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Article

Pharmacology of Brain Na⁺/Ca²⁺ Exchanger: From Molecular Biology to Therapeutic Perspectives

Division of Pharmacology, Department of Neuroscience, School of Medicine, Federico II University of Naples, Naples, Italy

Abstract

I. Introduction

II. Molecular Biology of Na+/Ca2+ Exchanger

III. Brain Distribution of Na+/Ca2+ Exchanger Isoforms

A. Cerebral Cortex

B. Hippocampus

C. Mesencephalon and Basal Ganglia

D. Cerebellum

E. Median Eminence

IV. Regulation of Na+/Ca2+ Exchanger Activity

A. Intracellular Ca2+ Concentrations

B. Intracellular Na+ Concentrations

C. Intracellular H+ Concentrations

D. ATP, Protein Kinase A, Protein Kinase C, and Phosphatidylinositol 4,5 Bisphosphate

E. Phosphoarginine

F. Redox Agents

G. Gaseous Mediator: NO

V. Pharmacological Modulation of Na+/Ca2+ Exchanger Activity

VI. Inhibitors

A. Inorganic Cations

B. Organic Derivatives

1. Peptides.

C. Heterocycles

D. Antisense Oligodeoxynucleotides versus Na+/Ca2+ Exchanger Isoforms

VII. Activators

A. Inorganic Cations

B. Redox Agents

C. Organic Compounds

VIII. Na+/Ca2+ Exchanger Intervention in Physiological Conditions

A. Na+/Ca2+ Exchanger: Hormonal and Neurotransmitter Release

B. Effect of Knocking Out Na+/Ca2+ Exchanger Genes

IX. Relevance of Na+/Ca2+ Exchanger Activity in Pathophysiological Conditions

A. Hypoxia-Anoxia

B. White Matter Degeneration after Spinal Cord Injury, Brain Trauma, and Optical Nerve Injury

C. Na+/Ca2+ Exchanger and Neuronal Apoptosis

D. Aging

E. Alzheimer's Disease

X. Conclusions and Future Perspectives

In the last two decades, there has been a growing interest in unraveling the role that the Na⁺/Ca²⁺ exchanger (NCX) plays in the function and regulation of several cellular activities. Molecular biology, electrophysiology, genetically modified mice, and molecular pharmacology have helped to delve deeper and more successfully into the physiological and pathophysiological role of this exchanger. In fact, this nine-transmembrane protein, widely distributed in the brain and in the heart, works in a bidirectional way. Specifically, when it operates in the forward mode of operation, it couples the extrusion of one Ca²⁺ ion with the influx of three Na⁺ ions. In contrast, when it operates in the reverse mode of operation, while three Na⁺ ions are extruded, one Ca²⁺ enters into the cells. Different isoforms of NCX, named NCX1, NCX2, and NCX3, have been described in the brain, whereas only one, NCX1, has been found in the heart. The hypothesis that NCX can play a relevant role in several pathophysiological conditions, including hypoxia-anoxia, white matter degeneration after spinal cord injury, brain trauma and optical nerve injury, neuronal apoptosis, brain aging, and Alzheimer's disease, stems from the observation that NCX, in parallel with selective ion channels and ATP-dependent pumps, is efficient at maintaining intracellular Ca²⁺ and Na⁺ homeostasis. In conclusion, although studies concerning the involvement of NCX in the pathological mechanisms underlying brain injury during neurodegenerative diseases started later than those related to heart disease, the availability of pharmacological agents able to selectively modulate each NCX subtype activity and antiporter mode of operation will provide a better understanding of its pathophysiological role and, consequently, more promising approaches to treat these neurological disorders.

I. Introduction

In the last two decades, there has been a growing interest in unraveling the role that the Na⁺/Ca²⁺ exchanger (NCX¹) plays in the function and regulation of several cellular activities. Biochemistry, molecular biology, electrophysiology, genetically modified mice, and molecular pharmacology have helped to delve deeper and more successfully into the physiological and pathophysiological role of this exchanger, as witnessed by more than 2000 papers written on this issue and published in the most internationally qualified journals (Philipson et al., 2002).

NCX was discovered and characterized in the late 1960s, when Baker et al. in the UK (Baker and Blaustein, 1968; Baker et al., 1969), Reuter and Seitz in Germany and Switzerland (Reuter and Seitz, 1968), and Martin and De Luca in the United States (Martin and De Luca, 1969) realized the presence of a countertransport mechanism that exchanged Na⁺ and Ca²⁺ ions across the plasma membrane of different excitable and nonexcitable cells. However, the most crucial advancement in NCX research was made in 1988 (Philipson et al., 1988) and 1990 (Nicoll et al., 1990), when Philipson and his colleagues purified and cloned the first isoform of this antiporter: NCX1. Remarkably, four years later, the same group of investigators cloned NCX2 (Li et al., 1994) and NCX3 (Nicoll et al., 1996b), two isoforms selectively expressed in the brain (Lee et al., 1994) and in the skeletal muscle (Nicoll et al., 1996b). Finally, in 1999, Philipson proposed a new topological model of the NCX1 exchanger (Nicoll et al., 1999) (Table 1).

The regulation of intracellular concentrations of Ca²⁺ and Na⁺ ions in excitable cells is a relevant physiological phenomenon that maintains cellular homeostasis. In fact, although cytosolic Ca²⁺ ions play a key role in signaling at the cytosolic and nuclear levels (Choi, 1988), Na⁺ ions play a major role in regulating cellular osmolarity, in inducing action potential (Lipton, 1999), and in transduction signaling (Yu et al., 1997). Indeed, the Na⁺/Ca²⁺ exchanger, in parallel with selective ion channels and ATP-dependent pumps, maintains the physiological cytosolic concentrations of these ions (Blaustein and Lederer, 1999). In particular, the Na⁺/Ca²⁺ exchanger can mediate Ca²⁺ and Na⁺ fluxes across the synaptic plasma membrane in a bidirectional way (Blaustein and Lederer, 1999; Philipson and Nicoll, 2000) (Table 2). The stoichiometry of NCX is generally accepted to be three Na⁺ ions/one Ca²⁺ ion; however, more recently, in addition to the major 3:1 transport mode, it has been demonstrated that ion flux ratio can vary from 1:1 to a maximum of 4:1, depending on the intracellular concentration of Na⁺ and Ca²⁺ ions (Fujioka et al., 2000; Hang and Hilgemann, 2004). In resting excitable cells, when intracellular Ca²⁺ concentrations ([Ca²⁺]_i) rise and the cells require the return of [Ca²⁺]_i to resting levels (Carafoli, 1985), this exchange transport mechanism couples the uphill extrusion of Ca²⁺ to the influx of Na⁺ ions into the cells down their electrochemical gradient. This mode of operation, defined as forward mode (Blaustein and Santiago, 1977), keeps the 10⁴-fold difference in Ca²⁺ concentrations across the cell membrane. In contrast, in other physiological circumstances, when intracellular Na⁺ concentrations ([Na⁺]_i) rise or membrane depolarization occurs, thus reducing the transmembrane Na⁺ electrochemical gradient, the Na⁺/Ca²⁺ exchanger mediates the extrusion of [Na⁺]_i and the influx of Ca²⁺ ions. This mode of operation is defined as reverse way (Baker and McNaughton, 1976; DiPolo, 1979) (Fig. 1; Table 2).

