Ca2+ Regulation of Ion Transport in the Na+/Ca2+ Exchanger*

The binding of Ca2+ to two adjacent Ca2+-binding domains, CBD1 and CBD2, regulates ion transport in the Na+/Ca2+ exchanger. As sensors for intracellular Ca2+, the CBDs form electrostatic switches that induce the conformational changes required to initiate and sustain Na+/Ca2+ exchange. Depending on the presence of a few key residues in the Ca2+-binding sites, zero to four Ca2+ ions can bind with affinities between 0.1 to 20 μm. Importantly, variability in CBD2 as a consequence of alternative splicing modulates not only the number and affinities of the Ca2+-binding sites in CBD2 but also the Ca2+ affinities in CBD1.


Structural Basis for Ca 2؉ Regulation in NCX
Structurally, NCX consists of an ␣-helical transmembrane domain and a large ϳ500-residue-long cytosolic loop ( Fig. 1A) (32,33). Residues 371-509 within this loop have been shown to bind Ca 2ϩ with high affinity (K d ϳ 140 -400 nM) (34,35). The solution structure of residues 371-509 of canine NCX1 (36) redefined the boundaries (Fig. 1B) of a module earlier described as the Calx-␤ domain (37). A single copy of the Calx-␤ domain is found in integrin ␤4 (37,38), and multiple copies are present in very large G protein-coupled receptors (39,40) and extracellular matrix proteins ranging from sponges to humans (41,42). A sequence identity of 27% for residues 501-650 with residues 371-500 suggested the presence of an unexpected, second Ca 2ϩ -binding domain (CBD) in the cytosolic exchanger loop (36). As for the first, the NMR structure of the second CBD was determined in the Ca 2ϩ -bound form. Both domains (CBD1 and CBD2) have the architecture of a ␤-sandwich, formed by two antiparallel ␤-sheets, with one ␤-sheet containing strands A, B, and E and the other containing strands C, D, F, and G (Fig. 1B). Each domain features a ␤-bulge in strand A, but only CBD2 displays a second ␤-bulge in strand G. In contrast, CBD1 possesses a cis-proline at a structurally similar position. The long FG loop in each of the individual domains (residues 468 -482 in CBD1 and residues 599 -627 in CBD2) is largely unstructured. A variable region comprising 39 -75 residues, depending on the splice form, precedes the FG loop in CBD2. Superposition of the ensemble of NMR structures of CBD1 and CBD2 (supplemental Fig. S1) shows the largest differences to be in the BC, CD, and FG loops as well as in parts of the first acidic segment (residues 446 -454 and 577-582, respectively). Ile-374/Phe-505, Phe-376/Phe-507, Tyr-422/Phe-553, Phe-431/Phe-562, Phe-456/Phe-587, and Leu-460/Ile-591 form the core of the two domains and are probably important for stability. Intriguingly, at the N-terminal side of the domains, the highly conserved Arg-396/Arg-527 (Fig. 1B) engages in a stacking interaction with the conserved Phe-431/Phe-562. Whereas Arg-527 in CBD2 clearly stabilizes the strictly conserved BC loop, the function of Arg-396 in CBD1 remains unclear. Subsequent x-ray structures of Ca 2ϩ -bound CBDs (CBD1, Protein Data Bank (PDB) code 2DPK (43) and codes 3E9T and 3EAD (44); and CBD2, code 2QVM (45)) have provided a more detailed view of the Ca 2ϩ -binding sites. The structures show that the largest contribution to the Ca 2ϩ -binding sites of the CBDs originates from two acidic segments, located in the EF loop and at the C terminus of the domains (Fig. 1, B-D), respectively. Interestingly, in CBD2, the EF loop is encoded by one of the mutually exclusive exons, A or B, which determines whether NCX1 can overcome Na ϩ -dependent inactivation. In both CBDs, Ca 2ϩ -binding sites are complemented by conserved glutamate (Glu-385/Glu-516) and aspartate (Asp-421/ Asp-552) residues in the AB and CD