Roles of Two Ca2+-binding Domains in Regulation of the Cardiac Na+-Ca2+ Exchanger*

We expressed full-length Na+-Ca2+ exchangers (NCXs) with mutations in two Ca2+-binding domains (CBD1 and CBD2) to determine the roles of the CBDs in Ca2+-dependent regulation of NCX. CBD1 has four Ca2+-binding sites, and mutation of residues Asp421 and Glu451, which primarily coordinate Ca2+ at sites 1 and 2, had little effect on regulation of NCX by Ca2+. In contrast, mutations at residues Glu385, Asp446, Asp447, and Asp500, which coordinate Ca2+ at sites 3 and 4 of CBD1, resulted in a drastic decrease in the apparent affinity of peak exchange current for regulatory Ca2+. Another mutant, M7, with 7 key residues of CBD1 replaced, showed a further decrease in apparent Ca2+ affinity but retained regulation, confirming a contribution of CBD2 to Ca2+ regulation. Addition of the mutation K585E (located in CBD2) into the M7 background induced a marked increase in Ca2+ affinity for both steady-state and peak currents. Also, we have shown previously that the CBD2 mutations E516L and E683V have no Ca2+-dependent regulation. We now demonstrate that introduction of a positive charge at these locations rescues Ca2+-dependent regulation. Finally, our data demonstrate that deletion of the unstructured loops between β-strands F and G of both CBDs does not alter the regulation of the exchanger by Ca2+, indicating that these segments are not important in regulation. Thus, CBD1 and CBD2 have distinct roles in Ca2+-dependent regulation of NCX. CBD1 determines the affinity of NCX for regulatory Ca2+, although CBD2 is also necessary for Ca2+-dependent regulation.

The Na ϩ -Ca 2ϩ exchanger (NCX) 2 is a plasma membrane protein that uses the electrochemical gradient of Na ϩ to extrude Ca 2ϩ from cells (1). NCX is particularly abundant in cardiac myocytes and helps restore intracellular Ca 2ϩ levels following excitation-contraction coupling (1). In addition to being transported substrates, cytoplasmic Na ϩ and Ca 2ϩ regulate NCX activity. Intracellular Na ϩ decreases exchanger activity by inactivating NCX (Na ϩ -dependent inactivation or I 1 ), whereas cytoplasmic Ca 2ϩ both stimulates activity and relieves the exchanger from the Na ϩ -dependent inactivation (2,3). By tuning exchanger activity, regulation by Na ϩ and Ca 2ϩ has fundamental roles in Ca 2ϩ homeostasis.
Regulatory Ca 2ϩ binds to two cytoplasmic Ca 2ϩ -binding domains (CBD1 and CBD2) located within the large intracellular loop of NCX, between transmembrane segments 5 and 6 (4 -9). Each CBD comprises a ␤-sandwich containing seven antiparallel ␤-strands with Ca 2ϩ -binding sites at one end of the ␤-sandwich (4,5,8,9) and an unstructured loop connecting ␤-strands F and G at the opposite end of the sandwich. In CBD1, there are sites for coordinating four Ca 2ϩ ions (Ca1-Ca4). Previous studies suggest that residues coordinating Ca 2ϩ ions at sites 3 and 4 of CBD1 (10) set the affinity of the exchanger for cytoplasmic Ca 2ϩ , whereas recent crystal and electrophysiological data show that binding of Ca1 is not required for exchanger regulation (11). No data are available on the role of Ca 2ϩ bound at site 2.
Although the structure of CBD2 is similar to that of CBD1, it only contains sites for two Ca 2ϩ ions (Ca1 and Ca2). Replacement of the residues responsible for coordinating Ca 2ϩ at its primary site (Ca1) completely abolished Ca 2ϩ regulation (4), indicating an important role for CBD2 in exchanger regulation. Ca 2ϩ bound at the secondary site (Ca2) appears to have no role in exchanger regulation (4).
Despite recent progress, an understanding of the mechanisms leading to activation of NCX by cytoplasmic Ca 2ϩ is still unresolved. It is well established that Ca 2ϩ activates the exchanger by decreasing the extent of the Na ϩ -dependent inactivation and also by directly increasing NCX activity (2,3). The relative contributions of CBD1 and CBD2 in the control of these mechanisms are unclear. To advance our understanding of Ca 2ϩ regulation of the NCX, we mutated residues that coordinate Ca 2ϩ in both domains and examined the effects on Ca 2ϩdependent regulation. Our findings indicate that only Ca 2ϩ sites 3 and 4 of CBD1 are important for Ca 2ϩ regulation and that CBD2 also contributes to this process.

