66 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 ATP-dependent 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 depletion 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 4,5-bisphosphate (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 loop^{48 69} or a loss-of-function 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⁺ (K_ATP) channels, in which ATP usually suppresses channel activity with a K_i of 1 mmol/L in intact cells.⁷⁵ In giant patches excised from oocytes expressing cloned K_ATP channels, the K_i for ATP is as low as 10 µmol/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 K_ATP 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 NCX1^{35 78} (Figure 2A). For this intracellular Ca²⁺–dependent activation, K_1/2 values of 0.1 to 0.4 µmol/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 "macropatches" excised from intact myocytes³⁶ ). In contrast, a much smaller K_1/2 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 K_1/2, because high ATP concentrations have been shown to reduce K_1/2 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 K_1/2 of 0.125 µmol/L, although similar regulation is not detected in mouse myocytes at [Ca²⁺]_i >0.1 µmol/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 (t_1/2 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 pH_i)^{77 83} but attenuated by micromolar intracellular Ca²⁺ (K_1/2 2 µmol/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 µmol/L with some variation in [Na⁺]_i around 10 mmol/L.^{2 6} The activation and deactivation of NCX activity by regulatory intracellular 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 (t_1/2 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 µmol/L) of intracellular Ca²⁺ occur in fast and slow phases, with an overall t_1/2 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 pH_i dependence, increasing monotonically from a near-zero activity at pH_i 6 to a high activity at pH_i 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 pH_i 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 V_max of NCX activity up to 2- to 3-fold with low affinity^{91 92 93 94} (K_1/2 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 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 pH_o 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

Top Abstract Introduction Cardiac NCX Regulation Structure and Function Pharmacology Concluding Remarks References

 
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 studies^{98 99 100} 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 analyses^{98 102} 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 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-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (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 low-resolution 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 ß repeats, which are conserved in the NCX family.¹⁰¹ Although the functions of these sequences are not clear, the ß-1 repeat almost overlaps the N-terminal portion of the Ca²⁺-regulatory site, which is reportedly required for high-affinity ⁴⁵Ca²⁺ binding.⁷⁸ The ß-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 ß-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 tissue-specific 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 pH_i. 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 (K_i 0.1 µmol/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 above-deleted 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. 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

Top Abstract Introduction Cardiac NCX Regulation Structure and Function Pharmacology Concluding Remarks References

 
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 reviews^{14 120} 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 on NCX3.⁹³ It is an amphiphilic molecule with a positively charged isothiourea group at neutral pH and is soluble at up to 100 µmol/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,^{121 122 123} 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 µmol/L, whereas it inhibits extracellular Na⁺–dependent Ca²⁺ efflux from these cells only weakly (IC₅₀ 30 µmol/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 µmol/L,^{108 122 123} but it is much less potently inhibitory to whole-cell inward NCX current (IC₅₀ 17 µmol/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 µmol/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, KB-R7943 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 site-directed 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 2B) alters drug sensitivity. Mutation at Gly833 causes a particularly large (30-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 µmol/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 µmol/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 µmol/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 µmol/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 µmol/L KB-R7943 in isolated guinea pig atria.¹²⁸ Thus, KB-R7943 at <5 µmol/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 µmol/L) and N-methyl-D-aspartate receptor channels (2 IC₅₀s 0.8 and 11 µmol/L), respectively, although the latter is in contradiction with another report.¹³¹ Furthermore, store-operated Ca²⁺ entry into cultured neurons and astrocytes is significantly inhibited by 10 µmol/L KB-R7943.¹³² All these possible side effects need to be taken into account if KB-R7943 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 µmol/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 µmol/L strophanthidin, 5 µmol/L KB-R7943 suppressed glycoside toxicity but preserved positive inotropy.¹²³ Similar effects by 30 µmol/L KB-R7943 were observed in spontaneously beating isolated guinea pig atria pretreated with 3 µmol/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 µmol/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 hearts^{133 134} ). 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 V_max 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-Cys-Phe-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 µmol/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 µmol/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 µmol/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

Top Abstract Introduction Cardiac NCX Regulation Structure and Function Pharmacology Concluding Remarks References

 
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 PIP₂; the roles of intracellular Ca²⁺ and Na⁺ in the beat-to-beat 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.

	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.

	Footnotes

 
Original received February 22, 2001; revision received March 15, 2001; accepted March 16, 2001.

	References

Top Abstract Introduction Cardiac NCX Regulation Structure and Function Pharmacology Concluding Remarks References

 

1. Bridge JHB, Smolley JR, Spitzer KW. The relationship between charge movements associated with I_Ca and I_Na-Ca in cardiac myocytes. Science. 1990;248:376–378.[Abstract/Free Full Text]
2. Bers DM. Calcium fluxes involved in control of cardiac myocyte contraction. Circ Res. 2000;87:275–281.[Free Full Text]
3. Sham JSK, Hatem SN, Morad M. Species differences in the activity of the Na⁺-Ca²⁺ exchanger in mammalian cardiac myocytes. J Physiol. 1995;488:623–631.[Medline] [Order article via Infotrieve]
4. Hasenfuss G. Alterations of calcium-regulatory proteins in heart failure. Cardiovasc Res. 1998;37:279–289.[Free Full Text]
5. Hove-Madsen L, Bers DM. Sarcoplasmic reticulum Ca²⁺ uptake and thapsigargin sensitivity in permeabilized rabbit and rat ventricular myocytes. Circ Res. 1993;73:820–828.[Abstract/Free Full Text]
6. Harrison SM, McCall E, Boyett MR. The relationship between contraction and intracellular sodium in rat and guinea pig ventricular myocytes. J Physiol. 1992;449:517–550.[Abstract/Free Full Text]
7. Crespo LM, Grantham CJ, Cannell MB. Kinetics, stoichiometry and role of the Na-Ca exchange mechanism in isolated cardiac myocytes. Nature. 1990;345:618–621.[Medline] [Order article via Infotrieve]
8. Sham JS, Cleeman L, Morad M. Functional coupling of Ca²⁺ channels and ryanodine receptors in cardiac myocytes. Proc Natl Acad Sci U S A. 1995;92:121–125.[Abstract/Free Full Text]
9. Sipido KR, Maes M, Van de Werf F. Low efficiency of Ca²⁺ entry through the Na⁺-Ca²⁺ exchanger as trigger for Ca²⁺ release from the sarcoplasmic reticulum: a comparison between L-type Ca²⁺ current and reverse-mode Na⁺-Ca²⁺ exchange. Circ Res. 1997;81:1034–1044.[Abstract/Free Full Text]
10. Litwin SE, Li J, Bridge JHB. Na-Ca exchange and the trigger for sarcoplasmic reticulum Ca release: studies in adult rabbit ventricular myocytes. Biophys J. 1998;75:359–371.[Abstract/Free Full Text]
11. Mattiello JA, Margulies KB, Jeevanandam V, Houser SR. Contribution of reverse-mode sodium/calcium exchange to contractions in failing human left ventricular myocytes. Cardiovasc Res. 1998;37:424–431.[Abstract/Free Full Text]
12. Tani M. Mechanisms of Ca²⁺ overload in reperfused ischemic myocardium. Annu Rev Physiol. 1990;52:543–559.[Medline] [Order article via Infotrieve]
13. Smith TW. Digitalis: mechanisms of