Crystal Structure of CBD2 from the Drosophila Na/Ca Exchanger: Diversity of Ca Regulation and Its Alternative Splicing Modification

Na⁺/Ca²⁺ exchangers (NCXs) promote the extrusion of intracellular Ca²⁺ to terminate numerous Ca²⁺-mediated signaling processes. Ca²⁺ interaction at two Ca²⁺ binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca²⁺ regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca²⁺ interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca²⁺ binding. The carboxylate residues that coordinate two Ca²⁺ in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca²⁺ regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca²⁺ binding site to change Ca²⁺ binding properties of CBD2 and (2) alternative splicing variation altering the protein domain-domain conformation to modulate the Ca²⁺ regulatory behavior.     

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.     

Structure and Functional Analysis of a Ca2+ Sensor Mutant of the Na+/Ca2+ Exchanger

The mammalian Na⁺/Ca²⁺ exchanger, NCX1.1, serves as the main mechanism for Ca²⁺ efflux across the sarcolemma following cardiac contraction. In addition to transporting Ca²⁺, NCX1.1 activity is also strongly regulated by Ca²⁺ binding to two intracellular regulatory domains, CBD1 and CBD2. The structures of both of these domains have been solved by NMR spectroscopy and x-ray crystallography, greatly enhancing our understanding of Ca²⁺ regulation. Nevertheless, the mechanisms by which Ca²⁺ regulates the exchanger remain incompletely understood. The initial NMR study showed that the first regulatory domain, CBD1, unfolds in the absence of regulatory Ca²⁺. It was further demonstrated that a mutation of an acidic residue involved in Ca²⁺ binding, E454K, prevents this structural unfolding. A contradictory result was recently obtained in a second NMR study in which Ca²⁺ removal merely triggered local rearrangements of CBD1. To address this issue, we solved the crystal structure of the E454K-CBD1 mutant and performed electrophysiological analyses of the full-length exchanger with mutations at position 454. We show that the lysine substitution replaces the Ca²⁺ ion at position 1 of the CBD1 Ca²⁺ binding site and participates in a charge compensation mechanism. Electrophysiological analyses show that mutations of residue Glu-454 have no impact on Ca²⁺ regulation of NCX1.1. Together, structural and mutational analyses indicate that only two of the four Ca²⁺ ions that bind to CBD1 are important for regulating exchanger activity.Cardiac contraction/relaxation relies upon Ca²⁺ fluxes across the plasma membrane (sarcolemma) of cardiomyocytes. Rapid Ca²⁺ influx (primarily through L-type Ca²⁺ channels) triggers the release of additional Ca²⁺ from the sarcoplasmic reticulum (SR),⁴ resulting in cardiomyocyte contraction. Removal of cytosolic Ca²⁺ by reuptake into the SR (through the SR Ca²⁺-ATPase) and expulsion from the cell (primarily through the Na⁺/Ca²⁺ exchanger, NCX1.1) results in relaxation (1). Altered Ca²⁺ cycling is observed in a number of pathophysiological situations including ischemia, hypertrophy, and heart failure (2). Understanding the function and regulation of NCX1.1 is thus of fundamental importance to understand cardiac physiology.NCX1.1 utilizes the electrochemical potential of the Na⁺ gradient to extrude Ca²⁺ in a ratio of three Na⁺ ions to one Ca²⁺ ion (3). In addition to transporting both Na⁺ and Ca²⁺, NCX1.1 is also strongly regulated by these two ions. Intracellular Na⁺ can induce NCX1.1 to enter an inactivated state, whereas Ca²⁺ bound to regulatory sites removes Na⁺-dependent inactivation and also activates Na⁺/Ca²⁺ exchange (3). These regulatory sites are located on a large cytoplasmic loop (∼500 residues located between transmembrane helices V and VI) containing two calcium binding domains (CBD1 and CBD2), which sense cytosolic Ca²⁺ levels. We have previously shown that Ca²⁺ binding to the primary site in CBD2 is required for full exchange regulation (4); CBD1, however, is a site of higher affinity and appears to dominate the activation of exchange activity by Ca²⁺.Both CBDs have an immunoglobulin fold formed from two antiparallel β sheets generating a β sandwich with a differing number of Ca²⁺ ions coordinated at the tip of the domain (4, 5). CBD1 binds four Ca²⁺ ions, whereas CBD2 binds only two Ca²⁺ ions. An initial NMR study revealed a local unfolding of the upper portion of CBD1 upon release of Ca²⁺ (6). In contrast, CBD2 did not display an unfolding response upon Ca²⁺ removal. A comparative analysis between CBDs revealed a difference in charge at residues in equivalent positions near the Ca²⁺ coordination site; Glu-454 in CBD1 is replaced by Lys-585 in CBD2. The unstructuring of CBD1 upon Ca²⁺ removal was alleviated by reversing the charge of the acidic residue (E454K) involved in Ca²⁺ coordination (6). Previously, we solved the structures of the Ca²⁺-bound and -free conformations of CBD2 and revealed a charge compensation mechanism involving Lys-585 (4). The positively charged lysine residue assumes the position of one of the Ca²⁺ ions upon Ca²⁺ depletion, permitting CBD2 to retain its overall fold (4). A similar phenomenon is predicted to take place in E454K-CBD1 mutant. In addition, Hilge et al. (6) showed that the E454K mutation of CBD1 decreases Ca²⁺ affinity to a level similar to that of CBD2 and suggested that the E454K mutation would cause the loss of primary regulation of NCX1.1 by CBD1.The significance of some of these observations is unclear as a recent NMR study (7) of CBD1 under more physiologically relevant conditions revealed no significant alteration in tertiary structure in the absence of Ca²⁺. It was hypothesized that Ca²⁺ binding induces localized conformational and dynamic changes involving several of the binding site residues. To clarify this issue, we solved the crystal structure of the E454K-CBD1 mutant and examined the functional effects of different CBD1 mutations in the full-length NCX1.1. The results indicate that charge compensation is indeed provided by the residue Lys-454 to replace one Ca²⁺, whereas the overall E454K-CBD1 structure is only slightly perturbed. The charge compensation, however, has no impact on Ca²⁺ regulation of NCX1.1.     

Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

In eukaryotic Na⁺/Ca²⁺ exchangers (NCX) the Ca²⁺ binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca²⁺ sensor (Ca3–Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca²⁺-driven interdomain switch has been described, albeit how it couples Ca²⁺ binding with signal propagation remains unclear. To resolve the dynamic features of Ca²⁺-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium ⁴⁵Ca²⁺ binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg²⁺-bound forms of CBD12 are highly flexible, whereas Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca²⁺ is observed on the globally derived D_max (maximal interatomic distance), although under comparable conditions a normal [Ca²⁺]-dependent allosteric regulation occurs. Low affinity sites (Ca1–Ca2) of CBD1 do not contribute to Ca²⁺-induced population shift, but the occupancy of these sites by 1 mm Mg²⁺ shifts the Ca²⁺ affinity (K_d) at the neighboring Ca3–Ca4 sites from ∼ 50 nm to ∼ 200 nm and thus, keeps the primary Ca²⁺ sensor (Ca3–Ca4 sites) within a physiological range. Thus, Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.     

Crystal Structures of Progressive Ca2+ Binding States of the Ca2+ Sensor Ca2+ Binding Domain 1 (CBD1) from the CALX Na+/Ca2+ Exchanger Reveal Incremental Conformational Transitions

Na⁺/Ca²⁺ exchangers (NCX) constitute a major Ca²⁺ export system that facilitates the re-establishment of cytosolic Ca²⁺ levels in many tissues. Ca²⁺ interactions at its Ca²⁺ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na⁺/Ca²⁺ exchange activity. The structure of the Ca²⁺-bound form of CBD1, the primary Ca²⁺ sensor from canine NCX1, but not the Ca²⁺-free form, has been reported, although the molecular mechanism of Ca²⁺ regulation remains unclear. Here, we report crystal structures for three distinct Ca²⁺ binding states of CBD1 from CALX, a Na⁺/Ca²⁺ exchanger found in Drosophila sensory neurons. The fully Ca²⁺-bound CALX-CBD1 structure shows that four Ca²⁺ atoms bind at identical Ca²⁺ binding sites as those found in NCX1 and that the partial Ca²⁺ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca²⁺ binding at CBD1. The structures also predict that the primary Ca²⁺ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu⁴⁵⁵, which coordinates the primary Ca²⁺ pair, produces dramatic reductions of the regulatory Ca²⁺ affinity for exchange current, whereas mutagenesis of Glu⁵²⁰, which coordinates the secondary Ca²⁺ pair, has much smaller effects. Furthermore, our structures indicate that Ca²⁺ binding only enhances the stability of the Ca²⁺ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca²⁺ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.     