View larger version (48K): [in this window] [in a new window]   FIG. 1. Schematic diagram of NCX and other ionic transporter mechanisms involved in [Na+]i and [Ca2+]i homeostasis in a pre- and postsynaptic structure.

Over the last five years, some exhaustive and basic reviews addressing the regulatory functions of the NCX have been published (Blaustein and Lederer, 1999; Egger and Niggli, 1999; Philipson and Nicoll, 2000; DiPolo and Beaugé, 2002), whereas reviews covering the pharmacological aspects of its modulation have been published only in 1997 and 1999 (Matsuda et al., 1997; Shigekawa and Iwamoto, 2001). Considering that a comprehensive review on the pharmacological modulation of brain NCX is still lacking, the present paper will particularly address the physiology and the molecular pharmacology of NCX in the brain and the possible therapeutic implications of its modulation.

II. Molecular Biology of Na⁺/Ca²⁺ Exchanger

The Na⁺/Ca²⁺ exchanger belongs to the superfamily of membrane proteins comprising the following members: 1) the NCX family, which exchanges three Na⁺ ions for one Ca²⁺ ion or four Na⁺ ions for one Ca²⁺ ion depending on [Na⁺]_i and [Ca²⁺]_i (Reeves and Hale, 1984; Fujioka et al., 2000; Hang and Hilgemann, 2004); 2) the Na⁺/Ca²⁺ exchanger K⁺-dependent family, which exchanges four Na⁺ ions for one Ca²⁺ plus one K⁺ ion (Schnetkamp et al., 1989; Lytton et al., 2002); 3) the bacterial family which probably promotes Ca²⁺/H⁺ exchange (Cunningham and Fink, 1996); 4) the nonbacterial Ca²⁺/H⁺ exchange family, which is also the Ca²⁺ exchanger of yeast vacuoles (Pozos et al., 1996); and 5) the Mg²⁺/H⁺ exchanger, an electrogenic exchanger of protons with Mg²⁺ and Zn²⁺ ions (Shaul et al., 1999). These membrane proteins are all peculiarly characterized by the presence of -repeats, the regions involved in ion translocation.

Regarding the NCX family, three dominant genes coding for the three different NCX1 (Nicoll et al., 1990), NCX2 (Li et al., 1994), and NCX3 (Nicoll et al., 1996b) proteins have been identified in mammals. These three genes appear to be dispersed, since NCX1, NCX2, and NCX3 have been mapped in mouse chromosomes 17, 7, and 12, respectively (Nicoll et al., 1996b). At the posttranscriptional level, at least 12 NCX1 and 3 NCX3 proteins are generated through an alternative splicing of the primary nuclear transcripts (Kofuji et al., 1994). These variants arise from a region of the large intracellular f loop, are encoded by six small exons defined A to F, and are used in different combinations in a tissue-specific manner (Nakasaki et al., 1993; Lee et al., 1994). To maintain an open reading frame, all splice variants must include either exon A or B, which are mutually exclusive (Quednau et al., 1997). Excitable tissues, such as those of the brain and heart, are usually characterized by the presence of exon A, whereas kidney, stomach, and skeletal muscle tissues comprise NCX with exon B (Quednau et al., 1997).

NCX1 is composed of 938 amino acids, in the canine heart, having a theoretical molecular mass of 120 kDa and containing nine transmembrane segments (TMS). NCX1 amino terminus is located in the extracellular space, whereas the carboxyl terminus is located intracellularly (Fig. 2). The nine transmembrane segments can be divided into an N-terminal hydrophobic domain, composed of the first five TMS (1–5), and into a C-terminal hydrophobic domain, composed of the last four TMS (6–9). These two hydrophobic domains are important for the binding and the transport of ions. The first (1–5) TMS are separated from the last four (6–9) TMS through a large hydrophilic intracellular loop of 550 amino acids, named the f loop (Nicoll et al., 1999). Although the f loop is not implicated in Na⁺ and Ca²⁺ translocation, it is responsible for the regulation of NCX activity elicited by several cytoplasmic messengers and transductional mechanisms, such as Ca²⁺ and Na⁺ ions, NO, phosphatidylinositol 4,5 bisphosphate (PIP2), protein kinase C (PKC), protein kinase A (PKA), phosphoarginine (PA), and ATP (Table 3). In the center of the f loop, a region of approximately 130 amino acids in length (371–508 amino acids) has been reported to exert a Ca²⁺ regulatory function. This region is characterized by a pair of three aspartyl residues and by a group of four cysteines (Nicoll et al., 1999; Qiu et al., 2001). At the N-terminal end of the f loop near the membrane lipid interface, an autoinhibitory domain, rich in both basic and hydrophobic residues and consisting of a 20-amino acid sequence (219–238), named exchange inhibitory peptide (XIP) (Matsuoka et al., 1997), has been identified. The f loop is also characterized by alternative splicing sites named 1-repeat and 2-repeat. These -repeats are characterized by similar regions comprising 60 to 70 amino acids for which no functional role has yet been proposed (Hilgemann, 1990).

View larger version (46K): [in this window] [in a new window]   FIG. 2. Molecular pharmacology of NCX. Putative sites of the molecular action of drugs interfering with NCX activity.

The NCX protein amino acid sequence found between TMS2 and TMS3 is called -1 repeat, whereas the one found between TMS7 and TMS8 is named -2 repeat. Both regions, -1 and -2 repeats, are located on the opposite site of the membrane and include two segments composed of 12 and 9 highly conserved residues separated by a nonconserved segment of 18 to 20 amino acids (Nicoll et al., 2002). Since the putative -helices of the -repeats are amphipathic, the hydrophilic faces of these helices may form a portion of the ion translocation pathway (Nicoll et al., 1996a). Interestingly, the -repeats form re-entrant loops of NCX1 that interact within the protein (Iwamoto et al., 2000; Qiu et al., 2001). Interestingly, the center of 2 repeat possesses a GIG sequence similar to the GYG sequence present in the P loop of K⁺ channels (Philipson and Nicoll, 2000). With electrophoretic gels and under nonreducing conditions, NCX1 migrates as a 120- and a 70-kDa band. The 120-kDa band represents the native protein, and the 70-kDa protein is a proteolytic fragment, which includes a large part of the f loop and retains an Na⁺/Ca²⁺ exchange activity (Saba et al., 1999; Van Eylen et al., 2001b).

Interestingly, NCX2 and NCX3 have been found only in the brain and in the skeletal muscle. These two gene products consist of 921 and 927 amino acids and are characterized by molecular masses of 102 and 105 KDa, respectively. In addition, NCX2 displays a 65% sequence identity with NCX1, whereas NCX3 possesses a 73% sequence identity with NCX1 and 75% sequence identity with NCX2 (Nicoll et al., 1996b). All three NCX gene products share the same membrane topology.

III. Brain Distribution of Na⁺/Ca²⁺ Exchanger Isoforms

The NCX1 gene is expressed in several tissues, including brain, heart, skeletal muscle, smooth muscle, kidney, eye, secretory, and blood cells, whereas transcripts encoded by the NCX2 and NCX3 genes have been found exclusively in neuronal and skeletal muscle tissues (Lee et al., 1994). In addition, NCX1 and NCX3 give rise to several splicing variants that appear to be selectively expressed in different regions and cellular populations of the brain (Quednau et al., 1997; Yu and Colvin, 1997).