loops, respectively. In CBD1, four Ca 2ϩ ions are bound in an arrangement reminiscent of C 2 domains (Fig. 1C) (43). In contrast, due to Lys-585 and the absence of acidic counterparts of Glu-454, Asp-499, and Asp-500 of CBD1, the Ca 2ϩ -binding sites of CBD2-AD (Fig. 1D), with AD denoting the presence of residues encoded FIGURE 1. Structural basis and organization of the two Ca 2؉ sensors (CBD1 and CBD2) in NCX. A, updated topology model of NCX consisting of a transmembrane domain and an ϳ500-residue-long cytosolic loop that harbors the two Ca 2ϩ -binding domains (CBD1 and CBD2). The variable region in CBD2 as a consequence of alternative splicing (AS) is shown in violet. The conserved ␣-repeat regions that define the cation/Ca 2ϩ exchanger superfamily are colored yellow. B, ribbon diagram displaying the ␤-sandwich architecture of Ca 2ϩ -bound CBD1. The structures of the CBDs redefined the previously described Calx-␤ motif by adding strands A and G (yellow). C and D, detailed views of the CBD1 (PDB code 2DPK) and CBD2 (code 2QVM) Ca 2ϩ -binding sites in the Ca 2ϩ -bound form. E, hypothetical model of NCX consisting of four domains: transmembrane domain (residues 1-217 and 727-903; gray), the CLD (residues 218 -370 and 651-726; blue), CBD1 (residues 371-500; red), and CBD2 (residues 501-650; green), with the numbering based on the canine NCX1 AD splice variant (NCX1.4). The ribbon diagram of CBD2 (upper right) displays the variable region encoded by the mutually exclusive exons A or B (blue) and the small cassette exons (yellow) at the opposite side of the CBD2 Ca 2ϩ -binding sites. F, interface of CBD1 and CBD2, displaying the crucial salt bridges and hydrogen bonds in the presence of Ca 2ϩ . Residues in parentheses refer to the numbering scheme of canine NCX1.
by exons A and D, show a reduced density of negative charges. Hence, CBD2-AD coordinates only two Ca 2ϩ ions (36,45), whereas Arg-578 and Cys-585 in exon B-encoded NCX1 splice variants prevent binding of Ca 2ϩ completely (29,46).
Comparison of 15 N heteronuclear single quantum coherence NMR spectra of the Ca 2ϩ -free and Ca 2ϩ -bound forms suggested that there is a substantial loss of structural integrity in the Ca 2ϩ -binding sites of CBD1 in the absence of Ca 2ϩ , but only a modest degree of disorder in the Ca 2ϩ -binding sites of CBD2-AD (36). These preliminary indications were later confirmed by an x-ray structure of the Ca 2ϩ -free form of CBD1 (PDB code 3E9T, chains C and D) (44), where no electron density was visible for residues corresponding to Asp-447-Glu-451 and Asp-498 -Asp-500. In the case of CBD2-AD, the NMR structure of the apo-form (PDB code 2KLS) (46) shows that Lys-585 crucially points into Ca 2ϩ -binding site II, where it forms a salt bridge with Asp-552 and thereby stabilizes the Ca 2ϩ -binding sites. Intriguingly, this NMR structure, determined at neutral pH, does not reflect a state as rigid and locked as the state suggested by an x-ray structure of supposedly Ca 2ϩfree CBD2-AD (PDB code 2QVK) (45) obtained from crystals grown at pH 4.9.
Using the CBDs as constraints resulted in a model for the intact exchanger (Fig. 1E) in which the Ca 2ϩ -binding sites of CBD1 are ϳ90 Å away from the transport Ca 2ϩ -binding site in the transmembrane domain (36). In contrast, the Ca 2ϩ -binding sites of CBD2 are close to a predicted third domain that shows homology to ␣-catenin (catenin-like domain (CLD)). The predicted oppositely oriented arrangement of the CBDs was subsequently confirmed by solution structures obtained from small-angle x-ray scattering (SAXS) (46) and by x-ray structures of CBD12 from Drosophila isoforms CalX1.1 and CalX1.2 (47), two-domain constructs containing CBD1 and CBD2.
