Calmodulin Interacts with the Sodium/Calcium Exchanger NCX1 to Regulate Activity

Changes in intracellular Ca2+ concentrations ([Ca2+]i) are an important signal for various physiological activities. The Na+/Ca2+ exchangers (NCX) at the plasma membrane transport Ca2+ into or out of the cell according to the electrochemical gradients of Na+ and Ca2+ to modulate [Ca2+]i homeostasis. Calmodulin (CaM) senses [Ca2+]i changes and relays Ca2+ signals by binding to target proteins such as channels and transporters. However, it is not clear how calmodulin modulates NCX activity. Using CaM as a bait, we pulled down the intracellular loops subcloned from the NCX1 splice variants NCX1.1 and NCX1.3. This interaction requires both Ca2+ and a putative CaM-binding segment (CaMS). To determine whether CaM modulates NCX activity, we co-expressed NCX1 splice variants with CaM or CaM1234 (a Ca2+-binding deficient mutant) in HEK293T cells and measured the increase in [Ca2+]i contributed by the influx of Ca2+ through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activity and decreased membrane localization. Without the mutually exclusive exon, the exchange activity was decreased and could be partially rescued by CaM1234. Point-mutations at any of the 4 conserved a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3rd or 4th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2nd and 3rd conserved a.a. residues in NCX1.3 decreased exchange activity. Taken together, our results demonstrate that CaM senses changes in [Ca2+]i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.


Introduction
The change in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) is an important signal that controls versatile cellular processes, and there are several mechanisms that maintain Ca 2+ homeostasis. At the plasma membrane, Ca 2+ -pumps and Na + gradient-dependent Ca 2+ transporters are the two main pathways for exporting Ca 2+ out of cells when [Ca 2+ ] i is elevated. In addition, the direction of the Na + gradient-dependent Ca 2+ transporters can be reversed to transport To maintain an open reading frame, exons A and B are mutually exclusive; the other exons can be used in a variety of combinations ( Fig 1C). The NCX1.1 splice variant has exons ACDEF; NCX1.3 has BD; and NCX1.4 contains AD. To characterize the importance of the mutually exclusive exons A and B, we constructed a clone containing only exon D (NCX1D). To characterize the importance of the CaMS, we deleted this region from NCX1.1 (NCX1.3ΔCaMS) and NCX1.3 (NCX1.3ΔCaMS). For pull-down assays, we cloned loop regions containing different exons but without the XIP (NCX1.1CL and NCX1.3CL). The V5 epitope at the C-terminus of these clones is for antibody recognition.

Transfection of HEK293T cells
For transient expression of the exchanger protein in HEK293T cells grown in a 12-well plate, we mixed plasmids (1 μg total, including 0.1 μg of GFP plasmid) with Lipofectamine 2000 according to the manufacturer's instructions. We used GFP fluorescence to identify transfected cells and performed experiments 24~36 hours after the transfection.

Calcium imaging
To elevate the intracellular Na + concentration, we incubated cells in Hank's Balanced Salt Solution (HBSS, 130 mM NaCl, 2 mM KCl, 2.2 mM CaCl 2 , 1 mM MgCl 2 , 5.6 mM glucose, and 10 mM HEPES, pH 7.3) containing the Na + /K + -ATPase inhibitor, ouabain (100 μM), and fura-2 AM (5 μM) for 40 min at RT. We then mounted the cells on the stage of a Nikon Ti inverted microscope; to activate the reverse mode NCX exchange activity (rNCX), we locally perfused cells with NMG buffer (130 mM N-methyl-D-glucosamine (NMG), 2.2 mM CaCl 2 , 1 mM MgCl 2 , 5.6 mM glucose, and 10 mM HEPES, pH 7.3) to reverse the Na + gradients. The buffer was puffed onto cells from a micropipette, with a tip opening of approximately 1 μm, positioned approximately 20 μm from a cell for 60 s at 3 psi, controlled by a Picospritzer III (Parker Instrument Inc., Parker Hannifin, Fairfield, NJ, USA). The excitation for fura-2 was provided by a DG4 (Sutter Inc., CA, U.S.A.) and emissions were collected by an EM-CCD camera (Photometrics, AZ, USA). The whole system was controlled by Nikon AIS Elements software. The fluorescence intensity ratios were converted into [Ca 2+ ] i by the previously reported protocol [19].

