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.     

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.     

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.     

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.     

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.     

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.     

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.     

Basic and editing mechanisms underlying ion transport and regulation in NCX variants

Structure-dynamic analysis of archaeal NCX (NCX_Mj) provided new insights into the underlying mechanisms of ion selectivity, ion-coupled alternating access, ion occlusion, and transport catalysis. This knowledge is relevant, not only for prokaryotic and eukaryotic NCXs, but also for other families belonging to the superfamily of Ca²⁺/CA antiporters. In parallel with the ion transport mechanisms, the structure-dynamic determinants of regulatory CBD1 and CBD2 domains have been resolved according to which the Ca²⁺-induced allosteric signal is decoded at the two-domain interface and "secondarily" modified by a splicing segment at CBD2. The exon-dependent combinations within the splicing segment control the number of Ca²⁺ binding sites (from zero to three) at CBD2, as well as the Ca²⁺ binding affinity and Ca²⁺ off-rates at both CBDs. The exon-dependent combinations specifically rigidify the local segments at CBDs, yielding the Ca²⁺-dependent activation (through Ca²⁺ binding to CBD1) and Ca²⁺-dependent alleviation of Na⁺-induced inactivation (through Ca²⁺ binding with CBD2). The exon-dependent synergistic interactions between CBDs characteristically differ in NCX1 and NCX3, thereby underscoring the physiological relevance of structure-controlled shaping of ion-dependent regulation in tissue-specific NCX variants. How the ion-dependent regulatory modules operate in conjunction with other regulators (PIP₂, palmitoylation, XIP, among the others) of NCX is an open question that remains to be determined.     

Mapping the in vitro interactome of cardiac sodium (Na+)‐calcium (Ca2+) exchanger 1 (NCX1)

The sodium (Na⁺)‐calcium (Ca²⁺) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca²⁺ homeostasis, serving as the primary mechanism for Ca²⁺ extrusion during relaxation. Dysregulation of NCX1 is observed in end‐stage human heart failure. In this study, we used affinity purification coupled with MS in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: (1) anti‐NCX1 against endogenous NCX1 and (2) anti‐His (where His is histidine) with His‐trigger factor‐NCX1_cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature, and 29 occurred in both screens. Ten novel protein partners (DYRK1A, PPP2R2A, SNTB1, DMD, RABGGTA, DNAJB4, BAG3, PDE3A, POPDC2, STK39) were validated for binding to NCX1, and two partners (DYRK1A, SNTB1) increased NCX1 activity when expressed in HEK293 cells. A cardiac NCX1 protein–protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where “cell communication” and “signal transduction” formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in “cardiovascular disease” which can be explored as novel drug targets in future research.     

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.     

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.     

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⁺.     

The role of calcium in the interaction between calmodulin and a minimal functional construct of eukaryotic elongation factor 2 kinase

Eukaryotic elongation factor 2 kinase (eEF‐2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF‐2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF‐2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM‐regulated kinases. We had previously identified a minimal construct of eEF‐2K (TR) that is activated similarly to the wild‐type enzyme by CaM in vitro and retains its ability to phosphorylate eEF‐2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1‐methyls of TR and Ile δ1‐ and Met ε‐methyls of CaM, as probes of their mutual interaction and the influence of Ca²⁺ thereon. We find that in the absence of Ca²⁺, CaM exclusively utilizes its C‐terminal lobe (CaM_C) to engage the N‐terminal CaM‐binding domain (CBD) of TR in a high‐affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca²⁺‐loaded N‐terminal lobe of CaM (CaM_N) at increased Ca²⁺ levels serves to enhance the affinity further. These latter interactions under Ca²⁺ saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca²⁺‐free state. Our data are consistent with a scenario in which Ca²⁺ enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM‐bound species without significantly modifying the conformation of TR within the final, active complex.     