In CalX1.1, the interface of CBD1 and CBD2 displays a relative paucity of interactions between the domains (Fig. 1F). Most importantly, Arg-584, the residue corresponding to the strictly conserved Arg-532 of canine NCX1, forms a strong salt bridge with Asp-552 (Asp-500) as well as two hydrogen bonds with Asp-517 (Asp-448) and Asp-552 (Asp-500), respectively. In addition, Arg-584 (Arg-532) is further stabilized via a hydrogen bond to Asn-615 (Asn-564). In the center of the interface, the side chain of His-553 (His-501) forms two hydrogen bonds with the backbone carbonyls of Leu-677 and Lys-679. At the opposite side of the interface, Glu-521 (Glu-452) maintains a bidentate salt bridge with the conserved Arg-673, located in helix 2 of CBD2. Finally, Phe-519 (Phe-450) at the tip of the EF loop may be involved in some limited van der Waals interactions with the side chains of His-553, Ile-674, and Ser-678. While the interface explains previously intriguing functions of the strictly conserved Asp-517 (Asp-448) (48) and Glu-521 (Glu-452), strikingly, the apparent structural importance of Arg-584 (Arg-532) in the CBD12 interface (46,47) is not in line with functional data for this residue (49).
A recent major development in our understanding of Na ϩ / Ca 2ϩ exchange was the determination of the structure of a prokaryotic NCX homolog from Methanococcus jannaschii (NCX_Mj) in the outward conformation (50). The 1.9 Å crystal structure revealed a largely symmetrical transmembrane domain, arranged in the form of two structurally similar halves of five ␣-helices with opposite topology ( Fig. 2A). Helices 2-5 and 7-10 are tightly packed, whereas helices 1 and 6 are somewhat separated from the bundle and tilted at an angle of ϳ45°w ith respect to the vertical axis of the membrane. In line with the proposed transport stoichiometry of one Ca 2ϩ ion in exchange for three Na ϩ ions, four cation-binding sites, one specific for Ca 2ϩ and three that probably bind Na ϩ , were identified in the protein core at the center of the membrane. Strikingly, all residues participating in ion binding originate from the ␣-repeat regions and are highly conserved in the NCX and Na ϩ / Ca 2ϩ -K ϩ exchanger branches of the cation/Ca 2ϩ exchanger superfamily (20). In particular, in the center, Glu-54 (Glu-113 in canine NCX1) and Glu-213 (Asp-814) coordinate the Ca 2ϩ ion via their carboxylate groups, whereas the backbone carbonyl oxygen atoms of Thr-50 and Thr-209 complement the binding site. In addition, the O⑀2 atoms of Glu-54 and Glu-213 contribute to the middle Na ϩ -binding site as well as to the Na ϩ -binding sites toward the extra-and intracellular sides, respectively. The latter two Na ϩ -binding sites share identical ligand chemistry and geometry, where Ser-77, Ala-206, Thr-209, and Ser-210 form the Na ϩ -binding site oriented toward the extracellular side, and Ala-47, Thr-50, Ser-51, and Ser-236 constitute the Na ϩ -binding site closer to the intracellular side. Finally, Asn-81 and Asp-240 complete the coordination scheme of the middle Na ϩ -binding site.
Two hydrophilic pathways permit independent access to the Na ϩ -and Ca 2ϩ -binding sites from the extracellular side. Making use of the symmetry of NCX_Mj by applying a molecular dyad defined by the middle Na ϩ ion and the Ca 2ϩ ion also enabled the construction of a feasible model for the inwardfacing conformation that reflects the access pathways from the cytosolic side. The crystal structure of the outward-facing conformation of NCX_Mj and the inward-facing model led to the proposal of a simple and rapid Na ϩ /Ca 2ϩ exchange mechanism that is in agreement with the high turnover rates found for NCX. Together, this work has provided another substantial piece of information to help solve the puzzle of the intact structure of mammalian NCX (Fig. 2B). Presently, residues 1-42, 218 -258, and 651-726 (numbering according to the AD splice variant of NCX1), which determine the relative positions of the domains with respect to each other, are the remaining missing pieces.