A. Cerebral Cortex

NCX1 isoforms are intensively expressed in the pyramidal neurons of layers III and V within the molecular layer of the cerebral motor cortex. This area, which contains the terminal dendritic field of the pyramidal cells, displays a more intense NCX1 immunoreactivity than that of NCX2. In contrast, the somatosensory cortical area seems to express preferentially NCX2 transcripts. Such anatomical distribution reveals that the upper neurons of the motor system and the terminal neurons of the somatosensory system preferentially express distinct NCX isoforms (Canitano et al., 2002; Papa et al., 2003).

B. Hippocampus

Within the hippocampus, the transcripts of the three NCX isoforms display an intense labeling of most neuronal populations. In particular, high levels of the three NCX genes have been detected in the granular cell layers of the dentate gyrus and in the pyramidal cells of CA1, CA2, CA3, and CA4 subfields (Papa et al., 2003). The three NCX protein isoforms also display high levels of expression within the hippocampus. Thus, in the orient and radiatum layers of the CA1, NCX3 protein is more intense than NCX1 and NCX2. NCX1 protein expression is particularly intense in the granule cell layer and in the hilum of the dentate gyrus, which constitutes the terminal field of the perforant pathway, the major excitatory input to the hippocampus originating from the enthorinal cortex. In the CA3 area, the NCX1 and NCX3 genes of the mossy fibers projecting from the granule cells located in the dentate gyrus are more intense than those of NCX2. This peculiar distribution suggests that distinct Na⁺/Ca²⁺ exchanger isoforms may play a crucial role in controlling the intracellular Na⁺ and Ca²⁺ homeostasis of the major afferent, intrinsic, and efferent hippocampal projections. Such circuitries are crucial for the synaptic plasticity phenomena, such as those involved in long-term potentiation (LTP) and long-term depression (Madison et al., 1991). Furthermore, NCX plays a major role in hippocampal ischemia. In fact, it has been demonstrated that under anoxic/glucopenic conditions, the inhibition of the Na⁺/Ca²⁺ exchanger influences the hippocampal electrophysiological responses (Schroder et al., 1999) and the excitatory neurotransmitter release (Amoroso et al., 1993; Trudeau et al., 1999).

C. Mesencephalon and Basal Ganglia

NCX isoforms are also expressed in crucial areas of the extrapyramidal control of motor coordination. In fact, NCX1 mRNA can be detected in the substantia nigra pars compacta, in which dopaminergic cell bodies are localized; the NCX1 protein isoform is present in the striatum, in which the terminal projection fields of dopaminergic nigrostriatal neurons are found (Canitano et al., 2002; Papa et al., 2003).

Interestingly, both the transcript and the protein, encoded by the three NCX genes, are abundantly expressed in the nucleus accumbens (Canitano et al., 2002; Papa et al., 2003), a brain region involved in the motivational control of motor coordination (Canitano et al., 2002; Papa et al., 2003).

D. Cerebellum

The analysis of the expression of Na⁺/Ca²⁺ exchanger transcripts and proteins in the cerebellum reveals their presence in the afferent projections and in the intrinsic neurons of this crucial brain region (Canitano et al., 2002; Papa et al., 2003). In particular, NCX1 protein is expressed in the excitatory mossy fibers that originate in the extracerebellar structures and that branch off to the granule cell layer, a site where the glomerular structure is formed by the mossy fiber terminals, the granule cell dendrites, and the Golgi cell axons (Canitano et al., 2002; Papa et al., 2003). Consistent with this regional brain distribution, functional studies, performed on cerebellar granule cells, suggest that [Ca²⁺]_i increase, induced by glutamate, may occur through the Na⁺/Ca²⁺ exchanger operating in the reverse mode (Kiedrowski et al., 1994).

E. Median Eminence

In the median eminence, a region devoid of neuronal perykaria and provided with nerve endings originating from neurons located in several hypothalamic regions, positive immunostaining for NCX1, NCX2, and NCX3 isoforms has been demonstrated (Papa et al., 2003). Since median eminence plays a relevant role in controlling dopamine (DA) and anterior pituitary hormone release (Annunziato et al., 1979), the presence of NCX1, NCX2, and NCX3 in this area suggests that NCX may exert an important function in the modulation of DA release and anterior pituitary hormone secretion.

The distribution of the three NCX isoforms in the mammalian brain may, therefore, give useful insights into unraveling the physiological and, possibly, the pathophysiological role played by the different NCX isoforms in the regulation of neuronal function.

IV. Regulation of Na⁺/Ca²⁺ Exchanger Activity

Several factors are involved in the regulation of Na⁺/Ca²⁺ exchanger activity: the two transported ions, Na⁺ and Ca²⁺; the intracellular pH; metabolic related compounds, ATP, PA, PIP2, PKA, and PKC; redox agents, hydroxyl radicals, H₂O₂, dithiothreitol (DTT), O₂^-·, Fe³⁺, Fe²⁺, Cu²⁺, OH°, glutathione reduced (GSH), and glutathione oxidized (GSSG); and the gaseous mediator, NO (Table 3).

A. Intracellular Ca²⁺ Concentrations

The site level at which [Ca²⁺]_i regulates NCX activity is different from the one required for Ca²⁺ transport (Levitsky et al., 1994). In fact, submicromolar concentrations (0.1–0.3 µM) of intracellular Ca²⁺ are needed to activate the antiporter (DiPolo, 1979; Hilgemann et al., 1992). Indeed, the removal of intracellular Ca²⁺ ions completely blocks NCX activity (Philipson and Nicoll, 2000). This regulatory function of low micromolar Ca²⁺ is more evident when the Na⁺/Ca²⁺ exchanger is working in the reverse mode. However, it is not completely clear how low µM Ca²⁺ can also regulate NCX when it operates in the forward mode (Matsuoka et al., 1995). The location of such regulatory site has been identified in the 134-amino acid-length region, situated in the center of the intracellular f loop. This region is characterized by a pair of three aspartyl residues and by a group of four cysteines (Matsuoka et al., 1995).

B. Intracellular Na⁺ Concentrations

In addition to the submicromolar intracellular Ca²⁺ regulatory site, an increase in [Na⁺]_i can also regulate the Na⁺/Ca²⁺ exchanger (Hilgemann, 1990). In particular, when intracellular Na⁺ increases, it binds to the transport site of the exchanger molecule, and after an initial fast outward Na⁺/Ca²⁺ current, an inactivation process occurs (Hilgemann et al., 1992). This inactivation process, very similar to the phenomenon occurring in voltage-dependent ionic channels, is named Na⁺-dependent inactivation. The region of the intracellular f loop, in which this regulatory site is located, has been identified in a 20-amino acid portion of the N-terminal part of the loop termed XIP (Matsuoka et al., 1997). Studies in vitro have characterized a negatively charged region of the intracellular f loop (445–455 amino acids) of the NCX protein that is able to cross link with synthetic XIP, thus suggesting that this amino acid sequence constitutes the binding site of XIP (Hale et al., 1997). On the other hand, since deletion mutagenesis of amino acids 562 to 685 results in an exchange activity that is no longer regulated by XIP (Matsuoka et al., 1993), it is likely that XIP interacts with residues 445 to 455 and with another region of the f loop located between residues 562 and 685. Indeed, this region is believed to be a Na⁺ regulatory site (Li et al., 1991). Regarding the mechanism by which XIP inhibits NCX activity, it has been proposed that when the XIP-binding site is ligand occupied, a conformational change is induced in the C-terminal portion of the f loop, thus resulting in the inhibition of the ion transport (Li et al., 1991). XIP is provided with relevant pharmacological implications. In fact, those exogenous peptides, having the same amino acid sequence as XIP, act as potent inhibitors of NCX activity (Li et al., 1991; Pignataro et al., 2004b). Interestingly, Ca²⁺ ions, at low micromolar concentrations, binding its regulatory site, decrease the extent of this Na⁺-dependent inactivation. In fact, mutations in the Ca²⁺ regulatory binding site alter the activation and inactivation kinetics of exchange currents by modulating Na⁺-dependent inactivation (Matsuoka et al., 1995).