EXPERIMENTAL PROCEDURES
Mutagenesis and RNA synthesis were performed as described previously (12). RNAs encoding for NCXs were injected into Xenopus laevis oocytes. Oocytes were kept at 18°C for 4 -7 days. Inside-out giant patch recordings of outward NCX currents were performed as described previously (12,13). Borosilicate glass pipettes of about 20 -30 m were utilized. Intracellular solutions were rapidly changed using a computercontrolled 20-channel solution switcher. Measurements were obtained using pipette solution (100 mM N-methylglucamine, 10 mM HEPES, 20 mM tetraethylammonium hydroxide, 0.2 mM niflumic acid, 0.2 mM ouabain, 8 mM Ca(OH) 2 (pH 7, adjusted with methanesulfonic acid)) and bath solution (100 mM CsOH or 100 mM NaOH, 20 mM tetraethylammonium hydroxide, 10 mM HEPES, 10 mM EGTA or HEDTA, and different Ca(OH) 2 concentrations to obtain the desired final free Ca 2ϩ concentrations (pH 7, using methanesulfonic acid)). Free Ca 2ϩ concentrations were calculated according to the WEBMAXC program (14) and confirmed with a Ca 2ϩ electrode.
Ca 2ϩ activation curves were obtained by perfusing solutions with different ion concentrations. Data were normalized to the maximum values and fitted to a Hill function. Each point is the average of between three and six experiments. Values are mean Ϯ S.E. PCLAMP (Axon Instruments, Burlingame, CA) software was used for acquisition and analysis. Data were acquired on line at 4 ms/point and filtered at 50 Hz using an 8-pole Bessel filter. Experiments were performed at 35°C and at a holding potential of 0 mV.

RESULTS
The crystal structures of the CBDs of the NCX have been resolved, and the regions involved in coordinating Ca 2ϩ are shown in Fig. 1. Our goal was to determine the regulatory roles of specific Ca 2ϩ ions that bind in CBD1 and CBD2. Residues mutated in this work are underlined. We utilized the giant patch technique (13) to characterize the response of mutant exchangers to cytoplasmic Ca 2ϩ . Outward exchange currents were elicited by rapidly applying Na ϩ (100 mM) into the bath at the intracellular surface of the patch. Ca 2ϩ (8 mM) was continuously present within the pipette at the extracellular surface. As can be seen in the traces recorded from the wild type exchanger (WT) ( Fig. 2A), exchange current peaks and then decays to a steady-state value when Na ϩ is applied to the intracellular surface. The decay is triggered by the high intracellular Na ϩ (Na ϩ -dependent inactivation or I 1 ). Increasing cytoplasmic Ca 2ϩ concentrations increases the peak currents and antagonizes I 1 (3).
Mutations within CBD1-First, the effects of mutations within CBD1 were analyzed. Fig. 2 shows examples of WT, D421A, and E451A exchanger currents recorded at different intracellular Ca 2ϩ concentrations. Asp 421 and Glu 451 are primarily involved in coordinating Ca 2ϩ at Ca1 and Ca2 of CBD1 (Fig.  1). Similar to WT, currents recorded from oocytes expressing these mutants peaked rapidly and then decayed over several seconds because of the Na ϩ -dependent inactivation. Peak currents from both WT and mutant exchangers were enhanced by raising the intracellular concentration of regulatory Ca 2ϩ . Because the onset of the Na ϩdependent inactivation is slow (3), the peak current reflects mainly the effects of Ca 2ϩ on exchanger activation. Peak currents as a function of Ca 2ϩ concentration for WT and mutant exchangers were fitted to a Hill function (Fig. 2B).