Ca 2؉ Binding Determinants of CBD1 and CBD2
The thermodynamic properties of the Ca 2ϩ -binding sites of CBD1 and CBD2 have been extensively investigated by isothermal titration calorimetry (46). Initial sequence comparison of all available Na ϩ /Ca 2ϩ exchanger sequences revealed strict conservation of the Ca 2ϩ -coordinating residues in CBD1 (Fig.  1C). The expected similarity of the Ca 2ϩ binding properties of the three known exchanger isoforms was confirmed by the titration of CBD1 of canine NCX1 and mouse NCX2 and NCX3 (46). However, these experiments did not allow their individual macroscopic binding constants to be confidently determined, but only the approximate range, 100 -600 nM. These affinities make CBD1 the primary Ca 2ϩ sensor and thus responsible for Ca 2ϩ activation in NCX (26,34,36).
In strong contrast to CBD1, sequence analysis of CBD2 has revealed that residue types at positions 552, 578, and 585, located around Ca 2ϩ -binding site II (Fig. 1D), can vary depending on the isoform and splice variant. As a consequence, the different isoforms and splice variants bind zero to three Ca 2ϩ ions with affinities ϳ5-50-times lower than those of CBD1 (for details, see Ref. 46).

Function of Residues Encoded by the Cassette Exons
An increasing amount of evidence indicates that besides the Ca 2ϩ -binding sites of CBD1 and CBD2, residues encoded by the cassette exons may contribute to ion transport regulation in NCX. The first sign that cassette exons can influence Ca 2ϩ affinities of the CBD1 Ca 2ϩ -binding sites came from isothermal titration calorimetry experiments (46) with CBD12-AD and CBD12-ACDEF, two-domain constructs representing the brain and heart splice variants. Although the individual CBD structures suggest that both constructs should bind six Ca 2ϩ ions, strikingly, the longer ACDEF variant appears to bind only two Ca 2ϩ ions with medium affinity, whereas CBD12-AD binds six Ca 2ϩ ions, as expected. Completely lacking the high affinity component of CBD1, particle-induced x-ray emission experiments (51) confirmed the coordination of four Ca 2ϩ ions in CBD12-ACDEF prior to the start of titration. Thus, residues encoded by the additional cassette exons C, E, and F, located at the opposite end of the CBD2 Ca 2ϩ -binding sites, apparently increase the CBD1 Ca 2ϩ affinities substantially. This is in line with the fact that the ACDEF splice variant is found predominantly in the heart, where the exchanger either is required to enter the activated state at considerably lower intracellular Ca 2ϩ concentrations or is even permanently activated (52) to guarantee high Ca 2ϩ fluxes.

Electrostatic Switches in CBD1 and CBD2
In analogy to C 2 domains (53), analysis of the electrostatic potentials of CBD1 and CBD2 in the absence and presence of Ca 2ϩ revealed dramatic differences and suggested that the CBD1 and CBD2 Ca 2ϩ -binding sites form electrostatic switches. In particular, in the absence of Ca 2ϩ , the CBD1 Ca 2ϩbinding sites display a strongly negative electrostatic potential due to the extensive cluster of aspartic and glutamic acid residues (Asp-421, Asp-446, Asp-447, Asp-448, Asp-498, Asp-499, Asp-500, Glu-385, Glu-451, and Glu-454) and the lack of any basic residues (Fig. 1C). This high density of negative charges not only is responsible for the loss of the structural integrity of the CBD1 Ca 2ϩ -binding sites in the absence of Ca 2ϩ but may also cause repulsion with a highly acidic conserved region consisting of four consecutive glutamate residues (Glu-622-Glu-625) at the beginning of helix 2 in CBD2. Upon binding of four Ca 2ϩ ions, both the negative potential and the repulsion are likely to be strongly reduced.