Model for the allosteric regulation of the Na+/Ca2+ exchanger NCX

The Na⁺/Ca²⁺ exchanger provides a major Ca²⁺ extrusion pathway in excitable cells and plays a key role in the control of intracellular Ca²⁺ concentrations. In Canis familiaris, Na⁺/Ca²⁺ exchanger (NCX) activity is regulated by the binding of Ca²⁺ to two cytosolic Ca²⁺‐binding domains, CBD1 and CBD2, such that Ca²⁺‐binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca²⁺‐regulation remains unclear. It was found previously that for NCX in the absence of Ca²⁺ the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca²⁺‐binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but also it should explain the distinctive behavior of Drosophila melanogaster's sodium/calcium exchanger, CALX, for which Ca²⁺‐binding to CBD1 inhibits Ca²⁺ exchange. Here, NMR spin relaxation and residual dipolar couplings were used to show that Ca²⁺ modulates CBD1 and CBD2 interdomain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca²⁺ regulation of the Na⁺/Ca²⁺ exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca²⁺‐binding to CBD1 controlling ion transport across the plasma membrane. Proteins 2016; 84:580–590. © 2016 Wiley Periodicals, Inc.     

Molecular insights on CALX-CBD12 interdomain dynamics from MD simulations,RDCs, and SAXS

Na⁺/Ca²⁺ exchangers (NCXs) are secondary active transporters that couple the translocation of Na⁺ with the transport of Ca²⁺ in the opposite direction. The exchanger is an essential Ca²⁺ extrusion mechanism in excitable cells. It consists of a transmembrane domain and a large intracellular loop that contains two Ca²⁺-binding domains, CBD1 and CBD2. The two CBDs are adjacent to each other and form a two-domain Ca²⁺ sensor called CBD12. Binding of intracellular Ca²⁺ to CBD12 activates the NCX but inhibits the NCX of Drosophila, CALX. NMR spectroscopy and SAXS studies showed that CALX and NCX CBD12 constructs display significant interdomain flexibility in the apo state but assume rigid interdomain arrangements in the Ca²⁺-bound state. However, detailed structure information on CBD12 in the apo state is missing. Structural characterization of proteins formed by two or more domains connected by flexible linkers is notoriously challenging and requires the combination of orthogonal information from multiple sources. As an attempt to characterize the conformational ensemble of CALX-CBD12 in the apo state, we applied molecular dynamics (MD) simulations, NMR (¹H-¹⁵N residual dipolar couplings), and small-angle x-ray scattering (SAXS) data in a combined strategy to select an ensemble of conformations in agreement with the experimental data. This joint approach demonstrated that CALX-CBD12 preferentially samples closed conformations, whereas the wide-open interdomain arrangement characteristic of the Ca²⁺-bound state is less frequently sampled. These results are consistent with the view that Ca²⁺ binding shifts the CBD12 conformational ensemble toward extended conformers, which could be a key step in the NCXs’ allosteric regulation mechanism. This strategy, combining MD with NMR and SAXS, provides a powerful approach to select ensembles of conformations that could be applied to other flexible multidomain systems.     

Dynamic features of allosteric Ca2+ sensor in tissue-specific NCX variants

The Na(+)-Ca(2+) exchanger (NCX) mediated Ca(2+) fluxes are essential for handling Ca(2+) homeostasis in many cell-types. Eukaryotic NCX variants contain regulatory CBD1 and CBD2 domains, whereas in distinct variants the Ca(2+) binding to Ca3-Ca4 sites of CBD1 results either in sustained activation, inhibition or no effect. CBD2 contains an alternatively spliced segment, which is expressed in a tissue-specific manner although its impact on allosteric regulation remains unclear. Recent studies revealed that the Ca(2+) binding to Ca3-Ca4 sites results in interdomain tethering of CBDs, which rigidifies CBDs movements with accompanied slow dissociation of "occluded" Ca(2+). Here we investigate the effects of CBD2 variants on Ca(2+) occlusion in the two-domain construct (CBD12). Mutational studies revealed that both sites (Ca3 and Ca4) contribute to Ca(2+) occlusion, whereas after dissociation of the first Ca(2+) ion the second Ca(2+) ion becomes occluded. This mechanism is common for the brain, kidney and cardiac splice variants of CBD12, although the occluded Ca(2+) exhibits 20-50-fold difference in off-rates among the tested variants. Therefore, the spliced exons on CBD2 affect the rate-limiting step of the occluded Ca(2+) dissociation at the primary regulatory sensor to shape dynamic features of allosteric regulation in NCX variants.     