The extrapolated values of the apparent Ca 2ϩ affinities were 0.86 M for the WT exchanger and 1.46 and 1.12 M for D421A and E451A, respectively, indicating that mutations at these sites slightly reduce the sensitivity of NCX for cytoplasmic Ca 2ϩ . In addition to activating the exchanger, increasing regulatory Ca 2ϩ releases NCX from Na ϩ -dependent inactivation (2,10). The effects of Ca 2ϩ on Na ϩ -dependent inactivation are analyzed by measuring fractional currents calculated as the ratio of the steady-state current to the peak current (fractional activity). Both WT and D421A and E451A mutant exchangers show a similar reduction in the extent of Na ϩ -dependent inactivation with increasing Ca 2ϩ (Fig. 2C), indicating that the mutations did not alter the effects of Ca 2ϩ on Na ϩ regulation. Overall, our data indicate that Ca1 and Ca2 of CBD1 contribute minimally to regulation of the exchanger by Ca 2ϩ .
The roles of residues coordinating Ca 2ϩ to sites 3 and 4 of CBD1 were also investigated. Residues Glu 385 , Asp 447 , Ile 449 , Glu 451 , Asp 498 , and Asp 500 coordinate Ca 2ϩ at site 3, whereas Asp 446 , Asp 447 , Asp 499 , and Asp 500 comprise site 4. Single mutants D447V, D498I, and D500V were previously shown to alter markedly the apparent affinity of the exchanger for cytoplasmic Ca 2ϩ (10). We now characterize the biophysical properties of two mutants E385A and double mutant D446A/ D447A and further investigate the effects of replacing Asp 500 with valine. Glu 385 exclusively coordinates Ca 2ϩ at site 3, whereas mutants D446A/D447A and D500V will perturb Ca 2ϩ binding at both sites 3 and 4. Fig. 3 shows representative outward current traces recorded at the indicated regulatory Ca 2ϩ concentrations from oocytes expressing the mutant exchangers. Activation of currents from E385A, double mutant D446A/ D447A, and D500V required higher intracellular Ca 2ϩ than did the WT exchanger. The dependence of the peak current on intracellular Ca 2ϩ for WT and exchanger mutants is shown in Fig. 3B. Mutations at these sites decreased the apparent Ca 2ϩ Next, we constructed and characterized an exchanger with mutations in 7 of the 10 acidic amino acids (designated M7, with the following residues mutated to alanine: 385, 421, 446, 447, 498, 499, and 500) that are directly involved in the coordination of Ca 2ϩ to CBD1. CBD1 of M7 should be incapable of binding Ca 2ϩ . Although it was not possible to quantify the apparent Ca 2ϩ affinity of peak currents because of lack of saturation, M7 retained Ca 2ϩ regulation (Fig. 3, A and B), unmasking a role for CBD2 in exchanger regulation.
The effects of cytoplasmic Ca 2ϩ on the Na ϩ -dependent inactivation of WT and exchanger mutants were then investigated. First, the fraction of inactivated mutant exchangers was quantified by measuring fractional activity. As shown in Fig. 3C, the fractional activity values measured for E385A, D446A/D447A, D500V, and M7 were significantly higher than that of WT (values are 0.12 Ϯ 0.04 for WT, 0.28 Ϯ 0.01 for E385A, 0.42 Ϯ 0.06 for D446A/D447A, 0.47 Ϯ 0.16 for D500V, and 0.35 Ϯ 0.07 for M7, measured in the presence of 1.4 M regulatory Ca 2ϩ ), indicating that the Na ϩ -dependent inactivation was less pronounced in the mutant exchangers.
To investigate Ca 2ϩ regulation further, we examined the Ca 2ϩ dependence of steady-state current. Ca 2ϩ influences steady-state current by both increasing exchanger activity and relieving NCX from the Na ϩdependent inactivation. For the WT exchanger, the dependence of the steady-state current on Ca 2ϩ is shifted to a much higher Ca 2ϩ than the peak current ( Fig. 4) (3). Fig. 4 also shows normalized peak and steady-state Ca 2ϩ activation curves for mutants E385A and D500V. The gap between the concentration dependences of peak and steadystate currents for regulatory Ca 2ϩ was significantly reduced in E385A mutant because of a decrease in Ca 2ϩ sensitivity of the peak current. Interestingly, for mutant D500V, the steady-state current is saturable and shows a nearly identical affinity as the peak current. In this mutant, the affinity of the peak current is simultaneously reduced, and the sensitivity of the steady-state current to cytoplasmic Ca 2ϩ is increased. The results emphasize the important role that Ca 2ϩ ions coordinated by sites 3 and 4 of CBD1 play in exchanger regulation.