A charge‐sensitive loop in the FKBP38 catalytic domain modulates Bcl‐2 binding

The Bcl‐2 inhibitor FKBP38 is regulated by the Ca²⁺‐sensor calmodulin (CaM). Here we show a hitherto unknown low‐affinity cation‐binding site in the FKBP domain of FKBP38, which may afford an additional level of regulation based on electrostatic interactions. Fluorescence titration experiments indicate that in particular the physiologically relevant Ca²⁺ ion binds to this site. NMR‐based chemical shift perturbation data locate this cation‐interaction site within the β5–α1 loop (Leu90–Ile96) of the FKBP domain, which contains the acidic Asp92 and Asp94 side‐chains. Binding constants were subsequently determined for K⁺, Mg²⁺, Ca²⁺, and La³⁺, indicating that the net charge and the radius of the ion influences the binding interaction. X‐ray diffraction data furthermore show that the conformation of the β5–α1 loop is influenced by the presence of a positively charged guanidinium group belonging to a neighboring FKBP38 molecule in the crystal lattice. The position of the cation‐binding site has been further elucidated based on pseudocontact shift data obtained by NMR via titration with Tb³⁺. Elimination of the Ca²⁺‐binding capacity by substitution of the respective aspartate residues in a D92N/D94N double‐substituted variant reduces the Bcl‐2 affinity of the FKBP38^35–153/CaM complex to the same degree as the presence of Ca²⁺ in the wild‐type protein. Hence, this charge‐sensitive site in the FKBP domain participates in the regulation of FKBP38 function by enabling electrostatic interactions with ligand proteins and/or salt ions such as Ca²⁺. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

The Na+/Ca2+exchanger in Alzheimer’s disease

As a pivotal player in regulating sodium (Na⁺) and calcium (Ca²⁺) homeostasis and signalling in excitable cells, the Na⁺/Ca²⁺ exchanger (NCX) is involved in many neurodegenerative disorders in which an imbalance of intracellular Ca²⁺ and/or Na⁺ concentrations occurs, including Alzheimer’s disease (AD). Although NCX has been mainly implicated in neuroprotective mechanisms counteracting Ca²⁺ dysregulation, several studies highlighted its role in the neuronal responses to intracellular Na⁺ elevation occurring in several pathophysiological conditions. Since the alteration of Na⁺ and Ca²⁺ homeostasis significantly contributes to synaptic dysfunction and neuronal loss in AD, it is of crucial importance to analyze the contribution of NCX isoforms in the homeostatic responses at neuronal and synaptic levels. Some studies found that an increase of NCX activity in brains of AD patients was correlated with neuronal survival, while other research groups found that protein levels of two NCX subtypes, NCX2 and NCX3, were modulated in parietal cortex of late stage AD brains. In particular, NCX2 positive synaptic terminals were increased in AD cohort while the number of NCX3 positive terminals were reduced. In addition, NCX1, NCX2 and NCX3 isoforms were up-regulated in those synaptic terminals accumulating amyloid-beta (Aβ), the neurotoxic peptide responsible for AD neurodegeneration. More recently, the hyperfunction of a specific NCX subtype, NCX3, has been shown to delay endoplasmic reticulum stress and apoptotic neuronal death in hippocampal neurons exposed to Aβ insult. Despite some issues about the functional role of NCX in synaptic failure and neuronal loss require further studies, these findings highlight the putative neuroprotective role of NCX in AD and open new strategies to develop new druggable targets for AD therapy.     

Differential regulation of the Na+-Ca2+ exchanger 3 (NCX3) by protein kinase PKC and PKA

Isoform 3 of the Na⁺-Ca²⁺ exchanger (NCX3) participates in the Ca²⁺ fluxes across the plasma membrane. Among the NCX family, NCX3 carries out a peculiar role due to its specific functions in skeletal muscle and the immune system and to its neuroprotective effect under stress exposure. In this context, proper understanding of the regulation of NCX3 is primordial to consider its potential use as a drug target. In this study, we demonstrated the regulation of NCX3 by protein kinase A (PKA) and C (PKC). Disparity in regulation has been previously reported among the splice variants of NCX3 therefore the activity of Ca²⁺ uptake and extrusion of the two murine variants was measured using fura-2-based Ca²⁺ imaging and revealed that both variants are similarly regulated. PKC stimulation diminished the Ca²⁺ uptake performed by NCX3 in the reverse mode, triggered by a rise in [Ca²⁺]_i or [Na⁺]_i, whereas an opposite response was observed upon PKA stimulation, with a significant increase of the Ca²⁺ uptake after a rise in [Ca²⁺]_i. The latter stimulation affected similarly the efflux capacity of NCX3 whereas Ca²⁺ extrusion capacity remained unaffected under activation of PKC. Next, using site-directed mutagenesis, the sensitivity of NCX3 to PKC was abolished by singly mutating its predicted phosphorylation sites T529 or S695. The sensitivity to PKC might be due to the influence of T529 phosphorylation on the Ca²⁺-binding domain 1. Additionally, we showed that stimulation of NCX3 by PKA occurred through residue S524. This effect may well participate in the fight-or-flight response in skeletal muscle and the long-term potentiation in hippocampus.     

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

The sodium (Na⁺)-calcium (Ca²⁺) exchanger 1 (NCX1) is an important regulator of intracellular Ca²⁺ homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na⁺/K⁺-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca²⁺ binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X_5–8Φ₁Φ₂-X_8–9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.     

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.