Binding of calcium is sensed structurally and dynamically throughout the second calcium‐binding domain of the sodium/calcium exchanger

We report the effects of Ca²⁺ binding on the backbone relaxation rates and chemical shifts of the AD and BD splice variants of the second Ca²⁺‐binding domain (CBD2) of the sodium–calcium exchanger. Analysis of the Ca²⁺‐induced chemical shifts perturbations yields similar K_D values of 16–24 μM for the two CBD2‐AD Ca²⁺‐binding sites, and significant effects are observed up to 20 Å away. To quantify the Ca²⁺‐induced chemical shift changes, we performed a comparative analysis of eight Ca²⁺‐binding proteins that revealed large differences between different protein folds. The CBD2 ¹⁵N relaxation data show the CBD2‐AD Ca²⁺ coordinating loops to be more rigid in the Ca²⁺‐bound state as well as to affect the FG‐loop located at the opposite site of the domain. The equivalent loops of the CBD2‐BD splice variant do not bind Ca²⁺ and are much more dynamic relative to both the Ca²⁺‐bound and apo forms of CBD2‐AD. A more structured FG‐loop in CBD2‐BD is suggested by increased S² order parameter values relative to both forms of CBD2‐AD. The chemical shift and relaxation data together indicate that, in spite of the small structural changes, the Ca²⁺‐binding event is felt throughout the molecule. The data suggest that the FG‐loop plays an important role in connecting the Ca²⁺‐binding event with the other cytosolic domains of the NCX, in line with in vivo and in vitro biochemical data as well as modeling results that connect the CBD2 FG‐loop with the first Ca²⁺‐binding domain of NCX. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Structural and dynamic aspects of Ca2+ and Mg2+ binding of the regulatory domains of the Na+/Ca2+ exchanger

Intracellular Ca2+ regulates the activity of the NCX (Na+/Ca2+ exchanger) through binding to the cytosolic CBD (Ca2+-binding domain) 1 and CBD2. In vitro studies of the structure and dynamics of CBD1 and CBD2, as well as studies of their kinetics and thermodynamics of Ca2+ binding, greatly enhanced our understanding of NCX regulation. We describe the fold of the CBDs in relation to other known structures and review Ca2+ binding of the different CBD variants from a structural perspective. We also report on new findings concerning Mg2+ binding to the CBDs and finally we discuss recent results on CBD1-CBD2 interdomain interactions.     

Collapsin Response Mediator Protein 2 (CRMP2) Interacts with N-Methyl-d-aspartate (NMDA) Receptor and Na+/Ca2+ Exchanger and Regulates Their Functional Activity

Collapsin response mediator protein 2 (CRMP2) is traditionally viewed as an axonal growth protein involved in axon/dendrite specification. Here, we describe novel functions of CRMP2. A 15-amino acid peptide from CRMP2, fused to the TAT cell-penetrating motif of the HIV-1 protein, TAT-CBD3, but not CBD3 without TAT, attenuated N-methyl-d-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca²⁺ dysregulation, suggesting the key contribution of CRMP2 in these processes. In addition, TAT-CBD3, but not CBD3 without TAT or TAT-scramble peptide, inhibited increases in cytosolic Ca²⁺ mediated by the plasmalemmal Na⁺/Ca²⁺ exchanger (NCX) operating in the reverse mode. Co-immunoprecipitation experiments revealed an interaction between CRMP2 and NMDAR as well as NCX3 but not NCX1. TAT-CBD3 disrupted CRMP2-NMDAR interaction without change in NMDAR localization. In contrast, TAT-CBD3 augmented the CRMP2-NCX3 co-immunoprecipitation, indicating increased interaction or stabilization of a complex between these proteins. Immunostaining with an anti-NCX3 antibody revealed that TAT-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. Indeed, the forward mode of NCX, evaluated in experiments with ionomycin-induced Ca²⁺ influx into neurons, was strongly suppressed by TAT-CBD3. Knockdown of CRMP2 with short interfering RNA (siRNA) prevented NCX3 internalization in response to TAT-CBD3 exposure. Moreover, CRMP2 down-regulation strongly attenuated TAT-CBD3-induced inhibition of reverse NCX. Overall, our results demonstrate that CRMP2 interacts with NCX and NMDAR and that TAT-CBD3 protects against glutamate-induced Ca²⁺ dysregulation most likely via suppression of both NMDAR and NCX activities. Our results further clarify the mechanism of action of TAT-CBD3 and identify a novel regulatory checkpoint for NMDAR and NCX function based on CRMP2 interaction with these proteins.     