Immunostaining
For antibody staining, we incubated cells in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 ) containing 4% paraformaldehyde for 20 min (shaded regions), Ca 2+ binding domains (CBD1 and CBD2), an alternative splicing site (AS), and the predicted CaM binding segment (CaMS). B. Sequence alignment of the CaM binding motif. The alignment includes the conserved 1-5-8-14 CaM binding sequences identified in human calcineurin (a.a. 405-424), human plasma membrane Ca 2+ -ATPase 4b (a.a. 1089-1108), human L-type Ca 2+ channel (a.a. 1601-1620), and human small conductance Ca 2+ -activated K + channel (a.a. 424-443), bovine/human NCX1.1 (a.a. 716-735), human NCX2 (a.a. 666-685), and human NCX3 (a.a. 663-681). Arrows indicates the conserved residues. C. The bovine NCX1 constructs. Empty box indicates the XIP (a.a. 216-270) and boxes filled with different patterns indicate the AS region with different exons. The filled circle indicates the putative CaMS and the empty circle at the C-terminus represents the V5 epitope tag. The number indicates the a.a residue in the NCX1.1. D. Immunostaining of NCX1.1 and NCX1.3 in non-permeabilized cells. HEK293T cells expressing NCX1.1 and NCX1.3 were stained with an antibody against the V5 epitope tagged at the C-terminus of these splice variants. Images were obtained using a Leica SP5 confocal microscope. Scale bar: 10 μm at RT. After a PBS wash, we placed the cells in PBS containing 0.5% Triton X-100 and 1% BSA for 1 hr. We then washed the cells with PBS and incubated them in PBS containing a primary mouse antibody against the V5 epitope (1:500 dilution) at 4°C overnight. After another PBS wash, we incubated the cells in PBS containing the secondary antibody (goat anti-mouse IGg) conjugated to Alexa Fluor 488 at a 1:2000 dilution for 1 hr at RT. To label F-actin and DNA, we placed the cells in PBS containing rhodamine-conjugated phalloidin (10 μM) and 4', 6-diamidino-2-phenylindole (DAPI) (0.5 μg/mL) for 20 min at RT. After a PBS wash, we visualized the fluorescence by imaging with a Leica SP5 confocal microscope. To keep the cell membrane intact, we did not fix the cells with paraformaldehyde until after binding the primary and secondary antibodies at 4°C. The incubation for primary antibody staining was 1 hr.
To visualize the plasma membrane, we coexpressed glycosylphosphatidylinositol (GPI)tagged green fluorescence protein (GFP-GPI) (a gift from Dr. You-Tzung Chen, National Taiwan University) with NCX1 alternative splice variants. To analyze the colocalization of GFP-GPI with NCX1 splice variants, we used a plug-in in ImageJ software to calculate the product of the differences from the mean (PDM) [20]. For each pixel, PDM = (red intensitymean red intensity) × (green intensity-mean green intensity) and we displayed positive image only. The more positive value indicates higher possibility in overlapping at that pixel.

Protein extraction
We resuspended transfected HEK293T cells in PBS and lysed the cells by sonication. We centrifuged the lysates at 1,000 × g for 30 min and collected the supernatant for another round of centrifugation at 100,000 × g for 2 hr. We collected the supernatant as the cytosolic fraction and the pellet as the membrane fraction.

GST pull-down assay
We purified the GST-fused CaM or CaM 1234 according to the previously published protocol [21] and used a Bradford-based protein assay kit (Bio-Rad, CA, USA) to estimate concentration. To pull down interacting proteins, we incubated GST-fused proteins or GST (1 mg) with GSH-Sepharose 4B beads (GE Healthcare, U.S.A.) following the protocol suggested by the manufacturer. We mixed the beads with cell lysates at 4°C overnight in a Binding Buffer (100 mM NaCl, 50 mM MgCl 2 , 10% Glycerol, 0.1% Nonidet P-40, 0.2% BSA, 25 mM HEPES, and 1 mM DTT) containing a Protease Inhibitor Cocktail (Set V, 1:100 dilution, CalBiochem, La Jolla, California, USA). The proteins bound to the beads were analyzed by SDS-PAGE followed by Western blot.