Mutations within CBD2-We next examined the effects of mutations within CBD2. Previously, we showed that three mutants within CBD2 (E516L, D578V, and E683V) lack Ca 2ϩ regulation (4). These three anionic residues all contribute directly to the binding of the primary Ca 2ϩ (Ca1) to CBD2. We refer to Ca1 of CBD2 as the primary Ca 2ϩ because Ca2 is bound loosely and appears to have no function (4). We hypothesized that introducing a positive charge at these sites might partially mimic a Ca 2ϩ ion and perhaps rescue Ca 2ϩ regulation. Fig. 5 shows representative outward NCX currents for WT and mutants E516R and E683R in the absence and presence of Ca 2ϩ . (Mutant D578R has been previously characterized and shown to be regulated by Ca 2ϩ (15).) In the absence of regulatory Ca 2ϩ , a substantial component of NCX current was present that was augmented by raising intracellular Ca 2ϩ . Thus, the E516R and E683R mutants partially retained Ca 2ϩ regulation of peak current in contrast to mutants in which neutral amino acids were used as replacements. Fig. 5B summarizes the effects of Ca 2ϩ on peak currents, indicating that the Ca 2ϩ -sensitive components of these mutants have apparent affinities for Ca 2ϩ similar to those of WT (K 1/2 micromolar values are 0.86, 0.72, and 0.6 for WT, E516R, and E683R, respectively). Fig. 5C shows a summary of the effects of Ca 2ϩ on fractional activity of the WT and mutant exchangers. For WT, the extent of Na ϩ -dependent inactivation decreases as Ca 2ϩ is elevated as indicated by increased fractional activity. As observed previously for mutants E516L, D578V, and E683V, introduction of a Lys 585 in CBD2 forms a salt bridge with Asp 552 and Glu 648 in the absence of Ca 2ϩ , conferring structural integrity to the Ca 2ϩ -free form (4). (Note that Glu 683 of NCX1.1 studied here is equivalent to Glu 648 of the splice variant NCX1.4 used in the crystallization studies.) Introduction of a negative charge at this location (K585E) slightly decreases the Ca 2ϩ sensitivity of the peak current (4). There is also a marked alteration in the effect of Ca 2ϩ on Na ϩ -dependent inactivation. The steady-state current becomes much more sensitive to intracellular Ca 2ϩ . This can be seen in Fig. 6A, where 20 M Ca 2ϩ completely removes the Na ϩ -dependent inactivation of the K585E mutant, whereas this is not true of the WT exchanger ( Figs. 2A and 3A; see also We inferred above that the low affinity Ca 2ϩ regulation of the CBD1 mutant M7 was due to CBD2. We tested that idea by introduction of the K585E mutation into the M7 background. If the K585E mutation does increase the affinity of CBD2 for Ca 2ϩ , then it might partially rescue the low apparent Ca 2ϩ affinity of M7. Indeed, this turns out to be the case. Addition of the mutation K585E into the M7 background resulted in an increase in apparent Ca 2ϩ affinity of the peak current (Fig. 6B).

Role of the F-G Loops of CBD1 and CBD2 in Ca 2ϩ
Regulation of the Exchanger-The structures of CBD1 and CBD2 have been determined by both NMR and crystal structure (4 -6, 8). Exceptions, however, are the long loops between ␤-strands F and G of both CBDs, which are unstructured. Although this F-G loop is well conserved among the CBD1 sequences of different exchangers, the corresponding amino acids of CBD2 vary substantially due to alternative splicing, resulting in CBD2s of varying length (16). To determine the functional role of these portions of the CBDs, we deleted residues 467-481 (CBD1) and 596 -633 (CBD2) of NCX1.1 (mutant ⌬(467-481)-(596 -633)) to determine the effects on Ca 2ϩ regulation. Fig. 7A shows outward currents recorded from oocytes expressing the exchanger carrying the double deletion. Similar to WT, exchange current peaked upon application of intracellular Na ϩ and then was inactivated due to the high intracellular Na ϩ . Raising the intracellular Ca 2ϩ concentration further activated the exchanger and decreased the extent of the Na ϩ -dependent inactivation. The fraction of steadystate to peak current values obtained for the WT and ⌬(467-481)-(596 -633) exchangers was not significantly different (  Fig. 7C. Both exchangers exhibit a similar increase in the peak current as regulatory Ca 2ϩ is raised. In summary, deletion of the F-G loops of CBD1 and CBD2 did not significantly alter the biophysical properties of the exchanger, indicating that these two unstructured regions do not play a fundamental role in secondary regulation of the exchanger by Ca 2ϩ .