Ionic regulation of the cardiac sodium-calcium exchanger

The Na⁺-Ca²⁺ exchanger (NCX) links transmembrane movements of Ca²⁺ ions to the reciprocal movement of Na+ ions. It normally functions primarily as a Ca²⁺ efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca²⁺ influx under certain conditions. Na⁺ and Ca²⁺ ions exert complex regulatory effects on NCX activity. Ca²⁺ binds to two regulatory sites in the exchanger's central hydrophilic domain, and this interaction is normally essential for activation of exchange activity. High cytosolic Na⁺ concentrations, however, can induce a constitutive activity that by-passes the need for allosteric Ca²⁺ activation. Constitutive NCX activity can also be induced by high levels of phopshotidylinositol-4,5-bisphosphate (PIP₂) and by mutations affecting the regulatory calcium binding domains. In addition to promoting constitutive activity, high cytosolic Na⁺ concentrations also induce an inactivated state of the exchanger (Na⁺-dependent inactivation) that becomes dominant when cytosolic pH and PIP₂ levels fall. Na+-dependent inactivation may provide a means of protecting cells from Ca²⁺ overload due to NCX-mediated Ca²⁺ influx during ischemia.     

Essential Role of the CBD1-CBD2 Linker in Slow Dissociation of Ca2+ from the Regulatory Two-domain Tandem of NCX1

In NCX proteins CBD1 and CBD2 domains are connected through a short linker (3 or 4 amino acids) forming a regulatory tandem (CBD12). Only three of the six CBD12 Ca²⁺-binding sites contribute to NCX regulation. Two of them are located on CBD1 (K_d = ∼0.2 μm), and one is on CBD2 (K_d = ∼5 μm). Here we analyze how the intrinsic properties of individual regulatory sites are affected by linker-dependent interactions in CBD12 (AD splice variant). The three sites of CBD12 and CBD1 + CBD2 have comparable K_d values but differ dramatically in their Ca²⁺ dissociation kinetics. CBD12 exhibits multiphasic kinetics for the dissociation of three Ca²⁺ ions (k_r = 280 s⁻¹, k_f = 7 s⁻¹, and k_s = 0.4 s⁻¹), whereas the dissociation of two Ca²⁺ ions from CBD1 (k_f = 16 s⁻¹) and one Ca²⁺ ion from CBD2 (k_r = 125 s⁻¹) is monophasic. Insertion of seven alanines into the linker (CBD12–7Ala) abolishes slow dissociation of Ca²⁺, whereas the kinetic and equilibrium properties of three Ca²⁺ sites of CBD12–7Ala and CBD1 + CBD2 are similar. Therefore, the linker-dependent interactions in CBD12 decelerate the Ca²⁺ on/off kinetics at a specific CBD1 site by 50–80-fold, thereby representing Ca²⁺ “occlusion” at CBD12. Notably, the kinetic and equilibrium properties of the remaining two sites of CBD12 are “linker-independent,” so their intrinsic properties are preserved in CBD12. In conclusion, the dynamic properties of three sites are specifically modified, conserved, diversified, and integrated by the linker in CBD12, thereby generating a wide range dynamic sensor.     

Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in Heart Failure

Cardiac sodium (Na⁺)-calcium (Ca²⁺) exchanger 1 (NCX1) is central to the maintenance of normal Ca²⁺ homeostasis and contraction. Studies indicate that the Ca²⁺-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca²⁺ binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met³⁶⁹ antibody identified a novel calpain cleavage site at Met³⁶⁹. Engineering NCX1-Met³⁶⁹ into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met³⁶⁹ inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met³⁶⁹ cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.     