C. Intracellular H⁺ Concentrations

H+ strongly inhibits NCX activity under steady-state conditions (Table 3) (Doering and Lederer, 1993). Changes in intracellular pH values, as little as 0.4, can induce a 90% inhibition of NCX activity. Since this H⁺ ion modulatory action is -chymotrypsin sensitive, the action site of the proton can be attributed to the antiporter's hydrophilic intracellular loop (Espinosa-Tanguma et al., 1993). Intriguingly, such inhibitory action depends on the presence of intracellular Na⁺ ions (Doering and Lederer, 1994). Hence, the action exerted by H+ ions is pathophysiologically relevant with regards to brain and heart ischemia. In fact, when intracellular H⁺ and Na⁺ ion homeostasis is deregulated, the anoxic conditions resulting in these cells may selectively interfere with the activity of the different NCX gene products.

D. ATP, Protein Kinase A, Protein Kinase C, and Phosphatidylinositol 4,5 Bisphosphate

Acting as a phosphoryl donor molecule, ATP may increase the activity of the exchanger in a number of ways (Blaustein and Santiago, 1977). Firstly, ATP directly participates in the NCX molecule phosphorylation process by PKA and PKC (Caroni and Carafoli, 1983). Secondly, it increases PIP2 production (Hilgemann and Ball, 1996). Finally, by activating G-protein-coupled receptors, via endogenous and exogenous ligands, ATP can stimulate the activity of the Na⁺/Ca²⁺ exchanger through the pathway involving PKC or PKA activation (DiPolo and Beaugé, 1998). The mechanism underlying the phosphorylating effect on the exchanger seems to be related to an increase in its affinity for both internal Ca²⁺ and external Na⁺ and to a decrease in its inhibition by internal Na⁺. Each of the NCX isoforms has distinctive putative phosphorylation sites, although their roles have not yet been elucidated (Linck et al., 1998). ATP cellular depletion inhibits NCX1 and NCX2 but does not affect NCX3 activity. The exchange activity of NCX1 and NCX3 is modestly increased by those agents that activate PKA and PKC (Linck et al., 1998). More recently, the mechanism by which PKA and PKC activate NCX has been clarified. In fact, it has been demonstrated that the regulation of PKA-induced phosphorylation is due to the existence of an NCX1 macromolecular complex that contains the kinase PKA holoenzyme. This holoenzyme consists of two PKA catalytic subunits and two identical PKA regulatory subunits (Schulze et al., 2003). Together with PKA, other critical regulatory enzymes are also associated with NCX1, including PKC and serine-threonine protein phosphatases, PP1 and PP2A (Schulze et al., 2003). Particularly a pathway involving PKC has been shown to stimulate NCX1 (Iwamoto et al., 1995, 1996a). In a more recent paper, it has been demonstrated that PKC-dependent regulation of NCX isoforms also involves NCX3 but not NCX2 (Iwamoto et al., 1998a). In the same paper, three phosphorylation sites in the NCX1 protein, Ser-249, Ser-250, and Ser-357, have been identified. Among these, Ser-250 is the amino acid that is predominantly phosphorylated (Iwamoto et al., 1998a).

The other mechanism by which ATP can activate NCX occurs through PIP2 production. This mechanism of activation is related to the relevant PIP2 influence on Na⁺-dependent inactivation of NCX. In fact, PIP2 directly interacts with the XIP region of the exchanger, thus eliminating its inactivation and stimulating NCX function. Indeed, exchangers with mutated XIP regions no longer respond to PIP2 or to PIP2 antibodies (Hilgemann et al., 1992; Hilgemann and Ball, 1996; He et al., 2000).

E. Phosphoarginine

PA, present in millimolar concentrations in the cytosol, activates NCX function in the forward mode of operation by an intracellular Mg²⁺- and Ca²⁺-dependent way (DiPolo et al., 1998, 2004). Such mechanism of activation is different from the one characterizing ATP. In fact, ATP and PA regulation is associated with different structures inside and outside the exchanger protein. Particularly, PA should interact with a new zone of NCX named the PA region, which is related to intracellular transport sites for Na⁺ and Ca²⁺ (DiPolo et al., 2004).

F. Redox Agents

In the last 15 years, several groups of investigators using different cellular models, such as cell-expressing cloned splicing variants of the brain, heart isoforms, cardiac sarcolemma vesicles, cells transiently transfected with NCX1 isoform, and giant excised patches, have found that the Na⁺/Ca²⁺ exchanger is sensitive to different combinations of redox agents (Reeves et al., 1986; Amoroso et al., 2000; Santacruz-Toloza et al., 2000). In particular, the stimulation of the exchange activity requires the combination of a reducing agent (DTT, GSH, or Fe²⁺) with an oxidizing agent (H₂O₂ and GSSG) (Santacruz-Toloza et al., 2000). The effects of both agents are mediated by metal ions (e.g., Fe²⁺). The antiporter's sensitivity to changes in the redox status can assume particular relevance during oxidative stress. In fact, in this condition, the modulation of reactive oxygen species (ROS) could affect the transport of Na⁺ and Ca²⁺ ions through the plasma membrane.

G. Gaseous Mediator: NO

The ubiquitous gaseous mediator NO seems to be involved in the modulation of NCX activity. In fact, Asano et al. (1995) provided evidence that NO, released by NO donors, is able to stimulate NCX in the reverse mode of operation in neuronal preparations and astrocytes through a cGMP-dependent mechanism. In contrast, in C6 glioma cells, the stimulatory action on NCX reverse mode of operation, elicited by the NO donor sodium nitroprusside (SNP), is not elicited by NO release but by the presence of iron in SNP molecule (Amoroso et al., 2000).

In addition, a direct relationship between the constitutive form of nitric oxide synthase (NOS), the enzyme involved in NO synthesis, and NCX has recently been demonstrated. Indeed, heat stress by inducing NOS phosphorylation causes NOS complexation with NCX, thus decreasing its activity (Kiang et al., 2003).