DISCUSSION
Regulatory Ca 2ϩ modulates the activity of the NCX by binding to two cytoplasmic domains encompassing residues 371-501 (CBD1) and 501-678 (CBD2) (4 -9, 17). CBD1 and CBD2 are both located within the large cytoplasmic loop of the exchanger between transmembrane segments 5 and 6 (7). Recently, the crystal structures of the exchanger CBDs have been resolved (4,8,9). CBD1 and CBD2 bind four and two Ca 2ϩ ions, respectively. The domains have similar structures comprising seven antiparallel ␤-strands with the Ca 2ϩ -binding sites within the connecting loops at one end of the structure. CBD1 may undergo large conformational changes upon unbinding and binding of Ca 2ϩ (5,18,19), although more constrained movements of CBD1 have also been reported (6). In contrast, CBD2 undergoes only minor structural rearrangements upon binding Ca 2ϩ (4). CBD2 of the exchanger of Drosophila melanogaster does not appear to bind Ca 2ϩ (9). Consistent with this observation, the Drosophila exchanger displays anomalous regulatory properties (20).
Binding of cytoplasmic Ca 2ϩ to these domains triggers two measurable molecular processes: Ca 2ϩ activates the exchange current and also rescues current from Na ϩ -dependent inactivation (1,21). Experimentally, these effects are investigated by measuring the initial peak of exchange current and the steady state current, respectively. Because Na ϩ -dependent inactivation occurs over several seconds, the effects of Ca 2ϩ on peak currents mainly reflect exchanger activation, whereas steady-state currents are a function of effects of Ca 2ϩ on both exchanger activation and rescue from the Na ϩ -dependent inactivation. The conformational changes that drive these two regulations are unclear as are the contributions of each CBD. To elucidate the roles of CBD1 and CBD2, we mutated strategic residues within these two domains, guided by the crystal structures, and we determined their effects on Ca 2ϩ regulation using electrophysiology. We first examined the effects of mutations within CBD1. Mutations of residues Asp 421 and Glu 451 minimally alter the Ca 2ϩ sensitivity of NCX. These residues primarily coordinate Ca1 and Ca2. Thus, our data indicate that the Ca1 and Ca2 sites within CBD1 are not crucial in conferring Ca 2ϩ regulation to the exchanger. These results are supported by recent structural and electrophysiological data showing that a mutant exchanger unable to bind Ca 2ϩ at position 1 displays a phenotype similar to the WT exchanger (11).
Recent studies investigating the kinetics and equilibrium properties of Ca 2ϩ binding to CBD1 and CBD2 detected two low affinity Ca 2ϩ sites in CBD1 (22). Our data would suggest that these sites are Ca1 and Ca2. Because the Ca 2ϩ -binding affinities for these sites are higher than 30 M (22), they may not be occupied under the conditions in which our electrophysiological experiments were performed.
In sharp contrast to the mutations that disrupt the Ca1 and Ca2 sites of CBD1, mutations of residues involved in forming the binding sites for Ca3 and Ca4 (E385A, D446V-D447V, and D500V) drastically alter the Ca 2ϩ sensitivity of NCX. The decrease of the apparent affinity for cytoplasmic Ca 2ϩ observed in these mutants emphasizes their important role in Ca 2ϩ regulation of the NCX.
A mutant of particular interest is D500V, which disrupts the binding of Ca 2ϩ to sites 3 and 4 of CBD1. As shown in Fig. 4, the Ca 2ϩ dose-response curves of the D500V peak and steady-state currents overlap. This observation reflects the fact that this mutation abolishes the sensitivity of the Na ϩ -dependent inactivation for Ca 2ϩ as shown in Fig. 3C. Only the activation of the exchanger transport by Ca 2ϩ is observed. D500V is the first single mutation within CBD1 known to alter the effects of Ca 2ϩ on Na ϩ -dependent inactivation therefore revealing an influence of CBD1 in this process.