Function and Regulation of the Na+-Ca2+ Exchanger NCX3 Splice Variants in Brain and Skeletal Muscle

Isoform 3 of the Na⁺-Ca²⁺ exchanger (NCX3) is crucial for maintaining intracellular calcium ([Ca²⁺]_i) homeostasis in excitable tissues. In this sense NCX3 plays a key role in neuronal excitotoxicity and Ca²⁺ extrusion during skeletal muscle relaxation. Alternative splicing generates two variants (NCX3-AC and NCX3-B). Here, we demonstrated that NCX3 variants display a tissue-specific distribution in mice, with NCX3-B as mostly expressed in brain and NCX-AC as predominant in skeletal muscle. Using Fura-2-based Ca²⁺ imaging, we measured the capacity and regulation of the two variants during Ca²⁺ extrusion and uptake in different conditions. Functional studies revealed that, although both variants are activated by intracellular sodium ([Na⁺]_i), NCX3-AC has a higher [Na⁺]_i sensitivity, as Ca²⁺ influx is observed in the presence of extracellular Na⁺. This effect could be partially mimicked for NCX3-B by mutating several glutamate residues in its cytoplasmic loop. In addition, NCX3-AC displayed a higher capacity of both Ca²⁺ extrusion and uptake compared with NCX3-B, together with an increased sensitivity to intracellular Ca²⁺. Strikingly, substitution of Glu⁵⁸⁰ in NCX3-B with its NCX3-AC equivalent Lys⁵⁸⁰ recapitulated the functional properties of NCX3-AC regarding Ca²⁺ sensitivity, Lys⁵⁸⁰ presumably acting through a structure stabilization of the Ca²⁺ binding site. The higher Ca²⁺ uptake capacity of NCX3-AC compared with NCX3-B is in line with the necessity to restore Ca²⁺ levels in the sarcoplasmic reticulum during prolonged exercise. The latter result, consistent with the high expression in the slow-twitch muscle, suggests that this variant may contribute to the Ca²⁺ handling beyond that of extruding Ca²⁺.     

Modulation of the cardiac Na+-Ca2+ exchanger by cytoplasmic protons: Molecular mechanisms and physiological implications

A precise temporal and spatial control of intracellular Ca²⁺ concentration is essential for a coordinated contraction of the heart. Following contraction, cardiac cells need to rapidly remove intracellular Ca²⁺ to allow for relaxation. This task is performed by two transporters: the plasma membrane Na⁺-Ca²⁺ exchanger (NCX) and the sarcoplasmic reticulum (SR) Ca²⁺‐ATPase (SERCA). NCX extrudes Ca²⁺ from the cell, balancing the Ca²⁺entering the cytoplasm during systole through L-type Ca²⁺ channels. In parallel, following SR Ca²⁺ release, SERCA activity replenishes the SR, reuptaking Ca²⁺ from the cytoplasm.The activity of the mammalian exchanger is fine-tuned by numerous ionic allosteric regulatory mechanisms. Micromolar concentrations of cytoplasmic Ca²⁺ potentiate NCX activity, while an increase in intracellular Na⁺ levels inhibits NCX via a mechanism known as Na⁺-dependent inactivation. Protons are also powerful inhibitors of NCX activity. By regulating NCX activity, Ca²⁺, Na⁺ and H⁺ couple cell metabolism to Ca²⁺ homeostasis and therefore cardiac contractility. This review summarizes the recent progress towards the understanding of the molecular mechanisms underlying the ionic regulation of the cardiac NCX with special emphasis on pH modulation and its physiological impact on the heart.     

Cloning,Expression, and Characterization of the Squid Na+–Ca2+ Exchanger (NCX-SQ1)

We have cloned the squid neuronal Na⁺–Ca²⁺ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na⁺–Ca²⁺ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca²⁺ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na⁺-dependent ⁴⁵Ca²⁺ uptake; the uptake was inhibited by injection of Ca²⁺ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na⁺-dependent inactivation, secondary activation by cytoplasmic Ca²⁺, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATPγS, in the presence of F⁻ (0.2 mM) and vanadate (50 μM), and both effects reversed on application of a phosphatidylinositol-4′,5′-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATPγS. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4′,5′-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5–10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na⁺ and Ca²⁺ transport reactions. Outward current transients associated with Na⁺ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca²⁺ extrusion were much larger. For NCX-SQ1, charge movements of Ca²⁺ transport could be defined in voltage jump experiments with a low cytoplasmic Ca²⁺ (2 μM) in the presence of high extracellular Ca²⁺ (4 mM). The rates of charge movements showed “U”-shaped dependence on voltage, and the slopes of both charge–voltage and rate–voltage relations (1,600 s⁻¹ at 0 mV) indicated an apparent valency of −0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.     