V. Pharmacological Modulation of Na⁺/Ca²⁺ Exchanger Activity

Since the time researchers discovered that cardiac glycosides increased cytosolic sodium by binding to the extracytoplasmatic face of the Na⁺/K⁺ ATPase pump -subunit (Eisner and Smith, 1991), a great deal of interest has been devoted to the pharmacological modulation of NCX. The reasons for this enormous interest lay in the hope of finding clinically effective drugs for those pathophysiological conditions in which a stimulation or an inhibition of the NCX might have achieved beneficial effects. Unfortunately, several drawbacks hampered the achievement of this goal: a lack of scientific knowledge on the specific role played by NCX activity in several physiological and pathophysiological conditions, such as neurological and cardiac diseases; a limited understanding of the role played by the different NCX gene products and splicing variants in the same cells; a lack of knowledge about the molecular biology and the regulatory sites of the NCX molecule; the poor availability of NCX selective activators and inhibitors incapable of interfering with other cellular ion transporting mechanisms, i.e., Na⁺ epithelial channels; K⁺ channels; plasma membrane store-operated Ca²⁺ channels; VGCC-, NMDA-, and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor-operated channels; Na⁺/H⁺ exchanger; Na⁺/K⁺ ATPase; and plasma membrane Ca²⁺ ATPase; and a lack of pharmacological probes able to selectively stimulate or inhibit the activity of each NCX gene product and their multiple splicing variants.

Although the specific scientific literature of the past two decades has been characterized by an overwhelming number of works on NCX, only few of them have dealt with its pharmacological modulation. Accordingly, field scientists attending the last international conference held in October 2001 in Banff, Alberta, Canada, adamantly advocated the need to develop new drugs that could specifically modulate NCX activity and, hence, help to evaluate the antiporter's pathophysiological and therapeutic role.

After the discovery of NCX activity in 1969, studies have reported that some compounds can interfere with this antiporter (Kaczorowski et al., 1989). In the last 35 years, in fact, several inorganic and organic compounds have been reported to activate or block the NCX activity (Fig. 2). However, the selectivity of this action has often been questioned (Matsuda et al., 2001; Reuter et al., 2002; Pignataro et al., 2004b). For this reason, the following section will review the major classes of pharmacological compounds that influence NCX activity and will critically examine the specificity of their action.

VI. Inhibitors

A. Inorganic Cations

1. Divalent Cations. Many divalent cations have been reported to block the Na⁺/Ca²⁺ exchanger (Iwamoto and Shigekawa, 1998b) (Fig. 2). This inhibitory effect can be due either to a direct action on the exchanger molecule or to the replacement of Ca²⁺ ions as a substrate for the antiporter.

Although Cd²⁺ (Hobai et al., 1997), Co²⁺ (Hilgemann, 1989), Zn²⁺ (Colvin et al., 2000), and Ni²⁺ (Kimura et al., 1987) are all NCX inhibitors, none of these cations can be considered as specific blockers. Some cations, like Ni²⁺, Mn²⁺, and Cd²⁺, may also function as substrates for the exchanger (Iwamoto and Shigekawa, 1998b). Among these divalent cations, Ni²⁺ is the element most commonly used for blocking NCX activity during electrophysiological measurements (Fujioka et al., 1998; Main et al., 1997). The use of Ni²⁺ as a NCX inhibitor is limited because its blocking concentrations are in the order of 2 to 5 mM, levels at which Ni²⁺ is also able to inhibit other membrane currents (Iwamoto and Shigekawa, 1998b). Interestingly, the IC₅₀ value of Ni²⁺ for NCX activity is increased 2- to 3-fold by membrane depolarization, suggesting that the affinity of Ni²⁺ for the inhibitory site is affected by membrane potential (Iwamoto and Shigekawa, 1998b). Regarding the mode selectivity, Ni²⁺ inhibits NCX in the reverse mode of operation (Iwamoto and Shigekawa, 1998b). The affinity of Ni²⁺ for NCX proteins differs in the three NCX gene products. In fact, NCX3 is 10-fold less sensitive to Ni²⁺ or Co²⁺ inhibition than NCX1 and NCX2 (Iwamoto and Shigekawa, 1998b). The inhibitory mechanism exerted by Ni²⁺ is apparently exerted by competing against extracellular Ca²⁺ for the external transport site (Iwamoto and Shigekawa, 1998b). In particular, using cysteine-substituted mutants it was possible to identify three amino acids residues influencing Ni²⁺ sensitivity of NCX1, Asp-130, at the level 1-repeat, Asp-825, and Glu-837 at the level of 2-repeats (Shigekawa et al., 2002) (Fig. 2). All these results underline the fact that the degree of sensitivity of the different NCX gene products to pharmacological agents may vary depending upon the type of NCX gene product involved.

2. Trivalent Cations. Among the different lanthanides, La³⁺ was one of the first inorganic cations to be tested as an effective NCX inhibitor in several excitable cells (Kwan and Putney, 1990; Amoroso et al., 1993, 1997). However, the K_i of La³⁺ for NCX inhibition was in the high micromolar range (500 µM), whereas its affinity for other Ca²⁺-transporting systems, such as VGCC and the plasma membrane Ca²⁺ ATPase, was much greater (Shimizu et al., 1997). Such discrepancy suggests that lanthanide concentrations capable of blocking NCX are also effective in inhibiting the other Ca²⁺-transporting mechanism.

Other lanthanides as Nd³⁺, Tm³⁺, and Y³⁺ can also inhibit the Na⁺/Ca²⁺ exchanger (Trosper and Philipson, 1983), but their potency is lower than that of La³⁺ (Trosper and Philipson, 1983). Interestingly, Gd³⁺, another lanthanide, has been shown to block VGCC without interfering with NCX activity (Canzoniero et al., 1993).

B. Organic Derivatives

1. Peptides. a. Endogenous Constrained Cyclic Peptides: Phe-Arg-Cys-Arg-Cys-Phe. Among one class of cyclic exapeptides found to inhibit cardiac NCX activity, Phe-Arg-Cys-Arg-Cys-Phe (FRCRCFa) is apparently the most potent, IC₅₀ value = 10 ± 3 µM (Khananshvili et al., 1996). This peptide is able to block both the forward and the reverse modes of Na⁺-Ca²⁺ exchange of all three NCX subtypes (Khananshvili et al., 1996). It has been proposed that the positively charged and conformationally constrained Arg-Cys-Arg-Cys structure might be a pharmacophore. Therefore, FRCRCFa may interact with two negatively charged domains. The first negative domain (amino acids 56–96) represents a major part of the short intracellular loop connecting TMS1 and TMS2; the second sequence (amino acids 723–733) is located on the NCX cytosolic side at the level of the f loop, where the action site of XIP effect is presumably located (Khananshvili et al., 1996). FRCRCFa rapidly (<20 ms) interacts with the inhibitory site of the exchanger molecule and prevents a specific conformational transition of ion/protein interaction without altering the interaction of the ions with the exchanger's transport sites (Khananshvili et al., 1995).