In comparison, the CBD2 electrostatic potential in the absence of Ca 2ϩ is considerably less negative due to the lower number of acidic residues and the presence of basic residues (Arg-547, Lys-583, and Lys-585) around Ca 2ϩ -binding site II. In addition, the different numbers and affinities of the Ca 2ϩbinding sites in the various isoforms and splice variants determine the strength of the electrostatic switch in CBD2.

Ca 2؉ Induces a Twist in the Hinge between CBD1 and CBD2
SAXS analysis of CBD12-AD and CBD12-ACDEF constructs in the absence and presence of Ca 2ϩ revealed a substantial conformational change upon Ca 2ϩ binding that leads to a compaction of the CBDs (46). Although in the intact exchanger this conformational change is probably constrained by the N-and C-terminal linker regions to the CLD, the SAXS analysis provided the first clue of the effects of Ca 2ϩ binding to the CBD1 Ca 2ϩ -binding sites and indicated that the conformational change induced may not be identical in CBD12-AD and CBD12-ACDEF. Indeed, superposition of the crystal structures of CBD12 from the Drosophila Na ϩ /Ca 2ϩ exchanger isoforms CalX1.1 and CalX1.2 shows a rotation by ϳ9°between them. Comparison of the interfaces of CalX1.1 and CalX1.2 reveals that the number of interactions between CBD1 and CBD2 is reduced in CalX1.2 with respect to CalX1.1. Most prominently, CalX1.2 apparently lacks the salt bridge between Glu-521 and Arg-673 (Fig. 1F), resulting in increased interdomain flexibility.

The Drosophila Exchanger Remains Enigmatic
CalX is unique among the characterized NCXs in that micromolar levels of Ca 2ϩ inhibit rather than activate Na ϩ /Ca 2ϩ exchange (54). With respect to the CBDs, there are at least three major differences between CalX and the other NCXs. First, CBD1 of CalX lacks most of the FG loop that is disordered in the isolated CBD1 structure of canine NCX1 (36,43). Similar to the FG loop of CBD2 at the CBD12 interface, the FG loop of CBD1 may rigidify in intact NCX and potentially interact with the CLD. Indeed, Hryshko and co-workers (55) could reinstall normal Ca 2ϩ activation in a CalX chimera that had Lys-404 -Tyr-580 substituted with the corresponding residues of NCX1, indicating that the trimmed FG loop in CBD1 might play a role in Ca 2ϩ inhibition of CalX. Second, CalX lacks three of the four conserved consecutive glutamate residues that might cause repulsion with the CBD1 Ca 2ϩ -binding sites in the absence of Ca 2ϩ . Third, CalX does not show Ca 2ϩ binding to CBD2 (PDB code 3E9U (56)) and is therefore incapable of forming an electrostatic switch in the second CBD. However, unlike in the exon B-encoded variants of NCX1, the region around the EF loop of the individual CBD2 of CalX is structured, although the same region is intriguingly disordered in the Ca 2ϩ -bound CBD12 structures (47).

Potential Mechanism
The combination of available structural and biophysical data with results from functional studies has resulted in the proposal of a dual electrostatic switch mechanism for Ca 2ϩ regulation in NCX (Fig. 3, A-C) (46). As intracellular Ca 2ϩ concentrations rise, Ca 2ϩ ions initially bind to the CBD1 Ca 2ϩ -binding sites. Increased rigidity and a substantial increase in the electrostatic potential of the Ca 2ϩ -binding sites thereby induce a conformational change that leads to a compaction of the CBDs as visualized by SAXS analysis and supported by FRET studies using full-length NCX and CBD12 constructs (57). As a consequence, the tension on the N-and/or C-terminal linker region to the CLD that relays Ca 2ϩ binding and release events to the transmembrane domain is most likely reduced. This initial activation step (Fig. 3B) is referred to as Ca 2ϩ -dependent activation and initiates Na ϩ /Ca 2ϩ exchange. Demonstrated by particleinduced x-ray emission analysis (46) and suggested by FRET measurements (57), the Ca 2ϩ concentration at which NCX becomes activated probably depends on the residues encoded by the cassette exons that modulate the affinities of the CBD1 Ca 2ϩ -binding sites. This modulation would, for instance, allow the appropriate response to single Ca 2ϩ transients produced by type 1 metabotropic glutamate receptors (mGluR1) at Purkinje cell synapses, whereas in hippocampal neurons, mGluR5 generates a radically different Ca 2ϩ signal in the form of an oscillatory pattern (8). Although there are no cell-specific expression data for exchanger splice variants in the brain, coexpression of NCX1.4 (encoded by exons A and D) with mGluR1 and of NCX1.5 (encoded by exons A, D, and F) with mGluR5 would make sense, as the latter probably features higher Ca 2ϩ affinities at the CBD1 Ca 2ϩ -binding sites and thereby could remain activated during the series of action potentials.