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 3^rd or 4^th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2^nd and 3^rd 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.     

Functional Differences in Ionic Regulation between Alternatively Spliced Isoforms of the Na+-Ca2+ Exchanger from Drosophila melanogaster

Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na⁺-Ca²⁺ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na⁺-Ca²⁺ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na⁺-Ca²⁺ exchange currents, where pipette Ca²⁺ _o exchanges for bath Na⁺ _i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na⁺ _i affinities and current–voltage relationships, significant differences were observed in their Na⁺ _i- and Ca²⁺ _i-dependent regulatory properties. Both isoforms underwent Na⁺ _i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na⁺ _i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na⁺ _i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na⁺-Ca²⁺ exchange activity by Ca²⁺ _i, but their responses to regulatory Ca²⁺ _i differed markedly. For both isoforms, the application of cytoplasmic Ca²⁺ _i led to a decrease in outward exchange currents. This negative regulation by Ca²⁺ _i is unique to Na⁺-Ca²⁺ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca²⁺ for all other characterized Na⁺-Ca²⁺ exchangers. For CALX1.1, Ca²⁺ _i inhibited peak and steady state currents almost equally, with the extent of inhibition being ≈80%. In comparison, the effects of regulatory Ca²⁺ _i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced (≈20–40% inhibition). For both exchangers, the effects of regulatory Ca²⁺ _i occurred by a direct mechanism and indirectly through effects on Na⁺ _i-induced inactivation. Our results show that regulatory Ca²⁺ _i decreases Na⁺ _i-induced inactivation of CALX1.2, whereas it stabilizes the Na⁺ _i-induced inactive state of CALX1.1. These effects of Ca²⁺ _i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na⁺-Ca²⁺ exchange proteins.     

Structure-dynamic and functional relationships in a Li+-transporting sodium‑calcium exchanger mutant

The cell membrane (NCX) and mitochondrial (NCLX) Na⁺/Ca²⁺ exchangers control Ca²⁺ homeostasis. Eleven (out of twelve) ion-coordinating residues are highly conserved among eukaryotic and prokaryotic NCXs, whereas in NCLX, nine (out of twelve) ion-coordinating residues are different. Consequently, NCXs exhibit high selectivity for Na⁺ and Ca²⁺, whereas NCLX can exchange Ca²⁺ with either Na⁺ or Li⁺. However, the underlying molecular mechanisms and physiological relevance remain unresolved. Here, we analyzed the NCX_Mj-derived mutant NCLX_Mj (with nine substituted residues) imitating the ion selectivity of NCLX. Site-directed fluorescent labeling and ion flux assays revealed the nearly symmetric accessibility of ions to the extracellular and cytosolic vestibules in NCLX_Mj (K_int?=?0.8–1.4), whereas the extracellular vestibule is predominantly accessible to ions (K_int?=?0.1–0.2) in NCX_Mj. HDX-MS (hydrogen-deuterium exchange mass-spectrometry) identified symmetrically rigidified core helix segments in NCLX_Mj, whereas the matching structural elements are asymmetrically rigidified in NCX_Mj. The HDX-MS analyses of ion-induced conformational changes and the mutational effects on ion fluxes revealed that the “Ca²⁺-site” (S_Ca) of NCLX_Mj binds Na⁺, Li⁺, or Ca²⁺, whereas one or more additional Na⁺/Li⁺ sites of NCLX_Mj are incompatible with the Na⁺ sites (S_ext and S_int) of NCX_Mj. Thus, the replacement of ion-coordinating residues in NCLX_Mj alters not only the ion selectivity of NCLX_Mj, but also the capacity and affinity for Na⁺/Li⁺ (but not for Ca²⁺) binding, bidirectional ion-accessibility, the response of the ion-exchange to membrane potential changes, and more. These structure-controlled functional features could be relevant for differential contributions of NCX and NCLX to Ca²⁺ homeostasis in distinct sub-cellular compartments.