b. The Molluscan Tetrapeptide Phe-Met-Arg-Phe and Its Related Peptides. Phe-Met-Arg-Phe (FMRFa) is a molluscan peptide having potent biological effects on excitable cells, such as cardiomyocytes and neurons. In addition, an endogenous peptide with an FMRFa sequence has been detected in the brain and in other organs of several vertebrates (Khananshvili et al., 1993; DiPolo et al., 1994; Van Eylen et al., 1994). FMRFa, along with other related peptides, blocks NCX activity (IC₅₀ values = 1 µM to 1 mM). Interestingly, the action of these molluscan peptides is reversible and is exerted both in the forward and in the reverse mode of NCX operation. As it stands, no evidence is available on the antiporter subtype selectivity of these molluscan peptides. The NCX inhibition exerted by these peptides is eliminated by trypsin preincubation, thus suggesting that its action occurs at the intracellular f loop level of the exchanger, viz., a trypsin-sensitive site (Khananshvili et al., 1993). However, since FMFRa, along with its related peptides, does not present clear homologies with the amino acid sequence of the XIP peptide, it is possible to speculate that these two peptides bind NCX molecule to two distinct sites. FMRFa, in effect, itself has a low inhibitory potency (IC₅₀ 750 µM) that completely resides in its carboxyl-terminal RFa portion (Khananshvili et al., 1993). Its inhibitory potency can increase by 300 to 500 times if the NH₂-terminal Phe is substituted by either Val or His (IC₅₀ value = 1–2 µM) (Khananshvili et al., 1993). This suggests the importance of potentiating the inhibitory activity of the NH₂ terminal portion. Moreover, since the inhibitory action of FMRFa and opioid derivatives on NCX activity is mutually exclusive, these two classes of compounds may act through the same molecular site (Khananshvili et al., 1993).

c. Callipeltin A. Callipeltin, a cyclic depsipeptide obtained from the New Caledonian Lithistida Sponge Callipelta sp., is a macrocyclic lacton comprising the following amino acids: Ala, Leu, and Thr (two residues) in the L configuration; Arg in the D configuration; two N-methyl amino acids, N-MeAla and N-MeGln; and a methoxy Tyr, 3,4-dimethyl-L-glutamine and a 4-amino-7-guanidino-2,3 dihydroxypentanoic acid, formally derived from L-Arg. Similarly to FRCRFa, the presence of two positively charged Arg residues confers to this New Caledonian sponge product a highly inhibitory property. The peptide's peculiar lipophilicity and resistance to proteolytic degradation can further explain its high inhibitory potency (IC₅₀ value = 0.85 µM) on NCX activity in the Na⁺_i-dependent Ca²⁺ uptake and its ability to induce a full NCX inhibition (Trevisi et al., 2000). Evidence on its ability to inhibit the exchanger forward mode of operation and on its NCX subtype selectivity is still lacking.

d. Endogenous Exchange Inhibitory Peptides. Since it was demonstrated that a 20-amino acid sequence (219–238) of the intracellular f loop of the exchanger molecule, XIP, played an autoinhibitory function through an Na⁺-dependent inactivating mechanism (Nicoll et al., 1990; Matsuoka et al., 1997) and that synthetic peptides with the same amino acid sequence could exert an inhibitory action on NCX function (Li et al., 1991; DiPolo and Beaugé, 1994), a great effort has been made to synthesize and to characterize the molecular pharmacology of different XIP analogs. XIP, an amphipathic molecule, potently inhibits Na⁺/Ca²⁺ exchange activity in both modes of operation with a K_i of 0.1 to 1.0 µM in a noncompetitive manner (Fig. 2) (Li et al., 1991). NCX1, NCX2, and NCX3 have homologous XIP regions. The three corresponding inhibitory peptides, XIP1, XIP2, and XIP3, have some residue variations, despite having well conserved sequences. For instance, XIP1 is provided with two basic residues at positions 17 and 19, whereas at these same positions, XIP2 has two neutral residues, a proline and a serine (He et al., 1997). Interestingly, whereas XIP1 is a good inhibitor of NCX1 activity, XIP2 and XIP3 exhibit only a weak inhibitory property on NCX1 (Linck et al., 1998). As previously mentioned, XIP probably interacts with the residues 445 to 455 of the f loop, previously described as a Ca²⁺ regulatory site, and with the residues 562 to 685, believed to be an Na⁺ regulatory site (Fig. 2). In regard to the mechanism by which XIP inhibits NCX activity, some authors have proposed that when the XIP-binding site is ligand occupied, a conformational change is induced in the C-terminal portion of the f loop, thus resulting in the inhibition of the ion transport (Li et al., 1991). Moreover, XIP can also open an inward current of unknown origin when applied to the extracellular medium.

The entire extent of XIP is important for maximal potency, although the major inhibitory components are found between the amino acid residues 5 and 16 (Fig. 3) (He et al., 1997). In this sequence, basic and aromatic residues are crucially important for the inhibitory function of XIP. However, since substitutions of Arg-12 and Arg-14 with Ala or Gln, respectively, dramatically decrease XIP potency, both residues also play a key role in possible charge-charge interactions (He et al., 1997). Finally, it should be underlined that although some residues vary in their position, the XIP region is well conserved in all the known NCX gene products (He et al., 1997).

View larger version (19K): [in this window] [in a new window]   FIG. 3. Structure of the 6-tyrosine glycosilated form of the exchange inhibitory peptide XIP.

Since XIP hardly penetrates the cell membrane because of its prevalent hydrophilia, an XIP bearing a molecule of glucose attached to the Tyr-6 residue has recently been synthesized (Fig. 3). By virtue of this strategy, the peptide more easily penetrates into the cell, since the glucose molecule, actively transported into the cell through the glucose transporters (glucose transporters 1 and 3), carries the attached peptide (Namane et al., 1992). Interestingly, this Tyr-6-glycosilated form of XIP (Fig. 2), intracerebroventricularly infused in male rats bearing permanent middle cerebral artery occlusion (pMCAO), caused a dramatic increase in infarct volume (Pignataro et al., 2004b). These results suggest that NCX plays a pivotal role in the mechanisms that lead to neuronal death under ischemic conditions. Therefore, a pharmacological modulation of its activity may represent one of the possible therapeutic strategies for stroke treatment.

C. Heterocycles

1. Amiloride Derivatives. The synthesis of amiloride and amiloride analogs as K⁺-sparing diuretics capable of inhibiting kidney epithelial Na⁺-channels was first described by Cragoe et al. (1967). Subsequently, these compounds were shown to inhibit other ion transport processes such as NCX, Na⁺/H⁺ exchanger, and VGCC (Murata et al., 1995). Since then, in an attempt to evaluate NCX activity, amiloride has been used as a probe to block NCX function (Sharikabad et al., 1997). However, two major drawbacks have limited its use. Firstly, millimolar concentrations are required for its NCX inhibitory activity; secondly, it lacks specificity, for it can also inhibit both the epithelial Na⁺ channel at micromolar concentrations and the Na⁺/H⁺ exchanger in the millimolar range. More recently, to overcome these hindrances, two classes of amiloride analogs have been developed (Fig. 4). The amiloride analogs of the first class, such as 5-[N-methyl-N-(guanidinocarbonylmethyl)] amiloride, bear substituent on the 5-amino nitrogen atom of the pyrazine ring (Taglialatela et al., 1988b). They lack inhibitory properties on the epithelial Na⁺ channel and the plasma membrane NCX, even though they display great effectiveness in inhibiting the Na⁺/H⁺ exchange in the 1 to 10 µM range (Taglialatela et al., 1988b, 1990a; Amoroso et al., 1990). The compounds of the second class, having no inhibitory effect on the Na⁺/H⁺ exchanger, bear substituents on the terminal guanidino nitrogen atom and behave as specific inhibitors (K_i = 1–10 µM) of the epithelial Na⁺ channels and NCX. Among these compounds, dimethylbenzamylamiloride (DMB), 3',4'-dichlorobenzamyl, and -phenylbenzamyl have been shown to be selective inhibitors of the Na⁺/Ca²⁺ exchanger in excitable cells, such as neurons, in which the kidney epithelial Na⁺ channels are not expressed (Taglialatela et al., 1988b, 1990a) (Figs. 2 and 4). In contrast, [N-(4-chlorobenzyl)]2,4-dimethylbenzamyl (CB-DMB) (Figs. 4 and 5) appears to be the most specific inhibitor of NCX activity (K_i = 7.3 µM), for it has no inhibitory properties against the Na⁺/H⁺ antiporter (K_i > 500 µM) and the epithelial Na⁺ channels (K_i > 400 µM) (Sharikabad et al., 1997).