Recently, several reports have suggested that Ca 2ϩ ions in sited I and II of CBD1 are not essential for Ca 2ϩ regulation (58,59). Although the impact of the Ca 2ϩ ions in sites III and IV (Fig. 1F) is without doubt most dominant due to their proximity to CBD2, the lack of Ca 2ϩ -binding sites I and II would substantially change the electrostatic potential at the CBD1 Ca 2ϩ -binding sites. The consequence would be a reduction in the repulsion and attraction between the CBD1 Ca 2ϩ -binding sites and CBD2 in the absence and presence of Ca 2ϩ , respectively, yet these predicted effects are not reflected in functional studies with NCX1 mutants D421A and E454K (58,59). Nevertheless, it remains intriguing why sites I and II should not be essential even though all Ca 2ϩ -coordinating residues in CBD1 are strictly conserved.
To sustain Na ϩ /Ca 2ϩ exchange, Ca 2ϩ ions probably have to also bind to CBD2 (Fig. 3C), a process that is isoform-and splice variant-specific. Functional (29,45,59) and thermodynamic (46) data for exchangers that bind zero to three Ca 2ϩ ions at the CBD2 Ca 2ϩ -binding sites, as well as distinct macroscopic Ca 2ϩ binding constants ranging from 250 nM to 20 M, strongly indicate that the number and affinities of Ca 2ϩ -binding sites in CBD2 determine the form of the steady-state exchange current.
For instance, exon B-encoded NCX1 splice variants become inactivated in a matter of seconds (29), whereas the NCX1 mutant K585E, which is comparable with the B variant of NCX3 and likely binds two Ca 2ϩ ions between the CD and EF loops, shows steady-state outward currents above their corresponding peak currents (45,59). At the same time, this contradicts suggestions that Ca 2ϩ ions between the CD and EF loops of CBD2 are not essential either (45), thus implying isoform-and splice variant-specific determinants to be irrelevant. In contrast, different properties of the CBD2 Ca 2ϩ -binding sites may enable optimal adaptation of Na ϩ /Ca 2ϩ exchange to meet special needs within the cell or a specific tissue.

NCX Inactivation
As cytosolic Na ϩ concentrations are low in normal functioning cells, it is unlikely that Na ϩ -dependent inactivation plays a substantial role under normal physiological conditions. It could, however, be beneficial by limiting NCX-mediated Ca 2ϩ influx under conditions such as ischemia, in which cytosolic Na ϩ levels are elevated and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) levels are low (52).
As early as 1968, Baker and Blaustein (60) reported that a drop in cytosolic pH could inhibit Na ϩ /Ca 2ϩ exchange. Extensive studies on the effect of pH were later performed on sarcolemmal vesicles of canine heart (61) and guinea pig ventricular cells (27,62,63). Prompted by the proton insensitivity of the transmembrane domain (62), experiments with the CBDs revealed that a mild reduction in cytosolic pH from 7.2 to 6.9 caused an up to 3-fold decrease in the Ca 2ϩ affinities of CBD1 (64). This implies that pH is an additional regulator of NCX activity, as it efficiently modulates Ca 2ϩ affinities of the CBD1 Ca 2ϩ -binding sites.