The recognition of a second CBD in NCX is recent, and the role of this domain is less studied. To gain insight into the function of CBD2 in controlling activity, we inactivated CBD1 by mutating 7 of the 10 amino acids that coordinate Ca 2ϩ binding to CBD1. This M7 exchanger was still Ca 2ϩ -regulated, though with decreased Ca 2ϩ affinity. As we predict CBD1 of M7 cannot bind Ca 2ϩ , the remaining Ca 2ϩ sensitivity must reflect the binding of Ca 2ϩ to CBD2. Because of competition between Ca 2ϩ and Na ϩ at the transport site, it was not possible to increase the intracellular Ca 2ϩ concentration sufficiently to determine the apparent affinity of CBD2 for Ca 2ϩ accurately. However, the apparent Ca 2ϩ affinity of CBD2 was increased and saturation was achieved when the mutation K585E of CBD2 was introduced into the M7 background. Lys 585 is near the Ca 2ϩ -binding sites of CBD2, and its replacement with a negative charge increases the apparent affinity for Ca 2ϩ to relieve Na ϩ -dependent inactivation (4) (Fig. 5). Introduction of the K585E mutation into the M7 background appears to increase the Ca 2ϩ affinity of CBD2, therefore conferring increased apparent Ca 2ϩ affinity to M7. The result confirms that the Ca 2ϩ regulation of M7 is due to CBD2. Alternatively, interactions between CBD1 and CBD2 may exist, and muta-tions in one CBD could affect the affinity of the other domain for Ca 2ϩ .
To investigate further the role of CBD2 in controlling the Ca 2ϩ regulation of the exchanger, two of the anionic amino acids (Glu 516 and Glu 683 ) that coordinate the primary Ca 2ϩ of CBD2 were replaced with positively charged lysines. Previously, we had shown that NCX mutants E516L, D578V, and E683V lack Ca 2ϩ regulation (4). Introduction of a positive charge at any of these positions may mimic the presence of Ca 2ϩ and therefore rescue Ca 2ϩ regulation. Electrophysiological characterization of NCX mutant exchangers E516R and E683R revealed that indeed these mutants were regulated by Ca 2ϩ with affinities similar to that of the WT exchanger. The mutant D578R has previously been shown to also be regulated by Ca 2ϩ (15). Thus, it appears that a specific conformation of CBD2 is required to permit Ca 2ϩ regulation to occur. This permissive conformation is provided by a positive charge at position 516 or 683 but not by substitution with a neutral amino acid. A functional CBD2 allows the Ca 2ϩ affinity for regulation to be set by CBD1.
The structures of the domains of the exchanger that bind Ca 2ϩ within the intracellular loop are known, with the exception of residues 467-481 and 596 -633 within the F-G loops of CBD1 and CBD2, respectively. Because of high flexibility, the structure of these regions in the mammalian NCX remains undetermined. Likewise, their functional roles, if any, are unknown. Of particular interest are amino acid residues 596 -633 (encoding exons C to F of NCX1.1) of CBD2 as this region varies greatly among exchanger isoforms due to alternative splicing (16). The use of alternative exons suggests a potential role of this region in exchanger regulation. For example, in the Drosophila NCX, which has anomalous inhibition by regulatory Ca 2ϩ , the amino acid sequence of the F-G loop is quite different from NCX1, and the region is structured forming two helices in close proximity to the ␤-barrel structure of CBD2 (9). This region  may influence either the binding of Ca 2ϩ to CBD1 or the transduction of the Ca 2ϩ regulatory signal (9). We explored the contribution of the F-G loops to regulation of NCX1.1 by deletion and by characterizing the resultant mutant with respect to activation by cytoplasmic Ca 2ϩ . Our data indicate that these portions of the CBD domains do not play any significant role in Ca 2ϩ regulation.
In summary, our results indicate that both CBD1 and CBD2 contribute to the Ca 2ϩ regulation of the NCX, although the exact roles of each domain are not completely resolved. Residues coordinating Ca 2ϩ sites 3 and 4 in CBD1 and the primary Ca 2ϩ -binding site (Ca1) in CBD2 are key in Ca 2ϩ regulation and are involved in controlling both I 1 and I 2 processes. Further effort is needed to determine how CBD1 and CBD2 communicate with one another. Likewise, further studies are required to understand the transduction of the binding of regulatory Ca 2ϩ to activation of the NCX.