View larger version (17K): [in this window] [in a new window]   FIG. 4. Chemical structures of the two classes of amiloride derivatives and their inhibitory concentrations on NCX and Na+/H+ exchanger activity.

View larger version (14K): [in this window] [in a new window]   FIG. 5. Chemical structures of the most widely used NCX blockers.

The amiloride derivatives are able to inhibit NCX activity either when the antiporter operates in the forward (Taglialatela et al., 1990b) or in the reverse mode of operation (Amoroso et al., 1997). Data on the subtype specificity of the K⁺-sparing diuretic derivatives are lacking. Amiloride derivatives are reversible inhibitors of the exchanger, and the inhibition is competitive with respect to the Na⁺ ion. It was hypothesized that these derivatives act as Na⁺ analogs, interacting at an Na⁺-binding site on the carrier, presumably, the region to which the third Na⁺ binds, and reversibly tie up the transporter in an inactive complex (Kaczorowski et al., 1985).

Because of their pharmacological properties, these amiloride derivative compounds have been shown to interfere with the release of neurotransmitters from the brain, under both physiological and pathophysiological conditions. As a result, DMB is able to prevent ouabain-induced DA release from tuberoinfudibular neurons (Taglialatela et al., 1990a). The same compound was demonstrated to increase anoxia-induced D-³H-aspartate release from hippocampal slices (Amoroso et al., 1993). Amiloride analogs have also enabled researchers to detect the NCX involvement in the process of anterior pituitary release; in fact, prolactin secretion, induced by the inhibition of the Na⁺/K⁺ ATPase, is remarkably blocked by DMB (Di Renzo et al., 1995). The role played by NCX in glial and neuronal damage, induced by anoxic conditions in vitro and by pMCAO in vivo, has also been revealed in studies performed with this class of agents. In fact, the blockade of NCX by CB-DMB enhances LDH release, induced by chemical hypoxia, and completely reverts the protective effect exerted by the removal of Na⁺ ions on glioma cells exposed to chemical hypoxia. Likewise, DMB and CB-DMB worsen delayed neuronal death, observed in cerebellar granule cell cultures after glutamate exposure (Andreeva et al., 1991). Moreover, studies of an in vivo model of cerebral ischemia have demonstrated that CB-DMB, intracerebroventricularly infused with an osmotic minipump for 24 h after the beginning of the pMCAO, increases the volume of the ischemic region (Pignataro et al., 2004b).

2. Diarylaminopropylamine Derivatives: Bepridil and Aprindine. Bepridil is a diarylaminopropylamine derivative having both antianginal and antiarrhythmic effects (Figs. 2 and 5). Bepridil has multiple inhibitory effects on ionic currents, including the L-type (Yatani et al., 1986) and T-type Ca²⁺ currents (Cohen et al., 1992), the delayed-rectifier K⁺ current, the transient outward current (Berger et al., 1989), as well as the K⁺ current, activated by intracellular Na⁺ (Mori et al., 1998). In addition to these pharmacological properties, bepridil can also block NCX activity with an IC₅₀ value of 8.1 µM (Watanabe and Kimura, 2001). This inhibitory action is dependent on the mode of operation of the antiporter. In fact, NCX operating in the forward manner is more strongly inhibited than in the reverse manner of action. Although this type of pharmacological inhibition is similar to that of 3',4'-dichlorobenzamil, it is different from the isothiourea derivative, KB-R7943 (2-[2-[4-(4-nitrobenzyloxy) phenyl] ethyl]isothiourea methanesulfonate) mode of action (see below), which inhibits, preferentially, NCX activity in the reverse mode (Watano et al., 1996). The site of action of bepridil may be located on the cytoplasmic side of the exchanger, since the intracellular treatment with trypsin attenuates its inhibitory action (Watanabe and Kimura, 2001).

In in vitro and in vivo models of hypoxia and ischemia, respectively, bepridil, by blocking NCX, enhances glial and neuronal injury elicited by chemical hypoxia and pMCAO, respectively (Amoroso et al., 2000; Pignataro et al., 2004b). In addition, in rats' striatal spiny neurons, bepridil has been used to investigate membrane potential changes induced by oxygen glucose deprivation or by excitatory amino acids (Calabresi et al., 1999). The results of these experiments, performed in preparations of brain slices, confirmed that the activation of NCX is able to exert a protective role during oxygen glucose deprivation (Calabresi et al., 1999). In contrast, this compound has been shown to improve the functional recovery of the spinal cord white matter after anoxia and traumatic compression (Li et al., 2000). Hence, NCX operating in the reverse mode seems to play a relevant role in cellular calcium overload and in reversible damage after the induction of anoxic and traumatic injury to the dorsal column white matter tracts. Aprindine, another diarylaminopropylamine derivative, belongs to the class of I-B antiarrhythmic agents widely used for treating atrial and ventricular tachyarrhythmia. This drug inhibits several ionic currents such as L-type Ca²⁺, Na⁺, and delayed rectifier K⁺ currents. Aprindine inhibits NCX current with an IC₅₀ value of 49 µM and with a Hill coefficient of 1.3. Its inhibitory NCX action site has not yet been clarified. However, since the deletion of amino acids 247 to 671 in the f loop does not interfere with its NCX inhibitory properties, it most likely acts at a locus different from that of amiodiarone (Watanabe et al., 2002) (see below).

3. Isothiourea Derivatives. KB-R7943, a 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate derivative (Figs. 2 and 5), was identified by Shigekawa's group (Iwamoto and Shigekawa, 1998b) because they screened a compound library for the inhibition of Na⁺-dependent Ca²⁺ uptake. A particular feature of this compound is that it inhibits the antiporter with a different potency, depending on NCX mode of operation. In fact, in intact cells, when the NCX operates in the reverse mode, the IC₅₀ value needed for NCX inhibition is 1.1 to 2.4 µM, whereas when NCX operates in the forward mode, the IC₅₀ value is much higher (IC₅₀ value > 30 µM) (Iwamoto et al., 1996b). In addition, this compound seems to have a different ability to block NCX activity depending on the gene product involved. In fact, NCX3 inhibition requires concentrations that are 3-fold lower than those necessary to inhibit NCX1 and NCX2 (Iwamoto and Shigekawa, 1998b).