In addition to its primary function as a Ca 2ϩ efflux mechanism, NCX may also mediate Ca 2ϩ influx, where high cytosolic Na ϩ concentrations induce a mode of exchange activity that does not require binding of Ca 2ϩ to the CBDs (52,65). Under these conditions, counteracting Na ϩ -dependent inactivation is overcome by high levels of PIP 2 , which keep NCX activated (Fig. 3D). Furthermore, it has been shown that PIP 2 strongly interacts with residues 218 -237 of NCX and that peptides encoding these residues can inhibit the exchanger (66). As a FIGURE 3. Hypothetical dual electrostatic switch mechanism of Ca 2؉ regulation in NCX. A, inactive Ca 2ϩ -free NCX in extended conformation. B, submicromolar Ca 2ϩ concentrations induce a conformational change via the electrostatic switch in CBD1 that results in a compaction of the CBDs and that probably reduces tension on the linker regions to the CLD. C, binding of Ca 2ϩ to CBD2 allows sustained Na ϩ /Ca 2ϩ exchange and removes counteracting Na ϩ -dependent inactivation. Whether Ca 2ϩ binding to CBD2 induces yet another conformational change or just promotes an interaction with the CLD remains elusive. D, depiction of the PIP 2 -activated state of NCX that is independent of Ca 2ϩ binding the CBDs.
result, this region, which also shows some similarity to the calmodulin-binding motif, is referred to as the exchanger inhibitory peptide region. Mutational studies of the exchanger inhibitory peptide region in intact NCX revealed a profound effect of changes in sequence with respect to the interaction with PIP 2 as well as Na ϩ -dependent inactivation (67).

NCX in Disease
NCX is of paramount importance in the heart, as, together with the sarcoplasmic reticulum Ca 2ϩ -ATPase, the exchanger removes ϳ99% (ϳ30% via NCX and ϳ70% via sarcoplasmic reticulum Ca 2ϩ -ATPase) of the Ca 2ϩ released to the cytosol (7). Dysregulation of this Ca 2ϩ balance can lead to cardiac arrhythmias, a leading cause of death in cardiovascular disease. In the failing heart and in patients with ischemia-reperfusion injury (68,69), in cases of salt-sensitive hypertension (70,71) or multiple sclerosis (72), NCX appears to operate predominantly in reverse mode, importing rather than exporting Ca 2ϩ . The resulting excessive Ca 2ϩ entry may cause the activation of Ca 2ϩ -dependent proteases such as calpain and caspases (73), subsequently leading to cell apoptosis. As the damaging role of NCX is related mostly to its reverse mode, benzyloxyphenyl derivatives (74) such as KB-R7943 (75,76), SEA0400 (77), and SN-6 (78), which specifically inhibit the reverse mode operation of NCX, have attracted a great deal of attention (for review, see Ref. 3). Although experiences with these inhibitors are overall rather positive, thorough testing in relevant preclinical models remains to be performed (79).

Concluding Remarks
Over the last 5 years, high resolution NMR and x-ray structures of a large part of the regulatory loop of NCX and the transmembrane domain of a prokaryotic NCX homolog have added a new dimension to our understanding of the regulation and function of this essential transporter. In addition, the role of different splice variants has become increasingly clear. The variability in CBD2 as a consequence of alternative splicing is now known to modulate not only the number and affinities of the Ca 2ϩ -binding sites in CBD2 but also the Ca 2ϩ affinities in CBD1 (46). Furthermore, the CBD12 SAXS solution structures have suggested a conformational change in the hinge between CBD1 and CBD2 upon Ca 2ϩ binding (46), whereas CBD12 x-ray structures have revealed the link between the CBD1 Ca 2ϩ -binding sites and CBD2 (47). This description of the interface includes hydrogen bonds and salt bridges with residues located in the FG loop of CBD2. Future efforts will aim to connect the regulatory effects of the CBDs to the ion-conducting transmembrane domain of NCX.