Data analysis
Data are presented as the Mean ± SEM from at least three different batches of cells and analyzed by one-way ANOVA with Fisher's post hoc test. Differences were considered significant when the p value was under 0.05. The number of cells (n) indicates the total number of cells tested.
To verify the orientation of the NCX1 C-terminus, we stained non-permeabilized HEK293T cells expressing NCX1.1 or NCX1.3 with an antibody against the V5 epitope, thereby preventing the antibody from diffusing into the cytosol (Fig 1D). Images show that the fluorescence signal localizes to the perimeter of the transfected cells when the cell membrane is intact. In contrast, cells expressing only the intracellular loop tagged with V5 did not show fluorescence staining in non-permeabilized cells, whereas fluorescence was visible after cells were permeabilized (S1 Fig). These results indicate that the C-terminus of NCX1 is extracellular.

CaM interacts with the NCX1 cytoplasmic loop
To determine whether NCX1 interacts with CaM, we used GST-CaM as the bait to pull down the cytoplasmic loops of NCX1.1 (NCX1.1CL: a.a. 288-805) and NCX1.3 (NCX1.3CL: a.a. 288-769) expressed in HEK293T cells (Figs 1C and 2). Western blots show that the antibody against the V5 epitope stains a protein with a molecular weight similar to the expected size (~70 kD) of NCX1.1CL or NCX1.3CL in the presence of Ca 2+ . In the absence of Ca 2+ or CaM, no stained band was visualized. Neither GST-CaM nor GST-CaM 1234 interacted with the cytoplasmic loop without the CaMS (NCX1.1CLΔCaMS and NCX1.3CLΔCaMS). These results suggest that CaM interacts specifically with the NCX1 cytoplasmic loop via the CaMS in a Ca 2+ -dependent manner.

CaMS deletion affects membrane localization
To characterize whether CaMS affects the localization of NCX1 splice variants, we expressed NCX1.1 and NCX1.3 with or without CaMS in HEK293T cells and stained the cells with an antibody against the V5 epitope (Fig 3). After fixation and permeabilization, the confocal images show that wild-type NCX1.1 and NCX1.3 are largely present at the plasma membrane compared with phalloidin staining, which illustrated the distribution of F-actin to the subplasmalemmal region. The line intensity profiles indicated that both NCX1.1 and NCX1.3 had an overlapping distribution with F-actin at the cell boundary. NCX1 mutants that lack the CaMS (NCX1.1ΔCaMS and NCX1.3ΔCaMS) localized to both the plasma membrane and the cytosolic region. Compared with the F-actin distribution shown by the line intensity profiles, both mutants without the CaMS localized mostly to the cytosolic side of the plasma membrane. To further confirm the membrane localization, we co-expressed the NCX1 splice variants and mutants with GFP-GPI, which encodes a GPI-anchored form of GFP that localized to the extracellular face of the plasma membrane, in HEK293T cells. We then stained the V5 epitope in non-permeabilized cells ( Fig 3B). Both NCX1.1 and NCX1.3 were present at the membrane surface and overlapped with GFP-GPI. Under the same exposure settings, both mutants without CaMS displayed lower membrane surface expression levels than those of the corresponding wild-type proteins. The PDM images showed that the distributions of both NCX1.1 and NCX1.3 correlated well with GFP-GPI at the plasma membrane; in contrast, a lower correlation was observed in cells expressing NCX1.1ΔCaMS or NCX1.3ΔCaMS. In permeabilized cells, the fluorescence signals of the stained V5 and GFP-GPI were concentrated at the plasma membrane for NCX1.1 and NCX1.3; in contrast, the fluorescence signals in permeabilized cells expressing NCX1.1ΔCaMS or NCX1.3ΔCaMS were mostly distributed in a cytosolic region Therefore, both F-actin and GFP-GPI staining revealed that NCX1 splice variants without the CaMS appeared to be localize to the cytosolic side of the membrane and partly on the cell membrane. These results suggest that CaMS plays an important role in the targeting of NCX1 splice variants to the plasma membrane.