KB-R7943 interacts with the exchanger molecule extracellularly at the -2 repeat, between the TMS7 and TMS8 of the exchanger and, more specifically, at the Val-820, Gln-826, and Gly-833 levels (Iwamoto et al., 2001; Shigekawa et al., 2002). Actually, site-directed mutagenesis and chimera exchange studies have recently demonstrated that when Val-820 and Gln-826 in NCX1 are replaced with Gly (V820G) and with Val (Q826V), two amino acids present in the same position in NCX3 molecule, the sensitivity of NCX1 to KB-R7943 increases by 3-fold (Iwamoto et al., 2001; Shigekawa et al., 2002). However, when Gly-833 is mutated, the exchanger becomes practically insensitive to the drug (Iwamoto et al., 2001). Furthermore, if the Ala-809 of NCX3 is substituted with Val, which is the corresponding NCX1 amino acid, the inhibitory property of KB-R7943 decreases by 3-fold. This evidence suggests that the three amino acids Val-820, Gln-826, and Gly-833, present in the 2-repeat region, play a critical role in the KB-R7943 molecular action (Fig. 2). Interestingly, its derivative KB-R7898 (2-[2-[4-(3,4-dichlorobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), in which two electron donor groups (-Cl) are inserted instead of one electron attractor group (-NO₂) and one -H atom, is able to block all three NCX gene products (NCX1, NCX2, and NCX3) (Shigekawa et al., 2002). Thus, this structure-activity relationship demonstrates that the structural modification in the benziloxy moiety is critical for its inhibitory activity.

In light of these peculiar pharmacological properties, in the last 7 years, KB-R7943 has aroused a great deal of interest among investigators working on NCX activity. It has been demonstrated that KB-R7943 exerts a neuroprotective effect in anoxic conditions. Specifically, in rat hippocampal slices, this drug preserves CA1 neurons from hypoxic-hypoglycemic injury (Schroder et al., 1999). This rescuing action has been attributed to the inhibition of the Na⁺/Ca²⁺ exchanger operating in the reverse mode. Analogously, in rats bearing pMCAO, KB-R7943 reduces infarct volume. This neuroprotective action of KB-R7943 can be theoretically attributed to its more selective inhibitory action on NCX3, since this isoform is consistently present in those brain regions involved in pMCAO-induced damage (Papa et al., 2003). However, this neuroprotective effect does not seem the result of NCX blockade, since other antiporter inhibitors, such as GLU-XIP, CB-DMB, and bepridil, do indeed aggravate brain injury (Tortiglione et al., 2002; Pignataro et al., 2004b). To explain the neuroprotective effect of KB-R7943 in pMCAO, it should be noted that this drug is also able to produce a remarkable and long-lasting hypothermic effect (Pignataro et al., 2004b), which itself exerts a relevant neuroprotective action in cerebral ischemia (Yanamoto et al., 2001). In addition, recent reports have shown that KB-R7943, besides its peculiar NCX blocking properties, also exerts an inhibitory effect on several other ionic transport mechanisms, such as L-type VGCC. Moreover, KB-R7943 inhibits receptor-operated ion channels, such as NMDA (Matsuda et al., 2001), whose blockade may also lead to neuroprotective actions (Paule et al., 2003). Finally, since this drug, at low concentrations, can depress Ca²⁺ transients in heart tubes from mouse embryos, whose NCX1 gene is knocked out, its specificity as an NCX inhibitor has been further questioned (Reuter et al., 2002).

4. Ethoxyanilines. In 2001, the newly synthesized 2-[4-[(2,5-difluorophenyl)methoxyl-phenoxy]-5-ethoxyaniline derivative, SEA0400 (2-[4-[(2,5-difluorophenyl)-methoxyl-phenoxy]-5-ethoxyaniline), was reported as being the most potent (IC₅₀ value = 5–92 nM) NCX inhibitor available at the time (Matsuda et al., 2001) (Figs. 2 and 5). More recent studies, however, demonstrated that it was also able to interfere with Ca²⁺ movement across the cell membrane (Reuter et al., 2002). This compound, similarly to KB-R7943, inhibits the antiporter's Na⁺ efflux-Ca²⁺ influx mode of operation. However, at variance with the isothiourea derivative, it predominantly blocks NCX1 (IC₅₀ value = 56 nM), it has a lower affinity for NCX2 (IC₅₀ value = 980 nM), and it has no effect on NCX3 (Iwamoto et al., 2004). It has recently been found that multiple mutations occurring at the Phe-213 level, located at the end of TMS5, significantly alter the exchanger's sensitivity to the SEA0400-induced blockade, whereas they do not modify KB-R7943 activity (Iwamoto et al., 2004). In addition, by replacing Gly-833, located at the 2-repeat level, with Cys, the SEA0400 and KB-R7943 sensitivity is eliminated. Therefore, whereas Phe 213 is crucial for SEA0400 inhibitory action, Gly-833 is a target for both SEA0400 and KB-R7943. On the other hand, the 2-repeat and TMS5 are not the only SEA0400 molecular action sites. In fact, mutants of the amino acids 224, 226, 228, and 231, belonging to the XIP region in which the Na⁺-dependent inactivation is completely eliminated, display a reduced sensitivity to SEA0400 and KB-R7943 inhibitory actions (Iwamoto et al., 2004). In contrast, the NCX1 mutants bearing Glu instead of Phe-223 (F223E), in which the Na⁺-dependent inactivation is accelerated, show an increased sensitivity to SEA0400 and KB-R7943. Such variable effects have led to the conclusion that the inhibitory effect of the isothiourea derivative and of the ethoxyaniline analog is better exerted during the Na⁺-dependent inactivation (Iwamoto et al., 2004).

On the basis of such potent and selective action, some studies have been performed to clarify the role played by NCX in anoxic/ischemic injury. This compound has been shown to attenuate dose dependently astrocyte damage induced by reperfusion injury in vitro and to reduce infarct volume after a transient middle cerebral artery occlusion in rat cerebral cortex and striatum (Matsuda et al., 2001). SEA0400 neuroprotective effect was attributed to its pharmacological capability of inhibiting prevalently NCX1 activity. However, the specificity of SEA0400 on NCX activity has recently been questioned, since it can also interfere with Ca²⁺ movement across the cell membrane, in the heart tubes of mouse embryos whose NCX1 was knocked out (Reuter et al., 2002). On the other hand, it should be considered that SEA0400 exerts its neuroprotective action in an animal model of cerebral ischemia in which the neuronal damage is mainly caused by the reperfusion period with a Ca²⁺ paradox-like injury accompanied by an exaggerated ROS production and apoptotic cell death (Matsuda et al., 2001). Therefore, the SEA0400 inhibition of Ca²⁺ transients could result in a neuroprotection.

5. Quinazolinone Derivatives. Most recently, in experiments designed to develop new and potent NCX inhibitors, a new compound belonging to quinazolinone family named SM-15811 (4-phenyl-3-[(N-benzyl)-4-pyperidine]-3,4-dihydro-2(1H)-quinazolinone) has been found to inhibit Na⁺-induced Fura-2-monitored Ca²⁺ increase. This drug is provided with a high affinity for the exchanger with an IC₃₀ value of 17 nM (Hasegawa et al., 2003).

6. Benzofuran Derivatives. Amiodarone is an antiarrhythmic agent, belonging to the class of benzofuran derivatives (Fig. 2) that is able to block several plasma membrane voltage-gated ionic channels (Watanabe and Kimura, 2000). So far, this compound has been found to inhibit bidirectional NCX current direction independently with an IC₅₀ value of 3.3 µM and a Hill c