CaMS deletion decreases exchange activity
To determine the importance of the CaMS on NCX1 activity, we expressed the splice variants in HEK293T cells and monitored [Ca 2+ ] i elevations induced by rNCX activity. To reverse the Na + gradient, we treated the cells with ouabain to inhibit the Na + -pump and locally perfused a single cell with NMG buffer, in which Na + was substituted with NMG. Fig 4A shows    HEK293T cells expressing NCX1 and mutants were treated with ouabain to elevate the intracellular Na + concentration. Cells were then locally perfused with NMG buffer for 1 min to induce reverse-mode exchange activity. The [Ca 2+ ] i was calibrated based on 913 ± 55 (n = 75) nM, respectively. Without the CaMS, the [Ca 2+ ] i increases were significantly reduced to 694 ± 77 (n = 25, p < 0.05) and 462 ± 67 (n = 32 p < 0.001) nM in cells expressing NCX1.1ΔCaMS and NCX1.3ΔCaMS, respectively (Fig 4B and 4C).
To characterize the importance of CaM in regulating exchange activity, we co-expressed the NCX1 splice variants with wild-type CaM or the Ca 2+ -binding deficient mutant, CaM 1234 , in HEK293T cells and monitored the rNCX activity. The results show that, on average, neither CaM nor CaM 1234 had a significant effect on NCX1.1 activity, with [Ca 2+ ] i increasing to 934 ± 88 (n = 36) and 1049 ± 108 (n = 37) nM, respectively. However, CaM 1234 , but not CaM, significantly enhanced the exchange activity of NCX1.1ΔCaMS to 899 ± 90 (n = 55, p < 0.05) nM.

Exons A and B are involved in CaM-mediated regulation
The difference between NCX1.1 and 1.3 are the mutually exclusive exons A and B, which are important for the Ca 2+ -chelating ability of the CBD2 motif [11,14]. To characterize the role of these exons in CaM-related regulatory effects, we constructed two NCX1 splice variants containing exons A and D (NCX1.4) or only exon D (NCX1D) for use in rNCX activity assays ( Fig  5). Immunostaining shows that both NCX1. 4  (named F1A, V5A, L8D, and L14D) [25] and monitored rNCX activity (Fig 6). Immunostaining for the V5 epitope at the C-termini of these mutants showed a concentrated distribution at the cell boundary, overlapping with F-actin. The line intensity profiles implied that these mutants were located at the cell membrane (S4 Fig). These mutants also showed rNCX activity;   The 4 conserved a.a. residues of the CaMS in NCX1.3 were individually mutated as F1A, V5A, L8D, and L14D to characterize the roles of these a.a. residues in modulating NCX1.3 activity (Fig 7). Immunostaining revealed that these NCX1.3 mutants were present at the cell boundary and that they overlapped with F-actin staining. The line intensity profiles also suggested a plasma membrane distribution for these mutants (S4 Fig). These mutants all displayed rNCX activity upon NMG perfusion, but NCX1.3 V5A and NCX1.3 L8D had lower [Ca 2+ ] i elevations than those of NCX1.3 F1A and NCX1.3 L14D . The average [Ca 2+ ] i changes in cells expressing NCX1.3 F1A and NCX1.3 L14D were 1160 ± 158 (n = 16) and 829 ± 90 (n = 49) nM, respectively, similar to that of NCX1.3 (902 ± 100 nM, Fig 4C). In contrast, the NCX1.3 V5A and NCX1.3 L8D had a significantly smaller [Ca 2+ ] i changes of 550 ± 56 (n = 45, p < 0,01) and 587 ± 59 (n = 51, p < 0,01) nM, respectively, compared with the wild type (Fig 4C). CaM and CaM 1234 coexpression did not affect the exchange activity of these mutants, except for NCX1.3 L8D , whose activity was slightly, but not significantly attenuated to 400 ± 78 nM (n = 25, p = 0.07) by CaM and significantly increased to 1013 ± 142 nM (n = 29, p < 0.001) by CaM 1234 . These results demonstrate that these conserved a.a. residues in the CaMS have differential effects on CaMmediated regulation.

Discussion
CaM is a multi-functional Ca 2+ -sensing protein that regulates a variety of physiological activities by interacting with different proteins [26,27,28]. Although CaM interacts with several Ca 2+ -related ion channels and transporters to regulate Ca 2+ homeostasis, it is not clear whether CaM interacts with NCX or NCKX, which are important for exporting Ca 2+ out of cells. This report demonstrates that the predicted 1-5-8-14 CaM binding motif in NCX1 splice variants is important for Ca 2+ transport and membrane localization; in addition, pull-down assays verify the interaction between CaM and NCX1 splice variants. Therefore, CaM may interact with the intracellular loop of NCX1 splice variants to regulate exchange activity.
Deleting the CaMS from NCX1 splice variants attenuates the exchange activity and interferes with the membrane targeting of these NCX1 splice variants. When co-expressed with the NCX1 splice variants without CaMS, the membrane targeting of GFP-GPI is also blocked, suggesting that the overexpression of NCX1 splice variants that lack the CaMS may block the secretory pathway and prevent other membrane proteins from reaching the plasma membrane. It is likely that the decrease in exchange activity is associated with the attenuated membrane expression level. In contrast, immunostaining of NCX1.1 and NCX1.3 mutants with point mutations in the CaMS show that these mutants localize mostly to the plasma membrane. Therefore, the whole CaMS is important for the correct targeting of NCX1 splice variants to the plasma membrane.
The binding of CaM to the 1-5-8-14 motif of the Ca 2+ -ATPase releases the auto-inhibitory domain and enhances transport activity [17]. For IP 3 receptors, an unidentified Ca 2+ -binding motif is thought to compete with CaM in binding to the endogenous 1-5-8-14 motif for channel activation [29]. Recently, a structural study characterizing the interaction between CaM and the 1-13-16 motif of the inositol 1,4,5-trisphosphate 3-kinase suggests that the recruitment of CaM to the kinase is determined by multiple interaction domains rather than a particular a.a residue [30]. In the present report, GST-CaM pull-down assays confirm that CaM undergoes a Ca 2+ -dependent interaction with the intracellular loop of NCX1 splice variants through the CaMS. In addition, the XIP at the N-terminus of the NCX1.1 intracellular loop may interact with a region at a.a. 566-679 of NCX1.1, which covers the CBD2 and the alternative splicing region [31,32]. It is possible that NCX1 splice variants have an endogenous 1-5-8-14 binding motif that competes with CaM to regulate the exchange activities, though this needs to be further verified. Since partial intracellular loop is used for the pulldown assay, we could not exclude the possibility that, in the absence of CaMS, CaM might bind to the NCX1 splice variants with a weak or transient association through the interactions with other motifs. The pulldown assay could not verify this weak interaction but the activity assays using full-length constructs could. Therefore, we postulate that XIP, CBDs, CaM, and CaMS may interact with each other to form a stable loop complex and support exchange activity [13,33].
The Ca 2+ -binding affinities of CBD1 and CBD2 are approximately 0.2 and 5 μM, respectively [13]; for CaM, the affinity is approximately 1 μM [34]. In addition, exon A in the CBD2 domain has the ability to bind Ca 2+ and stabilize the loop complex, whereas exon B lacks Ca 2+ binding ability and may destabilize the complex [35,36]. Therefore, the alternative splicing exons in CBD2 regulate the Ca 2+ binding affinities of CBD1/2 to meet the physiological requirements in different tissues under different [Ca 2+ ] i levels [36]. The intracellular loops of NCX1.1 and NCX1.4, which contain exon A, may have already obtained a stable loop complex, with CaM binding playing a minor role in regulating exchange activity, because overexpressed CaM and CaM 1234 had no effect on the exchange activity of these two constructs. In contrast, NCX1.1 mutants stained with phalloidin (Red), DAPI (Blue), and an antibody against the V5 epitope tag (Green) to visualize actin filaments, nuclear DNA, and the exchanger, respectively. Our results reveal that each conserved a.a. in the CaMS contributes differentially to the exchange activities of NCX1.1 and NCX1.3. For NCX1.1, mutating the first 2 conserved a.a. residues in the CaMS of NCX1.1 may directly destabilize the loop complex and down-regulate exchange activity. Although mutations in the 3 rd and 4 th conserved a.a. residues did not affect exchange activity, these mutations may weaken the loop complex, and the binding of overexpressed CaM, but not CaM 1234 , destabilizes the loop complex to reduce exchange activity. In summary, the first 2 conserved a.a. residues of CaMS in NCX1.1 may play a dominant role in stabilizing the structure to maintain exchange activity; the 3 rd and 4 th conserved a.a. residues may be involved in the access of CaM to this loop complex.
For the NCX1.3, which contains exon B, CaM binding may be required to stabilize the loop complex and support exchange activity. Mutating the 1 st and 4 th conserved a.a. residues in the CaMS of NCX1.3 does not affect exchange activity, suggesting that these mutations may not affect CaM binding. In contrast, the 2 nd and 3 rd conserved a.a. residues are the main residues involved in determining structural stability and CaM binding; therefore, mutations in these two residues decreased exchange activity. Since CaM 1234 does not bind to the cytosolic loop, it is not clear how CaM 1234 overexpression rescues the exchange activity of NCX1.3 L8D . It is possible that CaM 1234 transiently interacts with the loop complex and interferes with the interaction between endogenous CaM and the loop complex. How the CaMS interacts with other domains to modulate CaM binding will require further studies on the structure.
In intact cardiomyocytes, allosteric regulation of NCX1 activity by cytosolic Ca 2+ is highly cooperative, related to the binding of Ca 2+ to the CBD motifs, but exhibits slow activation kinetics, with a 20-50 msec lag-phase to reach maximal exchange activity [37,38]. CaM has a Ca 2+ affinity of approximately 1 μM, which is in between the affinities of CBD1 and CBD2. Hence, the cooperation among CaM, CBD1, and CBD2 in response to various [Ca 2+ ] i levels during excitation may help tune NCX1 activity. The lag in reaching maximal exchange activity may be due to the time required to form a stable loop complex among these Ca 2+ -binding motifs. Therefore, CaM is involved in regulating not only structural stability but also temporal control. In addition, NCX isoforms are extremely sensitive to mild cytosolic acidification; a pH decrease from 7.2 to 6.9 results in nearly 90% inactivation of NCX, called "proton block" [37,39]. Given that the protonation of CaM decreases its Ca 2+ affinity, pH decreases from 7.2 to 6.96 result in a decrease in pK Ca from 6.02 ± 0.04 to 5.51 ± 0.04 [34]. Therefore, the sensitivity of CaM to pH in terms of its Ca 2+ binding may be related to the mechanism behind the proton block of NCX activity. These correlations suggest a possible interaction between CaM and NCX1 splicing variants in regulating exchange activity under physiological conditions. The mutually exclusive exons A and B are important for exchange activity. Under high levels of [Ca 2+ ] i , exon A, but not B, releases the Na + -dependent inactivation of exchange activity [14]. Some pharmacological experiments also show differential sensitivities of NCX1.1 and NCX1.3 splice variants to fatty acids in regulating Ca 2+ homeostasis [40]. Without exon A, exchange activity of NCX1D is greatly reduced. Because the alternative splicing region is part of CBD2, different exon combinations may affect the Ca 2+ affinity of CBD2, which regulates the stability of the loop complex and exchange activity. Therefore, these domains in the intracellular loop of NCX1 splice variants may form a complex that depends on the Ca 2+ concentration and exon expressed.
The activation of ionotropic receptors in excitable cells allows the influx of Na + ions and depolarizes the membrane, which favors rNCX activity to transport Ca 2+ into the cytosol. This Ca 2+ influx triggers the Ca 2+ -induced Ca 2+ release pathway to maintain high [Ca 2+ ] i and support various physiological functions [41,42]. Therefore, the involvement of CaM in supporting rNCX activity may facilitate the fine-tuning of rNCX activity under different [Ca 2+ ] i levels to regulate Ca 2+ homeostasis. However, it is not clear whether CaM plays the same role in modulating forward exchange activity, and this question needs to be studied further.
NCX isoforms expressed in different tissues may contribute differentially in modulating Ca 2+ homeostasis [12,43,44]. In addition to the dynamic response of the CBD1/2 structure to [Ca 2+ ] i changes [36], our results demonstrate that the interaction among the alternative splicing region, CaM, and CaMS modulates NCX1 activity. Hence, different levels of [Ca 2+ ] i elevations will regulate NCX activity accordingly to modulate Ca 2+ homeostasis. NCX isoforms are involved in heart disease and brain damage, and this interaction may provide new insights for the selective pharmacological targeting of NCX isoforms.